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Correspondence to Martin E. Hemler: Martin_Hemler{at}dfci.harvard.edu
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3, 6, and ß4 subunits) and tetraspanins (CD9, CD81, and CD63) coexist in substantially overlapping complexes. Removal of ß4 palmitoylation sites markedly impaired cell spreading and signaling through p130Cas on laminin substrate. Also in palmitoylation-deficient ß4, secondary associations with tetraspanins (CD9, CD81, and CD63) were diminished and cell surface CD9 clustering was decreased, whereas core 6ß4–CD151 complex formation was unaltered. There is also a functional connection between CD9 and ß4 integrins, as evidenced by anti-CD9 antibody effects on ß4-dependent cell spreading. Notably, ß4 palmitoylation neither increased localization into "light membrane" fractions of sucrose gradients nor decreased solubility in nonionic detergents—hence it does not promote lipid raft association. Instead, palmitoylation of ß4 (and of the closely associated tetraspanin CD151) promotes CD151–6ß4 incorporation into a network of secondary tetraspanin interactions (with CD9, CD81, CD63, etc.), which provides a novel framework for functional regulation.
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Introduction |
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6ß4 integrin appears on epithelial and other types of cells, acts as a receptor for basement membrane laminin-5 and related laminin isoforms, and plays a key role during cell migration and tumorigenesis (Belkin and Stepp, 2000; Mercurio et al., 2001). In response to EGF receptor (EGFR) stimulation, 6ß4 disconnects from the intermediate filament cytoskeleton and becomes associated with the actin cytoskeleton in lamellipodia and membrane ruffles (Mercurio et al., 2001). During this process, EGFR signaling might activate the Src family kinase fyn, leading to phosphorylation of ß4 on tyrosine (Mainiero et al., 1996; Mariotti et al., 2001), or might activate conventional PKC, leading to ß4 phosphorylation on serine (Rabinovitz et al., 1999). Consistent with cooperative signaling between 6ß4 and growth factor receptors, 6ß4 has been suggested to physically associate with fyn (Mariotti et al., 2001), EGFR (Mariotti et al., 2001), ErbB2 (Gambaletta et al., 2000; Hintermann et al., 2001), c-met (Trusolino et al., 2001), and Ron (Santoro et al., 2003).
The laminin-binding integrins (6ß4, 3ß1, 6ß1, and 7ß1) not only form a distinct subgroup among integrins in terms of amino acid sequence similarity, but also show robust association with tetraspanin proteins (Hemler, 1998; Berditchevski, 2001). There are 32 mammalian tetraspanins, and at least a few of these are abundantly present on nearly all cell and tissue types. Tetraspanin proteins regulate cell motility, morphology, fusion, and signaling in the brain and immune system, on tumors, and elsewhere (Levy et al., 1998; Boucheix and Rubinstein, 2001; Hemler, 2001; Stipp et al., 2003b). Tetraspanins CD151, CD81, and CD9 can modulate 3ß1 and 6ß1 integrin–dependent neurite outgrowth, cell migration, and/or cell morphology (Yánez-Mó et al., 1998; Yauch et al., 1998; Stipp and Hemler, 2000; Kazarov et al., 2002; Zhang et al., 2002). Of particular relevance here, CD151 associates with 6ß4 to regulate kidney epithelial cell morphology (Yang et al., 2002), whereas CD9–6ß4 complexes may affect primary keratinocyte cell motility (Jones et al., 1996; Baudoux et al., 2000).
Associations of tetraspanins with each other are at least partly stabilized by palmitoylation. Mutation of CD9 palmitoylation sites impaired associations with tetraspanins CD81 and CD53 (Charrin et al., 2002), and loss of CD151 palmitoylation decreased association with other tetraspanins (CD81, CD63, and CD9), without affecting integrin 3ß1 association (Berditchevski et al., 2002; Yang et al., 2002). Palmitoylation of CD151 contributes to cell signaling (Berditchevski et al., 2002). In some proteins (e.g., G proteins and Src family kinases), palmitoylation leads to the diminished detergent solubility and lower protein density characteristic of lipid raft association (Dunphy and Linder, 1998; Resh, 1999). However, palmitoylation of tetraspanins CD9 and CD151 causes neither decreased protein density in sucrose gradients nor decreased detergent solubility (Berditchevski et al., 2002; Charrin et al., 2002; Yang et al., 2002).
The 6ß4 integrin, like other laminin-binding integrins, associates strongly with CD151 (Sterk et al., 2000, 2002). CD151 association with laminin-binding integrins is direct, occurs early in biosynthesis, and is resistant to disruption by nonionic detergents (Yauch et al., 2000; Berditchevski et al., 2001; Kazarov et al., 2002). Removal of CD151 palmitoylation sites did not disrupt the CD151–6ß4 complex in epithelial cells, but did strongly influence 6ß4 integrin–dependent cell morphology (Yang et al., 2002). In contrast to the primary (i.e., direct) associations of 3 and 6 integrins with CD151, there is an extended network of secondary (i.e., most likely indirect) associations with other tetraspanins (e.g., CD9, CD81, and CD63) that occur later in biosynthesis and are more sensitive to nonionic detergents (Berditchevski et al., 2001; Kazarov et al., 2002). These secondary-type associations are impaired upon removal of CD151 or CD9 palmitoylation sites (Berditchevski et al., 2002; Charrin et al., 2002; Yang et al., 2002).
The integrin ß4 subunit was recently shown also to be palmitoylated (Gagnoux-Palacios et al., 2003). Here, we provide evidence for palmitoylation of integrins 3, 6, and ß4, and show striking parallels between ß4 integrin and CD151 palmitoylations, in terms of effects on (1) core complex (CD151–6ß4) formation, (2) secondary complex formation (involving CD9, CD81, and CD63), (3) integrin density, (4) integrin solubility, (5) integrin-dependent cell morphology, and (6) integrin signaling. Our results are in sharp contrast to previous suggestions that ß4 palmitoylation promotes lipid raft and Src family kinase association (Mariotti et al., 2001; Gagnoux-Palacios et al., 2003). We suggest that integrin palmitoylation promotes, instead of lipid rafts, assembly of a novel type of signaling platform enriched for palmitoylated tetraspanins.
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3, and 6 integrin subunits (Fig. 1 A, lanes 3 and 4). [3H]palmitate-labeled integrin subunits from [3H]palmitate-labeled MDA-MB-231 breast carcinoma cells were more abundant on the cell surface (Fig. 1 A, lane 3) than intracellularly (Fig. 1 A, lane 4), and were not present in tetraspanin CD82 immunoprecipitations (Fig. 1 A, lanes 1 and 2). Integrin palmitoylation was confirmed by recovery of [3H]palmitate-labeled 3 (Fig. 1 B, lane 5), 6 (Fig. 1 B, lanes 6 and 8), and ß4 (Fig. 1 B, lane 6), but not 2 (Fig. 1 B, lanes 4 and 7), from stringent detergent (RIPA) lysates of A431 cells or B12 kidney epithelial cells. In A431 cells, 2, 3, and 6 integrin subunits were present at comparable levels, as indicated by cell surface labeling (Fig. 1 B, lanes 1–3), and in B12 cells, 2 and 6 were again at comparable levels (not depicted).
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6 and 3 (Fig. 1 C, bottom) revealed similar levels of total 6ß4 and 3ß1–CD151 complexes throughout the experiment. Palmitoylations of ß4 and CD151 were almost completely inhibited by a protein synthesis inhibitor (cycloheximide) and by a Golgi-disrupting agent (brefeldin A) (unpublished data). Hence, CD151 and ß4 palmitoylations both occur early in biosynthesis and undergo little subsequent turnover.
Results shown in Fig. 1 A suggested that additional palmitoylated proteins, including CD9, associate with palmitoylated CD151 and palmitoylated integrins. Indeed, the profiles of palmitoylated proteins associated with tetraspanins (CD9 and CD151) and integrins (6ß4 and 3ß1) from MDA-MB-231 cells are strikingly similar (Fig. 2 A). No [3H]palmitate-labeled proteins associated with 2 integrin, which itself is not palmitoylated (Fig. 2 A, lane 1). Palmitoylated protein profiles for CD9, CD151, 3ß1, and 6ß4 were similarly congruent in two additional cell lines (MCF-10A and A431; unpublished data). Immunoprecipitation of CD9, CD151, 3, and 6 from MDA-MB-231 cells (Fig. 2 B) or MCF-10A cells (Fig. 2 D) yielded, in each case, ß4, ß1, and CD9, as indicated by blotting. Immunoprecipitation of 3, 6, and CD9 from A431 cells again yielded, in each case, both ß4 and ß1 (Fig. 2 C). Immunoprecipitation of 2 yielded ß1 (as expected), but not ß4 or CD9 (Fig. 2, B and C). Likewise, immunoprecipitation of CD147 (a highly expressed control cell surface protein) did not yield ß1, ß4, or CD9 (Fig. 2 D). Results shown in Fig. 2 indicate that complexes containing palmitoylated CD9, CD151, 6ß4, and 3ß1 are substantially overlapping in three different cell lines.
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6 (Fig. 3 B, left). Wild-type and mutant ß4 were present at similar levels, as detected by anti-ß4 immunoblotting (Fig. 3 B, right; and Fig. 4 B, middle), flow cytometry (Fig. 4 A), cell surface biotinylation (Fig. 4 B, top), and by [35S]methionine labeling (not depicted). Mutant and wild-type ß4 associated similarly with their core partner, CD151 (Fig. 4 B, bottom), and brought similarly elevated amounts of CD151 and 6 to the cell surface (Fig. 4 A). Levels of cell surface 3 (Fig. 4 A) and ß1 (not depicted) also remained very similar when either wild-type or mutant ß4 was expressed.
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3, 6, and 7 cytoplasmic tails contain a single cysteine residue (Fig. 3 A) that is highly conserved in diverse animal species, but not in most other integrin chains. Upon replacement of this key residue in 3 with serine (C1067S mutation), or upon replacement of the entire 3 cytoplasmic tail and transmembrane with the tail and transmembrane of 5 (X3TC5), palmitoylation was completely lost (Fig. 3 C, lanes 4 and 5). In the same experiment, palmitoylation of wild-type human 3A and 3B was readily observed (Fig. 3 C, lanes 2 and 3). Despite loss of palmitoylation in the C1067S and X3TC5 mutants, these integrins retained association with palmitoylated tetraspanin CD151 (Fig. 3 C, lanes 4 and 5).
Effects of integrin ß4 palmitoylation on cell functions
We observed comparable hemidesmosome-like staining for GFP-tagged wild-type and mutant ß4 in A431 cells (unpublished data). Because a high level of endogenous ß4 precluded further functional studies in A431 cells, we switched to MDA-MB-435 cells, with minimal endogenous ß4, for studies of stably expressed wild-type and 7C/S ß4. On laminin-5, spreading of 7C/S cells was markedly impaired compared with that of cells with control vector or wild-type ß4 (Fig. 5 A, top). All cells spread equally well on vitronectin (Fig. 5 A, bottom). Quantitation of multiple experiments confirmed deficient 7C/S ß4 cell spreading on laminin-5 but not vitronectin substrate (Fig. 5 B). A marked defect in tyrosine phosphorylation of p130Cas was also observed for 7C/S ß4 cells (Fig. 5 C, lanes 7 and 12) compared with wild-type ß4 cells (Fig. 5 C, lanes 6 and 11), when plated on laminin-1 or laminin-5. No defect was seen on control ligand (vitronectin; Fig. 5 C, lanes 3 and 4), and minimal p130Cas phosphorylation was seen for cells in suspension (Fig. 5 C, lanes 1 and 2). In contrast to results with p130Cas, mutant and wild-type ß4 showed little difference in phosphorylation of FAK (unpublished data). In concert with cell spreading, p130Cas is typically phosphorylated by a mechanism dependent on Src family kinases (O'Neill et al., 2000). Consistent with this, the Src family kinase inhibitor PP2 (Hanke et al., 1996) abolished both p130Cas phosphorylation (see Fig. 7 A, lanes 8–10) and wild-type ß4–MDA-MB-435 cell spreading on laminin-5 (not depicted). Similar spreading and signaling defects were also seen for 7C/S ß4–transfected SK-MEL-5 melanoma cells (unpublished data).
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6ß1 dependent, whereas on laminin-5, 3ß1 also contributed (Fig. 6 C). On vitronectin, antibodies to laminin receptors had minimal inhibitory effect. The use of 6ß1, but not 6ß4, for adhesion is in accord with a previous study of untransfected and ß4-transfected MDA-MB-435 cells (Shaw et al., 1997). Expression levels for 3ß1 and 6ß1 were essentially unchanged, regardless of whether mutant or wild-type ß4 was present, as seen by flow cytometry (Fig. 4 A) and immunoblotting (not depicted).
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6 or ß4 (Fig. 8 C). In addition, there were no consistent differences in tyrosine phosphorylation of 7C/S and wild-type ß4 (unpublished data). Integrin 6ß4 also associates with ErbB2 (Hintermann et al., 2001), which in MDA-MB-435 cells forms dimers with ErbB3 (Adelsman et al., 1999). However, after heregulin treatment to induce ErbB phosphorylation, we observed no association of either wild-type or mutant ß4 with tyrosine-phosphorylated ErbB3 (Fig. 8 D) or ErbB2 (not depicted). Also, for mutant ß4 in MDA-MB-435 cells, we saw no diminution in ErbB3 tyrosine phosphorylation or ErbB3 association with the p85 subunit of PI 3-K (Fig. 8 E).
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3ß1 or 6ß4, but did alter secondary associations with other tetraspanins (Berditchevski et al., 2002; Yang et al., 2002). Likewise, removal of ß4 palmitoylation sites did not affect core association with CD151 (Fig. 4 B), but did markedly alter secondary associations with other tetraspanins (Fig. 9 A). As indicated by immunoprecipitations of 6 or CD151, association of CD9, cell surface CD9 (sCD9), CD81, and CD63 was impaired in 7C/S–MDA-MB-435 cells, in five different experiments. In control experiments, the levels of wild-type and 7C/S ß4 associated with 6 were unaltered, as were the total levels of CD151, CD9, CD81, and CD63 (Fig. 9 B). Similar levels of CD63 were seen by flow cytometry (Fig. 9 C, top).
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40% (unpublished data). These results indicate that cell surface tetraspanin organization is affected by ß4 palmitoylation.
Functional link between CD9 and ß4
In cells where CD9 associates with 6ß4, anti-CD9 antibodies can alter 6ß4-dependent cell motility (Jones et al., 1996; Baudoux et al., 2000). Here, we confirm a functional connection between CD9 and laminin-binding integrins in MDA-MB-435 cells. Addition of an anti-CD9 antibody (ALB6) stimulated cell spreading in 7C/S cells, and to an even greater extent in cells expressing wild-type ß4 (Fig. 10). Background spreading was observed in the presence of control anti-2 and anti-CD81 antibodies. As expected from the results shown in Fig. 5, spreading on laminin-5 was elevated for wild-type ß4 cells. The anti-CD9 mAb ALB6 did not promote spreading of either wild-type (Fig. 10) or 7C/S (not depicted) cells on vitronectin, and had no effect on static cell adhesion to laminin-1 or laminin-5.
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6ß4-dependent cell signaling and morphology. Fifth, immunoprecipitation of either CD151 or 6ß4 yielded strikingly similar patterns of palmitoylated proteins. Sixth, loss of palmitoylation from either molecule did not disrupt the core CD151–6ß4 complex. However, seventh, loss of palmitoylation from either CD151 (Berditchevski et al., 2002; Yang et al., 2002) or ß4 did markedly alter secondary associations with other tetraspanins. These observations reinforce the concept that the CD151–6ß4 core complex is similarly affected by palmitoylation on either CD151 or ß4.
Tetraspanin-enriched microdomains (TEMs)
It is well established that tetraspanin proteins (including CD151 and CD9) and their partners associate with each other in extended complexes known as the tetraspanin web (Trusolino et al., 2001), or TEMs (Berditchevski et al., 2002; Hemler, 2003). TEMs contain primary complexes (e.g., CD151–integrin [Serru et al., 1999; Yauch et al., 2000; Kazarov et al., 2002; Sterk et al., 2002] and tetraspanin homodimers [Kovalenko et al., 2004]) occurring through direct, protein–protein interactions. These are then brought together into extended secondary complexes, with tetraspanin palmitoylation playing a key role (Berditchevski et al., 2002; Charrin et al., 2002; Yang et al., 2002; Hemler, 2003). Given the abundance of evidence regarding CD151 palmitoylation, and the strong similarities between CD151 and ß4 (see previous section), it is not surprising that ß4 palmitoylation would also contribute to localization into TEMs. The evidence for this is as follows. First, in multiple cell lines, palmitoylated ß4 associates with other palmitoylated proteins (CD9, CD81, CD63, and others) in a pattern that is strikingly similar whether immunoprecipitated with antibodies to CD151, to CD9, or to integrins (6ß4 or 3ß1). The specificity of interaction among these particular components is emphasized by the exclusion of other abundant cell surface molecules (e.g., integrin 2ß1 and tetraspanin CD82). Consistent with our results, others have also shown that CD9 can associate with 6ß4 (Jones et al., 1996; Baudoux et al., 2000). Second, loss of ß4 palmitoylation diminished secondary associations with other tetraspanins (CD9, CD81, and CD63), just as seen previously upon removal of CD151 and CD9 palmitoylation sites (Berditchevski et al., 2002; Charrin et al., 2002; Yang et al., 2002). Third, not only was CD9 substantially dissociated from the palmitoylation-deficient 6ß4–CD151 complex, it also showed reorganization on the cell surface, which is consistent with reduced clustering. Pairs of monoclonal antibodies, diagnostic for cell surface clustering, have been described for other molecules, including CD147 (Koch et al., 1999) and CDw78 (Drbal et al., 1999). In each case, a lower affinity antibody (such as anti-CD9 mAb C9BB) showed more dependence on bivalent binding, and hence more sensitivity to cell surface clustering, compared with a higher affinity antibody (such as anti-CD9 mAb ALB6).
It is likely not a coincidence that the integrin subunits (3, 6, and 7) best able to interact with tetraspanins also contain palmitoylation sites. The integrin 3, 6, and 7 subunits contain single, membrane-proximal cysteine palmitoylation sites, conserved in orthologous subunits across a wide range of animal species. For 3 and 6, the key cysteine is present in both major (3A and 6A) and minor (3B and 6B) alternatively spliced forms. In contrast to the integrin 3, 6, and 7 subunits, the other 13 integrin subunits (except for E and 8) don't contain membrane-proximal cysteines.
Functional consequences
Removal of CD151 palmitoylation did not affect static cell adhesion but did alter 6ß4-dependent kidney epithelial cell morphology (Yang et al., 2002) and integrin-dependent signaling in fibroblasts (Berditchevski et al., 2002). Similarly, removal of ß4 palmitoylation did not alter cell surface expression levels, association with 6 or CD151, or cell adhesion to laminins, but did alter 6ß4-dependent spreading and associated signaling through p130Cas in MDA-MB-435 cells. Although ß4 has not previously been linked to p130Cas signaling, p130Cas is preferentially activated when cells are plated on laminin, compared with fibronectin (Gu et al., 2001). Inhibition by the Src family kinase inhibitor PP2 is consistent with cell spreading depending on Src family kinase phosphorylation of p130Cas (Nojima et al., 1996; Panetti, 2002). Presently, we cannot determine whether impaired cell spreading, due to the absence of ß4 palmitoylation, is a consequence or a cause of diminished Src kinase 
p130Cas signaling. Notably, Gagnoux-Palacios et al. (2003) have also reported altered Src family kinase–dependent signaling upon removal of ß4 palmitoylation sites. Upon removal of palmitoylation from CD151 (Berditchevski et al., 2002) or ß4, signaling through FAK was unaltered, consistent with cell adhesion being unaltered. Our results are in accord with previous evidence that FAK and p130Cas can sometimes be differentially regulated (Gu et al., 2001).
For several reasons, we suggest that ß4 palmitoylation–dependent recruitment of CD151–6ß4 into TEMs plays a major role in determining cell spreading and signaling. First, diminished spreading and signaling occurred in parallel with reduced secondary associations of 6ß4 with tetraspanins (CD9, CD81, and CD63) and reorganization of cell surface CD9. Second, CD9 is functionally connected to 6ß4, consistent with its physical association. As seen elsewhere, anti-CD9 antibodies inhibited the motility of cultured keratinocytes under conditions where motility is at least partially 6ß4-dependent (Jones et al., 1996; Baudoux et al., 2000). Consistent with this, we used an anti-CD9 antibody to stimulate MDA-MB-435 cell spreading, which is partly 6ß4 dependent, as evidenced by the negative effects of ß4 mutation. It was a little surprising that anti-CD9 antibody also stimulated spreading of MDA-MB-435 cells bearing mutant ß4. However, we note that removal of ß4 palmitoylation does not completely dissociate CD151–6ß4 from CD9 and other tetraspanins. There remain six palmitoylation sites in CD151, and one site in 6, that likely contribute to continued CD9 association. Third, an association with TEMs provides a mechanism to link CD151–6ß4 with several other molecules known to regulate cell morphology and signaling. These include other CD9-associated proteins such as EWI-2 (Stipp et al., 2003a; Kolesnikova et al., 2004), PKC (Zhang et al., 2001), and phosphatidylinositol 4-kinase (Yauch and Hemler, 2000). In this regard, CD9 has also been linked to signaling of c-kit and EGFR tyrosine kinases (Higashiyama et al., 1995; Shi et al., 2000; Anzai et al., 2002). It was suggested previously that CD9 regulation of 6ß4 might only be relevant when cells are migrating and/or when the 6ß4 is associated with the actin cytoskeleton rather than stably interacting with intermediate filaments within hemidesmosome-like structures (Baudoux et al., 2000). Indeed, tetraspanins CD9 and CD81 are not associated with the CD151–6ß4 complex in hemidesmosomes (Sterk et al., 2000), and mutation of ß4 palmitoylation sites did not affect hemidesmosome localization, as observed here and by Gagnoux-Palacios et al. (2003).
Our ß4 mutation had minimal effect on cell adhesion to laminin-1 or laminin-5. This result is consistent with previous results in which ß4 in MDA-MB-435 cells modulated downstream signaling but not initial cell adhesion to laminin-1 (Shaw et al., 1997). Furthermore, our ß4 mutant was deficient in spreading and signaling on both laminin-5 and laminin-1, even though only the former is a very good ligand for 6ß4. Hence, a mechanism is needed to explain how 6ß4 can exert substrate-dependent effects on cell morphology, signaling, and/or motility, even when it is not primarily responsible for cell adhesion. In this regard, we have demonstrated that multiple laminin-binding integrins can be linked together within overlapping complexes. Not only were the profiles of palmitoylated proteins associated with 6ß4 and 3ß1 strikingly similar, but also we observed 6ß4 and 3ß1 coimmunoprecipitations from multiple cell lines. Hence, we propose that 6ß4 can physically associate with 3ß1 (and likely also 6ß1), in the context of TEMs. This provides a mechanism for the close coordination of initial adhesion, mediated by 3ß1 (or 6ß1), with subsequent 6ß4-dependent spreading, signaling, or migration. Though novel, these results are not unexpected. It was previously established that 6ß4, 6ß1, and 3ß1 all can associate with CD151 (Berditchevski, 2001), and that CD151 may appear predominantly as a homodimer (Kovalenko et al., 2004), thus having the potential to link distinct integrins. Furthermore, all of these integrins were known to associate with additional tetraspanins (CD9, CD81, CD63, etc.), which can associate with each other (Boucheix and Rubinstein, 2001; Berditchevski et al., 2002; Hemler, 2003).
TEMs and lipid raft localization
Protein palmitoylation can lead to localization into lipid rafts and decreased solubility in nonionic detergents (Dunphy and Linder, 1998; Resh, 1999). Indeed, it was suggested elsewhere that ß4 palmitoylation promotes lipid raft localization, thus providing a mechanism for bringing ß4 into functionally important complexes with Src family kinases such as fyn and yes (Mariotti et al., 2001; Gagnoux-Palacios et al., 2003). However, we found no evidence for palmitoylation promoting either integrin or tetraspanin association with lipid rafts. First, in ß4-transfected A431 cells, the presence of ß4 palmitoylation was not associated with decreased density in either Brij 96 or Triton X-100 lysates. Second, neither the palmitoylated subset nor the cell surface–labeled subset of endogenous ß4 in A431 cells appeared in low density sucrose gradient fractions, even in mild Brij 96 detergent. Third, ß4 palmitoylation did not decrease detergent solubility in MDA-MB-435 cells. In fact, in a short-term detergent (Brij 96) solubility assay, ß4 palmitoylation increased solubility. Fourth, our ß4 results are in complete agreement with prior results regarding tetraspanin palmitoylation. Protein density was not affected by removal of palmitoylation from either CD151 or CD9 (Berditchevski et al., 2002; Charrin et al., 2002; Yang et al., 2002), and CD151 palmitoylation did not decrease detergent solubility (Yang et al., 2002). Fifth, we did not see changes in association of mutant ß4 with signaling proteins (EGFR, ErbB3/2, and fyn) that can be found in lipid rafts. Despite the potential of tetraspanins to associate with gangliosides and cholesterol, TEMs consistently remain distinct from lipid rafts. In contrast to lipid rafts, TEMs are resistant to cholesterol depletion, typically appear in the soluble phase upon extraction by nonionic detergents, and are not disrupted at 37°C (Hemler, 2003). An extensive proteomics analysis of lipid rafts did not reveal any tetraspanins or palmitoylated integrins (Foster et al., 2003). Conversely, we have identified, by mass spectrometry, numerous potential partners for tetraspanins and palmitoylated integrins, but this list does not contain typical raft-type proteins such as caveolins and GPI-linked proteins (unpublished data).
In conclusion, palmitoylation of ß4 integrin, as seen previously for CD151, plays a key role in integrin-dependent cell spreading and signaling, and in the maintenance of secondary interactions with additional tetraspanin proteins. We suggest that CD151–6ß4, CD151–3ß1, and CD151–6ß1 assemble with other tetraspanins into novel signaling platforms (TEMs). These provide a framework for the regulation of integrin-dependent postadhesion functions (spreading, signaling, and motility) and coordination of the functions and distributions of laminin-binding integrins at the cell–substrate interface.
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2 (A2-IIE10), 3 (A3-X8 and A3IVA5), 6 (A6-ELE); to tetraspanins CD9 (C9BB and DU-ALL-1), CD63 (6H1), CD81 (M38 and JS64), CD82 (M104), and CD151 (5C11 and 1A5); and to CD147 (8G6) were referenced previously (Kazarov et al., 2002; Yang et al., 2002). Other mAbs used were anti–integrin 6 (GoH3; BD Biosciences), anti-ß4 (AA3; provided by V. Quaranta, Vanderbilt University, Nashville, TN) anti-CD9 (ALB6; CHEMICON International), and anti-CD71 (OKT9; American Type Culture Collection). Also used were rabbit polyclonal antibodies to ß4 and ß1 integrins (CHEMICON International) and to the cytoplasmic domains of integrins 3A (Dipersio et al., 1995) and 6A (Ab 6843; a gift from V. Quaranta). mAbs to phosphotyrosine (4G10) and PI 3-K p85 were purchased from Upstate Biotechnology, and antibodies to EGFR, ErbB2, and ErbB3 were purchased from Transduction Laboratories. Antibodies to FAK, fyn, Src, and p130Cas were obtained from Santa Cruz Biotechnology, Inc., and anti-GFP was obtained from CLONTECH Laboratories, Inc.
L-[35S]methionine/L-[35S]cysteine and [3H]palmitic acid were purchased from NEN Life Science Products; brefeldin A (Sigma-Aldrich) was dissolved in DMSO or ethanol at 1–2 mg/ml and stored at –80°C. EGF and heregulin-ß1 were purchased from Upstate Biotechnology and R&D Systems, respectively. Sulfo-NHS-LC-Biotin and Sulfo-NHS-SS-Biotin were obtained from Pierce Chemical Co. PP2, a specific inhibitor for Src family kinases, was obtained from Calbiochem. Laminin-5 was a gift (Desmos Inc., San Diego, CA) and vitronectin was purchased from Becton Dickinson. Cell lines were grown in DME supplemented with 10% FCS (GIBCO BRL), 10 mM Hepes, and antibiotics (penicillin and streptomycin). B12 kidney epithelial cells, lacking 3 integrin, were a gift from J. Kreidberg (Children's Hospital Boston, Boston, MA; Kreidberg et al., 1996).
Integrin constructs and transfection
Point mutants (CS) were generated in human ß4 or 3 integrin subunits by PCR and subcloned into pcDNA3.1 (Invitrogen). To generate GFP-tagged ß4, a 2.2-kb NH2-terminal fragment of human ß4 was amplified and subcloned into EGFP-N1 vector via XhoI and EcoRI sites. Additional ß4 fragments (with or without CS point mutations) were subcloned and then were inserted via EcoRI and Kpn sites. ß4 and 3 sequences were confirmed by DNA sequencing at the Dana-Farber Cancer Institute core facility. Lipofectamine (GIBCO BRL) was used for transient and stable transfections. Human integrin wild-type and mutant subunits were stably expressed in the B12 cell line, a murine 3 integrin-null kidney epithelial cell line (Wang et al., 1999), using the Fugene 6 method (Roche Diagnostics Corporation). Integrin mutant X3TC5 is an 3 subunit with the transmembrane domain and cytoplasmic tail replaced by corresponding domains from 5 (Yauch et al., 1998).
Protein labeling, immunoprecipitation, and immunoblotting
For [3H]palmitate labeling, cell lines (at 80–90% confluence) were washed twice in PBS, serum-starved for 3–4 h, and then pulsed for 1–2 h in medium containing 0.2–0.3 mCi/ml [3H]palmitic acid plus 5% dialyzed FBS. For surface labeling with biotin, semiconfluent cells were cooled on ice, washed three times in PBS, incubated in PBS containing Sulfo-NHS-LC-Biotin at 0.1–0.2 mg/ml for 1 h at 4°C, and then washed with cold PBS containing 0.1 M glycine. Labeled cells were lysed in RIPA (1% Triton X-100, 1% deoxycholate, and 0.1% SDS) or in buffer containing 1% Brij 96 for 1 h at 4°C. Immunoprecipitation, immunoblotting, and detection of [3H]palmitate were described previously (Yang et al., 2002).
Immunofluorescence microscopy and flow cytometry
A431 cells were cultured overnight on 60-mm dishes with coverglass bottoms (MaTek Corp.). Cells were then transfected with GFP-tagged ß4 constructs, and after 48 h were rinsed in PBS and photographed (Axioskop; Carl Zeiss MicroImaging, Inc.) at a magnification of 100. For flow cytometry (FACSCalibur; Becton Dickinson), stably transfected semiconfluent MDA-MB-435 cells were detached and stained on ice with either control IgG or specific mAbs, followed by FITC-conjugated secondary antibody (Biosource International).
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Submitted: 16 April 2004
Accepted: 7 November 2004
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1.2-fold. Results (mean ± SD) are derived from at least two separate experiments, counting at least two representative fields in each experiment, with at least 50 cells/field. (C) MDA-MB-435 cells were suspended at 37°C for 30 min, and then either retained in suspension or plated on vitronectin (Vn), laminin-1, or laminin-5 for 45 min. Some samples (lanes 8–10) were preincubated with 1.0 µg/ml PP2 in DMSO for 20 min before plating. Cells were collected and lysed in RIPA, and p130Cas was immunoprecipitated. After SDS-PAGE, samples were blotted with antiphosphotyrosine mAb 4G10. To assess protein loading, the blot was stripped and reblotted with p130Cas-specific antibody. Similar results were seen in multiple experiments.
