Cancer Research UK Institute for Cancer Studies, The University of Birmingham, Edgbaston, Birmingham B15 2TT, UK
* Author for correspondence (e-mail: f.berditchevski{at}bham.ac.uk)
Accepted 22 July 2003
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Summary |
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Key words: CD82, EGFR, ErbB, Dimerization, Signalling
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Introduction |
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The receptor type tyrosine kinases of the ErbB family (EGFR/ErbB1, ErbB2/neu/HER2, ErbB3 and ErbB4) control various aspects of embryonic development, tissue differentiation and maintenance, and are implicated in tumour progression (Olayioye et al., 2000; Waterman and Yarden, 2001
; Yarden and Sliwkowski, 2001
). The ErbB receptors are activated by soluble and membrane-anchored ligands of EGF family, which bind to the extracellular part of the proteins and induce activation of the cytoplasmic kinase domain of the receptors. The diversity of signalling events and cellular responses induced by ErbB proteins are regulated at various levels including receptor compartmentalisation in lipid microdomains (Miljan and Bremer, 2002
), ligand-induced dimerization (Olayioye et al., 2000
; Yarden and Sliwkowski, 2001
) and intracellular transport of activated receptors (Waterman and Yarden, 2001
). Recent findings strongly suggested that there are multiple ErbB-containing signalling compartments within the plasma membrane. Furthermore, distribution between various surface compartments (controlled by the content of cholesterol and gangliosides in the plasma membrane) may have a dramatic effect on the early events in the ErbB-induced signalling, including ligand binding, ligandinduced dimerization and recruitment of the activated receptors to the clathrin-coated pits (Miljan and Bremer, 2002
; Chen and Resh, 2002
; Roepstorff et al., 2002
; Ringerike et al., 2002
). However, the cellular mechanisms that regulate compartmentalisation of the ErbB proteins on the cell surface remain unknown.
We have previously shown that tetraspanin CD82 is associated with EGFR and attenuates EGF-induced signalling (Odintsova et al., 2000). In this report we present evidence that identifies CD82 as a regulator of compartmentalisation and dimerization of the ErbB receptors. Furthermore, our data suggest that ganglioside GD1a is a mediator of the attenuating function of CD82 towards EGFR.
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Materials and Methods |
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Production of recombinant soluble proteins rs-LECL-CD82 and rs-EMMPRIN
rs-LECL-CD82 and rs-EMMPRIN were produced in 293T cells. pIg-CD82-Fc and pIg-EMMPRIN-Fc plasmids were constructed using a standard overlapping PCR protocol. The large extracellular loop of CD82 was `fused' in frame with the murine Ig kappa-chain secretion signal at the N terminus and cloned (HindIII-BamHI) into pIG-1 vector (a gift from C. Buckley, University of Birmingham, UK). The extracellular domain of EMMPRIN (17) containing the leader sequence was amplified and cloned (HindIII-BamHI) into pIG-1 vector. The cells were transfected with the pIG-CD82-Fc and pIg-EMMPRIN-Fc plasmids and the growth medium conditioned by the cells was collected 7-10 days later for purification. The recombinant soluble proteins were purified from the conditioned medium by affinity purification on protein A conjugated to agarose beads.
Immunoprecipitation and western blotting
The proteins were solubilised into the immunoprecipitation buffer containing 1% Brij98/PBS (or 1% Triton X-100/PBS), 2 mM phenylmethylsulphonyl fluoride, 10 µg/ml aprotinin, 10 µg/ml leupeptin for 2 hours at 4°C. The insoluble material was pelleted at 7000 g for 10 minutes. The cell lysates were then precleared by incubation for 2 hours at 4°C with agarose beads conjugated with goat anti-mouse antibodies (mIgG-beads; Sigma). Immune complexes were collected using appropriate mAbs prebound to mIgG-beads and washed four times with the immunoprecipitation buffer. The complexes were eluted from the beads with Laemmli sample buffer. Proteins were resolved in 6-10% SDS-PAGE, transferred to a nitrocellulose membrane and developed with the appropriate Ab. Protein bands were visualised using horseradish peroxidaseconjugated secondary antibodies (Sigma) and Enhanced Chemiluminescence reagent (Amersham Pharmacia Biochem).
Dimerization assay
Cells grown on two 100 mm2 tissue culture plates (7-8x106 cells) were washed three times with ice-cold PBS and incubated with
50-100 ng/ml 125I-ligand (EGF, TGF
, HRG) in serum-free DMEM/0.1% BSA for 2 hours at 4°C with regular rocking. Subsequently, the medium was discarded and cells were carefully washed with ice-cold PBS, to remove non-bound ligand. Then the cells were incubated with 0.5 mM non-permeable cross-linking reagent, bis(sulphosuccinimidyl) suberate [BS3(Pierce)], at 4°C for 1 hour. The reaction was quenched by further incubation with 50 mM Tris-HCl, pH 7.5. The cells were washed with ice-cold PBS and lysed overnight in lysing buffer based on 1% Triton X-100. Cell lysates were precleared by incubation for 2 hours at 4°C with mIgG-beads. Immune complexes were collected on protein A- or mIgG-beads that were preincubated with monoclonal or polyclonal antibodies against respective ErbB receptors. After washing with the immunoprecipitation buffer the complexes were eluted from the beads with Laemmli sample buffer, loaded onto a 6% polyacrylamide gel and subjected to SDS-PAGE. The gel was dried and exposed to X-ray film (Kodak) at 70°C.
Fractionation in sucrose gradient
Cells were lysed for 10 minutes in ice-cold 1% Brij98/MES (25 mM, pH 6.5) buffer supplemented with 100 µM Na3VO4, 5 mM NaF, 10 mM Na4P2O7, 10 µg/ml aprotinin, 10 µg/ml leupeptin and 2 mM PMSF. Lysates (derived from 9x106 cells) were passed successively twenty times through the 25G hypodermic needles and 800 µl of lysate was mixed with two volumes of 2 M sucrose solution. This layer was overlaid with 200 µl layers of decreasing concentration of sucrose from 0.9 M to 0.2 M. Samples were centrifuged at 100,000 g for 16-18 hours at 4°C in a Beckman SW60 rotor, and 9 fractions each of 200 µl were then collected from the top of gradient. The pellet was suspended in 200 µl of the lysis buffer. Equal amounts of each fraction were mixed with 4x Laemmli loading buffer and the proteins were resolved in 10% SDS-PAGE.
Immunofluorescence staining
Cells were grown on glass coverslips in complete media for 24-36 hours. Spread cells were fixed with 2% paraformaldehyde/PBS for 10-15 minutes. Staining with primary and fluorochrome-conjugated secondary antibodies was carried out as previously described (Berditchevski and Odintsova, 1999). Staining was analysed using a Nikon Eclipse E600 microscope. Images were acquired using a Leica DC200 digital camera and subsequently processed using a DC200 image processing programme.
Flow cytometry
Cells were incubated with saturating concentrations of primary mouse mAbs for 45 minutes at 4°C, washed twice and then labelled with fluorescein isothiocyanate (FITC)-conjugated goat anti-mouse immunoglobulin. Stained cells were analysed using Coulter Epics programme (Becton Dickinson).
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Results |
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Soluble large extracellular loop of CD82 does not interfere with EGF-induced dimerization
The crystal structure of ligand-bound EGFR indicates that the extracellular domain plays a critical role in homodimerization of the receptor (Ogiso et al., 2002; Garrett et al., 2002
). The structure revealed that ligand binding generates a conformational change in the receptor extracellular domain and leads to the exposure of a `dimerization arm' that subsequently interacts with a similar part on a dimerization partner (Ogiso et al., 2002
; Garrett et al., 2002
). Although, the proposed model does not offer a mechanism for the formation of heterodimers involving the ligand-less ErbB2, it has been recently established that the dimerization loop of this protein is maintained in the constitutively `open' configuration thus making it a preferable dimerization partner for other ErbB receptors (Garrett et al., 2003
). Given the crucial role played by the tetraspanin large extracellular loop (LECL) in regulation of the biological activities of the associated protein partners (Baudoux et al., 2000
; Berditchevski, 2001
; Boucheix and Rubinstein, 2001
; Hemler, 2001
) we hypothesised that this part of the protein may be important in conferring the negative effect of CD82 on the EGFR-ErbB2 dimerization. To test this hypothesis we carried out dimerization experiments in the presence of a recombinant soluble protein containing LECL of CD82 (rs-LECL-CD82). Firstly, we established that rs-LECLCD82 (produced in 293T cells) could be immunoprecipitated with a panel of four anti-CD82 mAbs (Fig. 1C). This suggests that overall folding of LECL-CD82 is similar to that of a native protein. For the control experiments we also produced a recombinant soluble protein containing the extracellular domain of EMMPRIN (rs-EMMPRIN), an unrelated transmembrane protein (Biswas et al., 1995
; Berditchevski et al., 1997
). Cells were pre-incubated with rs-LECL-CD82 (or rs-EMMPRIN) and subsequently stimulated with 125I-EGF in the presence of the recombinant-soluble proteins (Fig. 1D). As expected, in the control experiments (i.e. no recombinant soluble protein added) a number of EGFR-ErbB2 dimers formed in HB2/CD82 cells was significantly lower than in HB2/ZEO cells (Fig. 1D, compare lanes 1 and 5 in top and bottom panels). However, we also found that the presence of rs-LECL-CD82 (or rs-EMMPRIN) did not affect the formation of EGFR-ErbB2 dimers in either HB2/ZEO or HB2/CD82 cells (Fig. 1D, top and bottom panels, lanes 2-4 and 6-8). Although these results do not rule out completely a possible involvement of LECL-CD82 in the dimerization they indicate that the other part(s) of the protein may be critical for interference with the dimerization process.
CD82 does not affect spontaneous dimerization of ErbB receptors
There is evidence that in some cells ErbB proteins undergo spontaneous dimerization in the absence of ligand (Yu et al., 2002). This can prime the receptors to more efficient ligand binding and, in turn, facilitate ligand-induced dimerization. To investigate whether expression of CD82 affects ligandindependent dimerization, serum-starved HB2/ZEO and HB2/CD82 cells were pre-treated with a membraneimpermeable chemical crosslinker and EGFR- and ErbB2-containing complexes were purified using an immunoprecipitation protocol. Formation of EGFR-ErbB2 dimers was subsequently examined by western blotting. As illustrated in Fig. 2 there was a small number of the pre-formed ErbB homodimers in both HB2/ZEO and HB2/CD82 cells (detected as proteins of a higher molecular mass in the EGFR and ErbB2 immunoprecipitates; lanes 1, 2 and 7, 8). However, the densitometric analysis of the signals showed no apparent differences between the cell lines. Furthermore, no pre-formed EGFR-ErbB2 heterodimers were detected in either HB2/ZEO or HB2/CD82 cells (Fig. 2, lanes 3, 4 and 5, 6). These data indicate that CD82 only affects ligand-induced dimerization.
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CD82 is associated with ErbB3 and ErbB2 but does not interfere with formation of ErbB2-ErbB3 dimers
To examine whether CD82 influences ligand-induced dimerization of the ErbB receptors, other than EGFR, we carried out the dimerization assay using a pair of newly established cell lines, MCF-7/CD82 and MCF-7/ZEO. Parental MCF-7 cells express high levels of ErbB2 and ErbB3 but no EGFR or CD82. Thus, by stimulating the transfectants with 125I-HRG, a well-established ligand for ErbB3, we were able to examine the effect of the tetraspanin on formation of the ErbB2-ErbB3 heterodimers. MCF-7/ZEO and MCF-7/CD82 cells pre-incubated with the labelled ligand were treated with BS3 and ErbB complexes were recovered from the cellular lysates using either anti-ErbB2 or anti-ErbB3 Abs (expression levels of both receptors are illustrated in Fig. 3A). As illustrated in Fig. 3 formation of 125I-HRG-ErbB dimer complexes was detected in both cell lines (the upper protein band in the ErbB2 and ErbB3 immunoprecipitates). However, the amount of immunoprecipitated dimers was similar for MCF/ZEO and MCF-7/CD82 cells (Fig. 3B, compare lanes 1 and 2, and 3 and 4). These results indicated that although CD82 has a negative role in formation of EGFR-ErbB2 dimers, ligand-induced dimerization of ErbB3 with ErbB2 is not affected by this tetraspanin.
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To examine the mechanisms underlying functional selectivity of CD82 towards ligand-induced formation of various ErbB heterodimers we analysed the association of CD82 with ErbB2 and ErbB3 in HB2/CD82 and MCF7/CD82 cells. Fig. 4 illustrates that ErbB3 could be coimmunoprecipitated by anti-CD82 antibodies from protein lysates prepared from both cell lines (Fig. 4B, lanes 2, 8). In addition, we found that CD82 forms complexes with ErbB3 in T47D cells, which express both of these proteins endogenously (Fig. 4B, lane 5). In contrast, the association of CD82 with ErbB2 was cell-type specific (Fig. 4B): it could be detected in MCF-7/CD82 cells but not in HB2/CD82 or T47D cells (Fig. 4B, top panels, lanes 2, 8 and 5, respectively). In the control experiments we showed that anti-CD82 mAb efficiently precipitated the protein from all cell lines (Fig. 4B, lower panels, lanes 2, 5, 8). Furthermore, the level of expression of ErbB2 in T47D and HB2/CD82 is similar or higher of that detected in MCF-7/CD82 cells (Fig. 4A). These results indicate that the assembly of CD82-ErbB2 and CD82-ErbB1 complexes is controlled by distinct mechanisms. Although the molecular mechanisms that underlie these differences remain to be established one possibility is that EGFR exerts a negative effect on the formation of the CD82-ErbB2 complex (as observed for HB2/CD82 and T47D cells). Previously we reported that CD82/EGFR complex was destabilised in the presence of EGF (Odintsova et al., 2000). In contrast, we have found that stability of ErbB2-CD82 and ErbB3-CD82 complexes in MCF-7/CD82 cells is not affected after HRG treatment (Fig. 4C). Similar amounts of ErbB3 and ErbB2 were precipitated by anti-CD82 mAb from unstimulated or HRG-treated MCF-7/CD82 cells (Fig. 4C, compare lanes 1 and 2, 4 and 5, 6). These data demonstrate that ligand binding has a differential effect on the stability of various CD82-ErbB complexes. Taken together, our results demonstrate that the association itself does not predicate the negative effect of CD82 on ligand-induced dimerization of ErbB receptors.
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CD82 changes membrane compartmentalisation of EGFR
Recent findings suggest that there are multiple EGFR-containing signalling compartments within the plasma membrane (Mineo et al., 1999; Waugh et al., 1999
; Miljan and Bremer, 2002
). Furthermore, membrane compartmentalisation has a significant effect on the ligand-induced dimerization and signalling potential of activated EGFR. To investigate whether expression of CD82 changes the membrane microenvironment of EGFR and ErbB2 we fractionated HB2/CD82 and HB2/ZEO cellular lysates in a sucrose gradient. The distribution of proteins in the gradient fractions was assessed by immunoblotting. Our data have shown that EGFR from HB2/ZEO cells is distributed almost evenly between the gradient fractions 5, 6, 7, 8 and 9 (Fig. 5): densitometric measurements of the blots indicate that the combined amount of EGFR in these fractions is over 85% of the total amount of the protein detected in all fractions of the gradient. The amount of the receptor detected in fraction 4 is only 8.5% of the total protein and no signal was detected in the first three fractions of the gradient. However, we have consistently observed (in three independent experiments) that in HB2/CD82 cells the distribution of EGFR has been shifted to the left towards light fractions of the gradient. Over 50% of EGFR was concentrated in fractions 4, 5 and 6 (Fig. 5), with fraction 4 containing the largest amount of the receptor (over 20%). Furthermore, approximately 7% of the total protein could be detected in the third fraction of the gradient. CD82 has a similar effect on the fractional distribution of ErbB2: the protein from HB2/ZEO cells was abundant in fractions 5 and 6 (
24% and
22% of the total amount, respectively). No signal was detected in the gradient fraction 3 and less than 9% of the protein was found in fraction 4. In contrast, similar amounts (
20%) of ErbB2 from HB2/CD82 cells were detected in both fraction 4 and 5 of the gradient. In addition, more than 8% of the protein was found in fraction 3 of the gradient. Given the fact that ErbB2-CD82 complex is not detectable in HB2/CD82 cells (Fig. 4B), we concluded that the alteration of floating characteristics of ErbB2 was independent of its association with the tetraspanin. In fact, no CD82 was detected in the fraction 3 and less than 5% of the protein was found in fraction 4 of the gradient (Fig. 5). Thus, we propose that the expression of CD82 causes global changes in physico-chemical characteristics of the plasma membrane, which has a general impact on the organisation of various microdomains. Indeed, the fractional distribution of caveolin, a cholesterol-associated membrane protein that is not linked to tetraspanin microdomains, was also affected by CD82 (Fig. 5). In the control experiments we found that the fractional distribution of EMMPRIN was not influenced by the expression of CD82 (Fig. 5). These results suggest that CD82 only affects membrane compartmentalisation of proteins that are associated with lipid rafts.
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CD82 increases surface expression of gangliosides GM1 and GD1a and changes distribution of EGFR and GD1a on the plasma membrane
To further illustrate differences in membrane compartmentalisation of EGFR we analysed surface distribution of the receptor by immunofluorescence staining. In the majority of HB2/ZEO cells (>70%) fine clusters of EGFR were evenly distributed over the cell surface (Fig. 6A). In contrast, in more than 80% of HB2/CD82 cells EGFR was more concentrated towards the cell periphery (Fig. 6B). Gangliosides play an important role in regulation of signalling through EGFR (Miljan and Bremer, 2002). We examined whether the effect of CD82 on compartmentalisation and dimerization of EGFR correlates with surface distribution of gangliosides. Flow cytometry experiments have shown that out of four gangliosides that are known to influence EGF-dependent signalling (i.e. GM3, GM1, GD1a and GT1b) (Zurita et al., 2001
; Miljan and Bremer, 2002
; Mirkin et al., 2002
), only GM1 and GD1a could be detected on the surface of HB2/ZEO and HB2/CD82 cells (Table 1). Notably, expression of both GM1 and GD1a was significantly higher in HB2/CD82 cells with more dramatic difference observed for GD1a (
2.8-fold increase). This observation was confirmed further in immunofluorescence experiments (Fig. 6C,D). Interestingly, in HB2/CD82 cells a significant proportion of GD1a clusters re-localised towards cell margins and contained CD82 (Fig. 6D,G,H) and EGFR (Fig. 6I,J). In contrast, cellular distribution of ganglioside GM1 was similar in HB2/ZEO and HB2/CD82 cells (Fig. 6E,F). We observed similar changes in the surface expression (
2.5 fold) and distribution of GD1a in MCF-7/CD82 cells (Fig. 7 compare A and B). Furthermore, a significant proportion of GD1a in these cells was co-localised with CD82 (Fig. 7B-D). Taken together these results confirm that CD82 causes re-distribution of EGFR and GD1a, and suggest that ganglioside GD1a may function as mediators of activity of the tetraspanin towards the receptor.
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Discussion |
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The effect of CD82 on dimerization correlates with redistribution of EGFR and ErbB2 in cells (detected by sucrose gradient fractionation and immunofluorescence staining). We propose that this observation is indicative of a novel function of CD82 as a regulator of compartmentalisation of proteins associated with the plasma membrane. Furthermore, our results show that the effect of CD82 on compartmentalisation is not limited to the tetraspanin-associated proteins (e.g. compartmentalisation of caveolin is also affected in HB2/CD82 cells). Not only does this observation distinguish CD82 from CD151 (a tetraspanin that specifically regulates compartmentalisation of the integrin 3ß1) (Berditchevski et al., 2002
), but it also suggests that the elevated expression of CD82 (and consequent changes in compartmentalisation) may significantly affect the function of a broad range of transmembrane receptors. In this regard, it has been previously reported that the elevated expression of CD82 in T cells stimulated integrin-mediated cell-cell adhesion (Shibagaki et al., 1999
). Although the effect of the tetraspanin on compartmentalisation of integrins has not been analysed, recent results clearly demonstrated that localisation in lipid rafts has a key role in regulation of integrin functions (Leitinger and Hogg, 2002
).
Compartmentalisation of EGFR in microdomains on the surface membrane has a significant impact on the early events associated with the receptor activation. These include ligandbinding, dimerization and re-localisation of activated receptors into clathrin-coated pits (Mineo et al., 1999; Carpenter, 2000
). Although the mechanistic link between signalling via EGFR and its localisation in microdomains is not established, one possibility is that localisation in microdomains affects its spatial orientation relative to other ErbB proteins, an important factor in ligand-induced dimerization of the receptors. Recent results clearly established that lipid microenvironment plays an important role in signalling via EGFR (Miljan and Bremer, 2002
; Chen and Resh, 2002
; Pike and Casey, 2002
; Roepstorff et al., 2002
; Ringerike et al., 2002
). For example, EGFR has been found in the cholesterol-enriched microdomains, and pharmacological depletion of cholesterol profoundly increased ligand-induced dimerization and activation of the receptor (Chen and Resh, 2002
; Pike and Casey, 2002
; Roepstorff et al., 2002
; Ringerike et al., 2002
). EGF-induced signalling is also affected by gangliosides (reviewed by Miljan and Bremer, 2002
). Various gangliosides were shown to interact with the extracellular domain of EGFR and either inhibit or stimulate ligand-induced activation of the receptor (Miljan et al., 2002
). This association is dependent on glycosylation of EGFR (Wang et al., 2001b
), which in turn, regulates the conformation of the extracellular domain and ligand-induced dimerization of the receptor (Bishayee, 2000
; Tsuda et al., 2000
; Fernandes et al., 2001
). In this regard, we found that expression of CD82 had a marked effect on surface expression of gangliosides GM1 and GD1a (Table 1 and Fig. 6). It remains to be established how CD82 affects expression of gangliosides. One possibility is that CD82 targets one of the glycosyltransferases responsible for biosynthesis of gangliosides. In cells the activity of various glycosyltransferases, residents of Golgi apparatus can be regulated at the transcriptional (Kolter et al., 2002
) and posttranscriptional levels (Yu and Bieberich, 2001
). For example, both N-acetylgalactosaminyltransferase (GalNAcT) and sialyltransferase IV (SAT IV), an enzyme that converts GM1 into GD1a, are regulated by phosphorylation (Yu and Bieberich, 2001
). Furthermore, it has been established that the activity of GalNAcT is regulated via the cAMP-dependent mechanisms. However, SAT IV is associated with a 33kD isoform of 14-3-3 proteins (Gao et al., 1996
), well established participants of various signalling pathways (Yaffe, 2002
). Alternatively, CD82 may affect the activity (or expression level) of membrane sialidases. Interestingly, it has been reported that elevated expression of Neu3, a lipid raftassociated sialidase, enhanced signalling via EGFR (Wang et al., 2001a
). Finally, it is possible that CD82 stabilises GD1a and GM1 on the cell surface by suppressing shedding of these gangliosides from the plasma membrane or their internalisation (Dolo et al., 2000
).
Several lines of evidence suggest that GD1a plays a major role in the attenuating activity of CD82 towards EGFR in HB2 cells. Firstly, expression of CD82 specifically affected surface distribution of GD1a whereas distribution of GM1 has not changed. Secondly, whereas a significant proportion of GD1a clusters also contained EGFR, co-localisation of EGFR with GM1 was less apparent. Finally, it has been recently demonstrated that GD1a and not GM1 can inhibit EGF-induced signalling in intact cells (Mirkin et al., 2002). Further experiments will be needed to establish the connection between the GD1a-enriched microdomains and a modulatory effect of CD82 on other transmembrane receptors (e.g. integrins, T-cell receptor complex). In this regard it is important to emphasize that localisation of transmembrane receptors in lipid-ordered microdomains may have opposing effects on the receptors' activity. Whereas disruption of the cholesterol-enriched microdomains potentiates signalling via receptor tyrosine kinases (Chen and Resh, 2002
; Pike and Casey, 2002
; Roepstorff et al., 2002
; Ringerike et al., 2002
), it inhibits the signalling function of the FAS/CD95 complex (Hueber et al., 2002
). Similarly, in various cell types both GD1a and CD82 can either negate (Van Brocklyn et al., 1993
; Odintsova et al., 2000
; Mirkin et al., 2002
) or potentiate (Zuberbier et al., 1995
; Shibagaki et al., 1999
; Lang et al., 2001
) transmembrane signalling triggered by different types of the receptors.
How can CD82 influence membrane compartmentalisation of GD1a and EGFR? Firstly, CD82 may directly regulate the distribution of these molecules. Being palmitoylated on multiple sites (Charrin et al., 2002; Yang et al., 2002
; Berditchevski et al., 2002
) CD82 has an inherent tendency to coalesce in the lipid-ordered microdomains assembled within the inner leaflet of the plasma membrane. Inevitably, this would have an affect on distribution and dynamics of CD82-associated molecules (e.g. EGFR). Interestingly, it has been reported that when added exogenously to colorectal cancer cells ganglioside GM3 could form complexes with the tetraspanin CD9 (Ono et al., 1999
). Furthermore, it has been proposed that this association is critical for anti-migratory function of CD9 (Ono et al., 2001
). Although physical interaction between CD82 and GD1a is yet to be established, our data show that this ganglioside is present in the CD82-containing clusters on the cell surface. This observation suggests that CD82 may function as a molecular linker that physically connects GD1a-enriched microdomains of the outer leaflet with lipid-ordered domains of the inner leaflet of the membrane. Secondly, CD82 is known to associate with various protein kinase C isoforms (Zhang et al., 2001
), enzymes that have a short-term influence on the dynamics of glycolipid domains in the plasma membrane (Pitto et al., 1999
) and regulate recruitment of cellular proteins into lipid-ordered microdomains (Parolini et al., 1999
). Finally, CD82 is associated with and may modulate the activity of CD36 (E.O. and F.B., unpublished results), a transmembrane protein that functions as a fatty acid and cholesterol transporter (Febbraio et al., 2001
). This may have a general impact on the lipid content of the plasma membrane and, consequently, distribution of GD1a and EGFR on the cell surface.
In summary, we have shown that CD82 attenuates the activity of EGFR by redistributing the receptor into the GD1a-containing microdomains on the cell surface. Not only do these results link the function of CD82 with a particular ganglioside, but they also provide a new mechanistic insight into the role of the tetraspanin in a signalling process.
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Acknowledgments |
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References |
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---|
Baudoux, B., Castanares-Zapatero, D., Leclercq-Smekens, M., Berna, N. and Poumay, Y. (2000). The tetraspanin CD9 associates with the integrin alpha6beta4 in cultured human epidermal keratinocytes and is involved in cell motility. Eur. J. Cell Biol. 79, 41-51.[Medline]
Berditchevski, F. (2001). Complexes of tetraspanins with integrins: more than meets the eye. J. Cell Sci. 115, 4143-4151.[Medline]
Berditchevski, F. and Odintsova, E. (1999). Characterization of integrin/tetraspanin adhesion complexes: role of tetraspanins in integrin signalling. J. Cell Biol. 146, 477-492.
Berditchevski, F., Chang, S., Bodorova, J. and Hemler, M. E. (1997). Generation of monoclonal antibodies to integrin-associated proteins. Evidence that alpha3beta1 complexes with EMMPRIN/basigin/OX47/M6. J. Biol. Chem. 272, 29174-29180.
Berditchevski, F., Odintsova, E., Sawada, S. and Gilbert, E. (2002). Expression of the palmitoylation-deficient CD151 weakens the association of alpha 3 beta 1 integrin with the tetraspanin-enriched microdomains and affects integrin-dependent signalling. J. Biol. Chem. 277, 36991-37000.
Bishayee, S. (2000). Role of conformational alteration in the epidermal growth factor receptor (EGFR) function. Biochem. Pharmacol. 60, 1217-1223.[CrossRef][Medline]
Biswas, C., Zhang, Y., DeCastro, R., Guo, H., Nakamura, T., Kataoka, H. and Nabeshima, K. (1995). The human tumor cell-derived collagenase stimulatory factor (renamed EMMPRIN) is a member of the immunoglobulin superfamily. Cancer Res. 55, 434-439.[Abstract]
Boucheix, C. and Rubinstein, E. (2001). Tetraspanins. Cell. Mol. Life Sci. 58, 1189-1205.[Medline]
Carpenter, G. (2000). The EGF receptor: a nexus for trafficking and signalling. BioEssays 22, 697-707.[CrossRef][Medline]
Charrin, S., Manié, S., Qualid, M., Billard, M., Boucheix, C. and Rubinstein, E. (2002). Differential stability of tetraspanin/tetraspanin interactions: role of palmitoylation. FEBS Lett. 516, 139-144.[CrossRef][Medline]
Chen, X. and Resh, M. D. (2002). Cholesterol depletion from the plasma membrane triggers ligand-independent activation of the epidermal growth factor receptor. J. Biol. Chem. 277, 49631-49637.
Dolo, V., Li, R., Dillinger, M., Flati, S., Manela, J., Taylor, B. J., Pavan, A. and Ladisch, S. (2000). Enrichment and localization of ganglioside G(D3) and caveolin-1 in shed tumor cell membrane vesicles. Biochim. Biophys. Acta 1486, 265-274.[Medline]
Febbraio, M., Hajjar, D. P. and Silverstein, R. L. (2001). CD36: a class B scavenger receptor involved in angiogenesis, atherosclerosis, inflammation, and lipid metabolism. J. Clin. Invest. 108, 785-791.
Fernandes, H., Cohen, S. and Bishayee, S. (2001). Glycosylation-induced conformational modification positively regulates receptor-receptor association: a study with an aberrant epidermal growth factor receptor (EGFRvIII/DeltaEGFR) expressed in cancer cells. J. Biol. Chem. 276, 5375-5383.
Gao, L., Gu, X. B., Yu, D. S., Yu, R. K. and Zeng, G. (1996). Association of a 14-3-3 protein with CMP-NeuAc:GM1 alpha 2,3-sialyltransferase. Biochem. Biophys. Res. Com. 224, 103-107.[CrossRef][Medline]
Garrett, T. P., McKern, N. M., Lou, M., Elleman, T. C., Adams, T. E., Lovrecz, G. O., Zhu, H. J., Walker, F., Frenkel, M. J., Hoyne, P. A. et al. (2002). Crystal structure of a truncated epidermal growth factor receptor extracellular domain bound to transforming growth factor alpha. Cell 110, 763-773.[Medline]
Garrett, T. P., McKern, N. M., Lou, M., Elleman, T. C., Adams, T. E., Lovrecz, G. O., Kofler, M., Jorissen, R. N., Nice, E. C., Burgess, A. W. and Ward, C. W. (2003). The crystal structure of a truncated ErbB2 ectodomain reveals an active conformation, poised to interact with other ErbB receptors. Mol. Cell 11, 495-505.[Medline]
Graus-Porta, D., Beerli, R. R., Daly, J. M. and Hynes, N. E. (1997). ErbB2, the preferred heterodimerization partner of all ErbB receptors, is a mediator of lateral signalling. EMBO J. 16, 1647-1655.
Hemler, M. E. (2001). Specific tetraspanin functions. J. Cell Biol. 155, 1103-1107.
Hueber, A. O., Bernard, A. M., Herincs, Z., Couzinet, A. and He, H. T. (2002). An essential role for membrane rafts in the initiation of Fas/CD95-triggered cell death in mouse thymocytes. EMBO Rep. 3, 190-196.
Kolter, T., Proia, R. L. and Sandhoff, K. (2002). Combinatorial ganglioside biosynthesis. J. Biol. Chem. 277, 25859-25862.
Lang, Z., Guerrera, M., Li, R. and Ladisch, S. (2001). Ganglioside GD1a enhances VEGF-induced endothelial cell proliferation and migration. Biochem. Biophys. Res. Com. 282, 1031-1037.[CrossRef][Medline]
Leitinger, B. and Hogg, N. (2002). The involvement of lipid rafts in the regulation of integrin function. J. Cell Sci. 115, 963-972.
Miljan, E. A. and Bremer, E. G. (2002). Regulation of growth factor receptors by gangliosides. Sci. STKE. 2002, RE15.[Medline]
Miljan, E. A., Meuillet, E. J., Mania-Farnell, B., George, D., Yamamoto, H., Simon, H. G. and Bremer, E. G. (2002). Interaction of the extracellular domain of the epidermal growth factor receptor with gangliosides. J. Biol. Chem. 277, 10108-10113.
Mineo, C., Gill, G. N. and Anderson, R. G. (1999). Regulated migration of epidermal growth factor receptor from caveolae. J. Biol. Chem. 274, 30636-30643.
Mirkin, B. L., Clark, S. H. and Zhang, C. (2002). Inhibition of human neuroblastoma cell proliferation and EGF receptor phosphorylation by gangliosides GM1, GM3, GD1A and GT1B. Cell Prolif. 35, 105-115.[CrossRef][Medline]
Odintsova, E., Sugiura, T. and Berditchevski, F. (2000). Attenuation of EGF receptor signalling by a metastasis suppressor tetraspanin KAI-1/CD82. Curr. Biol. 10, 1009-1012.[CrossRef][Medline]
Ogiso, H., Ishitani, R., Nureki, O., Fukai, S., Yamanaka, M., Kim, J. H., Saito, K., Sakamoto, A., Inoue, M., Shirouzu, M. and Yokoyama, S. (2002). Crystal structure of the complex of human epidermal growth factor and receptor extracellular domains. Cell 110, 775-787.[Medline]
Olayioye, M. A., Neve, R. M., Lane, H. A. and Hynes, N. E. (2000). The ErbBH signalling network: receptor heterodimerization in development in cancer. EMBO J. 19, 3159-3167.
Ono, M., Handa, K., Withers, D. A. and Hakomori, S. (1999). Motility inhibition and apoptosis are induced by metastasis-suppressing gene product CD82 and its analogue CD9, with concurrent glycosylation. Cancer Res. 59, 2335-2339.
Ono, M., Handa, K., Sonnino, S., Withers, D. A., Nagai, K. and Hakomori, S. (2001). GM3 ganglioside inhibits CD9-facilitated haptotactic cell motility: Coexpression of GM3 and CD9 is essential in the downregulation of tumor cell motility and malignancy. Biochem. 40, 6414-6421.[CrossRef][Medline]
Parolini, I., Topa, S., Sorice, M., Pace, A., Ceddia, P., Montesoro, E., Pavan, A., Lisanti, M. P., Peschle, C. and Sargiacomo, M. (1999). Phorbol ester-induced disruption of the CD4-Lck complex occurs within a detergent-resistant microdomain of the plasma membrane. Involvement of the translocation of activated protein kinase C isoforms. J. Biol. Chem. 274, 14176-14187.
Pike, L. J. and Casey, L. (2002). Cholesterol levels modulate EGF receptormediated signalling by altering receptor function and trafficking. Biochemistry 41, 10315-10322.[CrossRef][Medline]
Pitto, M., Palestini, P., Ferraretto, A., Flati, S., Pavan, A., Ravasi, D., Masserini, M. and Bottiroli, G. (1999). Dynamics of glycolipid domains in the plasma membrane of living cultured neurons, following protein kinase C activation: a study performed by excimer-formation imaging. Biochem. J. 344, 177-184.[CrossRef][Medline]
Ringerike, T., Blystad, F. D., Levy, F. O., Madshus, I. H. and Stang, E. (2002). Cholesterol is important in control of EGF receptor kinase activity but EGF receptors are not concentrated in caveolae. J. Cell Sci. 115, 1331-1340.
Roepstorff, K., Thomsen, P., Sandvig, K. and van Deurs, B. (2002). Sequestration of epidermal growth factor receptors in non-caveolar lipid rafts inhibits ligand binding. J. Biol. Chem. 277, 18954-18960.
Shibagaki, N., Hanada, K., Yamaguchi, S., Yamashita, H., Shimada, S. and Hamada, H. (1998). Functional analysis of CD82 in the early phase of T cell activation: roles in cell adhesion and signal transduction. Eur. J. Immunol. 28, 1125-1133.[CrossRef][Medline]
Shibagaki, N., Hanada, K., Yamashita, H., Shimada, S. and Hamada, H. (1999). Overexpression of CD82 on human T cells enhances LFA-1 / ICAM1-mediated cell-cell adhesion: functional association between CD82 and LFA-1 in T cell activation. Eur. J. Immunol. 29, 4081-4091.[CrossRef][Medline]
Tsuda, T., Ikeda, Y. and Taniguchi, N. (2000). The Asn-420-linked sugar chain in human epidermal growth factor receptor suppresses ligand-independent spontaneous oligomerization. Possible role of a specific sugar chain in controllable receptor activation. J. Biol. Chem. 275, 21988-21994.
Van Brocklyn, J., Bremer, E. G. and Yates, A. J. (1993). Gangliosides inhibit platelet-derived growth factor-stimulated receptor dimerization in human glioma U-1242MG and Swiss 3T3 cells. J. Neurochem. 61, 371-374.[Medline]
Wang, X., Rahman, Z., Sun, P., Meuillet, E., George, D., Bremer, E. G., Al Qamari, A. and Paller, A. S. (2001a). Ganglioside modulates ligand binding to the epidermal growth factor receptor. J. Invest. Dermatol. 116, 69-76.
Wang, X. Q., Sun, P., O'Gorman, M., Tai, T. and Paller, A. S. (2001b). Epidermal growth factor receptor glycosylation is required for ganglioside GM3 binding and GM3-mediated suppression [correction of suppression] of activation. Glycobiology 11, 515-522.
Wang, Z., Zhang, L., Yeung, T. K. and Chen, X. (1999). Endocytosis deficiency of epidermal growth factor (EGF) receptor-ErbB2 heterodimers in response to EGF stimulation. Mol. Biol. Cell 10, 1621-1636.
Waterman, H. and Yarden, Y. (2001). Molecular mechanisms underlying endocytosis and sorting of ErbB receptor tyrosine kinases. FEBS Lett. 490, 142-152.[CrossRef][Medline]
Waugh, M. G., Lawson, D. and Hsuan, J. J. (1999). Epidermal growth factor receptor activation is localized within lowbuoyant density, non-caveolar membrane domains. Biochem. J. 337, 591-597.[CrossRef][Medline]
Yaffe, M. B. (2002). How do 14-3-3 proteins work? Gatekeeper phosphorylation and the molecular anvil hypothesis. FEBS Lett. 513, 53-57.[CrossRef][Medline]
Yang, X., Claas, C., Kraeft, S.-K., Chen, L. B., Wang, Z., Kriedberg, J. A. and Hemler, M. E. (2002). Palmitoylation of tetraspanin proteins: modulation of CD151 lateral interactions, subcellular distribution, and integrin-dependent cell morphology. Mol. Biol. Cell 13, 767-781.
Yarden, Y. and Sliwkowski, M. X. (2001). Untangling the ErbB signalling network.: Nat. Rev. Mol. Cell Biol. 2, 127-137.[CrossRef][Medline]
Yu, R. K. and Bieberich, E. (2001). Regulation of glycosyltransferases in ganglioside biosynthesis by phosphorylation and dephosphorylation. Mol. Cell Endocrinol. 177, 19-24.[CrossRef][Medline]
Yu, X., Sharma, K. D., Takahashi, T., Iwamoto, R. and Mekada, E. (2002). Ligand-independent dimer formation of epidermal growth factor receptor (EGFR) is a step separable from ligand-induced EGFR signalling. Mol. Biol. Cell 13, 2547-2557.
Zhang, X. A., Bontrager, A. L. and Hemler, M. E. (2001). TM4SF proteins associate with activated PKC and Link PKC to specific beta1 integrins. J. Biol. Chem. 276, 25005-25013.
Zuberbier, T., Pfrommer, C., Beinholzl, J., Hartmann, K., Ricklinkat, J. and Czarnetzki, B. M. (1995). Gangliosides enhance IgE receptordependent histamine and LTC4 release from human mast cells. Biochim. Biophys. Acta 1269, 79-84.[CrossRef][Medline]
Zurita, A. R., Maccioni, H. J. and Daniotti, J. L. (2001). Modulation of epidermal growth factor receptor phosphorylation by endogenously expressed gangliosides. Biochem. J. 355, 465-472.[CrossRef][Medline]
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