INDICE



INDEX

◆ INTRODUZIONE

◆ Plasma membrane p. 3

◆ Raft function p. 12

◆ Glycosphingolipids p. 13

◆ Functions of gangliosides p. 19

◆ Gangliosides in disease pathogenesis p. 22

◆ The ErbB family receptors and their ligands p. 26

◆ Receptor trafficking to novel sites and receptor fragments p. 33

◆ Embryogenesis and organ development p. 37

◆ ErbB pathology p. 38

MATERIALS AND METHODS

◆ Cell culture p. 41

▪ EGF stimulation of HC11 cells p. 41

▪ Gangliosides depletion p. 41

▪ Treatment with exogenus ganglioside GM3 of HC11 p. 41

◆ Antibodies p. 42

◆ Analysis of ErbB2-GM3 colocalization by scanning confocal microscopy p. 42

◆ Isolation and analysis of lipid raft fractions p. 43

◆ Immunoprecipitation experiments from membrane fractions p. 43

◆ Cell lysis, immunoprecipitation and immunoblot analyses p. 44

◆ Extraction, purification and analysis of gangliosides p. 45

◆ Extraction, purification and analysis of gangliosides from the

immunocomplexes p. 45

◆ Lysis and alkaline phosphatase treatment p. 46

AIM OF THE STUDY p. 47

RESULTS

First Part: Molecular interaction between ErbB2, EGFR and gangliosides

◆ Ganglioside profiles in HC11 cells p. 49

◆ Inhibition of gangliosides biosynthesis by D-PDMP p. 49

◆ ErbB2 expression levels in normal and D-PDMP treated cells p. 50

◆ EGFR expression levels in normal and D-PDMP treated cells p. 53

◆ Dimerization and tyrosine-phosphorylation of ErbB2 and EGFR in

EGF stimulated HC11 cells p. 55

◆ Molecular interaction of gangliosides with ErbB2 and EGFR heterodimers p. 57

◆ ErbB2 and EGFR expression levels in ganglioside depleted HC11 cells

after addition of exogenus ganglioside GM3 p. 58

◆ Dimerization and tyrosine-phosphorylation of ErbB2 and EGFR in

Ganglioside depleted HC11 cells p.60

Second Part: ErbB2 and EGFR localization in HC11 plasma membrane

◆ ErbB2-GM3 colocalization in mammary epithelial HC11 cells p. 62

◆ ErbB2 preferential association with lipid raft fraction in HC11 cells p. 62

◆ EGFR preferential association with lipid raft fractions in HC11 cells p. 66

◆ Profile distribution of raft markers in plasma membrane fractions of

HC11 cells p. 66

DISCUSSION p. 68

REFERENCES p. 74

INTRODUCTION

Plasma Membrane

All living cells, prokaryotic and eukaryotic, have a plasma membrane that encloses their contents and serves as a semi-porous barrier to the outside environment. The membrane acts as a boundary, holding the cell constituents together and keeping other substances from entering. The plasma membrane is permeable to specific molecules, however, and allows nutrients and other essential elements to enter the cell and waste materials to leave the cell. Small molecules, such as oxygen, carbon dioxide, and water, are able to pass freely across the membrane, but the passage of larger molecules, such as amino acids and sugars, is carefully regulated. According to the accepted current theory, known as the fluid mosaic model, the plasma membrane is composed of a double layer (bilayer) of lipids, oily substances found in all cells. Most of the lipids in the bilayer can be more precisely described as phospholipids, that is, lipids that feature a phosphate group at one end of each molecule. Phospholipids are characteristically hydrophilic ("water-loving") at their phosphate ends and hydrophobic ("water-fearing") along their lipid tail regions. In each layer of a plasma membrane, the hydrophobic lipid tails are oriented inwards and the hydrophilic phosphate groups are aligned so they face outwards, either toward the aqueous cytosol of the cell or the outside environment. Phospholipids tend to spontaneously aggregate by this mechanism whenever they are exposed to water. Within the phospholipid bilayer of the plasma membrane, many diverse proteins are embedded, while other proteins simply adhere to the surfaces of the bilayer (Fig.1). Some of these proteins, primarily those that are at least partially exposed on the external side of the membrane, have carbohydrates attached to their outer surfaces and are, therefore, referred to as glycoproteins. The positioning of proteins along the plasma membrane is related in part to the organization of the filaments that comprise the cytoskeleton, which help anchor them in place. The arrangement of proteins also involves the hydrophobic and hydrophilic regions found on the surfaces of the proteins: hydrophobic regions associate with the hydrophobic interior of the plasma membrane and hydrophilic regions extend past the surface of the membrane into either the inside of the cell or the outer environment . Plasma membrane proteins function in several different ways. Many of the proteins play a role in the selective transport of certain substances across the phospholipid bilayer, either acting as channels or active transport molecules. Others function as receptors, which bind information-providing molecules, such as hormones, and transmit corresponding signals based on the obtained information to the interior of the cell.

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FIG.1: Plasma membrane structural component

Membrane proteins may also exhibit enzymatic activity, catalyzing various reactions related to the plasma membrane. Since the 1970s, the plasma membrane has been frequently described as a fluid mosaic, which is reflective of the discovery that oftentimes the lipid molecules in the bilayer can move about in the plane of the membrane (1). However, depending upon a number of factors, including the exact composition of the bilayer and temperature, plasma membranes can undergo phase transitions which render their molecules less dynamic and produce a more gel-like or nearly solid state. Cells are able to regulate the fluidity of their plasma membranes to meet their particular needs by synthesizing more of certain types of molecules, such as those with specific kinds of bonds that keep them fluid at lower temperatures. The presence of cholesterol and glycolipids, which are found in most cell membranes, can also affect molecular dynamics and inhibit phase transitions. Recently the classical fluid bilayer model was challenged by the concept of lipid islands termed “rafts” supported by findings of detergent insoluble membrane fragments enriched in certain lipids like sphingomyelin (SM) and cholesterol (CHOL) (2). Lipid rafts are localized regions of elevated cholesterol and glycosphingolipid content within cell membranes (FIG. 2). The fatty-acid side chains of the phospholipids present in lipid rafts tend to be more highly saturated than those in the surrounding membrane. This allows close packing with the saturated acyl chains of sphingolipids, and probably leads to phase separation. Due to the presence of cholesterol, a liquid-ordered domain is formed that exhibits less fluidity than the surrounding plasma membrane. This tight packing of lipids and phase separation is probably responsible for the signature property of lipid rafts: their insolubility in nonionic detergents (2). Caveolae are small plasma-membrane invaginations that can be viewed as a subset of lipid rafts. Like lipid rafts, caveolae have a high content of cholesterol and glycosphingolipids; however, caveolae are distinguished from lipid rafts by the presence of the cholesterol-binding protein caveolin-1 (4) that appears to be responsible for stabilizing the invaginated structure of caveolae (5, 6). The presence within lipid rafts (and caveolae) of a variety of membrane proteins involved in cell signaling (7, 8) has led to the consensus that these lipid domains play an important role in the process of signal transduction.

A model for the organization of lipid-rafts shown sphingolipids associate laterally with one another, probably trough weak interactions between the carbohydate heads af the glycosphingolipids. The sphinholipids head groups occupy larger excluded areas in the plane of the exoplasmic leaflet than do their predominantly saturated lipid hydrocarbon chains. Any volds between associating sphingolipids are filled by cholesterol molecules wich function as sapacers. The close-packed sphingolipid-cholesterol clusters as assemblies within the exoplasmic leaflet, where the intervening fluid regions are occupied by unsaturated phosphatidylcholine molecules. Glycospingolipids usually carry a long fatty acid which is amide-bonded to the sphingosine base and can interdigitate with the cytoplasmic leaflet of the bilayer (9). As cholesterol is present in both leaflets, it functions as a spacer in the cytoplasmic leaflet as well, filling voids created by interdigitating fatty acid chains. The nature of phospholipids occupying the cytoplasmic side of the microdomain is unknown, but they will probably also be carrying mainly saturated fatty acid chains to optimize packing. Individual lipids may move in and cut of the rafts, explaining why sphingolipid-cholesterol clustering is difficult to detect spettroscopically.

A variety of proteins have been found to be enriched in lipid rafts and/or caveolae. This includes caveolins, flotillins,

GPI-linked proteins, low molecular weight and heterotrimeric G proteins, src family kinases, EGF receptors, platelet-derived growth factor (PDGF) receptors, endothelin receptors, the phosphotyrosine phosphatase syp, Grb2, Shc, MAP kinase (MAPK), protein kinase C, and the p85 subunit of PI 3-kinase (8, 10-18).

The mechanisms through which raft association occurs seem to be variable. Caveolin, an intrinsic membrane protein, is a cholesterol binding protein and is probably concentrated in caveolae because of its ability to bind this sterol (4).

By contrast, GPI-anchored proteins, src family kinases, and endothelial nitric oxide synthase appear to localize to lipid rafts as a result of lipid modifications (19, 20). For some transmembrane proteins, the membranespanning domain appears to mediate the partitioning of the protein into cholesterol-enriched membrane domains (21). Surveys of the behavior of other transmembrane proteins (TM) have suggested that the association of proteins with cholesterol-enriched domains is influenced by the length of their transmembrane domains (22). Extracellular carbohydrate-containing motifs have also been implicated in directing the association of TMs with cholesterol- enriched domains (23, 24). More recently, Yamabhai and Anderson (25) demonstrated that sequences in the most membrane-proximal portion of the extracellular domain of the EGF receptor target it to lipid rafts, independent of any carbohydrate modifications of the sequence. Thus, a variety of mechanisms appear to be employed for localizing proteins to lipid rafts.

Many receptor tyrosine kinases, including the EGF receptor, the PDGF receptor, and the insulin receptor, have been shown to be localized to lipid rafts (26-30).

Receptor tyrosine kinases are transmembrane proteins and the mechanisms through which transmembrane proteins are localized to rafts are varied and less well understood. For some transmembrane proteins, the membrane-spanning domain appears to mediate the partitioning of the protein into cholesterol-enriched membrane domains. The association of proteins with cholesterol-enriched domains is influenced by the length of their transmembrane domains. Extracellular carbohydrate-containing motifs have also been implicated in directing the association of transmembrane proteins with cholesterol-enriched domains (31).

Of the growth factor receptors, only the EGF receptor has been examined for the presence of targeting sequences that would direct the receptor to lipid rafts. EGF receptors lacking the entire cytoplasmic domain as well as EGF receptors in which the transmembrane domain was exchanged with that of the LDL receptor still localized to lipid rafts. By contrast, a receptor in which the entire extracellular domain had been deleted failed to be target to rafts (32). The targeting signal was further localized to a 60 amino acid membrane proximal sequence. However, when just this sequence was deleted from the extracellular domain of the EGF receptor, the receptor still localized to rafts suggesting that multiple raft localization signals may be present. Like the data on the adenylate cyclase, these findings suggest that the EGF receptor may become enriched in lipid rafts by binding to a resident raft lipid or protein.

A caveolin-interacting motif that is thought to mediate the association of caveolin with a variety of proteins has been identified (33).This motif, ψXψXXXXψ or ψXXXXψXXψ where ψ is a hydrophobic amino acid, is sufficiently loose in specificity so that many proteins can be identified as containing one of these motifs. The insulin receptor, the PDGF receptor and the EGF receptor all contain such a motif and these sequences have been proposed to mediate the effects of caveolin on the function of these receptors (34). This sequence might also potentially mediate the localization of the receptors to caveolae. However, as might be expected from the hydrophobic nature of these sequences, they are located in the internal core of the protein kinase domain in all three proteins and would be unavailable for interaction with caveolin. Thus, it is unlikely that these promiscuous motifs either interact with caveolin or serve to target receptor tyrosine kinases to caveolae.

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Although it is clear that rafts and caveolae are important for growth factor signaling, our understanding of the system is incomplete. Fig. 3 depicts a scheme through which some of the differences in the behavior of various growth factor receptors can be understood. It is based on a distinction between receptors that are localized to lipid rafts (e.g., the EGF receptor) and those that are targeted to caveolae (e.g., the insulin receptor). For the raft-localized growth factor receptor, its function is suppressed by the raft environment, either due to effects of fluidity, membrane thickness or the clustering of receptors or other proteins. Binding of the ligand leads to receptor autophosphorylation and signaling is mediated through complex formation. The complex is simple and transient, and signaling is down-regulated by rapid receptor internalization. For the caveolae-localized receptor, the invaginated structure provides an optimal environment for receptor function, possibly due to lipid composition, membrane curvature or the clustering of receptors. Within this environment, the growth factor receptor can form a more complicated, more stable signaling complex. This is due to the ability of phosphorylated caveolin to bind some receptor substrates and the ability of IRS-1 (or other similar molecules) to scaffold other substrates. The entire structure is stabilized by the caveolar coat, a function that results in reduced endocytosis and a more prolonged signal. Upon cholesterol depletion, the raft-localized receptor is relieved of the inhibitory effects of the raft environment and signals more effectively, partially due to enhanced kinase activity and partially due to an increase in the sites that are phosphorylated. By contrast, the caveolar receptor is now localized in a ‘caveolar remnant’ that contains caveolin but is not in an appropriate configuration to scaffold a signaling complex. Receptor function is normal but access of the receptor to exogenous substrates is limited due to steric factors. Thus, all down stream signaling is impaired. Sphingolipid–cholesterol rafts are insoluble in the detergent Triton X-100 at 4 °C, in which they form glycolipid-enriched complexes (35). Because of their high lipid content, these detergent-insoluble, glycolipid-enriched complexes (DIGs) float to a low density during gradient centrifugation (36), which enables any associated proteins to be identified and distinguishes DIGs from other detergent insoluble complexes. Milder detergents such as octylglucoside will solubilize lipid rafts (37). Glycosphingolipids are insoluble by themselves, and sphingomyelin is resistant to detergent extraction in the presence of cholesterol (37). One problem with Triton X-100 extraction is that the original subcellular locations of DIGs are unknown. Confusion has been created because sphingolipid rafts in apical transport vescicles, in caveolae, and even in plasma membranes from cells devoid of morphologically recognizable caveolae, all form DIGs after Triton X-100 extraction (38). In retrospect, the mere isolation of DRM (detergent resistant membrane) from cells does not prove that there were co-existing ordered and disordered domains present prior to detergent treatment. Instead, it is an indication that a membrane has a composition that is close to one in which ordered domains would be stable. Nevertheless, despite its limitations, insolubility in detergent remains a useful tool. In studies of cells, the relative level of lipids and proteins in DRM provides a initial estimate of their affinity of molecules for ordered domains (39). Changes in DRM association under different physiological conditions are likely to be even more significant than absolute levels of association. In model membranes, detergent insolubility can be particularly useful because over a range of experimental temperatures detergent-insoluble domains arising apparently exclusively from Lo domains (cholesterol-containing state was eventually named the liquid ordered state) can be isolated when Lo and Ld (phospholipid bilayers could contain solid-like gel state domains co-existing with fluid state domains, named liquid disorder domain ) domains do co-exist prior to detergent addition. Development of improved detergent insolubility methods would be of great value, and solubilization by various detergents has been compared (40) and (41). Detergents that can be used to isolate ordered domains at 37°C should be especially useful. As noted above, Brij family detergents have been proposed for this purpose (42). However, Brij detergents tend to be less strongly solubilizing than Triton X-100 and may not always fully dissolve non-raft domains (40-41, 43 ). Non-detergent methods for isolating ordered domains from cell membranes may have a similar limitation. In the case of Lubrol detergents insolubility patterns that differ from those with Triton X-100 have been suggested to be indicative of the presence of multiple types of lipid rafts with different levels of solubilization resistance (44). Alternatives are that Lubrol and Triton have differing abilities to fully dissolve disordered domains or differentially extract molecules from ordered domains (41). In either case, differential extractability by different detergents may contain important information.

Can detergents with improved solubilization properties be found? Surveys of different detergents have yet to identify major differences that would favor substitution of TX-100 with another detergent in all cases (40-41). An alternative strategy that has not been exploited is to try mixtures of stronger and weaker detergents (e.g., TX-100 and Brij). The degree of association of a protein with insoluble membranes as the fraction of TX-100 in the detergent mixture was increased (at 37°C) might be a useful parameter for evaluating relative raft affinity. One of the most important properties of lipid rafts is that they can include or exclude proteins to variable extents. Proteins with raft affinity include glycosylphosphatidylinositol (GPI)-anchored proteins (45,46), doubly acylated proteins, such as Src-family kinases or the α-subunits of heterotrimeric G proteins, transmembrane proteins, cholesterol-linked and palmitoylated proteins such as Hedgehog (47). GPI-anchored proteins or proteins that carry hydrophobic modifications probably partition into rafts owing to preferential packing of their saturated membrane anchors. It is not yet clear why some transmembrane proteins are included into rafts, but mutational analysis has shown that amino acids in the transmembrane domains near the exoplasmic leaflet are critical (21). Palmitoylation can increase a protein’s affinity for rafts, but it is not sufficient for raft association (48). It is likely that a given protein can associate with rafts with different kinetics or partition coefficients. For instance, a monomeric transmembrane protein may have a short residency time in rafts, spending most of its time outside rafts. But when the same protein is crosslinked or otherwise oligomerized, its affinity for rafts increases (49). Whereas cholesterol is synthesized in the endoplasmic reticulum (ER), sphingolipid synthesis and head-group modification are completed largely in the Golgi (50). As these data predict, cholesterol–sphingolipid rafts first assemble in the Golgi.Movement of lipid rafts out of the Golgi seems to be mainly towards the plasma membrane, as vesicles going back to the ER contain little sphingomyelin and cholesterol. The inclusion of proteins into rafts is important for polarized delivery to the cell surface in many cell types (3,51,52). Lipid raft trafficking does not end with surface delivery — rafts are continuously endocytosed (53) from the plasma membrane. From early endosomes, rafts either recycle directly back to the cell surface or return indirectly through recycling endosomes, which could also deliver rafts to the Golgi (54). The most important role of rafts at the cell surface may be their function in signal transduction. It is well established that, in the case of tyrosine kinase signaling, adaptors, scaffolds and enzymes are recruited to the cytoplasmic side of the plasma membrane as a result of ligand activation (55). One way to consider rafts is that they form concentrating platforms for individual receptors, activated by ligand binding. If receptor activation takes place in a lipid raft, the signaling complex is protected from non-raft enzymes such as membrane phosphatases that otherwise could affect the signaling process. In general, raft binding recruits proteins to a new micro-environment, where the phosphorylation state can be modified by local kinases and phosphatases, resulting in downstream signaling. Signaling in a variety of hematopoietic cells (including basophils) involves tyrosine phosphorylation of conserved sequences in the cytoplasmic domains of cell-surface receptors by Src-family kinases. This mechanistic similarity suggests that rafts are important in signaling in other cells as well as in basophils. For example, antibody-mediated crosslinking of the cell-surface transmembrane protein CD20 triggers signaling in B cells and tumor cells and recruits the protein into DRMs (56). Cell-surface receptors do not always associate with DRMs, possibly implying that they do not associate with rafts. However, in vitro studies have shown that detergent insolubility is not always a perfect measure of the lo phase (45). Receptors could have a moderate affinity for rafts in vivo and still not associate stably with DRMs. In fact, although the studies described above show that Fc²RI associates with rafts, detergent insolubility of the receptor is somewhat difficult to detect (57). Additional indirect evidence suggests that transmembrane receptors associate with rafts during signaling. For instance, activation of T cells through the T cell receptor (TCR) is impaired in cells that are defective for GPI anchor synthesis (58). Cross-linking of GPI-anchored proteins in these cells can stimulate signaling, in a manner that may involve rafts. Thus recruitment of the TCR to rafts via association with GPI-anchored proteins may enhance its signaling. It is also interesting to note that depletion of cellular cholesterol can inhibit signaling in mast cells (59) and T cells (60), possibly by affecting the structure of rafts. Several aspects of raft structure and function still need to be explained. One important area is raft composition and the question of whether more than one kind of raft exists on the cell surface of different cell types (61,62). Not only do we need to identify raft-associated proteins, but we also have to determine the lipid composition in both the exoplasmic and cytoplasmic leaflets of rafts. As detergent extraction undoubtedly leads to raft aggregation, it is not easy to isolate individual rafts or ligand-activated raft clusters in such a way that their native state is preserved.

Raft function

In principle, targeting of proteins to rafts might affect function in either of two ways. First, concentration of proteins in rafts could facilitate interactions between them. (Similarly, segregation of raft and non-raft proteins could separate them during sorting.) Second, the ordered lipid environment might directly affect function, possibly by altering protein conformation. There are no clear examples of this second possibility, although cholesterol concentration and bilayer width can affect transmembrane helix orientation and helix- helix interaction (63, 64). In addition, cholesterol depletion (which can disrupt raft function) alters the function of a raft associated potassium channel (65). Several approaches have been taken to investigate raft function. One is to show that a protein is enriched in DRMs. This is consistent with a role for rafts in the function of that protein but does not prove it. More suggestive is showing that several proteins that must interact to function all redistribute and co localize with each other when one raft component is experimentally clustered. Another approach is to show that disrupting the association of a protein with rafts disrupts function. For example, mutation of palmitoylation sites on two proteins (Lck and LAT, described below) simultaneously abolished DRM association and affected function. Finally, raft disruption by depletion of cholesterol (or occasionally sphingolipids) can affect function. Although this method is useful, cholesterol depletion may have pleiotropic effects on membrane structure and lipid-protein interactions in addition to disrupting rafts. The strongest evidence for the involvement of rafts in function is provided when several approaches point to the same conclusion. This is well illustrated in the case of signaling in hematopoietic cells, particularly T cells and basophils. Before examining this topic in detail, we will briefly mention other processes in which rafts have been implicated. Rafts were first proposed to mediate sorting in the trans-Golgi network, especially in polarized epithelial cells and neurons (2, 3, 66-68). Recent results suggest that rafts may also be important in sorting in the endocytic pathway (69). Rafts can serve as docking sites for certain pathogens and toxins (70). In addition, they may be important in the aberrant amyloid precursor protein processing that contributes to Alzheimer’s disease (8, 71). Integrin receptors may also function in rafts. Several integrins have been found in DRMs (8, 45, 72, 73).

In one study, integrins, the integrin-associated protein IAP (which can regulate integrin function), and heterotrimeric G proteins formed a stable cholesterol-dependent complex that was enriched in DRMs (72). Finally, rafts polarize to the front of adenocarcinoma cells migrating in a chemotactic gradient (74). In these cells, development of front-rear polarity, which is required for directed migration, is abolished by cholesterol depletion.

Glycosphingolipids

Sphingolipids have long been known as cell components. Their name was given by J.L.W. Thudichum in 1884, because of their enigmatic nature. In the second half of the past century, when the basic lipid metabolic pathways were unraveled, the catabolic and biosynthetic interrelations of sphingolipids became clear.

In particular, ceramide and sphingosine were found to be intermediates in the metabolism of the more complex phospho- and glycosphingolipids (75, 76). More recently, with the discovery of the sphingolipid signalling pathway (77, 78) the interest in these lipids was renewed. Very soon ceramide, and then sphingosine and other related compounds became established as lipid second messengers, or metabolic signals (79, 80).

Sphingolipids can be found in all eucaryotic cells, where they are especially plentiful in the plasma membrane and related cell membranes, such as Golgi membranes and lysosomes. The basic building block of sphingolipids is sphingosine, (2S, 3R, 4E)-2-amino-4-octadecene-1,3-diol in current systematic nomenclature. According to an older, but extensively used, notation, its configuration is d-erythro-trans (Fig. 5). There are currently over 300 known sphingolipids with distinct headgroups (82) and all contained a long chain (sphingoid) base backbone. In mammalian cells, the most common sphingoid base is D-erythro-sphingosine; double bond at the 4, 5 carbon–carbon bond of the sphinganine backbone results in the formation of ceramide (N-acylsphingosine).

More complex sphingolipids are formed when polar head groups are added at position 1 of ceramide (e.g., sphingomyelin, ganglioside GM3). Thus, the presence of free sphingosine in cells is believed to be primarily derived from turnover of more complex sphingolipids (Fig. 6) or from dietary sources (Fig. 7), rather than as a biosynthetic intermediate as is the case with sphinganine.

Other sphingosine-related molecules are also found in sphingolipids instead of the parent compound, for example the saturated dihydrosphingosine, or the saturated phytosphingosine (containing a third OH group in C4). The C1-phosphate ester of sphingosine is also an important molecule, commonly denoted as sphingosine-1-phosphate, also known to exist in cell membranes (Fig. 5).

[pic]

FIG.5: Molecular structures of simple sphingolipids

Ceramides have been found in nature with fatty acids containing from 2 to 28 carbon atoms, either saturated or monounsaturated, in some cases containing an OH group in either C′2 (α-hydroxy fatty acids) or in the terminal carbon atom (ω-hydroxy fatty acids). Occasionally, a second fatty acid can be esterified to an OH group of sphingosine or related base. Ceramides constitute the hydrophobic backbones of all the complex sphingolipids: sphingomyelin, cerebrosides, gangliosides, etc (fig.6). Free ceramides are only found in large amounts in the skin stratum corneum (81). They exist in much smaller proportions in cell membranes, in which they occur primarily as intermediates in the metabolism of the more complex sphingolipids, and where they play an important role in cell signalling (79, 80).

De novo biosynthesis of the sphingoid bases is essential because dietary sphingolipids are mostly catabolized; however, careful control of the de novo pathway is critical because many of its intermediates are highly bioactive. Enzymes of the biosynthetic pathway are all membrane-bound and have intriguing features. Serine palmitoyltransferase, the first enzyme of the pathway, is a pyridoxal 5_-phosphate-dependent decarboxylase comprised of two subunits, one of which has been shown to be defective in human sensory neuropathies. (Dihydro)ceramide synthase catalyzes the formation of an amide linkage between a long chain (typically saturated) fatty acid and the amino group of the sphingoid bases and is the target of a family of mycotoxins (fumonisons) that cause a wide spectrum of disease symptoms (neurotoxicity, hepatotoxicity, and nephrotoxicity, pulmonary edema, cancers of liver, kidney, and esophagus, birth defects, and possibly other disorders). And finally, dihydroceramide desaturase and hydroxylase, which introduce the 4-trans double bond (to produce sphingosine) and the C-4 hydroxyl of phytosphingosines, respectively. The desaturase reaction is critical because it converts an essentially innocuous precursor (dihydroceramide) into a highly bioactive product (ceramide and downstream species such as sphingosine and their derivatives).

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FIG.6: Basic pathways of ceramide metabolism and interrelationship of regulatory pathways mediated by bioactive lipids.

Ceramide can be formed de novo or from hydrolysis of SM or complex glycolipids (horizontal plane in diagram). In turn, ceramide may be converted to sphingosine (Sph) or serve as a substrate for SM synthesis (generating DAG) or glycolipids. Each of the major bioactive lipids is then capable of interacting with specific targets leading to specific responses (vertical plane). PalCoA, palmitoyl-CoA; DHS, dihydrosphingosine; DHCer, dihydroceramide; SK, sphingosine kinase; PC, phosphatidylcholine; PA, phosphatidic acid; SMS, SM synthase; DGK, DAG kinase; SL, sphingolipid.

Sphingolipids vary especially in the different substituents at the 1-hydroxyl position, ranking from the simplest being a phosphate group, yielding ceramide-1-phosphate, to a L-glycosidically linked glucose or galactose substituent, yielding the cerebrosides glucosylceramide and galactosylceramide, or even higher glycosylated ceramide species, the glycosphingolipids (GSL). Sulfatides are derived from galactosylceramide by the addition of a sulfate group in ester linkage to the 3-position of the sugar residue. Gangliosides are a subclass of the GSLs as they contain a sialic acid (mostly N-acetyl neuraminic acid) moiety in the carbohydrate head group. In sphingomyelin, the 1-hydroxy group of ceramide is linked to a phosphorylcholine moiety. A final group of sphingolipids consists of N-deacylated derivatives, like 1-galactosylsphingosine (psychosine), glucosylsphingosine, sphingosine-1-phosphate, or sphingosyl phosphorylcholine (SPC or lysosphingomyelin). These lytic and membrane-labilizing `lyso' sphingolipids are found in very low concentrations in the cell, but may turn out to play an important role as signaling molecules and in pathophysiological conditions.

The amount and composition of GSLs of a cell varies in a species-dependent manner. Furthermore, GSLs show a cell-type-specific pattern on the cell surface, which changes with the differentiation state of the cell and with viral or oncogenic transformation (83).

The first steps of GSL biosynthesis leading to the formation of ceramide are catalyzed by membrane- bound enzymes active at the cytosolic face of the endoplasmic reticulum (84).

The next steps of GSL formation are localized in the Golgi apparatus and involve stepwise glycosylation of ceramide (85, 86). Thus the question of ceramide transport from the ER to the Golgi arises. Transport via vesicular membrane flow (87) as well as non-vesicular transport (88, 89) are described in the literature. The enzymes catalyzing the first two glycosylation steps (glucosyltransferase and galactosyltransferase I) are both accessible from the cytosolic side of the Golgi (90). The precise localization of ceramide glucosyltransferase is, however, not yet clear and could be on the Golgi and/or a pre- Golgi compartment (90-93). Also, the cytosolic topology of galactosyltransferase I is not unambiguously clarified. The finding that mutant Chinese hamster ovary cells, with an intact enzyme but lacking the translocator for UDP-Gal into the Golgi lumen, have greatly reduced levels of LacCer (94) strongly suggests a luminal topology for galactosyltransferase I. The sequential addition of further monosaccharide and sialic acid residues to the growing oligosaccharide chain, yielding GM3, GD3, and more complex gangliosides, is catalyzed by membrane-bound glycosyltransferases, which are restricted to the luminal face of the Golgi membranes (90, 95-99) (Fig. 7). It is, however, an open question how LacCer and/or GlcCer, the starting materials, are transferred from the cytosolic to the luminal side of the Golgi membranes. Similarly, it is unclear how ceramide formed at the cytosolic surface of ER membranes reaches the luminal side of early Golgi membranes for sphingomyelin biosynthesis (100,101).

Only a small number of glycosyltransferases are involved in the further elongation of the oligosaccharide chains of LacCer, GM3, GD3 and GT3, thereby generating the manifold members of the different ganglioside series (Fig. 7). Brady and co-workers (86, 102) suggested very early that the same GalNAc- and galactosyltransferases might be involved in mono- and disialoganglioside synthesis. Years later is proved for the first time that almost all glycosyltransferases involved in ganglioside biosynthesis are rather unspecific for their acceptors. Competition experiments performed in rat liver Golgi demonstrated that they catalyze the transfer of the same sugar molecule to analogous glycolipid acceptors, which differ in the number of sialic acid residues bound to the inner galactose of the oligosaccharide chain (103-105); they also use neoglycolipids with changed hydrophobic anchors (106). For example sialyltransferase V generates in vitro GD1c, from GMlb, GT1a from GD1a, GQlb from GT1b, and Gplc from GQlc and even GT3 from GD3 (105). Information on the localization of the individual glycosyl-transferases within the Golgi stack was derived from metabolic studies in cultured cells that took advantage of the use of transport inhibitors (107-109). The most impressive uncoupling of ganglioside biosynthesis was observed in primary cultured neurons in the presence of brefeldin A (110). The results suggested that GM3 and GD3 are synthesized in early Golgi compartments, whereas complex gangliosides such as GMla, GDla, GDlb, GTlb, and GQlb are formed in a late Golgi compartment or even in the trans Golgi network, beyond the brefeldin A-induced block. These findings were later confirmed in Chinese hamster ovary cells (111). These indirect pieces of evidence obtained by the use of drugs in cultured cells were supported by Golgi subfractionation studies in rat liver (112, 113) as well as in primary cultured neurons (114). As expected, ganglioside biosynthetic enzymes were also detected at low levels in the organelle of their biogenesis, namely in highly purified fractions of the ER of rat liver (115). Despite an abundance of studies, detailed localization of GSL biosynthesis as well as mechanisms of intracellular transfer of GSL are still to be elucidated.

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Fig.7: Schematic view of biosynthetic pathways of complex gangliosides. All reaction steps are catalyzed by membranous glycosyltransferases in the lumen of the Golgi apparatus.

Functions of gangliosides

Since their discovery in the 1940s, gangliosides have been associated with a number of biological processes, such as growth, differentiation, an toxin uptake. Hypotheses about regulation of these processes by gangliosides are based on indirect observations and lack a clear definition of their mechanism within the cell. The first insights were provided when a rduction in cell proliferation in the presence of gangliosideswas attributed to inhibition of epidermal growth factor receptor (EGFR). Since that initial finding, most, if not all, growth factor receptors have been described as regulated by gangliosides. The differential distribution of gangliosides in various tissues and the complexity of their structures is a strong indication that they play important roles in carrying out specific functions in these tissues. More specifically, sequential changes are observed in developing brain [60] and each of the regions of the adult brain is characterized by a specific ganglioside constitution [48, 61, 62]. From these observations, no particular role can be assigned to an individual ganglioside but the general concept emerges that gangliosides contribute to cell-cell recognition or cell-matrix interactions, either through lectin-

like proteins or by carbohydrate-carbohydrate interactions [63], that are important in development and tissue organization.

Numerous investigations have demonstrated that gangliosides can affect cell–cell interactions (120), differentiation (121), proliferation (122), and neurite outgrowth (123) in a variety of cell types.

The ganglioside composition of murine and human neuroblastoma cell lines has been characterized and glycolipids with diverse chemical structures have been found to be present in their cell membranes.

Changes in cellular ganglioside composition were associated with altered growth properties (124). Furthermore, exogenus addition of gangliosides lost upon oncogenic trasformation could reduce proliferation of a tumor cell line in culture (125). These data provided a correlation between cellular growth and gangliosides. In another experimenal system, exogenus administration of gangliosides to neuro-2A neurobastoma cells stimulated neurite sprouting and enhanced axonal elongation (126) These data suggest that gangliosides are involved in differentiation and that they might aid in neural repair. At the time these first observation were made it was not known how these different functions could be regulated by gangliosides. The finding that growth factor receptor activity can be modulated by gangliosides(127) suggested a way by which glycosphingolipids could cause changes in growth and differentiation. The original finding demonstrated that fibroblast growth factor (FGF) stimulated cell growth was suppressed by exogenus addition of saturating amounts of gangliosides through the inibition of FGFR activity. The PDGFR (128) and the EGFR (129) activities were also regulated by gangliosides, and the number of growth factor receptors found to be modulated in some way by gangliosides has been growing ever since. Thus, gangliosides can now be appreciated for their structural role in plasma membrane organization into lipid microdomains, and their regulatory role in signaling processes mediated by growth factor receptors (130). Three distinct mechanism of growth factor receptor modulation by gangliosides emerge on the basis of data for the FGFR, the PDGFR and the EGFR. These mechanistic models focus on , respectively, ganglioside-ligand interaction (excess GM1 probably inhibits the FGFR through competition with the physiological ligand, whereas physiological levels of GM1 can aid in the activation of the FGFR), gangliosides regulation of receptor dimerization (gangliosides may prevent unliganded activationof both EGFR and PDGFR through inhibition of spontaneous dimerization) and gangliosides modulation of receptor activity and subcellular localization (gangliosides may function to stimulate receptor deactivation by contributing to a change in the equilibrium between active and inactive states). In addition although further studier are required, gangliosides modulation of the receptor activation state may be achieved through subcellular localization to GEMs.

An example of the modulation activity of gangliosides on tyrosine kinase receptors is what happend to PDGFR. Platelet-Derived Growth Factor Receptor-b (PDGFRb) which binds homodimers of the platelet-derived growth factor B (PDGF-BB). The ligand-activated protein tyrosine kinase of PDGFRb phosphorylates and activates a number of signal transduction proteins. Pretreatment of cells with GM1 inhibits PDGF-BB activation of PDGFRb through a mechanism involving inhibition of receptor dimerization. GM1 significantly inhibits PDGFR-associated signaling events such as phosphatidyl inositol 3 kinase activity but does not appear to affect PDGF-mediated activation of MAP kinase (ERK2) activity. We have also studied the PDGFRa, which is structurally related to PDGFRb.

Preliminary studies show that pretreatment of cells with several different gangliosides significantly prevents the PDGF-mediated activation of PDGFRa in Swiss 3T3 cells. Thus, both PDGFRa and PDGFRb are modulated in a similar fashion by gangliosides. We also describe a chimeric receptor in which the cytoplasmic tail of PDGFRb was fused to the extracellular and transmembrane regions of TrkA, the receptor for Nerve Growth Factor (NGF). Unlike PDGFRb GM1 stimulates NGF-mediated TrkA tyrosine phosphorylation, and GM1 treatment enhanced the NGF-mediated tyrosine phosphorylation of this chimeric molecule.

These results support the idea that gangliosides interact with the extracellular portion of tyrosine kinase receptors.

Another possible function of glycosphingolipids (GSLs)’ is at the of cell-cell interactions. This assumption is based on observations of dramatic changes in GSL composition in association with differentiation, development, and oncogenesis and on inhibition of cell recognition occurring in morphogenesis and histogenesis by specific types of GSLs or their oligosaccharide sequences. The specific carbohydrate structures differentially expressed in each cell type and each stage of development are assumed to be recognized by complementary molecule(s) expressed at the surface of the counterpart interacting cell. The recognition molecules complementary to carbohydrates have been assumed to be proteins such as endogenous lectins and glycosyltransferases. Recently, an alternative possibility has been proposed that the molecule recognizing cell surface carbohydrate (Lex) per se is carbohydrate (Lex) (116), and the recognition system involves a homotypic interaction. In contrast, there are evidence for specific interaction between two GSLs, GM3 and Gg3, and demonstrate clear heterotypic interaction between two cell types expressing GM3 and Gg3, respectively.

It ‘s just been demonstrate that gangliosides are involved in several biologic processes of keratinocytes and keratinocyte- derived cell lines in vitro, not only in cell adhesion and differentiation but also in cell proliferation, migration, and apoptosis, at least in part by affecting transmembrane signaling (117-118). For example Keratinocytes2 and the keratinocyte-derived SCC12 cell line share three major gangliosides, GM3, GD3, and GT1b (119), all components of the “b” pathway of ganglioside biosynthesis. This content of membrane gangliosides is dictated by the expression of enzymes that promote synthesis and metabolism, particularly glycosyltransferases and ganglioside-specific sialidase. The effect of gangliosides and ganglioside depletion on cell spreading and on signaling that may impact on spreading, particularly FAK and Src signaling ( component af focal adhesion sites ) are studied and results show that depletion of specific gangliosides stimulates cell spreading, whereas increased membrane content of gangliosides inhibits it. The cell spreading inhibited by GT1b overexpression or induced by anti-GT1b antibody is mediated exclusively by the FAK/Src/PI3K pathway, whereas the stimulation of spreading induced by anti- GD3 antibody appears to involve the PKC pathway as well.

So in this way GT1b and GD3 allowed differential inhibition of signaling pathways, leading to apoptosis and inhibition of spreading by different mechanisms.

Gangliosides are important messengers of the adaptive responses to stress such as apoptosis. Under stress conditions that dramatically increase their intracellular concentration, gangliosides can initiate the induction of an apoptotic program. Thus, a myriad of crucial cellular responses may be influenced or controlled by gangliosides and ultimately result in either cell growth and division, differentiation, or cell death. Nonetheless, the molecular mechanisms underlying many of these ganglioside-mediated responses remain largely unknown.

Many signal transduction events occur at the plasma membrane and are thought to proceed within caveolae or lipid rafts; these events are greatly influenced by the concentration and subtype of gangliosides. Evidence that gangliosides directly perturb membrane composition and

permeability or affect the function of membrane components remains circumstantial. However, it is becoming increasingly clear that gangliosides also play crucial roles in subcellular compartments such as the ER and mitochondria.1 At these sites, gangliosides influence often opposite cell fate decisions (e.g., proliferation versus apoptosis), and this action appears to depend on their local concentration, structural characteristics, and sugar modifications.

Underlying many ganglioside-mediated effects is a change in intracellular calcium levels (Ca2P).4,5 Cytosolic Ca2P concentration of resting cells is maintained at low levels by the concerted action of specialized channels, a Ca2P pump, Ca2P-dependent enzymes, and Ca2P-binding proteins. These mechanisms are localized in the cytosol, ER, and mitochondria, the three compartments that control the traffic of Ca2P across the plasma membrane or into intracellular stores. As Ca2P regulates a plethora of physiological processes, it is not surprising that perturbation of Ca2P homeostasis is a potent inducer of an ER stress response that, in turn, dictates the fate of the cells.

Gangliosides in Disease Pathogenesis

The importance of gangliosides in cellular integrity and homeostasis is made apparent by the many catastrophic pathogenic conditions (e.g., neurodegenerative diseases and cancer) associated with the abnormal expression, degradation, or distribution of these molecules. Regulation of the metabolism of gangliosides in specific cell types is also imperative, as indicated by the numerous human genetic diseases known as GSL storage diseases or glycosphingolipidoses 13–15 (Table 1). These monogenic disorders of metabolism that belong to the large group of the lysosomal storage diseases (LSDs) result from deficiency of any one of the lysosomal enzymes involved in GSL degradation and consequent accumulation of undigested GSLs or their intermediates in lysosomes. An account of the cellular consequences of ganglioside accumulation in lysosomes is given in Figure 8.

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FIG.8: Genetic defect in GM1-gangliosidosis. Lack of b-galactosidase leads to expansion of the lysosomal system and massive accumulation of GM1-ganglioside in neurons of GM1 gangliosidosis mouse model. The presence of numerous vacuoles in the cytoplasm represents the typical histological finding in LSDs (electron microscopy picture)

Glycosphingolipidoses represent one of the most frequent causes of neurodegeneration and mental retardation in children. These diseases are complex and, in most cases, present with a progressive and severe neurodegenerative course and a broad spectrum of systemic abnormalities (131, 132).

The neurologic symptoms include mental retardation or dementia, motor dysfunction, sensory deficits, increased startle response, and seizures. In some disorders, cerebellar signs predominate, whereas in others, the cerebral cortex, basal ganglia, or spinal cord neurons are the most affected. The variations in symptoms associated with different glycosphingolipidoses may reflect differences in the metabolic needs of individual cell types that depend on the selective nature of the primary defect. Pathophysiologic studies of patients and animal models have identified changes in neuronal connectivity in the cerebral cortex, including degeneration of axons and synapses of inhibitory neurons (axon swelling or ‘spheroids’), regrowth of dendrites (ectopic dendrites or meganeurites), and formation of new synapses of pyramidal neurons (133-135). Many of these phenomena have been attributed to ganglioside storage, albeit the underlying molecular effectors are still unknown. Neuronal cell death and demyelination occur in some of these LSDs (136-138) and are often accompanied by astrogliosis and microgliosis that appear mostly in areas of severe neuronal vacuolation (139-141). The presence of reactive astrocytes is indicative of an elicited neuroinflammatory response, and the biochemical heterogeneity and adaptive plasticity may reflect differences in microenvironmental cues, such as the combination of cytokines, growth factors, adhesion molecules, and other signals emanating from injured neurons, activated microglia, endothelial cells, and vascular components (142-144).

In the central nervous system (CNS), gangliosides constitute 10–12% of the total lipid content, and in the mammalian brain, GM1 ganglioside is the most abundant glycolipid. The ganglioside storage diseases (or gangliosidoses) include the GM1 storage disorder, GM1-gangliosidosis, and the GM2 storage disorders, Tay–Sachs disease, Sandhoff disease, and GM2-gangliosidosis AB variant (Table 1) (145-146). Given the abundance of gangliosides in the CNS, the clinical and pathologic manifestations of these LSDs are characteristic of generalized CNS disorders. Gangliosidoses are severe neurosomatic conditions that occur mainly in infants, although milder forms with later onset and longer survival also occur in adolescents and adults (145-146). The severe forms are characterized primarily by growth retardation, progressive neurologic deterioration due to extensive brain atrophy, visceromegaly, and skeletal dysplasia (147-148).

In most glycosphingolipidoses, the predominant storage is one particular GSL, for which the corresponding enzyme has the highest affinity (Table 1). Hence, GM1- and GM2-gangliosides accumulate primarily in GM1- and GM2-gangliosidoses. GlcCer and galactosylceramide are the primary storage products in Gaucher disease and Krabbe disease, respectively (131). In some LSDs, gangliosides accumulate secondarily to the primary storage material that may not necessarily be a GSL (149). For example, secondary storage of gangliosides GM2 and GM3 in brain tissue has been reported in the following diseases: Niemann–Pick type A/B disease, in which the primary storage material is sphingomyelin; Niemann–Pick type C disease, where the primary storage material is cholesterol (150); and mucopolysaccharidosis types I (151) and III, (152) in which the primary storage materials are dermatan and heparan sulfate, respectively. As GSLs act as second messengers, their secondary accumulation may be the result of GSL-mediated activation of a signal transduction pathway(s) that controls the synthesis of these molecules.

Conceivably, the secondary accumulation of gangliosides may contribute synergistically with the primary storage product to elicit pathogenesis.

In addition to the altered intracellular concentration or distribution of gangliosides that occur as a consequence of lysosomal storage, the presence of free pools of gangliosides in the blood plasma, cerebrospinal fluid (CSF), and other body fluids could be part of the molecular mechanisms of disease.

Under physiologic conditions, a constant exchange appears to occur between cell-associated and non-cell-associated gangliosides. However, increased turnover of cell membranes resulting from cell degeneration or cell growth leads to an increased release of gangliosides into the extracellular milieu by a process called ‘shedding’.

Several studies have suggested that gangliosides in the CSF are shed from plasma membranes of neural cells.

Degenerative processes in the CNS have been linked to increased shedding of membrane fragments into the intercellular space.For example, ganglioside GD3, which is present only in trace amounts in the normal adult brain, is expressed at high levels in activated microglia and in reactive astrocytes. GD3 is released by primary murine microglial cells under neuroinflammatory conditions and is directly responsible for the induction of apoptosis in oligodendrocytes (GD3-mediated apoptotic response is discussed in detail below). Increased GD3 expression has also been detected in brain tissue from patients with various neurodegenerative disorders such as Creutzfeld–Jacob disease and multiple sclerosis (153-154). Moreover, elevated CSF levels of GM3 and to a lesser extent GD3 have been associated with pronounced dysfunction of the blood–brain barrier (155). On the basis of these observations, it is thought that altered intracellular expression of GM3 and GD3 and their release into the extracellular milieu occur in other neurodegenerative an neuroinflammatory conditions including some LSDs.

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The ErbB family of receptors and their ligands

A fundamental requirement of biological systems is the ability to translate cues from the extracellular environment into cellular responses. Receptor tyrosine kinases (RTKs) mediate the transduction of many of these signals and are involved in such diverse processes as cell proliferation, differentiation, migration, survival, and death. RTKs are single-pass transmembrane proteins with an extracellular ligand-binding domain and an intracellular tyrosine kinase domain. Ligand binding induces homo- or heterodimerization of the receptor, which is essential for tyrosine kinase activity. The recruitment of target proteins to the activated receptor complex then initiates a signaling cascade that regulates downstream transcriptional programs. ErbB family receptor tyrosine kinases (RTKs) ErbB1 (also known as human epidermal growth factor [EGF] receptor 1 [HER1] or EGF receptor [EGFR]), ErbB2 (also known as HER2 or Neu), ErbB3, and ErbB4 consist of an extracellular ligand-binding domain, a single transmembrane domain, an uninterrupted tyrosine kinase domain, and a cytoplasmic tail.

There are at least 16 different EGF family ligands that bind ErbB receptors. The ligands can be grouped into three categories: (i) those that bind to ErbB1 alone (EGF, transforming growth factor a, and amphiregulin), (ii) those that bind to ErbB3 and ErbB4 (neuregulin 1 [NRG1] and NRG2), and (iii) those that bind to ErbB1 and ErbB4 (betacellulin, heparin-binding EGF, NRG3, and epiregulin) (156, 157).

EGF family members share a conserved receptor-binding motif, and soluble forms are usually produced from bioactive, integral membrane precursors (158) In vertebrates, the different EGF family members bind to the various ErbB receptors with differing degrees of preference. For example, EGF, TGF-α, and AR bind to EGFR, whereas HB-EGF, EPR, and BTC bind to both EGFR and ErbB4. NRG-1 and NRG-2 bind to ErbB3 and ErbB4, while NRG-3 and NRG-4 bind to ErbB4, but not to ErbB3. Similar to other allosteric enzymatic systems, oligomerization of ErbB proteins is essential for their activation. Early work demonstrated that minimal oligomerization: namely, dimer formation, is sufficient for enzyme stimulation (159-160). Furthermore, the extracellular ligand acts as an allosteric regulator of the cytoplasmic enzyme, simply by inducing receptor/enzyme oligomerization. Thus, the transmembrane topology of ErbB proteins allows an allosteric mechanism for signal transduction, that bypasses the need for vertical propagation of conformational changes across the plasma membrane. That dimerization is not limited to homodimer formation, but also includes heterodimerization of ErbB proteins, was shown by demonstrating that ErbB2 can heterodimerize with the EGF-receptor (161-162) and NRG-receptors (163). The driving force for homo- as well as heterodimer formation is the higher stability of the ternary complex formed between a ligand and two receptors, as compared with a monomeric receptor. In other words, receptor dimers have higher ligand affnity when compared with the corresponding receptor monomers (164-165). It was later demonstrated that at least nine different homo- and heterodimers of ErbB proteins exist but their formation displayed a distinct hierarchy (166). In this network, ErbB2 plays a major coordinatory role, as each liganded direct receptor appears to prefer ErbB2 as its heterodimeric partner. This preference is further biased upon overexpression of ErbB2, as seen in many types of human cancer cells. For two reasons, ErbB2-containing heterodimers are characterized by extremely high signaling potency. First, due to the ability of ErbB2 to remarkably reduce the rate of ligand dissociation, signaling by growth factors is prolonged and enhanced by this oncoprotein (166-167).

Second, because ErbB2 can efficiently signal through MAP-kinases, its presence enhances mitogenic, and perhaps also other types of cellular signals (168).

Thus, ErbB2 overexpression in tumor cells is thought to confer a selective advantage due to better utilization of stroma-derived EGF-like growth factors.

Unlike homodimers whose biological activities are relatively weak, heterodimers appear to be more potent. This is best exemplified by the ability of each ErbB protein to transform a normal fibroblast into a cancer cell: co-expression of two ErbB proteins, either ErbB1 and ErbB-2, or each of the two NRG receptors together with ErbB2 or ErbB1, drives cellular transformation more efficiently than by each singly expressed protein (169-170). In model cellular systems whose growth depends on an interleukin, co-expression of two ErbB proteins confers a superior proliferative effect, consistent with the synergistic transforming potential (171-172). A graded range of mitogenic signals is thus formed in which homodimers of the kinase-defective receptor, ErbB3, are completely inactive and heterodimers between ErbB3 and ErbB2 are the most mitogenic. The ErbB2/ErbB3 heterodimer exemplifies the role of heterodimer formation, not only in signal diversification but also in achieving better control; formation of the most potent combination requires both a ligand for ErbB-3, namely NRG, and a heterodimerizing partner, ErbB2.

As I just said, EGF family members vary in their ability to activate distinct ErbB heterodimers, which may partly account for differences in their bioactivities. Each receptor homo- or heterodimer has a different set of tyrosine autophosphorylation sites, which serve as docking sites for specific SH2-containing proteins and recruit different combinations of signaling molecules such as the p85 subunit of phosphoinositide 39-kinase (PI 3-kinase), phospholipase Cg1, Src family kinases, protein tyrosine phosphatases, SH2 domain containing tyrosine phosphatases 1 and 2, Shc and Grb2, Grb7, Grb10, c-Cbl, Nck, Crk, Eps8, and Eps15 (173; 174 175) (Fig. 9).

ErbB receptors also induce tyrosine phosphorylation of proteins involved in cell adhesion signaling such as the focal adhesion kinase (176), Crk-associated substrate (Cas) (177.)., paxillin (178), cortactin (179), and catenins (180). It is likely that different ErbB dimers recruit or activate different sets of signaling molecules. For example, the p85 subunit of PI 3-kinase is thought to associate only with ErbB3 (181), c-Cbl with ErbB1 (182), and Chk with ErbB2 (183). c-Src associates with both ErbB1 and ErbB2, though it appears that c-Src prefers ErbB2 over ErbB1 (184). Very little is known about how ErbB homodimers and heterodimers differ in their biological properties, and it is also not known whether heterodimers possess unique signaling properties compared to homodimers. Unliganded state, ErbB1, ErbB3 and ErbB4 exist in a ‘tethered’ intramolecular conformation, in which the dimerization motif is unable to mediate monomer–monomer interactions. By contrast, ligand-bound ErbB1 and unliganded ErbB2 exhibit a dimerization competent conformation in which the dimerization arm (a 10-residue sequence) is exposed on the receptor surface (Fig. 10b). Ectodomain contacts between two monomers occur mainly through this dimerization arm, although other contact regions exist.

Mutagenesis of the arm (185-186) or an adjacent loop (187) abrogates receptor activation but not ligand binding. In the absence of bound ligand, this arm is tethered by intramolecular contacts, preventing intermolecular association of monomers. Introduction of mutations thought to release the tethered arm do increase ligand binding affinity but do not provoke receptor dimerization or activation (188-189). This indicates that ligand-induced exposure of the dimerization arm is necessary, but not sufficient, for receptor dimerization. Ligand-induced dimerization is postulated to mediate kinase activation by positioning two cytoplasmic domains such that transphosphorylation can occur. A recent paper (190) has indicated that kinase activation is, not surprisingly, a more intricate process. All protein kinase structures show that the kinase is divided into two lobes, termed N and C, that cooperate to form the active site. Based on the analysis of intermolecular contacts observed in crystals of the isolated kinase domain and directed mutagenesis of the contact residues in the full-length receptor expressed in cells, the authors conclude that initiation of the active conformation of the one kinase domain is explained within the context of an asymmetric kinase dimer.

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Fig.9: Schematic representation of the ErbB signaling network. Three layers of diversity generation are proposed: multiple ligands with specificity to distinct receptors, nine receptor combinations, and various sets of cytoplasmic signaling proteins (most of them containing SH2 domains). The plasma membrane is shown as a gray horizontal bar and the various ErbB proteins as bilobular structures with helical transmembrane domains. The defective kinase of ErbB-3 is crossed. Receptor phosphorylation sites are shown by encircled P letters. The various signaling proteins underlying each receptor dimer are listed in vertical boxes. Receptor heterodimers that can be induced by either EGF (E) or NDF/neuregulin (N) are presented with two boxes. Note that only some of the known ErbB ligands are represented and they dffer in their dimer recruitment abilities.

In this mechanism the C-lobe of one kinase allosterically activates a second kinase molecule by contacting the N-lobe of that kinase and repositioning the activation loop such that catalysis is facilitated (Fig. 10c).

Left unresolved from the current data is the ultimate structure of the dimer in which both kinase domains are active. This structure addresses two other questions about ErbB kinase activation. One is the ErbB3 conundrum. The ErbB3 kinase domain is enzymatically inactive because of point mutations (relative to other ErbB kinases) and its heterodimerization with ErbB2 is considered essential for ligand-bound ErbB-3 to activate signaling. However, if reciprocal transphosphorylation is required, this cannot occur when one kinase monomer is inactive. The model predicts that the C-lobe of the ErbB3 kinase is capable of interacting with the N-lobe of an ErbB2 kinase and allosterically initiating its activation. A second explanation derived from this structure deals the use of ErbB1-specific kinase inhibitors, such as Iressa, for treatment of patients with lung cancer. Surprisingly, in a large trial, only a small fraction of these patients responded to treatment with Iressa. Only later was it recognized that patients who have mutations in the ErbB1 kinase domain were responsive to the drug, whereas those without mutations were not (191). These mutations promote survival signaling when placed in a mouse background and are oncogenic (192-193). The study by Zhang et al. (190) shows that the ErbB1 L834R mutation, which occurs in the kinase domain activation loop, increases the catalytic activity of the isolated kinase domain and suggests that this mutant intramolecularly destabilizes the inactive conformation of the kinase domain, probably allowing the inhibitor to bind more readily.

However, the mutant still requires ligand stimulation for maximal activity. The dimerization model reveals that because the mutation partially facilitates the active conformation, the mutant receptor should be more sensitive to clinically used kinase inhibitors, such as Iressa, that preferentially bind to the active state of the kinase. The biological activity of these clinically important ErbB1 kinase domain mutations must also be interpreted in light of the influence of other ErbB receptors present in tumor cells. Evidence has been presented that ErbB3 is required for survival signaling by these mutants (194), as ErbB1 is by itself a poor activator of PtdIns-3 kinase. In addition, oncogenic kinase domain mutations in ErbB2 provoke activation of ErbB1 and resistance to certain ErbB-1 kinase inhibitors (195).

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FIG.10: ErbB receptor dimerization and activation. (a) A general scheme for ligand-dependent dimerization and activation of an ErbB receptor. (b) The contribution of the dimerization arm to receptor association within the ectodomain. Before ligand binding, the arm is sequestered within a monomer by interactions with subdomain IV. Ligand binding alters this interaction such that the arm is now exposed to facilitate dimerization by intermonomer associations between dimerization arms. From the data available, the likely consequence of ectodomain dimerization is the asymmetric interaction of kinase domains such that activation occurs (c). P, phosphorylation; Y, tyrosine

As these mutations are in the N-lobe of the ErbB2 kinase, the model for allosteric activation of the ErbB1 kinase by the C-lobe of ErbB2 does not, however, provide a ready explanation.

Although these structural studies are certainly enlightening, there is as yet no clear picture of how these dimerization and activation mechanisms operate together in the context of the membrane-bound intact receptor. Also unaccounted for in any of the crystal structures are the transmembrane domain (TM), the cytojuxtamembrane region (JM) and the C-terminal domain (CT), which are present in each of the ErbB receptors (Fig.11). There is evidence that the unphosphorylated _225-residue CT domain of ErbB-1 negatively influences receptor activation and, when tyrosine is phosphorylated, the CT is displaced from the kinase domain. The JM region in other kinases, such as c-Kit, is known to contact the kinase domain and contains residues that, when mutated, activate kinase activity (196).

In ErbB receptors there are non-tyrosine phosphorylation sites in the 35-residue JM region. Threonine 654, a protein kinase C (PKC) site in the JM region of ErbB1, is known to modulate kinase activity by a mechanism that has not been investigated. In addition, the JM region of each ErbB contains an unusually high number of basic residues that are reported to associate with acidic phospholipids, bind calmodulin, and influence dimerization (197-199). The potential involvement of the TM domains is illustrated by the Neu mutation in the ErbB2 TM that provokes constitutive dimerization and kinase activation.

Finally, although the mechanistic focus is, at this point, on receptor dimerization, ligand-dependent receptor tetramerization occurs and might be part of the receptor activation process (200). Given the presence of multiple negative regulators (phosphatases, ubiquitin ligases, adaptors) in the ErbB system (201-202), it is plausible that kinase activation also involves release from negative intermolecular constraints. The negative regulator RALT (Mig6, gene 33), a protein with no other known function, binds to all ErbB kinase domains and attenuates kinase activity (203). RALT expression is induced by ErbB growth factors and might therefore act as a feedback inhibitor to reduce kinase activation. Depletion or knockout of RALT activates ErbB receptors and facilitates tumor formation (204). Similarly, targeted overexpression of RALT in the skin blocks ErbB-1 signaling and generates a phenotype similar to a hypomorphic Egfr allele (205). Although RALT seems to interact directly with ErbB kinase domains, the contact regions and mechanism of kinase inhibition have not been reported.

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FIG.11: Using the numbering for ErbB-1, this diagram illustrates the structural features of each ErbB extracellular and intracellular region.

Receptor trafficking to novel sites and receptor fragments

Transmembrane receptors, including most RTKs, are predominantly localized in specialized regions of the plasma membrane called lipid rafts, which may or may not be associated with caveolins. Signaling and adapter proteins are also associated with lipid rafts to facilitate signaling from these domains. In addition to signal transduction, ligand activation of RTKs promotes internalization of the activated receptors via clathrin-dependent or –independent pathways (Fig. 11).( 206) Clathrin-dependent endocytosis is known to occur outside the lipid rafts in the bulk plasma membrane, necessitating that activated RTKs (including EGFR, PDGFR, IR, and IGF-1R) must exit from lipid raft–signaling platforms to be internalized via the clathrin mediated pathway. However, new studies on EGFR suggest lipid rafts may also coordinate the assembly of nascent clathrin-coated vesicles (CCVs) for the internalization of activated receptors, offering seamless integration of signaling and internalization activities.

Among RTKs, the endocytic trafficking of EGFR has been studied in the greatest mechanistic detail and serves as a useful paradigm when elucidating the pathways for other RTKs (Figure 11).(207.) Activated EGFR recruits the SH2 domain–containing signaling molecule Grb2 (Growth factor receptor– binding protein 2) that, in turn, mediates the binding of EGFR to the E3 ubiquitin ligase c-Cbl to start the cascade of events leading to internalization and degradation.( 208-209) Cbl-interacting protein of 85k (CIN85) may then be recruited to disabled2 (DAB2) and endophilin and drive clathrin assembly and budding.(210-211). Ubiquitylation by c-Cbl is an important reversible modification that generates further docking sites for endocytic adapter proteins with ubiquitin interacting motifs (UIMs), such as the adaptor proteins EGFR pathway substrate-15 (Eps15) and epsin(212).

Bridging to clathrin is, in turn, mediated by the clathrin adaptor protein-2 (AP2) or epsin.(213) Thus, the initial assemblies of endocytic machinery components at the plasma membrane are triggered in parallel with EGFR signaling and facilitate formation of nascent coated pits.(214.) In this regard, it is noteworthy that EGFR/ErbB2 heterodimerization prolongs plasma membrane signaling after ligand activation by impairing clathrin assembly, most likely in an hsp90-dependent manner.(215-216) Thus, based on the EGFR/ErbB family of receptor paradigm, there is a close coupling between RTK signaling and membrane trafficking. Analogous to EGFR, other RTKs, such as PDGFR,(217) keratinocyte growth factor receptor (KGFR), (218) FGFR,(219.) IR, (220) and IGF-1R, (221) when activated by their respective ligands, are also internalized via the clathrin-mediated pathway. Some of the proteins involved in clathrin-mediated endocytosis, such as Cbl, Grb, and Shc, are shared by most of these receptors, although some may use different signaling and adaptor proteins than EGFR. For instance, clathrin-mediated endocytosis of KGFR is not mediated by Eps15 (223). Differences in adaptor proteins are likely to be important for scaffolding signaling molecules and defining intracellularfates such as transport to the nucleus, recycling, or degradation. For a number of key receptors in the vascular system, these important details of membrane trafficking remain to be elucidated. Following internalization, CCVs bearing EGFR shed the clathrin and deliver their cargo to early endosomes (Fig. 11). Early endosome delivery is regulated by the Rab5 GTPase, whose activity is, itself, subject to regulation by EGFR signaling (223-224). Bifurcation of RTK trafficking pathways occurs in early endosomes, allowing for recycling or degradation. Endocytosed RTKs destined for degradation remain ubiquitylated are sorted into luminal vesicles of multivesicular bodies (MVBs) and targeted to lysosomes for degradation by acid-dependent proteases. MVB sorting depends on clustering of cargo within specific phosphoinositide- and clathrin containing membrane domains. Such cargo clustering requires ubiquitin signal recognition by a large protein complex consisting of HRS (Hepatocyte growth factor–Regulated tyrosine kinase Substrate), TSG101 (Tumor Susceptibility Gene-101), and ESCRT (Endosomal Sorting Complex Required for Transport) proteins that are recruited through specific phosphoinositide- and ubiquitin-binding domains.16 The ubiquitin signals used in MVB sorting require active c-Cbl (212). Hence, c-Cbl function has been implicated for both internalization and late endosomal sorting, which finally leads toward degradation. Alternatively, a subset of endocytosed RTKs may recycle back to the plasma membrane, remain associated with and actively signal from endosomes, or even translocate into the nucleus Internalized receptors generally recycle from early endosomes back to the plasma membrane in a Rab11-dependent manner (225). Recycling of EGFR/ErbB2 heterodimers, for instance, is promoted by ligand dissociation in the mildly acidic early endosomes and loss of the ubiquitin signal (207). Active recruitment of various adaptor and signaling proteins such as Grb2, Shc, phospholipase C (PLC)-_1, and phosphatidylinositol 3-kinase (PI3K) by activated EGFR and PDGFR on endosomes indicate that ligand-bound RTKs can actively recruit proteins and signal after internalization (226).

Furthermore, EGFR-signaling scaffolds continue to be remodeled even en route to degradation with specific mitogen-activated protein kinase (MAPK)-scaffolding proteins and nuclear-signaling proteins recruited to select only endosomes (227-228). RTKs and associated proteins have frequently been found localized in the nucleus following ligand stimulation, raising the exciting possibility that direct links between RTKs and the nucleus may exist in parallel with signal transduction cascades (229-230)

A variation of the endocytic pathway is proposed as part of the mechanism for the translocation of activated intact ErbB-1 and ErbB-2 to the nucleus (231) where these receptors are implicated in the induction of specific genes, such as cyclin D, iNOS, c-myb and COX-2 (232) (Figure 2). The presence of ErbB-3 in the nucleus has also been described (233).

There are two outstanding issues in these studies. The first is the absence of a demonstrable mechanism to extract these transmembrane molecules from a lipid bilayer into the nucleoplasm, as non-membranous molecules. The second is whether nuclear translocation is required for EGF action. The only evidence for this latter point is a mutant ErbB-1 receptor that exhibits neither nuclear translocation nor induction of certain target genes, such as iNOS, following the addition of EGF (232). As the mutations used are within the basic region of the JM, pleiotropic effects are possible. Importantly, control experiments show that this mutant retains the ability to mediate EGF-dependent activation of Erk and induction of an Elk reporter. This result is consistent with a nuclear ErbB1 requirement for iNOS induction by EGF, but does not prove the issue. In the absence of more mechanistic and direct information, this issue will continue to evoke skepticism. A different scenario exists for the ligand-dependent relocalization of ErbB4 to the nucleus. In this case, growth factor-dependent secretase cleavage liberates the ErbB4 intracellular domain (ICD, also known as membrane bound m80 or soluble s80) from the plasma membrane, allowing it to localize to the cytoplasm and nucleus (234) (Figure 2). The ICD associates with several transcription factors (235) and it seems that the ICD is required to shuttle transcription factors into the nucleus or to regulate transcription factor function. There is less compelling evidence that the ICD functions as an essential part of a transcription complex at target gene promoters.

Consistent with the ability of ErbB4 to regulate STAT5, a key protein in mammary development, mammary cell studies suggest that ErbB4 cleavage might be part of a mammary differentiation program (236-237). Paradoxically, the ICD can also provoke cell death (238) or cell proliferation in mammary cells (239). The cell death response to the ICD is suggested to be a consequence of ICD translocation to the mitochondria. These studies, however, involve exogenous expression of the ICD, with the attendant concern that the level of expression could drive an artifactual response. Although it is clear that ErbB4 has an important function in mammary differentiation (240), evidence for the requirement of the ICD fragment is only supportive at this time. ErbB-4 has been shown to regulate neural development and more recently to regulate astrogenesis in the developing mouse cerebral cortex (241). Mechanistically, the experiments demonstrate that the ICD fragment associates with a TAB-2–N-COR transcriptional repression complex. The function of the ICD in this system is to act as a carrier of TAB-2 and NCOR into the nucleus, reminiscent of data showing that the ICD is required for STAT5 nuclear localization in mammary cells (242). Similarly, another group reports the association of the ICD with ETO-2, which is usually present with N-COR in a repression complex (243). The ICD fragment is an active kinase that can form dimers (244).

The ICD phosphorylates tyrosine residues in Mdm2 (245) and increases p53 levels, which might be responsible, in part, for ErbB4-dependent apoptotic and differentiation responses. Surprisingly, no other ICD phosphorylation substrates have been reported.

It has been known for many years that in some cells secreted ErbB1 or ErbB3 ectodomain fragments are generated by a discrete mRNA; however, no function has been attributed to these fragments. In addition, ErbB2 fragments are generated by several mechanism(s) that are not yet clearly understood. Many studies have reported that an ectodomain fragment of ErbB2 is found in the fluids of cancer patients (246). This ectodomain fragment can be produced by proteolysis (247) or an aberrant transcriptional mechanism (248). As the fragment can attenuate the activity of ErbB receptors, its clinical use is being explored (249). Additionally, a 95-kDa ErbB2 cytoplasmic domain fragment is produced by two distinct mechanisms: ectodomain cleavage or an alternate reading frame mechanism (250). Although there is a clinical association of disease progression with this fragment (251), its cellular function is not known, nor is it known if this fragment is present in the nucleus.

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FIG.12: Endocytic pathways and regulatory factors governing vesicular trafficking of RTK(s). RTKs are internalized via 2 major pathways: a clathrin-dependent pathway and a caveolar pathway

Embryogenesis and organ development

Targeted inactivation of components of the ErbB signaling network, and expression patterns of ErbB receptors and their ligands highlighted the importance of short-range ligand-receptor interactions especially in mid-gestation inductive processes. Apparently, the network is involved primarily in two types of interactions: mesenchyme-epithel crosstalk and neuronal e¡ects on target cells, including muscle, astroglia, oligodendrocytes and Schwann cells (252). NRG is synthesized by mesenchymal or neuronal cells which influence the differentiation, proliferation and migration of adjacent epithelial or non- neuronal cells, respectively. An essential role in mid-gestation was indicated by embryonic lethality of ErbB2-, ErbB4- and NRG-deficient mice at around day 10 post-fertilization due to aberrant cardiac development (253). Moreover, the non-redundant part played by the NRG receptors and ErbB2 emphasizes that the functional complex is a heterodimer. The trabeculae, a finger-like extension of the ventricular myocardium fails to develop, and thus the mutant heart is characterized by irregular beat, an enlarged common ventricle and reduced blood flow. Since ErbB4 is expressed in the underlying muscular portion of the ventricle and atrium (myocardium) and NRG is expressed in the endothelial ventricular lining, it seems that NRG activates trabeculea formation by the ErbB-4-expressing myocardium, thereby initiating ventricular differentiation. In addition to cardiac disorders, ErbB4-deficient mice displayed severe defects in the development of the cranial sensory ganglia following migration from the neural crest, thus suggesting a unique role for ErbB4. Targeting of the ErbB1 gene demonstrated a pivotal role during epithelial cell development, consistent with expression profiles of ErbB1, EGF and TGFK in lung epithelium and in the gastrointestinal tract (254). Mutant mice displayed impaired epithelial development in several organs, resulting in different phenotypes ranging from peri-implantation death to live progeny suffering from abnormalities in multiple organs, depending on the genetic background. Knockout of one the ErbB1 ligands, namely TGFK, suggested that each ligand plays a distinct role during development. Thus, TGFK-disrupted mice displayed only part of the defects observed in ErbB1 null mice, namely eye abnormalities and derangement of hair follicles (255). An example of a post-birth function of the ErbB network, which is obscured by lethality of gene-targeted mice, is provided by analyses of ErbB receptors in the developing mammary gland. ErbB1 levels parallel the increase in DNA synthesis in the mammary gland during pregnancy and decline immediately before the onset of lactation . EGF treatment of mammary glands, both in vitro and in vivo, resulted in ductal and alveolar epithelial differentiation and in the suppression of the accumulation of milk fat droplets in the alveoli during mid- to late pregnancy, while overexpression of TGFK in mammary glands resulted in earlier alveolar development (256). Thus, two different cell populations may function as targets of ErbB-1 ligands during distinct stages of mammary gland development. In vitro treatment of mammary glands with NRG induced formation of lobuloalveolar structures and increased appearance of milk-producing cells (257). The NRG isoform K2 is highly expressed during the process of lobuloalveolar morphogenesis at pregnancy, and its levels markedly decrease during lactation and involution, while no expression is detected during the virginal period (258). Interestingly, a switch between ErbB3 and ErbB4 expression was observed in the developing mammary gland, suggesting that the two receptors play different roles in mammary morphogenesis (259).

In addition, ErbB overexpression is associated with tumorigenesis of the breast, ovaries, brain, and prostate gland (260-262). Experiments in transgenic mice and cell culture models clearly indicate that ErbB receptors and their ligands can promote the development and progression of mammary tumorigenesis (263).

ErbB pathology

The relevance of ErbBs to important pathologies is evidenced by recent papers, as described here. ErbB1 is known to be transactivated by a substantial variety of heterologous agonists, such as ligands that activate Gprotein- coupled receptors (GPCRs) (264-265). In many cases, this requires the increased cleavage of an ErbB1 ligand from its plasma membrane precursor, such that the diffusible ligand then dimerizes and activates ErbB-1 (Box 1). This ‘transactivation’ pathway allows GPCRs to influence cell growth and proliferation. One such transactivator is angiotensin II (AngII), which promotes a variety of lesions in the kidney that manifest as chronic renal disease. Lautrette et al. (266) have now shown that the capacity of AngII to promote kidney lesions in mice requires ErbB-1 transactivation. In this instance, AngII promotes the cleavage of protransforming growth factor (proTGF) by the transmembrane protease ADAM17 (TACE), resulting in increased levels of free TGF-a and activation of ErbB-1. Similarly, adrenergic receptor transactivation of ErbB1, mediated by the release of heparin-binding (HB)-EGF, is reported to be a significant factor in vasoconstriction and hypertension (267). Although a role for ErbB4 in neural development is clear, it was a surprising finding that ErbB4 and its ligand neuregulin-1 (NRG-1) are involved in the pathogenesis of schizophrenia. The initial evidence came from human population studies that identified the NRG-1 gene as one of a small number of candidate genes for this disorder (268). The schizophrenia haplotypes are mostly in the 50 region of the NRG-1 gene and might influence transcription factor association (269); however, this has not been well characterized. Subsequent evidence has appeared that connects NRG-1 with ErbB4 in schizophrenia (270). Tissue studies show a large amount of activated ErbB4 in schizophrenic brain tissue and an increased level of ErbB4 association with PSD-95, an adaptor protein that interacts with the C-terminus of ErbB4 and is known to mediate protein–protein scaffolding in neural synapses. One target of the ErbB4–PSD-95 complex is the N-methyl-D-aspartate (NMDA) receptor and hypofunction of this receptor is implicated in schizophrenia. Although NRG-1 decreases NMDA receptor activation in normal brain tissue, the effect is greater in schizophrenic tissue, indicating that NRG-activated ErbB-4 might mediate the hypofunction of NMDA receptors in schizophrenia. It is interesting to note behavioral studies concluding that mice heterozygous for the NRG-1 or ErbB4 gene exhibit behaviors akin to mouse models of schizophrenia (271).

ErbB1 and ErbB2 have recently been identified as receptors for viruses and bacteria, respectively. ErbB-1 is used by cytomegalovirus to enter target cells and ErbB2 functions as a receptor for Mycobacterium leprae, the causative agent of leprosy. In each of these cases, strong evidence is presented that the ErbB in question is activated by pathogen binding to the cell surface and that this activation is required for biological activity of the pathogen. In the case of cytomegalovirus, evidence is presented to show that envelope glycoprotein gB interacts directly with ErbB1 (272). The data for M. leprae directly associating with ErbB2, however, are less convincing, although consistent with that conclusion (273). This could be an important issue because, despite intensive efforts, no direct ligand for ErbB2 has been identified.

Overexpression of tyrosine kinase receptors has a deleterious effect on normal cell growth, leading to the induction of transformation (274). The ErbB1 receptor provided one of the first links between an activated oncogene and human tumor biology. Human ErbB1 is highly homologous to the viral oncogene v-erbB, which is carried by an avian retrovirus.

ErbB1 is also overexpressed in a variety of human tumors and it may undergo oncogenic conversion by gene rearrangements, resulting in large amino-terminal deletions. For example, ErbB1 overexpression is associated with non-small cell lung carcinoma and is correlated with high metastatic rate, poor differentiation and short patient survival time (275). Amplification and overexpression of ErbB2 have been reported for breast carcinoma, where high levels of ErbB2 were correlated with poor prognosis in node-positive patients (262).

ErbB2 is amplified and/or overexpressed in both non-invasive and invasive ductal breast carcinoma, reflecting its importance in the early as well as progressive stages of tumor development. In the rat, a single point mutation in the transmembrane domain of ErbB2 results in a transforming ability, even at low expression levels, probably due to constitutive kinase activity. Analyses of other types of tumors indicated that overexpression of ErbB2 may be present in most carcinomas, including lung adenocarcinomas and gastric and cervical carcinomas (276).

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Table 3: Summary of the effect of EGF family growth factors knockout (KO) in mice

MATERIALS AND METHODS

Cell culture

The HC11 mouse mammary epithelial cell line, clonally derived from the COMMA-D mouse mammary cell line [23], was a kind gift of E. Garattini (Institute for Pharmacological Research “Mario Negri”, Milan, Italy). HC11 cells were routinely maintained in RPMI-1640 medium containing 8% heat-inactivated newborn calf serum (NCS), 2 mM L-glutamine, 50 µg/ml gentamycin and 5 μg/ml insulin from bovine pancreas (HC11 routine growth medium) (SIGMA, St. Louis, MO, USA).

EGF stimulation of HC11 cells.

HC11 cells were transferred in HC11 routine growth medium supplemented with 10 nM EGF (SIGMA, St. Louis, MO, USA) and incubated at 37°C. After 15 min of stimulation, cells were washed twice with ice cold phosphate buffered saline (PBS) and lysed in culture dishes for further experiments.

Gangliosides depletion

To induce endogenous ganglioside biosynthesis inhibition, both HC11 cells were grown to confluence in the respective routine growth medium supplemented with 30 μM [D]-PDMP ((±)treo-1-fenil-2-decanoilamino-3-morfolino-1-propanol) for 5 days. Il PDMP è un inibitore competitivo della glucosilceramide sintasi, enzima che catalizza il trasferimento di un residuo di glucosio alla molecola del ceramide, prima tappa della via biosintetica dei gangliosidiA 4 mM stock solution of [D]-PDMP in sterile water was prepared immediately before use. After two washes with PBS, the cells were analysed to determine the ganglioside content and expression levels of ErbB2 and EGFR.

Treatment with exogenous ganglioside GM3 of HC11

Treatment with exogenous ganglioside GM3 was performed on HC11 cells grown in the presence of 30 μM [D]-PDMP for 5 days. On the 5th day, confluent cells were transferred in a serum-free growth medium supplemented with 125 μM ganglioside GM3 and incubated at 37 °C for short (5 min) or long (1 h) times. Ganglioside GM3 from bovine brain (Alexis Biochemicals, San Diego, CA, USA) was solved in ethanol, dried under nitrogen flow and dissolved, at the moment of use, in serum-free medium by sonication in water bath at the concentration of 1 mg/ml. The cells, harvested and exhaustively washed with PBS, were tested for p185c-neu and EGFR expression in Western blot experiments.

Antibodies

The C18 rabbit polyclonal anti-ErbB2 IgG antibody (Santa Cruz Biotechnology Inc., Santa Cruz, CA, USA) and the Immobilized rProtein ATM (RepliGen Corporation, Cambridge, MA, USA) were employed in the immunoprecipitation experiments. The 1005 rabbit polyclonal anti-EGFR IgG antibody (Santa Cruz Biotechnology Inc., Santa Cruz, CA, USA) and the C18 anti-ErbB2 antibody were used in the subsequent western blot analyses. The PY20 mouse monoclonal anti-phosphotyrosine IgG2b antibody (Transduction Laboratories, Lexington, KY, USA) was utilized to investigate the tyrosine-phosphorylated form of both ErbB2 and EGFR receptors. Ganglioside GM3 was immunodetected both in TLC-immunostaining and in western blotting analyses by the GMR6 mouse anti-GM3 IgM antibody (Seikegaku Corporation, Tokyo, Japan). Bound primary antibodies were visualized by the proper secondary horseradish peroxidase (HRP)-linked antibodies (GE Healthcare, Little Chalfont, UK) and immunoreactivity assessed by chemiluminescence.

Analysis of ErbB2–GM3 colocalization by scanning confocal microscopy

HC11 cells, treated or not with [D]-PDMP and GM3, were fixed in situ with 4% paraformaldehyde in NaCl ⁄ Pi for 30 min at room temperature and then permeabilized with 0.5% TX-100 in NaCl ⁄ Pi for 30 min at room temperature. Cells were labeled with rabbit anti-ErbB2 polyclonal serum (C18, Santa Cruz Biotechnology, Santa Cruz, CA) for 1 h at 4°C, followed by the addition (30 min at 4°C) of fluorescein isothiocyanate (FITC)-conjugated anti-(rabbit IgG) serum (Calbiochem, La Jolla CA). After three washes in NaCl ⁄ Pi, cells were incubated with GMR6 anti-GM3 monoclonal serum (Seikagaku Corp., Chuo-ku, Tokyo, Japan) for 1 h at 4 _C, followed by three washes in

NaCl ⁄ Pi and the addition (30 min at 4°C) of Texas red-conjugated goat anti-(mouse IgM) serum (Sigma). In parallel experiments, cells were stained with anti-GM3 monoclonal serum before fixing the cells. Alternatively, control experiments were performed omitting the monoclonal antibody from the immunolabeling procedure. After washing as above, cells were mounted upside down onto a glass slide in 5 lL of glycerol ⁄ Tris ⁄ HCl (6:4, v:v), pH 9,2. As a control, cells were mounted in glycerol ⁄ NaCl ⁄ Pi (6:4, v:v), pH 7,4 and the results were virtually the same. The images were acquired using a high-resolution x63 objective through a confocal laser scanning microscope Zeiss LSM 510 (Zeiss, Oberkochen, Germany) equipped with argon and HeNe ion lasers. The green (FITC) and red (Texas Red) fluorophores were excited simultaneously at 488 and 543 nm. Acquisition of single FITC-stained samples in dual-fluorescence scanning configuration did not show contribution of green signal in red. Images were collected at 512 x 512 pixels.

Isolation and analysis of lipid-raft fractions

GEM fractions from HC11 cells, treated or not with EGF (10 nm for 15 min at 37°C), [D]-PDMP (30 µm for 5 days at 37°C), [D]-PDMP and EGF, [D]-PDMP and GM3 (125 µm for 5 min at 37°C), or [D]-PDMP and GM3 plus EGF, were isolated as described previously [27]. Briefly, 2x108 cells were suspended in 1 mL of lysis buffer, containing 1% TX-100, 10 mm Tris⁄HCl pH 7,5, 150 mm NaCl, 5 mm EDTA, 1 mm Na3VO4, and 75 U aprotinin, and allowed to stand for 20 min at 4°C. The cell suspension was mechanically disrupted by Dounce homogenization (10 strokes). The lysate was centrifuged for 5 min at 1300 g to remove nuclei and large cellular debris. The supernatant fraction (postnuclear fraction) was subjected to sucrose density gradient centrifugation, i.e. the fraction was mixed with an equal volume of 85% sucrose (w ⁄ v) in lysis buffer (10 mm Tris⁄HCl pH 7,5, 150 mm NaCl, 5 mm EDTA). The resulting diluents was placed at the bottom of a linear sucrose gradient (5–30%) in the same buffer and centrifuged at 200 000 g for 16–18 h at 4°C in a SW41 rotor (Beckman Institute, Palo Alto, CA). After centrifugation, the gradient was fractionated, and 11 fractions were collected starting from the top of the tube. All steps were performed at 0–4°C. The amount of protein in each fraction was first quantified by Bio-Rad protein assay (Bio-Rad Laboratory GmbH, Munchen, Germany). Finally, fractions were subjected western blot, immunoprecipitation experiments or ganglioside extraction.

Immunoprecipitation experiments from membrane fractions

Briefly, TX-100-insoluble (fractions 4–6) or TX-100-soluble (fractions 10–11) fractions from HC11 cells, untreated or treated with 10 nm EGF (Sigma) for 15 min at 37°C were lyzed in lysis buffer (10 mm Tris ⁄ HCl (pH 8,0), 150 mm NaCl, 1% Nonidet P-40, 1 mm phenylmethylsulfonyl fluoride, 10 µg of leupeptin x mL-1). Cell-free lysates were mixed with protein G–acrylic beads and stirred by a rotary shaker for 2 h at 4°C to preclear nonspecific binding. After centrifugation (500 g for 1 min), the supernatant was immunoprecipitated with the rabbit polyclonal anti-(Shc IgG) serum (Transduction Laboratories) plus protein G–acrylic beads. A rabbit IgG isotypic control (Sigma) was employed. Immunoprecipitates were subjected to western blot analysis with the rabbit anti-ErbB2 polyclonal serum (Santa Cruz Biotechnology). Immunoreactivity was assessed by chemiluminescence reaction using the ECL western blotting detection system (Amersham).

Cell lysis, immunoprecipitation and immunoblot analyses

HC11 cells, stimulated or not with EGF (see previous paragraph), were washed twice with ice cold PBS and lysed in culture dishes in complete RIPA buffer (1% Nonidet P-40, 0,5% sodium deoxycholate, 0,1% SDS, 100 (g/ml PMSF, 50 KIU/ml aprotinin, 1 mM Na3VO4 in PBS) for 20 min at 4°C on a rotary shaker. Collected crude lysates were allowed to stand for another hour on ice and then centrifuged 10 min at 10,000 g at 4°C to remove nuclei and large cell debris. An aliquot of each sample supernatant (1 mg total proteins in 1 ml complete RIPA buffer) was immunoprecipitated with the anti-ErbB2 antibody. The immunocomplexes, collected by addition of Immobilized rProtein ATM, were washed four times with complete RIPA buffer, loaded onto 7,5% SDS-PAGE, transferred onto nitrocellulose membrane (Bio-Rad, Richmond, CA, USA) with the Bio-Rad Transfer Blot Apparatus at 150 mA for 16 hours at 4°C in 25 mM Tris HCl, 190 mM glycine, 20% methanol and 0,05% SDS as transfer buffer and, finally, subjected to western blot analyses with anti-EGFR, anti-ErbB2, anti-phosphotyrosine, anti-GM3 antibodies.

Briefly, nitrocellulose membranes were blocked for 1 hour in washing buffer (10 mM Tris HCl, pH 7,5, 150 mM NaCl, 0,1% Tween 20®, T-TBS) added with 5% blocking reagent (ECL System, GE Healthcare, Little Chalfont, UK) for anti-EGFR and anti-ErbB2 antibodies, or with 1% bovine serum albumin (BSA) for anti-GM3 and anti-phosphotyrosine antibodies and then incubated with the specific primary antibody for 1 hour at room temperature (2 hours for anti-GM3) in the appropriate blocking buffer. After six 5 min washes in T-TBS, the blots were incubated for 1 hour at room temperature with the proper horseradish peroxidase (HRP)-linked secondary antibodies in the suitable blocking buffer. After six 5 min washes with T-TBS, membranes were finally developed with the ECL Western blotting detection reagents (GE Healthcare, Little Chalfont, UK) following manufacturer’s instructions. Immunoreactive proteins were visualized by autoradiography on X-OMAT AR film (Kodak, Rochester, NY, U.S.A.).

Manufacturer-specified protocols (30 min at 50°C in 2% SDS, 62,5 mM Tris HCl pH 6,8, 100 mM mercaptoethanol; ECL manual, GE Healthcare, Little Chalfont, UK) were used to strip the nitrocellulose membranes and to re-probe the blots with other antibodies.

Extraction, purification and analysis of gangliosides

Gangliosides were extracted and purified from wet cell pellets of HC11 at different degrees of confluence, treated or not with EGF and/or [D]-PDMP and/or exogenous GM3 (see previous paragraphs). Cell pellets were extracted in chloroform/methanol, partitioned with water and purified by dialysis. Total gangliosides were run on a high-performance thin-layer chromatography (HP-TLC) plate (Merck GmbH, Darmstadt, Germany) in a chloroform/methanol/0.2% aqueous CaCl2 (C/M/W, 60:40:9, vol/vol/vol) solvent system. Gangliosides were visualized by the Ehrlich's reagent (0.6 g p-dimethylaminobenzaldehyde, 80 ml ethyl alcohol, 20 ml 37% HCl). The single ganglioside species were identified on the basis of their HP-TLC mobility in comparison to pure reference standards (Alexis Biochemicals).

Extraction, purification and analysis of gangliosides from the immunocomplexes

Gangliosides were extracted and purified from the lyophilised immunoprecipitates obtained with the anti-ErbB2 Ab from control and EGF-stimulated HC11 cell lysates, according to previously described methods.

In brief, lyophilised immunoprecipitates were extracted with three different chloroform/methanol mixtures 1:1, 1:2, 2:1 (vol:vol, vol:vol, vol:vol), partitioned with the theoretical upper phase (TUP, chloroform/methanol/water, 47:48:1, vol:vol:vol) and then with water and purified by dialysis against distilled water. Equal volumes of ganglioside fractions from each sample, together with pure reference ganglioside standards (Alexis Biochemicals, San Diego, CA, USA), were loaded onto a high-performance thin-layer chromatography (HP-TLC) plate (Merck GmbH, Darmstadt, Germany) and run in a mixture of chloroform/methanol/0.2% aqueous CaCl2 (60:40:9, vol:vol:vol), as solvent system. Gangliosides were colorimetrically visualized by spraying with the resorcinol reagent (200 mg resorcinol, 80 ml 37% HCl, 1 ml 25 mM CuSO4), under carefully controlled conditions (120°C for 15 min). Each ganglioside was identified by comparing its HP-TLC mobility (Rf) with the Rf of the pure reference standards.

For the steady identification of ganglioside GM3, one tenth of the volume used for the HP-TLC assays was used to perform TLC-immunostaining with the anti-GM3 antibody.

A known amount of standard ganglioside GM3 (corresponding to 500 ng sialic acid) and equal volumes of the ganglioside fractions from control and EGF-stimulated cells were loaded onto an aluminium-baked TLC plate (Merck GmbH, Darmstadt, Germany) and run in the solvent system described above. The TLC plate was then dried and impermeabilized in 0.1% polyisobuthylmetacrylate in n-exane, sank for 2 hours at room temperature in blocking buffer (1% BSA in PBS) and incubated with the anti-GM3 Ab in blocking buffer for 2 hours at room temperature. After three 5 min washes in blocking buffer, the TLC plate was incubated with the proper HRP-linked secondary antibody in the same buffer for 1 hour at room temperature, washed as above described and subjected to the ECL detection reagents. Immunoreactive species were visualized by autoradiography on X-OMAT AR film.

Lysis and alkaline phosphatase treatment

Control and treated with PDMP/EGF HC11 cells, washed with PBS, are mechanically removed from culture plates and subjected to lysis for 30 min in ice with CIAP buffer(25 mM Hepes pH 7,8, 300 mM NaCl, 1.5 mM MgCl2 1%Triton X-100, 0.1 mM DTT, 0.1 mM ZnCl2, PMSF e aprotinin) The lysate are centrifugated for 15min at 10,000 g at 4°C, to remove nucleus and cellular fragments; surnatants collected.

To analize the ErbB2 and EGFR phosphorylation state in ganglioside depleted cells stimulated or not with EGF, protein lysates (100µg) are subdued , utilizing different experimental conditions, to calf intestinal alkaline phosphatase (CIAP, Amersham) treatment. CIAP belongs to a group of hydrolasic enzymes that catalize the removal of groups phosphate from aminoacid residues as serine, threonine and tyrosine.

The same quantity of protein is incubated with 5 or 10 CIAP units for 30 min or 1 h at 37°Cand is important that all samples are incubated in the same volume of reaction buffer (25 mM Hepes pH 7,8, 300 mM NaCl, 1,5 mM MgCl2, 1%Triton X-100, 0,1 mM DTT, 0,1 mM ZnCl2, and protease inhibitors).To block the reaction samples are putted in ice, added an identical acetone volume and then placed at -20°C to precipitate proteins. After 16 h, samples are centrifugated at 12000 g at 4°C for 30 min. Precipitates are resuspended in loading buffer and subjected to SDS-PAGE.

AIM OF THE STUDY

Gangliosides are a large and heterogeneous family of sialic acid containing glycosphingolipids, ubiquitous components of all eukaryotic cell membranes.

They are mainly located in the outer leaflet of the plasma membrane, where because of their amphiphilic properties and peculiar composition of the hydrophobic tails, they can spontaneously undergo lateral phase separation in the membrane layer. They can partially segregate together with cholesterol and specific signaling transduction proteins into unique more or less stable clusters or microdomains, indicated as “glycolipid-enriched domains” (GEM), “lipid rafts” or “caveolae”, contributing to define the membrane structure and organization and to modulate the function of the proteins located in the plasma membrane.

One of the most important role that they play is regulation of cell growth and differentiation through the modulation of the activity of most of the membrane tyrosine kinase receptors includined the ErbB family member EGFR. ErbB family comprises 4 receptors called ErbB1 (or EGFR), ErbB2, ErbB3 and ErbB4.

Whereas EGFR, ErbB3, and ErbB4 have activated by specific ligands (EGF), the tyrosine kinase receptor ErbB2 is a specific ligandless receptor, structurally related to EGFR or ErbB1; it can be activated by the binding of ligands to other members of the ErbB, including EGF and neuregulins, only in the context of ErbB heterodimers. It has been ascertained that ErbB2 is the preferred heterodimerization partner for all other ErbB proteins and that ErbB2 containing heterodimers have attributes that prolong and enhance downstream signaling and their outputs.

Data concerning the structural and mechanicistic aspect required for EGF-dependent “trans-activation” of EGFR and ErbB2 in ErbB2/EGFR heterodimers are growing rapidly; on the contrary, the role of cell membrane components for determining local differences in the relative densities on the plasma membrane of receptors and for modulating their expression, activation and metabolic fate has not yet been investigated. Our work was dedicated to investigate the eventual functional relationship between gangliosides, ErbB2 and EGFR so to define if gangliosides can be considered determinant factors in the modulation of EGFR/ErbB2 heterodimerization, activation and metabolism.

In this study we have used as cellular model, normal mammary epithelial HC11cells, where EGFR and ErbB2 are physiologically expressed.

To investigate the EGFR and ErbB2 behaviour in association with gangliosides the researches were carried out in two subsequent step. The first one has been directed to demonstrate the existence of a correlation between EGFR, ErbB2 and gangliosides expression levels, and in particular, to verify the existence of a specific ganglioside mostly involved in modulation of EGFR/ErbB2 activation.

To achieve this aim we have analized the expression of the receptors (by western blot) and gangliosides (by HP-TLC) in HC11 cells at two days of confluence a before and after EGF stimulation, and before and after treatment with D-PDMP, an inhibitory of glycosphingolipid biosynthesis.

Subsequently we have investigated the molecular interaction between EGFR, ErbB2 and gangliosides by ErbB2 immunoprecipitation and subsequently, analysed by western blot with anti-EGFR, anti-phosphotyrosine, and anti-ErbB2 antibodies. Furthermore, the eventual ganglioside species associated to the receptors were analysed after lipid extraction and partitioning of the immunoprecipitates by HPTLC and immunostaining.

Finally, as literature data report that gangliosides are located preferentially in particular membrane structure called lipids rafts, we tryied to localize our receptors on the cell membrane and in particular to verify if they are in lipids rafts. This aim was achieved by scanning confocal microscopy and cell membrane sucrose gradient fractionation experiments.

RESULTS

First Part: Molecular interaction between ErbB2, EGFR and gangliosides

Ganglioside profiles in HC11 cells

In order to investigate the functional relationship between gangliosides and the two tyroine kinase receptors EGFR and ErbB2, my study was initially directed to define the ganglioside profile in normal mouse mammary epithelial cells HC11 cells. As Kornilova et al. have been demonstrated that ErbB2 and EGFR expression levels is dependent on cell density and the presence of EGF in the culture medium (277), the ganglioside profile was analysed in HC11 cells grown in a culture medium supplemented or not with 10 nM EGF, at different degrees of confluence.

The ganglioside pattern HC11 cells, grown in culture medium without EGF, are shown in Fig. 13, Panel A and the ganglioside profile of EGF-stimulated HC11 cells in Fig 13 Panel B. Four ganglioside species, identified as GM3, GM2, GD2 and GD1a on the basis of their HP-TLC mobility in comparison to pure reference standards, are detectable in EGF not stimulated cells (Panel A): GM2 is the most abundant species, followed by GM3, GD1a and GD2. However, by comparing lane 1 to lane 2, significative variations in the ganglioside distribution are detectable: the amount of GM3 increases moving to cell confluence, whereas the amount of any other species (GM2, GD1a and GD2) decreases.

After EGF exposure, GM2 remains the major species and its amount, as well as GD1a and GD2, progressively decreases moving from not-confluent (lane 1) to confluent (lane 2) and to 2-day confluent cells (lane 3). In contrast, GM3 is not detectable in any of the cell samples. These results indicate that both the cell density and the EGF stimulation affect the ganglioside content and distribution. The ganglioside GM3 is the lipid species whose content varies more drastically.

Inhibition of gangliosides biosynthesis by D-PDMP

The treatment of cell coltures with the specific inhibitors of glycosphingolipid biosynthesis represent a largely used experimental approach to identify functions of endogenous GSLs. In particular, Inokuchi and Radin have demonstrated that an analog of ceramide, D-threo-1-phenyl-2-decanoylamino-3-morpholino-1-propanol (D-PDMP),exhibit competitive inhibition for the UDP-glucose:N-acylsphingosine glucosyltransferase (EC. 2.4.1.80), resulting in the cell depletion of all GluCer derived gangliosides species. It should be noted that PDMP does not inhibit UDP-galactose:N-acylsphingosine galactosyltransferase and beta-glucocerebrosidase and that only one of the four PDMP isomers, D-threo isomer (1R, 2R), was able to inhibit GlcCer synthesis. Therefore, to obtain the endogenus ganglioside depletion HC11 cells were treated with the ceramide analogue [D]-PDMP. As expected, the treatment with this selective inhibitor.

As expected, the treatment with 30µM D-PDMP for 5 days causes the complete disappearance of all the ganglioside species present in the HC11 cells (Fig. 13; compare lane 2 with lane 1 in Panel C).

ErbB2 expression levels in normal and D-PDMP treated cells

Normal and gangliosides-depleted HC11 cells before and after EGF stimulation were then analized to examine the expression levels of ErbB2 by immunoblotting analyses on total cell lysates of 2 day confluent cells, using specific anti-ErbB2 antibody (see Materials and methods). As clearly evidenced in Fig.14 EGF stimulation results in appreciable decrease of ErbB2 level of normal HC11 cells (Fig. 14 compare lane 1 with lane 2). In addition, the ErbB2 protein in EGF-stimulated HC11 normal cells (Fig. 14, lanes 2) shows a slower electrophoretic mobility with respect to the protein in untreated cells (Fig. 14, lane 1). To ascertain that the up-shifted protein was the tyrosine-phosphorylated form of ErbB2, as previously reported by Kornilova et al. (277), immunoprecipitation experiments with anti-erbB2 antibody followed by immunodetection with anti phosphotyrosine antibody were performed. The single immunoreactive band, observed only in EGF-treated HC11 cells (Fig. 14, compare lane 6 with lane 5) and showing the same mobility shift detected in lane 2 confirms that the different gel migrations are due to the tyrosine phosphorylation of the receptor. In contrast, whereas in EGF not-stimulated ganglioside-depleted HC11 cells (Fig. 14, lane 3), ErbB2 is expressed in the not-phosphorylated form at similar levels to normal cells (lane 1), the EGF exposure of ganglioside-depleted HC11 cells results in a drastical increase of the protein level (Fig. 14, compare lane 4 with lane 1). ErbB2 is apparently present only in the phosphorylated form. As the anti-ErbB2 antibody used recognizes both phosphorylated and not-phosphorylated forms of ErbB2, the increment of the up-shifted protein is not explainable by a mere increase of its phosphorylation.

To ascertain that the content of ErbB2 protein in [D]-PDMP and EGF-treated HC11 cells is really increased, labeling experiments with L-[35S]-methionine added to cells in culture followed by immunoprecipitation with anti-ErbB2 antibody were performed.

The results are shown in Fig. 14, lanes 7 and 8: a more intensive signal, corresponding to ErbB2 phosphorylated form, is detectable in [D]-PDMP and EGF-treated HC11 cells (lane 8) with respect to [D]-PDMP-treated cells (lane 7).

Therefore, the depletion of gangliosides in HC11 cells affects the levels of the tyrosine phosphorylated ErbB2, suggesting an involvement of gangliosides in the ErbB2 metabolism.

[pic]

FIG.13: HP-TLC analysis of total gangliosides from HC11 cells at different degree of confluence.

Panel A: HC11 cells grown in medium without EGF.

Lane 1: not confluent (70–80% of confluence) cells;

Lane 2: 2-day confluent cells.

Panel B: HC11 cells grown in medium with 10 nM EGF for 15 min.

Lane 1: not confluent (70–80% of confluence) cells;

Lane 2: confluent cells;

Lane 3: 2-day confluent cells.

Panel C: HC11 cells 2 day confluent

Lane 1: cells not treated with 30(M [D]-PDMP;

Lane 2: cells treated with 30(M [D]-PDMP for 5 days.

The cromatographic positions of pure reference ganglioside standards are indicated.

[pic]

FIG.14: Analysis of ErbB2 protein in [D]-PDMP-treated or not HC11 cells.

Panel A: Western blot with anti-ErbB2 antibody on cell protein extracts from 2 days confluent HC11 cells.

Lane 1: cells growth in medium without D-PDMP and not EGF stimulated;

Lane 2: cells growth in medium without D-PDMP and EGF stimulated;

Lane 3: cells growth in medium with [D]-PDMP and not EGF stimulated;

Lane 4: cells growth in medium with [D]-PDMP and not EGF stimulated.

Panel B: Western blot with antiphosphotyrosine antibody on anti-erbB2 immunoprecipitated samples prepared from 2-day confluent HC11 cells.

Lane 5: not EGF stimulated cells;

Lane 6: EGF stimulated cells.

Panel C: immunoprecipitation of L-[35S]-methionine labeled proteins with anti-erbB2 antibody from 2-day confluent cells cultured in medium with [D]-PDMP

Lane 7: not EGF stimulated cells;

Lane 8: EGF stimulated cells.

The position of ErbB2 is indicated at the left side of Panel A; the arrow at the right side of Panel B indicates the tyrosine-phosphorylated form of ErbB2.

EGFR expression levels in normal and D-PDMP treated cells

Analogous immunoblotting analyses were performed on the total cell lysates previously used in order to examine EGFR expression levels. Results of the western blot analysis with a specific anti EGFR antibody are reported in Fig. 15.

Whereas EGFR expression in D-PDMP-treated HC11 cells is rather similar to that of normal cells (Fig. 15, compare lane 3 to lane 1), EGF stimulation causes significative alterations in the EGFR expression profile. In fact whereas in normal cells, EGF treatment shows a sensitive decrease of the EGFR levels and more interestingly, the appearance of a protein with lower molecular mass, EGF treatment of gangliosides depleted cells results in the presence of three different immunoreactive proteins, possibly due to a different EGFR phosphorylation degree. This hypothesis has been examined by treatment of protein lysates from EGF stimulated ganglioside depleted cells with calf alkaline phosphatase (CIAP). The equal total protein amount were been treated with CIAP for different time and concentration (see “Materials and Methods”) and analysed by western blot anti-EGFR antibody. As evidenced in Fig.16, the CIAP treatment results in disappearance of the molecular species with minor electrophoretical mobility, in EGF stimulated cells (Fig. 16, compare lane 2 with lanes 4, 6, 8 and 10) and in appearance of a molecular species with lower molecular weight in the EGF not stimulated cells (Fig. 16, compare lane 1 to lanes 3, 5, 7, 9) indicating that the absence of ganglioside modifyies the EGFR phosphorylation profile independently of EGF stimulation.

[pic]

FIG.16: Expression profile of EGFR after alkaline phosphatase treatment in ganglioside depleted HC11 cells

U: CIAP unit;

T: incubation reaction time.

Dimerization and tyrosine-phosphorylation of ErbB2 and EGFR in EGF-stimulated HC11 cells

It has been largely demonstrated that in cells co-expressing both ErbB2 and EGFR, EGF and other EGFR-agonists preferentially stimulate the formation of ErbB2/EGFR heterodimers in wich cross-phosphorylation occurs (279).

Therefore to ascertain that ErbB2 and EGFR are dimerization partners in the HC11 cell line too, the experiments of co-immunoprecipitation of both receptors from the total cell lysates of normal and EGF-stimulated cells with the specific anti-ErbB2 antibody were performed. The immunocomplexes subsequently were analysed by western blot with the anti-EGFR antibody and, as positive control, with the anti-ErbB2 antibody. Results are presented in Fig. 17, Panels A and B, respectively. As shown in Fig. 17, Panel A, both in control (lane 1) and in EGF stimulated (lane 2) cells a protein with molecular mass of approximately 170 kDa, corresponding to EGFR, is detectable. Similarly, in Fig. 17, Panel B, an immunoreactive species with molecular mass of about 185 kDa, corresponding to ErbB2, is detectable both in control (lane 1) and in EGF stimulated (lane 2) cells.

The 185-kDa molecular species detected in EGF-stimulated cells (Fig. 17 Panel B, lane 2) shows a slower electrophoretic mobility if compared with the species identified in control cells (Fig. 17, Panel B, lane 1). As previously demonstrated, the slight decrease of electrophoretic mobility of ErbB2 is due to tyrosine-phosphorylation of the receptor. A species showing molecular mass much higher than those corresponding to EGFR and ErbB2 monomers, but specifically immunoreactive to anti-EGFR and anti-ErbB2 Abs, is evident in samples from EGF stimulated cells (lanes 2 of Fig. 17, Panels A and B, respectively). This molecular species may represent stable SDS-resistant ErbB2/EGFR heterodimers or, possibly, oligomers. In addition, we have investigated the tyrosine-phosphorylation of ErbB2 and EGFR, by immunoblotting with the antiphosphotyrosine antibody. To this analysis we used the same blots probed with the anti-EGFR and anti-ErbB2 antibodies, after careful stripping the nitrocellulose membrane. Results are shown in Fig. 17, Panel C.

As expected, no tyrosine-phosphorylated band is evident in control cells (lane 1), whereas three molecular species with molecular masses consistent to those of EGFR, ErbB2 and ErbB2/EGFR high molecular complexes are clearly distinguishable after EGF stimulation (lane 2). Altogether these data indicate that ErbB2 and EGFR tightly, although transiently, interact even in the absence of the specific ligand and that EGF is indispensable to give stability to receptor aggregates and to trigger their tyrosine-phosphorylation (302).

FIG.17: Western blot analyses of the ErbB2 immunocomplexes.

Panels A: western blot analyses with anti-EGFR

Panels B: western blot analyses with anti-ErbB2

Panels C: western blot analyses with anti-phosphotyrosine (PY20)

Panels D: western blot analyses with anti-GM3 antibodies.

Lanes 1: immunocomplexes from control cells;

Lanes 2: immunocomplexes from EGF-stimulated cells.

The asterisk positioned at the right side of Panel B indicates tyrosine-phosphorylated ErbB2. The arrows at the right side of each panel indicate high molecular mass species specifically immunoreactive to the used antibodies.

[pic]

FIG.18: Analyses of ganglioside fractions of the ErbB2 immunocomplexes.

Panel A: HP-TLC analysis.

Lane 1: ganglioside fraction of control cells;

Lane 2: ganglioside fraction of EGF stimulated cells;

Lane 3: pure reference standard gangliosides (from top to bottom: GM3, GM2, GD1a, GD2). Panel B: TLC-immunostaining analysis.

Lane 1: pure reference standard GM3;

Lane 2: ganglioside fraction of control cells;

Lane 3: ganglioside fraction of EGF stimulated cells.

Molecular interaction of gangliosides GM3 with ErbB2 and EGFR heterodimers

To assess a possible molecular association of gangliosides with ErbB2 and or EGFR, the immunocomplexes from EGF-stimulated or not cells were lyophilized and subjected to lipid extraction and partitioning; the resulting ganglioside fractions were analysed by HP-TLC. As shown in Fig. 18, Panel A, a unique ganglioside species, with retardation factor (Rf) comparable to the pure reference standard GM3 is clearly evident in EGF-stimulated cells (lane 2), whereas neither GM3 nor other ganglioside species are detectable in HC11 control cells (lane 1). To definitely identify the ganglioside detected in EGF-stimulated cells as GM3, one tenth of the same ganglioside fractions were further analysed by TLC-immunostaining with the specific anti-GM3 Ab. As shown in Fig. 18, Panel B, an immunoreactive species is evident in EGF-stimulated cells (lane 3), but not in EGF not treated cells (lane 2).

In addition although the data clearly demonstrate that GM3 co-immunoprecipitates with ErbB2 and EGFR exclusively after EGF stimulation; to address the specific association of GM3 with both or either the receptors, immunocomplexes from control and EGF-stimulated cells were loaded onto SDS-PAGE and sequentially analysed by western blotting with anti phosphotyrosine antibody and, after accurate stripping of the membrane, with the specific anti-GM3 antibody. Results are shown in Fig. 17, Panel D. No immunoreactive species is detectable in control cells (lane 1); on the contrary, two distinct anti-GM3 reactive species are clearly identifiable following EGF stimulation (lane 2). The slower molecular species displays an electrophoretic mobility almost similar to that of the ErbB2/EGFR aggregates previously detected by western blot with anti-EGFR, anti-ErbB2 and antiphosphotyrosine antibodies (see Fig. 17, lanes 2, Panels A, B and C, respectively). Interestingly, the molecular species having faster electrophoretic mobility seems to move almost like EGFR rather than ErbB2 (compare its electrophoretic mobility with that of tyrosine-phosphorylated EGFR of Fig. 17, Panel C, lane 2), indicating the preferential and SDS-resistant molecular association of GM3 with EGFR rather than ErbB2 monomers (302).

ErbB2 and EGFR expression levels in ganglioside depleted HC11 cells after addition of exogenous ganglioside GM3

To determine if GM3 is involved in determining the D-PDMP effects on ErbB2 and EGFR expression GM3, exogenous GM3 was additioned to the medium of ganglioside depleted cells. Results of Western blot analyses of the protein extracts from cells grown in different conditions are reported in Fig. 19.

The addition of D-PDMP in combination with EGF to growth medium drastically enhances the content of phosphorylated-ErbB2 respect to cells treated with EGF (Fig. 19, Panel A; compare lane 3 with lane 2). This increment is completely reversed by the GM3 addition in the growth medium and the control levels are restored (Fig. 19, Panel A; compare lane 4 with lane 2). In addition, this decrease does not seem to depend upon time of treatment with the exogenous ganglioside (Fig. 19, Panel A; compare lane 4 with lane 5). However, the addition of the exogenous GM3 is also able to reverse the D-PDMP effect on EGFR content consequent to EGF stimulation of D-PDMP-treated HC11 cells (Fig. 19, Panel B), indicating that the GM3 effect is for only ErbB2 and suggesting that the modulation of tyrosine-phosphorylated ErbB2 levels through GM3 could involve EGFR too.

FIG.19: Analysis of ErbB2 and EGFR proteins in HC11 cells after treatment with exogenous ganglioside GM3. Cell protein extracts were prepared from normal HC11 cells and from HC11 cells treated with [D]-PDMP, exogenous GM3 and EGF in different combination.

Panel A: Western blot anti-ErbB2

Panel B: Western blot anti-EGFR

Lanes 1: proteins from normal cells;

Lanes 2: proteins from EGF stimulated cells;

Lanes 3: proteins from D-PDMP treated and EGF stimulated cells;

Lanes 4: proteins from D-PDMP treated, EGF stimulated cells and treated for 5 min with exogenous GM3 ;

Lanes 5: proteins from D-PDMP treated, EGF stimulated cells and treated for 1h with exogenous GM3 ;

Dimerization and tyrosine phosphorylation of ErbB2 and EGFR in ganglioside depleted HC11cells

Experiments with anti-EGFR or anti-ErbB-2 antibodies were also performed on D-PDMP treated cells. Results of western blot analyses of immunoprecipitates carried out with anti-EGFR anti-ErbB2 and anti-phosphotyrosine antibodies are in Fig. 20.

Although anti EGFR antibody is able to immunoprecipitate EGFR in both EGF stimulated and EGF not treated cells (Fig. 20, panel A lanes 3 and 4), no ErbB2 protein is detectable in these EGFR immunocomplexes (Fig. 20, Panel A lanes 7 and 8).

However, analyses of immunocomplexes obtained after ErbB2 immunoprecipitation (Fig. 20 Panel B) show that ErbB2 is able to form heterodimers with EGFR in both EGF stimulated or not cells (Fig.20 Panel B, lanes 2 and 3 for EGFR and lanes 5 and 6 for ErbB2). Whereas a band corresponding to phosphorylated ErbB2 is present only in EGF stimulated cells, the EGFR is also phosphorylated in the absence of EGF (Fig. 20, Panel A lanes 9 and 19, Panel B lanes 7 and 8) further supporting our hypothesis that gangliosides effect on ErbB2 metabolism are mediated by EGFR.

Second Part: ErbB2 and EGFR localization in HC11 plasma membrane

ErbB2–GM3 colocalization in mammary epithelial HC11 cells

The possible association of ErbB2 with GM3, has been also investigated, in collaboration with Prof. M. Sorice, by scanning confocal microscopy experiments. Cells were labeled with anti-ErbB2 polyclonal serum and then with anti-GM3 monoclonal serum. Results are reportein Fig. 21.

Analysis of GM3 expression and distribution in normal EGF not stimulated HC11 cells (Fig. 18 A) reveal that GM3 staining appeared uneven over the cell surface, similar to that seen on ErbB2 molecule fluorescence. A merged image of the two staining clearly revealed orange areas, resulting from the overlap of green and red fluorescence, which corresponded to colocalization areas. As expected, no staining was observed in D-PDMP treated cells labeled with anti-GM3 serum (Fig. 21, Panel B);in addition the lack of immunolabeling demonstrates the effect of D-PDMP on the depletion of gangliosides and the specificity of the anti-GM3 serum. The distribution of ErbB2 appeared more diffuse compared with control untreated cells. By contrast, overlain areas were reverted when exogenous GM3 was added to ganglioside-depleted HC11 cells (Fig. 21 Panel C).

Scatter-plot diagrams showed how the dual labels are colocalized. Figure 18 D shows a colocalization area that is evident in untreated HC11 cells. In cells treated with [D]-PDMP and then with GM3 a major colocalization index is evident, because the blue area is larger and more directed towards the diagonal line.

ErbB2 preferential association with lipid-raft fractions in HC11 cells

To evaluate the distribution of ErbB2 in raft fractions of HC11 cells, treated or not with D-PDMP, EGF or D-PDMP and EGF, all fractions obtained by sucrose gradient were analyzed by western blot (Fig. 22). The results revealed that in non-EGF-stimulated cells ErbB2 was present mainly in fractions 5 and 6, but also in fractions 7–11 (Fig. 22 Panel A), indicating that ErbB2 is preferentially present in raft fractions. EGF stimulation did not seem to appreciably modify this distribution (Fig. 22 Panel B). Interestingly, D-PDMP treatment induced a drastic cell-surface redistribution of ErbB2 (Fig. 22 Panel C). Indeed, the receptor became completely Triton soluble and was present exclusively in fractions 10 and 11.

[pic]

FIG.21: Scanning confocal microscopy analysis of GM3–ErbB2 association on HC11 cells.

Panel A: Untreated HC11 cells.

Panel B: HC11 cells treated with 30 µM [D]-PDMP for 5 days.

Panel C: HC11 cells treated with 30 µM [D]-PDMP for 5 days and then with 125 µM GM3 for 5 min.

Panel D: Two-dimensional scatter plot analysis of the dual labeled fluorochromes (pseudocolor) GM3–ErbB2. Diagrams show the pixel intensity distribution of a dual-channel section. The x-axis represents intensity from the red channel; the y-axis represents intensity from the green channel; a colocalization area is evident in untreated HC11 cells. In cells treated with [D]-PDMP and then with GM3 a major colocalization index is evident because the blue area is larger and more directed towards the diagonal line. Figure shows analysis of about 40 cells. ErbB2–raft association in HC11 cells.

Identical profile redistribution of ErbB2 was also evident in HC11 cells treated with [D]-PDMP and EGF (Fig. 22 Panel D), indicating that EGF is not determinant in defining the retention of ErbB2 into the lipid rafts.

The effect of D-PDMP treatment was partially abolished by addition of exogenous GM3 to ganglioside- depleted HC11 cells. In fact, after D-PDMP incubation, followed by GM3 treatment, a significant proportion of ErbB2 returned to fractions 4 and 5 (Fig. 22 Panel E). In addition to better clarify the functional role of the association between ErbB2 and lipid rafts, the distribution of phospho-ErbB2 in sucrose-gradient fractions obtained from HC11 cells in the absence or presence of treatment with EGF, D-PDMP and D-PDMP ⁄GM3 has been also analyzed. Although, as expected, virtually no phosphorylated ErbB2 was detected in all the fractions obtained from control cells (Fig. 23 Panel A), after triggering with EGF (Fig. 23 Panel B), phosphorylated ErbB2 was found in both the TX-100-insoluble fractions and the TX-100-soluble fractions. Interestingly, in cells treated with D-PDMP and EGF (Fig. 23 Panel D) a band corresponding to phosphorylated ErbB2 was detected in fractions 10 and 11, whereas in ganglioside-depleted cells and cells treated with D-PDMP and GM3 no ErbB2 phosphorylation was observed (Fig. 23 Panel C and E respectively). These findings support the view that GM3 is mainly involved in retaining ErbB2 in lipid raft domains, but that it is not involved in ErbB2 phosphorylation.

[pic]

FIG. 22: ErbB2 distribution in HC11 sucrose gradient membrane fractions.

Panel A: Untreated HC11 cells;

Panel B: HC11 cells treated with EGF;

Panel C: HC11 cells treated with D-PDMP;

Panel D: HC11 cells treated D-PDMP and then with 10 nM EGF;

Panel E: HC11 cells treated with D-PDMP and then with GM3 for 5 min.

[pic]

FIG.23: Analysis of the distribution of phosphorylated ErbB2 in HC11 sucrose gradient membrane fractions.

Panel A: Untreated HC11 cells;

Panel B: HC11 cells treated with EGF;

Panel C: HC11 cells treated with D-PDMP;

Panel D: HC11 cells treated with D-PDMP and then with EGF;

Panel E: HC11 cells treated with D-PDMP and then with GM3 for 5 min.

EGFR preferential association with lipid-raft fractions in HC11 cells

Because in HC11 cells, the two proteins strictly interact resulting in the formation of ErbB2 ⁄EGFR heterodimers as previously reported, the raft fractions of HC11 cells analyzed previously, were also examined for the distribution of EGFR.

EGFR is present in fractions 5 and 6, but also in fractions 7–11 (Fig. 24 Panel A). In EGF-stimulated cells, movement of the receptor to TX-100-soluble fractions was observed (Fig. 24 Panel B). After D-PDMP treatment, the receptor became completely Triton soluble and is present exclusively in fractions 10 and 11 (Fig. 24 Panel C). After D-PDMP incubation, followed by GM3 treatment, a proportion of EGFR returns to fractions 4–6 (Fig. 24 Panel D).

Profile distribution of raft markers in plasma membrane fractions of HC11 cells

To confirm the correct sucrose density gradient separation of TX-100-insoluble and TX-100-soluble fractions, we analyzed both ganglioside profile of sucrose-gradient fractions from HC11 cells and the distribution pattern of the known raft protein flotillin-2. Gangliosides were extracted in chloroform ⁄ methanol ⁄ water and separated by HPTLC. Resorcinol-positive bands were identified on the basis of their HPTLC mobility, compared with standard reference molecules. Three main resorcinol positive bands, having a retardation factor (Rf) analogous to GM3, GM2 (the most prominent) and GD1a, respectively, were detected (Fig. 25 Panel A). The observation that the main band comigrates with GM2 is not surprising, because this molecule is the main ganglioside constituent in these cells. All the ganglioside bands were exclusively detectable in fractions 4–6, which, under our experimental conditions, correspond to lipid rafts. The analysis was performed loading fraction samples by volume. Because the protein content of TX-100-soluble fractions 10 and 11 was much higher than that of TX-100-insoluble fractions 4–6 (not shown), we can observe that flotillin-2 was consistently enriched in rafts (TX-100-insoluble fractions) (Fig. 25 Panel B).

[pic]

FIG.24: EGFR distribution in HC11 sucrose gradient membrane fractions.

Panel A: Untreated HC11 cells;

Panel B: HC11 cells treated with EGF;

Panel C: HC11 cells treated with D-PDMP;

Panel D: HC11 cells treated with D-PDMP and then with GM3 for 5 min

[pic]

FIG.25: Ganglioside distribution in HC11 sucrose gradient membrane fractions.

Panel A: Gangliosides were extracted in chloroform ⁄ methanol ⁄ water from each fraction and analyzed by HPTLC. St: standard gangliosides GM3, GM2, GM1, GD1a, GD1b, GT1b.

Panel B: Western blotting with anti-(flotillin-2) polyclonal serum in the same fractions.

DISCUSSION

Cell surface gangliosides exist in specialised plasma membrane lipid domains, such as caveolae and lipid rafts, where they play a key role in the modulation of transmembrane signalling. It is known that the specific inhibition of cellular ganglioside synthesis, that conceivably abolishes ganglioside domain formation, as well as the enrichment of cell membrane ganglioside by incubation with exogenous gangliosides, that possibly promotes ganglioside domain formation, can affect several cell events. Particularly, extensive studies support the involvement of gangliosides in the regulation of cell differentiation and growth, and in oncogenesis through the modulation of the activation of several GFRs, including EGFR, PDGFR, TrkA and insulin receptors (127).

It is well known that the EGFR and the structural related protein ErbB2, the ligandless member of the receptor tyrosine-kinase ErbB family, play a fundamental role in pathological and physiological processes in many tissues, including development of mammary tumors and differentiation of mammary gland (259). Many studies investigated the features of homo- or heterodimerization and tyrosine-phosphorylation of ErbB2 and EGFR in response to EGF, the subsequent signalling cascade and their endocytotic pathway (280). However, the factors that can regulate these events are not clearly defined yet.

In this context the main objective of researches carried out during these three years of my PhD was the investigation of the eventual functional relationship between gangliosides, ErbB2 and EGFR so to define if gangliosides can be considered determinant factors in the modulation of EGFR/ErbB2 heterodimerization, activation and metabolism.

The study was performed in HC11 cells, a normal mouse mammary epithelial cell line constitutively co-expressing both ErbB2 and EGFR, where ErbB2 and EGFR expression and activation are depending on the cell confluence degree and on the presence of EGF in the culture medium. In fact, previous immunoblotting analyses (data not presented in this work, see ref. 281) on cell extracts from HC11 cells at different degree of confluence grown in the medium with or without EGF indicated that, in absence of EGF, the increase of cell confluence up-regulates ErbB2 levels; on the contrary, the presence of EGF down-regulates them. In addition, the EGF stimulation results into a rapid phosphorylation of ErbB2. However, changes in the ErbB2 content are not specific for this receptor, because levels of EGFR also vary. Similarly to ErbB2, the EGFR content in non-EGF stimulated cells increases with increasing of cell confluence, whereas it dramatically decreases after exposure to EGF. All these our preliminary observations were in complete agreement with results of Kornilova et al. (277), but, interestingly, the drastic decrease of ErbB2 and EGFR expression levels we observed was obtained after a shirt period of EGF treatment: fifteen minutes of 10 nM EGF stimulation induced changes in the ErbB2 levels identical to those described by Kornilova et al. (277) in HC11 cells grown continuously in the presence of 1.6 nM EGF. On the other hand, the modifications we observed after fifteen minutes of EGF treatment could find a likely explanation through time-dependent internalisation experiments performed by the same authors (277), who demonstrated, by analysing the subcellular distribution of the two receptors, that EGFR is found quite completely in the lysosome fraction after 5 min of EGF treatment and completely degraded after 30 min treatment. The same fate was demonstrated to be followed by ErbB2.

As the treatment of a cell line with D-PDMP, that blocks endogenous ganglioside synthesis, is a known and sound approaches for studying various functional roles of gangliosides, during my PhD we investigated the modulating action of gangliosides on ErbB2 and EGFR activation and metabolism by immunoblotting analyses on cell extracts from ganglioside-depleted HC11 cells. In our study, the quite complete disappearance of all the endogenous gangliosides has been ascertained by HPTLC analysis of the total ganglioside fraction from HC11 cells grown for 5 days in a medium containing 30 (M D-PDMP.

Immunoblotting analyses in D-PDMP-treated HC11 cells stimulated or not with EGF show that the ganglioside depletion promotes significant alterations in the phosporylation levels of ErbB2 respect to normal cells. Moreover, ErbB2 phosphorylation, detectable through changes in its gel electrophoretic mobility, remains EGF-dependent both in the presence and in the absence of gangliosides. On the contrary, in the ganglioside-depleted cells the EGFR phosphorylation occurs also in the absence of the ligand EGF but, more interestingly, the EGF stimulation promotes the appearance of a new molecular species, specifically immunoreactive to anti-EGFR antibody, with a Mr slightly higher than those ones detected in not [D]-PDMP-treated cells. It results entirely CIAP-sensitive suggesting that this new species can arise from an ‘iper’-phosphorylation of the EGFR.

Altogether these findings indicate that gangliosides promote drastic modifications in the phosphorylation levels of ErbB2 and simultaneously induce changes in the phosphorylation of egfr

To investigate if gangliosides are directly involved in the molecular association between ErbB2 and EGFR, experiments of co-immunoprecipitations with anti-EGFR and anti-ErbB2 antibodies were performed. Particularly, samples from normal (not ganglioside-depleted) HC11 cells, stimulated or not with EGF, were subjected to experiments of co-immunoprecipitation by anti-ErbB2 antibody and, subsequently, analysed by western blot with anti-EGFR, anti-phosphotyrosine, and anti-ErbB2 antibodies. Furthermore, the eventual ganglioside species associated to the receptors were analysed after lipid extraction and partitioning of the immunoprecipitates by HPTLC and immunostaining.

Our results indicate that in normal HC11 cells ErbB2 co-immunoprecipitates with EGFR, both in control and in EGF-stimulated cells, suggesting the existence of a close relationship between the receptors even in the absence of the proper activating stimuli; however EGF is indispensable to trigger tyrosine-phosphorylation of both ErbB2 and EGFR. Noteworthy, species with higher molecular mass, immunoreactive to anti-EGFR, anti-phosphotyrosine and anti-ErbB2 antibodies and displaying detergent and reducing agent resistance, are clearly visible in EGF-stimulated cells. These species may represent supra-molecular complexes of phosphorylated ErbB2/EGFR, either heterodimers or oligomers, indicating the strong stabilizing effect of the ligand on the activated receptors. Analogous effect, ascribed to the treatment with agonists, has been observed studying the dimerization and oligomerization of G-protein coupled receptors, like the D2 dopamine receptor and the 5-HT1B and 5-HT1D serotonin receptors (292). Moreover, it has been well demonstrated, at least for EGFR, its ability to form not only homo-/hetero-dimers but also higher order association of receptors after stimulation by the ligand EGF (293); time-resolved phosphorescence anisotropy decay experiments performed by Jovin and collegues (294) established that the EGF-bound EGFR was aggregated in clusters containing 10-50 receptors on the surface of A431 cells. As in our experiments EGFR co-immunoprecipitated with ErbB2, we can not exclude the possible organization of mixed clusters of receptor populations (i.e. ErbB2 and EGFR) at the HC11 cell surface.

Moreover, although GM3 accounts for a low content of the various gangliosides expressed by HC11 cells, the HP-TLC analyses of the total ganglioside fraction extracted from the immunoprecipitates displayed the presence of a unique ganglioside, with Rf analogous to the standard GM3, exclusively in EGF-stimulated cells. The identity of this species was assessed by TLC-immunostaining with anti-GM3 antibody. These results strongly support our hypothesis that gangliosides, and GM3 in particular, can be really involved in the EGFR/ErbB2 heterodimerization through a specific interaction with either or both the receptor molecules.

Literature data indicate that EGFR glycosylation is essential for GM3 binding and GM3-mediated suppression of EGFR activation (295). More recently, the extracellular domain of the human recombinant EGFR was demonstrated to be able to directly bind GM3, in a site that seems different from the EGF-binding site; results indicate that GM3 has the highest affinity among many other ganglioside species, suggesting the importance of the characteristic extracellular saccharidic chain of GM3 molecule (283). It has also been suggested that gangliosides, like GD1a and GM1, may act by altering the membrane topology and favouring a ligand-independent EGFR dimerization, termed pre-dimerization, that in turn enhances the efficiency of binding and signalling once stimulated by the growth factor. No data concerning a possible ErbB2/GM3 interaction are available.

To verify if GM3 specifically interacts with either or both the receptor molecules the immunocomplexes were analysed by western blotting with the specific anti-GM3 antibody; this approach is currently used to investigate the association of co-immunoprecipitated gangliosides and proteins (295, 296). Our results indicate that no immuno-reactive species was detectable in control cells, whereas the existence of two distinct immuno-reactive species was evident in EGF-stimulated cells. Comparison of their electrophoretic mobility with those of ErbB2, EGFR and ErbB2/EGFR higher molecular complexes indicated that GM3 joins with the tyrosine-phosphorylated ErbB2, in a stable manner, only within the ErbB2/EGFR complexes; moreover it is preferentially, or perhaps more stably, associated to the phosphorylated EGFR monomer rather than to the phosphorylated ErbB2 monomer.

Altogether collected data lead us to conclude that if the ganglioside GM3 plays a role in modulating ErbB2/EGFR heterodimer metabolism, its influence is not directly exerted on ErbB2, but it is mediated by molecular interaction with EGFR.

Experiments of co-immunoprecipitations with anti-EGFR and anti-ErbB2 antibodies, followed by western blot with anti-EGFR, anti-phosphotyrosine, and anti-ErbB2 antibodies, were also performed on cell extracts from ganglioside-depleted HC11 cells, stimulated or not with EGF. A tight and transient ligand-independent interaction between EGFR and ErbB2 can be established also in the absence of gangliosides; in fact, two molecular species with Mr consistent to those of EGFR and ErbB2 are clearly distinguishable in ErbB2-immunocomplexes from ganglioside-depleted cells both before and after EGF-stimulation. However, the immonoblotting with anti-EGFR antibodies of ErbB2 immunocomplexes shows the presence of the only ‘iper’-phosphorylated EGFR form, indicating that the absence of gangliosides promotes the formation of heterodimers between ErbB2 and the ‘iper’-phosphorylated EGFR.

Therefore, our data support the hypothesis that the absence of gangliosides (particularly GM3) affect the EGFR phosphorylation state giving rise to an ’iper’-phosphorylated receptor that maintain the levels of ErbB2 phosphorylated higher respect to those observed in normal cells. Further and deeper investigations are now necessary to ascertain how the gangliosides promote the EGFR ‘iper’-phosphorylation, but also to verify what are the consequences of the presence of this EGFR ‘iper’-phosphorylated on the activity and metabolism of the heterodimer.

It is well known that both ErbB2 and EGFR have a putative caveolin binding motif within the conserved kinase domains in their cytoplasmic tail and that they can be located in caveolae (33) and in lipid rafts (47). Therefore, to further support the molecular association between ErbB2/EGFR/GM3 and to investigate the involvement of gangliosides in determining ErbB2 and EGFR membrane localization, preliminary confocal microscopy analysis and subsequent membrane fractionation experiments were also carried out in collaboration with Prof. M. Sorice (università La Sapienza, Roma).

The laser scanning confocal microscopy observations revealed co-localization areas between GM3, a well known marker of lipid rafts, and ErbB2. This finding is in agreement and extends previous observations about the surface distribution of ErbB2, which was mostly excluded from clathrin coated pits on the cell plasma membrane, and it gives further support to the conclusions of Nagy and colleagues (284), who hypothesized the association of ErbB proteins (ErbB2 and ErbB3) with these microdomains by quantitative fluorescence microscopy in SKBR-3 breast cancer cells. In addition, in CHO-K1 cells the expression of GD3 affected, to some extent, the plasma membrane distribution of endogenous ErbB2 (285).

The preferential distribution of ErbB2 in lipid rafts was also clearly demonstrated by our membrane fractionation experiments, which also revealed that EGF is not able to modify the receptor localization. However, the analysis revealed that ErbB2 is not exclusively associated with the raft fractions. This finding is consistent with the observations of Hommelgaard et al. (286), prompting to hypothesize that ErbB2 is in a dynamic equilibrium with lipid rafts in the membrane protrusions so that, at a single time point, only a fraction of ErbB2 is directly interacting with the raft gangliosides. This transient ErbB2-gangliosides interaction could potentially regulate the function of ErbB2 (heterodimerization, signaling and metabolic fate) (287).

The key role played by gangliosides in defining the distribution of ErbB2 into signaling specialized plasma membrane domains was demonstrated by treatment of HC11 cells with D-PDMP. The ganglioside depletion, due to the inhibition of endogenous ganglioside synthesis, showed to have striking effects upon the plasma membrane localization of ErbB2. Indeed, ErbB2 underwent a complete redistribution within the high-density TX-100-soluble fractions of the plasma membrane, indicating, by a novel approach, that gangliosides play a key role in the retention of this protein in lipid rafts. These findings are strongly supported by the observation that D-PDMP does not destroy the organization of lipid rafts, since cholesterol as well as caveolin-1 were still detectable in TX-100 insoluble fractions after treatment with D-PDMP. In addition, treatment with exogenous ganglioside GM3 of ganglioside-depleted HC11 cells induced the return of a significant proportion of ErbB2 in raft fractions. However, both ErbB2 localized in TX-100-soluble and in TX-100-insoluble fractions were phosphorylated.

These data strengthen the view that ganglioside (and in particular GM3) can play an important role in ErbB2 membrane localization but not in its phosphorylation, suggesting that gangliosides might influence the signaling transduction pathways after EGF stimulation by compartmentalizing the receptor in different membrane domains.

The same plasma membrane fractions was also used to analyze EGFR distribution. Conflicting results have been reported in the literature on the presence of EGFR within lipid rafts (288). It may depend on the cell type, the use of different detergents and, mainly, the ganglioside composition of the cells. The latter may also be influenced by cell cycle and/or cell density (281). Zurita et al. (285) found EGFR mainly in TX-100-soluble fractions in CHO-K1 cells, although they observed that EGFR and GD3 co-localized on the cell surface. In the same vein, Wang et al. demonstrated that endogenous overexpression of GM3 promotes coimmunoprecipitation of GM3 with EGFR (289). On the other hand, it has been demonstrated that GM3 can specifically interact with the purified recombinant extracellular domain of EGFR (283) and that this tyrosine-kinase receptor contains a structural domain with targeting information for lipid domains (290).

Our results displayed variations in the distribution profiles rather similar to that of ErbB2. Indeed, in control cells, EGFR is mainly enriched in TX-100 insoluble fractions, whereas treatment with [D]-PDMP and [D]-PDMP/EGF shifts the receptor towards the TX-100 soluble fractions, confirming the straight correlation between the two receptors. However, after stimulation with EGF, we observed a movement of EGFR to Triton-soluble fractions, in agreement with previous studies demonstrating that EGFR is initially concentrated in caveolae within lipid rafts, but rapidly move out of this membrane domain in response to EGF (288). The inefficient movement of ErbB2 out of these microdomains may be related to its impaired internalization by clathrin-coated pits (291).

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FIG

FIG.2: Structure of lipids raft

TM: transmembrane proteins; PC: Phosphstidylcholine;

PE: phosphatidylethanolamine; PS: Phosphatidylserine;

PI: Phosphatidylinositol; CHOL: Cholesterol

GANG: Gangliosides

FIG.3: Model for the role of lipid rafts and caveolae in growth factor receptor function

Panel A: Receptors are localized to either flat lipid rafts (blue receptor) or invaginated caveolae (green receptor).

Panel B: Upon cholesterol depletion, receptors in rafts increase function due to the release of constraints on receptor activity. Receptors in caveolae are retained in the residual elements of the caveolar coat. While receptor function is normal, downstream signaling is blocked due to steric inhibition and the inability to assemble a signaling complex.

FIG.4: Initial signaling events in raft for:

a) IgE receptor (FcεRI)

b) T-cell antigen receptor (TCR)-mediated signaling

Table 1:Glycosphingolipid storage diseases in man and animal models.

Table 2: Summary of the effect of ErbB receptors knockout (KO)in mice

D

FIG.20: Western blot analysis with anti-EGFR, anti-ErbB2 and anti-phosphotyrosine (anti-PY20) in D-PDMP treated cells.

Panel A: Analysis of proteins from total cell lysates and EGFR immunocomplexes with different western blot:

• Anti-EGFR

Lanes 1: proteins from normal cells;

Lanes 2: proteins from EGF stimulated cells;

Lanes 3: EGFR immunocomplexes from normal cells;

Lanes 4: EGFR immunocomplexes from EGF stimulated cells.

• Anti-ErbB2

Lanes 5: proteins from normal cells;

Lanes 6: proteins from EGF stimulated cells;

Lanes 7: EGFR immunocomplexes from normal cells;

Lanes 8: EGFR immunocomplexes from EGF stimulated cells.

• Anti-phosphotyrosine (PY20)

Lanes 9: EGFR immunocomplexes from normal cells;

Lanes 10: EGFR immunocomplexes from EGF stimulated cells.

Panel B: Analysis of proteins from total cell lysates and ErbB2 immunocomplexes with different western blot:

• Anti-EGFR

Lanes 1: proteins from normal cells;

Lanes 2: ErbB2 immunocomplexes from normal cells;

Lanes 3: ErbB2 immunocomplexes from EGF stimulated cells.

• Anti-ErbB2

Lanes 4: proteins from normal cells;

Lanes 5: ErbB2 immunocomplexes from normal cells;

Lanes 6: ErbB2 immunocomplexes from EGF stimulated cells.

• Anti-phosphotyrosine (PY20)

Lanes 7: ErbB2 immunocomplexes from normal cells;

Lanes 8: ErbB2 immunocomplexes from EGF stimulated cells.

;

•

Anti-PY20

Anti-ErbB2

Anti-EGFR

1 2 3 4 5 6 7 8 9 10

A

Anti-PY20

Anti-ErbB2

Anti-EGFR

1 2 3 4 5 6 7 8

B

1 2 3 4 5

EGFR

ErbB2

B

A

EGFR

ErbB2

[pic]

- - 30 30 60 60 30 30 60 60

- - 5 5 5 5 10 10 10 10

T(min)

CIAP(u)

EGF

EGFR

9

7

8

2

6

5

4

3

1

- + - + - + - + - +

10

FIG.15: Western blot with anti-EGFR antibody on cell protein extracts. The lysates were from HC11 cells harvested from 2-day confluent cells

Lane 1: proteins from normal cells;

Lane 2: proteins from EGF stimulated cells;

Lane 3: proteins from D-PDMP treated cells;

Lane 4: proteins from D-PDMP treated and EGF stimulated cells.

2

4

3

1

EGFR

[pic]

D-PDMP

EGF

[pic]

8

7

6

5

-

-

ErbB2

D-PDMP

EGF

C

A

B

2d conf

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