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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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Anti-CD9 antibody stimulates cell spreading. MDA-MB-435 cells spread on the indicated substrates for 45 min, in the presence of mAb ALB6 (anti-CD9) or control antibodies (2E10, anti–integrin α2; and M38, anti-CD81). Spreading was assessed as described in the Fig. 5 legend (**, P < 0.02 vs. controls; *, P < 0.06 vs. controls). Error bars indicate SEM, and dashes indicate that no antibody was added.
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fig10: Anti-CD9 antibody stimulates cell spreading. MDA-MB-435 cells spread on the indicated substrates for 45 min, in the presence of mAb ALB6 (anti-CD9) or control antibodies (2E10, anti–integrin α2; and M38, anti-CD81). Spreading was assessed as described in the Fig. 5 legend (**, P < 0.02 vs. controls; *, P < 0.06 vs. controls). Error bars indicate SEM, and dashes indicate that no antibody was added.

Mentions: In cells where CD9 associates with α6β4, anti-CD9 antibodies can alter α6β4-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 β4 (Fig. 10). Background spreading was observed in the presence of control anti-α2 and anti-CD81 antibodies. As expected from the results shown in Fig. 5, spreading on laminin-5 was elevated for wild-type β4 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.


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)

Anti-CD9 antibody stimulates cell spreading. MDA-MB-435 cells spread on the indicated substrates for 45 min, in the presence of mAb ALB6 (anti-CD9) or control antibodies (2E10, anti–integrin α2; and M38, anti-CD81). Spreading was assessed as described in the Fig. 5 legend (**, P < 0.02 vs. controls; *, P < 0.06 vs. controls). Error bars indicate SEM, and dashes indicate that no antibody was added.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2172609&req=5

fig10: Anti-CD9 antibody stimulates cell spreading. MDA-MB-435 cells spread on the indicated substrates for 45 min, in the presence of mAb ALB6 (anti-CD9) or control antibodies (2E10, anti–integrin α2; and M38, anti-CD81). Spreading was assessed as described in the Fig. 5 legend (**, P < 0.02 vs. controls; *, P < 0.06 vs. controls). Error bars indicate SEM, and dashes indicate that no antibody was added.
Mentions: In cells where CD9 associates with α6β4, anti-CD9 antibodies can alter α6β4-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 β4 (Fig. 10). Background spreading was observed in the presence of control anti-α2 and anti-CD81 antibodies. As expected from the results shown in Fig. 5, spreading on laminin-5 was elevated for wild-type β4 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.

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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