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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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Loss of β4 palmitoylation sites causes tetraspanin reorganization. (A) MDA-MB-435 cells stably expressing wild-type (Wt) or 7C/S β4 were surface labeled with biotin and lysed in 1% Brij 96, and then the α6β4–CD151 complex was immunoprecipitated using anti-α6 mAb GoH3 or anti-CD151 mAb 5C11. Panels were then blotted for CD9 (mAb C9BB; first and third panels), cell surface CD9 (avidin blotting; second panel), CD81 (mAb M38; fourth panel), or CD63 (mAb 6H1; fifth panel). (B) From an anti-α6 immunoprecipitation, surface labeled β4 was detected (avidin blotting; first panel). CD151 was immunoprecipitated (mAb 5C11) and detected (mAb 1A5; second panel), CD9 was immunoprecipitated (mAb ALB6) and blotted (mAb C9BB; third panel), CD81 was immunoprecipitated and blotted (mAb M38; fourth panel), and CD63 was immunoprecipitated and blotted (mAb 6H1; fifth panel). (C) MDA-MB-435 cells stably expressing mock, Wt, or 7C/S β4 were analyzed using anti-CD63 (mAb 6H1) and the indicated anti-CD9 antibodies. Histograms are from Wt β4 cells (thick line), 7C/S β4 cells (dashed line), and background autofluorescence (dotted line). Data are representative of three independent experiments.
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fig9: Loss of β4 palmitoylation sites causes tetraspanin reorganization. (A) MDA-MB-435 cells stably expressing wild-type (Wt) or 7C/S β4 were surface labeled with biotin and lysed in 1% Brij 96, and then the α6β4–CD151 complex was immunoprecipitated using anti-α6 mAb GoH3 or anti-CD151 mAb 5C11. Panels were then blotted for CD9 (mAb C9BB; first and third panels), cell surface CD9 (avidin blotting; second panel), CD81 (mAb M38; fourth panel), or CD63 (mAb 6H1; fifth panel). (B) From an anti-α6 immunoprecipitation, surface labeled β4 was detected (avidin blotting; first panel). CD151 was immunoprecipitated (mAb 5C11) and detected (mAb 1A5; second panel), CD9 was immunoprecipitated (mAb ALB6) and blotted (mAb C9BB; third panel), CD81 was immunoprecipitated and blotted (mAb M38; fourth panel), and CD63 was immunoprecipitated and blotted (mAb 6H1; fifth panel). (C) MDA-MB-435 cells stably expressing mock, Wt, or 7C/S β4 were analyzed using anti-CD63 (mAb 6H1) and the indicated anti-CD9 antibodies. Histograms are from Wt β4 cells (thick line), 7C/S β4 cells (dashed line), and background autofluorescence (dotted line). Data are representative of three independent experiments.

Mentions: Removal of CD151 palmitoylation sites did not affect core association with α3β1 or α6β4, but did alter secondary associations with other tetraspanins (Berditchevski et al., 2002; Yang et al., 2002). Likewise, removal of β4 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 α6 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 β4 associated with α6 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).


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)

Loss of β4 palmitoylation sites causes tetraspanin reorganization. (A) MDA-MB-435 cells stably expressing wild-type (Wt) or 7C/S β4 were surface labeled with biotin and lysed in 1% Brij 96, and then the α6β4–CD151 complex was immunoprecipitated using anti-α6 mAb GoH3 or anti-CD151 mAb 5C11. Panels were then blotted for CD9 (mAb C9BB; first and third panels), cell surface CD9 (avidin blotting; second panel), CD81 (mAb M38; fourth panel), or CD63 (mAb 6H1; fifth panel). (B) From an anti-α6 immunoprecipitation, surface labeled β4 was detected (avidin blotting; first panel). CD151 was immunoprecipitated (mAb 5C11) and detected (mAb 1A5; second panel), CD9 was immunoprecipitated (mAb ALB6) and blotted (mAb C9BB; third panel), CD81 was immunoprecipitated and blotted (mAb M38; fourth panel), and CD63 was immunoprecipitated and blotted (mAb 6H1; fifth panel). (C) MDA-MB-435 cells stably expressing mock, Wt, or 7C/S β4 were analyzed using anti-CD63 (mAb 6H1) and the indicated anti-CD9 antibodies. Histograms are from Wt β4 cells (thick line), 7C/S β4 cells (dashed line), and background autofluorescence (dotted line). Data are representative of three independent experiments.
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fig9: Loss of β4 palmitoylation sites causes tetraspanin reorganization. (A) MDA-MB-435 cells stably expressing wild-type (Wt) or 7C/S β4 were surface labeled with biotin and lysed in 1% Brij 96, and then the α6β4–CD151 complex was immunoprecipitated using anti-α6 mAb GoH3 or anti-CD151 mAb 5C11. Panels were then blotted for CD9 (mAb C9BB; first and third panels), cell surface CD9 (avidin blotting; second panel), CD81 (mAb M38; fourth panel), or CD63 (mAb 6H1; fifth panel). (B) From an anti-α6 immunoprecipitation, surface labeled β4 was detected (avidin blotting; first panel). CD151 was immunoprecipitated (mAb 5C11) and detected (mAb 1A5; second panel), CD9 was immunoprecipitated (mAb ALB6) and blotted (mAb C9BB; third panel), CD81 was immunoprecipitated and blotted (mAb M38; fourth panel), and CD63 was immunoprecipitated and blotted (mAb 6H1; fifth panel). (C) MDA-MB-435 cells stably expressing mock, Wt, or 7C/S β4 were analyzed using anti-CD63 (mAb 6H1) and the indicated anti-CD9 antibodies. Histograms are from Wt β4 cells (thick line), 7C/S β4 cells (dashed line), and background autofluorescence (dotted line). Data are representative of three independent experiments.
Mentions: Removal of CD151 palmitoylation sites did not affect core association with α3β1 or α6β4, but did alter secondary associations with other tetraspanins (Berditchevski et al., 2002; Yang et al., 2002). Likewise, removal of β4 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 α6 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 β4 associated with α6 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).

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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