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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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Palmitoylated integrins associate with each other and with multiple palmitoylated tetraspanin proteins. (A) MDA-MB-231 cells were pulsed with [3H]palmitate, lysed in 1% CHAPS, and then integrins (α2, α6, and α3) and tetraspanins (CD9 and CD151) were immunoprecipitated using mAbs A2-IIE10, GoH3, A3-X8, Du-All, and 5C11, respectively. Question mark indicates unknown protein. (B) MDA-MB-231 cells were immunoprecipitated as in A, and then samples were blotted for β4 (rabbit pAb), β1 (rabbit pAb), and CD9 (C9BB), as indicated. (C) A431 cells were lysed and immunoprecipitated as in A, and then samples were blotted for β4 and β1 (as in B). Note that the band in the α2 lanes migrating slightly below the β4 band (B and C) is an Ig background band, which does not appear in the α2 lane when [3H]palmitate labeling is used (A). (D) Indicated proteins were immunoprecipitated from MCF-10A cells, as in A, and then blotted as in B. As a negative control, abundant cell surface protein CD147 was immunoprecipitated using mAb 8G6. White lines (A and D) indicate that intervening lanes have been spliced out.
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fig2: Palmitoylated integrins associate with each other and with multiple palmitoylated tetraspanin proteins. (A) MDA-MB-231 cells were pulsed with [3H]palmitate, lysed in 1% CHAPS, and then integrins (α2, α6, and α3) and tetraspanins (CD9 and CD151) were immunoprecipitated using mAbs A2-IIE10, GoH3, A3-X8, Du-All, and 5C11, respectively. Question mark indicates unknown protein. (B) MDA-MB-231 cells were immunoprecipitated as in A, and then samples were blotted for β4 (rabbit pAb), β1 (rabbit pAb), and CD9 (C9BB), as indicated. (C) A431 cells were lysed and immunoprecipitated as in A, and then samples were blotted for β4 and β1 (as in B). Note that the band in the α2 lanes migrating slightly below the β4 band (B and C) is an Ig background band, which does not appear in the α2 lane when [3H]palmitate labeling is used (A). (D) Indicated proteins were immunoprecipitated from MCF-10A cells, as in A, and then blotted as in B. As a negative control, abundant cell surface protein CD147 was immunoprecipitated using mAb 8G6. White lines (A and D) indicate that intervening lanes have been spliced out.

Mentions: 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 (α6β4 and α3β1) from MDA-MB-231 cells are strikingly similar (Fig. 2 A). No [3H]palmitate-labeled proteins associated with α2 integrin, which itself is not palmitoylated (Fig. 2 A, lane 1). Palmitoylated protein profiles for CD9, CD151, α3β1, and α6β4 were similarly congruent in two additional cell lines (MCF-10A and A431; unpublished data). Immunoprecipitation of CD9, CD151, α3, and α6 from MDA-MB-231 cells (Fig. 2 B) or MCF-10A cells (Fig. 2 D) yielded, in each case, β4, β1, and CD9, as indicated by blotting. Immunoprecipitation of α3, α6, and CD9 from A431 cells again yielded, in each case, both β4 and β1 (Fig. 2 C). Immunoprecipitation of α2 yielded β1 (as expected), but not β4 or CD9 (Fig. 2, B and C). Likewise, immunoprecipitation of CD147 (a highly expressed control cell surface protein) did not yield β1, β4, or CD9 (Fig. 2 D). Results shown in Fig. 2 indicate that complexes containing palmitoylated CD9, CD151, α6β4, and α3β1 are substantially overlapping in three different cell lines.


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)

Palmitoylated integrins associate with each other and with multiple palmitoylated tetraspanin proteins. (A) MDA-MB-231 cells were pulsed with [3H]palmitate, lysed in 1% CHAPS, and then integrins (α2, α6, and α3) and tetraspanins (CD9 and CD151) were immunoprecipitated using mAbs A2-IIE10, GoH3, A3-X8, Du-All, and 5C11, respectively. Question mark indicates unknown protein. (B) MDA-MB-231 cells were immunoprecipitated as in A, and then samples were blotted for β4 (rabbit pAb), β1 (rabbit pAb), and CD9 (C9BB), as indicated. (C) A431 cells were lysed and immunoprecipitated as in A, and then samples were blotted for β4 and β1 (as in B). Note that the band in the α2 lanes migrating slightly below the β4 band (B and C) is an Ig background band, which does not appear in the α2 lane when [3H]palmitate labeling is used (A). (D) Indicated proteins were immunoprecipitated from MCF-10A cells, as in A, and then blotted as in B. As a negative control, abundant cell surface protein CD147 was immunoprecipitated using mAb 8G6. White lines (A and D) indicate that intervening lanes have been spliced out.
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fig2: Palmitoylated integrins associate with each other and with multiple palmitoylated tetraspanin proteins. (A) MDA-MB-231 cells were pulsed with [3H]palmitate, lysed in 1% CHAPS, and then integrins (α2, α6, and α3) and tetraspanins (CD9 and CD151) were immunoprecipitated using mAbs A2-IIE10, GoH3, A3-X8, Du-All, and 5C11, respectively. Question mark indicates unknown protein. (B) MDA-MB-231 cells were immunoprecipitated as in A, and then samples were blotted for β4 (rabbit pAb), β1 (rabbit pAb), and CD9 (C9BB), as indicated. (C) A431 cells were lysed and immunoprecipitated as in A, and then samples were blotted for β4 and β1 (as in B). Note that the band in the α2 lanes migrating slightly below the β4 band (B and C) is an Ig background band, which does not appear in the α2 lane when [3H]palmitate labeling is used (A). (D) Indicated proteins were immunoprecipitated from MCF-10A cells, as in A, and then blotted as in B. As a negative control, abundant cell surface protein CD147 was immunoprecipitated using mAb 8G6. White lines (A and D) indicate that intervening lanes have been spliced out.
Mentions: 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 (α6β4 and α3β1) from MDA-MB-231 cells are strikingly similar (Fig. 2 A). No [3H]palmitate-labeled proteins associated with α2 integrin, which itself is not palmitoylated (Fig. 2 A, lane 1). Palmitoylated protein profiles for CD9, CD151, α3β1, and α6β4 were similarly congruent in two additional cell lines (MCF-10A and A431; unpublished data). Immunoprecipitation of CD9, CD151, α3, and α6 from MDA-MB-231 cells (Fig. 2 B) or MCF-10A cells (Fig. 2 D) yielded, in each case, β4, β1, and CD9, as indicated by blotting. Immunoprecipitation of α3, α6, and CD9 from A431 cells again yielded, in each case, both β4 and β1 (Fig. 2 C). Immunoprecipitation of α2 yielded β1 (as expected), but not β4 or CD9 (Fig. 2, B and C). Likewise, immunoprecipitation of CD147 (a highly expressed control cell surface protein) did not yield β1, β4, or CD9 (Fig. 2 D). Results shown in Fig. 2 indicate that complexes containing palmitoylated CD9, CD151, α6β4, and α3β1 are substantially overlapping in three different cell lines.

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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