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Palmitoylation supports assembly and function of integrin-tetraspanin complexes.

Yang X, Kovalenko OV, Tang W, Claas C, Stipp CS, Hemler ME - J. Cell Biol. (2004)

Bottom Line: There is also a functional connection between CD9 and beta4 integrins, as evidenced by anti-CD9 antibody effects on beta4-dependent cell spreading.Notably, beta4 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 beta4 (and of the closely associated tetraspanin CD151) promotes CD151-alpha6beta4 incorporation into a network of secondary tetraspanin interactions (with CD9, CD81, CD63, etc.), which provides a novel framework for functional regulation.

View Article: PubMed Central - PubMed

Affiliation: Dana-Farber Cancer Institute and Department of Pathology, Harvard Medical School, Boston, MA 02115, USA.

ABSTRACT
As observed previously, tetraspanin palmitoylation promotes tetraspanin microdomain assembly. Here, we show that palmitoylated integrins (alpha3, alpha6, and beta4 subunits) and tetraspanins (CD9, CD81, and CD63) coexist in substantially overlapping complexes. Removal of beta4 palmitoylation sites markedly impaired cell spreading and signaling through p130Cas on laminin substrate. Also in palmitoylation-deficient beta4, secondary associations with tetraspanins (CD9, CD81, and CD63) were diminished and cell surface CD9 clustering was decreased, whereas core alpha6beta4-CD151 complex formation was unaltered. There is also a functional connection between CD9 and beta4 integrins, as evidenced by anti-CD9 antibody effects on beta4-dependent cell spreading. Notably, beta4 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 beta4 (and of the closely associated tetraspanin CD151) promotes CD151-alpha6beta4 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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β4 palmitoylation does not affect association with EGFR, fyn, or ErbB3. (A) A431 cells stably expressing wild-type (Wt) or mutant β4–GFP proteins were serum-starved for ∼12 h, stimulated with (or without) 50 ng/ml EGF for 10 min, and lysed in RIPA, and then β4 was immunoprecipitated using anti-GFP pAb. Samples were blotted for phosphotyrosine (mAb 4G10) and β4 (mAb anti-GFP). Del1, β4 deleted after the third fibronectin repeat (aa 1582); Del2, β4 deleted after the second fibronectin repeat (aa 1412). (B) Samples were prepared as in A, and then EGFR and fyn were immunoprecipitated and blotted for phosphotyrosine, using mAb 4G10. (C) A431 transfectants were stimulated and lysed as in A; α2, α6, and β4 immunoprecipitations were performed; and samples were blotted for fyn (using pAb). W, wild-type β4; 7, 7C/S β4. (D) MDA-MB-435 transfectants were treated with (or without) 100 ng/ml heregulin for 10 min and lysed in RIPA, and then α6β4 was immunoprecipitated using mAb GoH3. ErbB3 was blotted for phosphotyrosine (top) and β4 was blotted using anti-β4 pAb (bottom). (E) MDA-MB-435 cell lysate was prepared in D, and ErbB3 was immunoprecipitated. Samples were blotted for phosphotyrosine, total ErbB3, and p85 subunit of PI 3-K.
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fig8: β4 palmitoylation does not affect association with EGFR, fyn, or ErbB3. (A) A431 cells stably expressing wild-type (Wt) or mutant β4–GFP proteins were serum-starved for ∼12 h, stimulated with (or without) 50 ng/ml EGF for 10 min, and lysed in RIPA, and then β4 was immunoprecipitated using anti-GFP pAb. Samples were blotted for phosphotyrosine (mAb 4G10) and β4 (mAb anti-GFP). Del1, β4 deleted after the third fibronectin repeat (aa 1582); Del2, β4 deleted after the second fibronectin repeat (aa 1412). (B) Samples were prepared as in A, and then EGFR and fyn were immunoprecipitated and blotted for phosphotyrosine, using mAb 4G10. (C) A431 transfectants were stimulated and lysed as in A; α2, α6, and β4 immunoprecipitations were performed; and samples were blotted for fyn (using pAb). W, wild-type β4; 7, 7C/S β4. (D) MDA-MB-435 transfectants were treated with (or without) 100 ng/ml heregulin for 10 min and lysed in RIPA, and then α6β4 was immunoprecipitated using mAb GoH3. ErbB3 was blotted for phosphotyrosine (top) and β4 was blotted using anti-β4 pAb (bottom). (E) MDA-MB-435 cell lysate was prepared in D, and ErbB3 was immunoprecipitated. Samples were blotted for phosphotyrosine, total ErbB3, and p85 subunit of PI 3-K.

Mentions: As mentioned in the Introduction, integrin β4 can associate with activated EGFR-type growth factor receptors and Src family kinases. However, in EGF-stimulated A431 cells, we observed no difference in levels of tyrosine-phosphorylated EGFR associating with GFP-tagged 7C/S and wild-type β4 (Fig. 8 A, top, lanes 2 and 4). In control experiments, deletion of portions of the β4 tail did diminish association with tyrosine-phosphorylated EGFR (Fig. 8 A, top, lanes 6 and 8) and EGFR protein (Fig. 8 A, middle, lanes 5–8). Tyrosine-phosphorylated EGFR was not detected in the absence of EGF stimulation (Fig. 8 A, top, lanes 1, 3, 5, and 7) or when β4 was not immunoprecipitated (Fig. 8 A, lane 9). Comparable amounts of β4 were recovered in each lane (Fig. 8 A, bottom, lanes 1–8). As expected (Mariotti et al., 2001), an abundance of tyrosine-phosphorylated EGFR was recovered in association with the Src family kinase fyn (Fig. 8 B), but no fyn was recovered in association with either mutant or wild-type β4 that had been immunoprecipitated with antibodies to either α6 or β4 (Fig. 8 C). In addition, there were no consistent differences in tyrosine phosphorylation of 7C/S and wild-type β4 (unpublished data). Integrin α6β4 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 β4 with tyrosine-phosphorylated ErbB3 (Fig. 8 D) or ErbB2 (not depicted). Also, for mutant β4 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).


Palmitoylation supports assembly and function of integrin-tetraspanin complexes.

Yang X, Kovalenko OV, Tang W, Claas C, Stipp CS, Hemler ME - J. Cell Biol. (2004)

β4 palmitoylation does not affect association with EGFR, fyn, or ErbB3. (A) A431 cells stably expressing wild-type (Wt) or mutant β4–GFP proteins were serum-starved for ∼12 h, stimulated with (or without) 50 ng/ml EGF for 10 min, and lysed in RIPA, and then β4 was immunoprecipitated using anti-GFP pAb. Samples were blotted for phosphotyrosine (mAb 4G10) and β4 (mAb anti-GFP). Del1, β4 deleted after the third fibronectin repeat (aa 1582); Del2, β4 deleted after the second fibronectin repeat (aa 1412). (B) Samples were prepared as in A, and then EGFR and fyn were immunoprecipitated and blotted for phosphotyrosine, using mAb 4G10. (C) A431 transfectants were stimulated and lysed as in A; α2, α6, and β4 immunoprecipitations were performed; and samples were blotted for fyn (using pAb). W, wild-type β4; 7, 7C/S β4. (D) MDA-MB-435 transfectants were treated with (or without) 100 ng/ml heregulin for 10 min and lysed in RIPA, and then α6β4 was immunoprecipitated using mAb GoH3. ErbB3 was blotted for phosphotyrosine (top) and β4 was blotted using anti-β4 pAb (bottom). (E) MDA-MB-435 cell lysate was prepared in D, and ErbB3 was immunoprecipitated. Samples were blotted for phosphotyrosine, total ErbB3, and p85 subunit of PI 3-K.
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fig8: β4 palmitoylation does not affect association with EGFR, fyn, or ErbB3. (A) A431 cells stably expressing wild-type (Wt) or mutant β4–GFP proteins were serum-starved for ∼12 h, stimulated with (or without) 50 ng/ml EGF for 10 min, and lysed in RIPA, and then β4 was immunoprecipitated using anti-GFP pAb. Samples were blotted for phosphotyrosine (mAb 4G10) and β4 (mAb anti-GFP). Del1, β4 deleted after the third fibronectin repeat (aa 1582); Del2, β4 deleted after the second fibronectin repeat (aa 1412). (B) Samples were prepared as in A, and then EGFR and fyn were immunoprecipitated and blotted for phosphotyrosine, using mAb 4G10. (C) A431 transfectants were stimulated and lysed as in A; α2, α6, and β4 immunoprecipitations were performed; and samples were blotted for fyn (using pAb). W, wild-type β4; 7, 7C/S β4. (D) MDA-MB-435 transfectants were treated with (or without) 100 ng/ml heregulin for 10 min and lysed in RIPA, and then α6β4 was immunoprecipitated using mAb GoH3. ErbB3 was blotted for phosphotyrosine (top) and β4 was blotted using anti-β4 pAb (bottom). (E) MDA-MB-435 cell lysate was prepared in D, and ErbB3 was immunoprecipitated. Samples were blotted for phosphotyrosine, total ErbB3, and p85 subunit of PI 3-K.
Mentions: As mentioned in the Introduction, integrin β4 can associate with activated EGFR-type growth factor receptors and Src family kinases. However, in EGF-stimulated A431 cells, we observed no difference in levels of tyrosine-phosphorylated EGFR associating with GFP-tagged 7C/S and wild-type β4 (Fig. 8 A, top, lanes 2 and 4). In control experiments, deletion of portions of the β4 tail did diminish association with tyrosine-phosphorylated EGFR (Fig. 8 A, top, lanes 6 and 8) and EGFR protein (Fig. 8 A, middle, lanes 5–8). Tyrosine-phosphorylated EGFR was not detected in the absence of EGF stimulation (Fig. 8 A, top, lanes 1, 3, 5, and 7) or when β4 was not immunoprecipitated (Fig. 8 A, lane 9). Comparable amounts of β4 were recovered in each lane (Fig. 8 A, bottom, lanes 1–8). As expected (Mariotti et al., 2001), an abundance of tyrosine-phosphorylated EGFR was recovered in association with the Src family kinase fyn (Fig. 8 B), but no fyn was recovered in association with either mutant or wild-type β4 that had been immunoprecipitated with antibodies to either α6 or β4 (Fig. 8 C). In addition, there were no consistent differences in tyrosine phosphorylation of 7C/S and wild-type β4 (unpublished data). Integrin α6β4 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 β4 with tyrosine-phosphorylated ErbB3 (Fig. 8 D) or ErbB2 (not depicted). Also, for mutant β4 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).

Bottom Line: There is also a functional connection between CD9 and beta4 integrins, as evidenced by anti-CD9 antibody effects on beta4-dependent cell spreading.Notably, beta4 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 beta4 (and of the closely associated tetraspanin CD151) promotes CD151-alpha6beta4 incorporation into a network of secondary tetraspanin interactions (with CD9, CD81, CD63, etc.), which provides a novel framework for functional regulation.

View Article: PubMed Central - PubMed

Affiliation: Dana-Farber Cancer Institute and Department of Pathology, Harvard Medical School, Boston, MA 02115, USA.

ABSTRACT
As observed previously, tetraspanin palmitoylation promotes tetraspanin microdomain assembly. Here, we show that palmitoylated integrins (alpha3, alpha6, and beta4 subunits) and tetraspanins (CD9, CD81, and CD63) coexist in substantially overlapping complexes. Removal of beta4 palmitoylation sites markedly impaired cell spreading and signaling through p130Cas on laminin substrate. Also in palmitoylation-deficient beta4, secondary associations with tetraspanins (CD9, CD81, and CD63) were diminished and cell surface CD9 clustering was decreased, whereas core alpha6beta4-CD151 complex formation was unaltered. There is also a functional connection between CD9 and beta4 integrins, as evidenced by anti-CD9 antibody effects on beta4-dependent cell spreading. Notably, beta4 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 beta4 (and of the closely associated tetraspanin CD151) promotes CD151-alpha6beta4 incorporation into a network of secondary tetraspanin interactions (with CD9, CD81, CD63, etc.), which provides a novel framework for functional regulation.

Show MeSH