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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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Sucrose density gradient analyses of β4 integrins and integrin–tetraspanin complexes. (A) A431 cells (expressing either wild-type [Wt] or 7C/S β4–GFP) were lysed in 1% Brij 96 or 1% Triton X-100, and then fractionated on 5–35–45% discontinuous sucrose density gradients at 4°C (Claas et al., 2001). 50-μl aliquots of total lysate from each fraction were blotted for β4, using anti-GFP mAb, or for caveolin-1, using rabbit pAb. (B) Untransfected A431 cells were pulsed with [3H]palmitate, surface labeled with biotin, lysed in 1% Brij 96, and then fractionated as in A. From each of the 12 fractions, 50 μl was used for immunoprecipitation of CD151 (mAb 5C11), and [3H] labeling was detected (top), or CD151 was immunoprecipitated and proteins that were surface labeled with biotin were detected by avidin blotting (middle). Also, 50-μl aliquots of total lysate from each fraction were blotted for CD71 (transferrin receptor; bottom).
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fig7: Sucrose density gradient analyses of β4 integrins and integrin–tetraspanin complexes. (A) A431 cells (expressing either wild-type [Wt] or 7C/S β4–GFP) were lysed in 1% Brij 96 or 1% Triton X-100, and then fractionated on 5–35–45% discontinuous sucrose density gradients at 4°C (Claas et al., 2001). 50-μl aliquots of total lysate from each fraction were blotted for β4, using anti-GFP mAb, or for caveolin-1, using rabbit pAb. (B) Untransfected A431 cells were pulsed with [3H]palmitate, surface labeled with biotin, lysed in 1% Brij 96, and then fractionated as in A. From each of the 12 fractions, 50 μl was used for immunoprecipitation of CD151 (mAb 5C11), and [3H] labeling was detected (top), or CD151 was immunoprecipitated and proteins that were surface labeled with biotin were detected by avidin blotting (middle). Also, 50-μl aliquots of total lysate from each fraction were blotted for CD71 (transferrin receptor; bottom).

Mentions: We observed comparable hemidesmosome-like staining for GFP-tagged wild-type and mutant β4 in A431 cells (unpublished data). Because a high level of endogenous β4 precluded further functional studies in A431 cells, we switched to MDA-MB-435 cells, with minimal endogenous β4, for studies of stably expressed wild-type and 7C/S β4. On laminin-5, spreading of 7C/S cells was markedly impaired compared with that of cells with control vector or wild-type β4 (Fig. 5 A, top). All cells spread equally well on vitronectin (Fig. 5 A, bottom). Quantitation of multiple experiments confirmed deficient 7C/S β4 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 β4 cells (Fig. 5 C, lanes 7 and 12) compared with wild-type β4 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 β4 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 β4–MDA-MB-435 cell spreading on laminin-5 (not depicted). Similar spreading and signaling defects were also seen for 7C/S β4–transfected SK-MEL-5 melanoma cells (unpublished data).


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)

Sucrose density gradient analyses of β4 integrins and integrin–tetraspanin complexes. (A) A431 cells (expressing either wild-type [Wt] or 7C/S β4–GFP) were lysed in 1% Brij 96 or 1% Triton X-100, and then fractionated on 5–35–45% discontinuous sucrose density gradients at 4°C (Claas et al., 2001). 50-μl aliquots of total lysate from each fraction were blotted for β4, using anti-GFP mAb, or for caveolin-1, using rabbit pAb. (B) Untransfected A431 cells were pulsed with [3H]palmitate, surface labeled with biotin, lysed in 1% Brij 96, and then fractionated as in A. From each of the 12 fractions, 50 μl was used for immunoprecipitation of CD151 (mAb 5C11), and [3H] labeling was detected (top), or CD151 was immunoprecipitated and proteins that were surface labeled with biotin were detected by avidin blotting (middle). Also, 50-μl aliquots of total lysate from each fraction were blotted for CD71 (transferrin receptor; bottom).
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fig7: Sucrose density gradient analyses of β4 integrins and integrin–tetraspanin complexes. (A) A431 cells (expressing either wild-type [Wt] or 7C/S β4–GFP) were lysed in 1% Brij 96 or 1% Triton X-100, and then fractionated on 5–35–45% discontinuous sucrose density gradients at 4°C (Claas et al., 2001). 50-μl aliquots of total lysate from each fraction were blotted for β4, using anti-GFP mAb, or for caveolin-1, using rabbit pAb. (B) Untransfected A431 cells were pulsed with [3H]palmitate, surface labeled with biotin, lysed in 1% Brij 96, and then fractionated as in A. From each of the 12 fractions, 50 μl was used for immunoprecipitation of CD151 (mAb 5C11), and [3H] labeling was detected (top), or CD151 was immunoprecipitated and proteins that were surface labeled with biotin were detected by avidin blotting (middle). Also, 50-μl aliquots of total lysate from each fraction were blotted for CD71 (transferrin receptor; bottom).
Mentions: We observed comparable hemidesmosome-like staining for GFP-tagged wild-type and mutant β4 in A431 cells (unpublished data). Because a high level of endogenous β4 precluded further functional studies in A431 cells, we switched to MDA-MB-435 cells, with minimal endogenous β4, for studies of stably expressed wild-type and 7C/S β4. On laminin-5, spreading of 7C/S cells was markedly impaired compared with that of cells with control vector or wild-type β4 (Fig. 5 A, top). All cells spread equally well on vitronectin (Fig. 5 A, bottom). Quantitation of multiple experiments confirmed deficient 7C/S β4 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 β4 cells (Fig. 5 C, lanes 7 and 12) compared with wild-type β4 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 β4 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 β4–MDA-MB-435 cell spreading on laminin-5 (not depicted). Similar spreading and signaling defects were also seen for 7C/S β4–transfected SK-MEL-5 melanoma cells (unpublished data).

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