Limits...
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.

Show MeSH

Related in: MedlinePlus

β1-dependent cell adhesion is unaffected by β4 mutation. (A) To assess cell adhesion, MDA-MB-435 cells were plated on specific substrates for 30 min, and then nonadherent cells were removed by washing and adherent cells were stained using Wright-Giemsa and then quantitated at OD 590 using an ELISA 96-well plate reader. Starting with 70,000 cells/well, ∼70% cell adhesion corresponds to OD 590 = 0.3. (B) Cells were incubated with ∼10 μg/ml of the indicated mAbs at RT for 15 min before being plated on laminin-5. Adhesion was determined as in A. (C) Effects of indicated antibodies on adhesion of wild-type (Wt) β4 cells was determined as in A and B. Results shown are the means of triplicate determinations ± SD.
© Copyright Policy
Related In: Results  -  Collection


getmorefigures.php?uid=PMC2172609&req=5

fig6: β1-dependent cell adhesion is unaffected by β4 mutation. (A) To assess cell adhesion, MDA-MB-435 cells were plated on specific substrates for 30 min, and then nonadherent cells were removed by washing and adherent cells were stained using Wright-Giemsa and then quantitated at OD 590 using an ELISA 96-well plate reader. Starting with 70,000 cells/well, ∼70% cell adhesion corresponds to OD 590 = 0.3. (B) Cells were incubated with ∼10 μg/ml of the indicated mAbs at RT for 15 min before being plated on laminin-5. Adhesion was determined as in A. (C) Effects of indicated antibodies on adhesion of wild-type (Wt) β4 cells was determined as in A and B. Results shown are the means of triplicate determinations ± SD.

Mentions: The spreading defect does not arise because of diminished cell adhesion. In a 30-min static cell adhesion assay, all cells adhered similarly to laminin-1, laminin-5, and vitronectin (Fig. 6 A). Adhesion to laminin-5 (Fig. 6 B) or laminin-1 (not depicted) was dependent on β1 rather than β4 integrins, as indicated by nearly complete blocking with an anti-β1 monoclonal antibody. On laminin-1, adhesion was almost entirely α6β1 dependent, whereas on laminin-5, α3β1 also contributed (Fig. 6 C). On vitronectin, antibodies to laminin receptors had minimal inhibitory effect. The use of α6β1, but not α6β4, for adhesion is in accord with a previous study of untransfected and β4-transfected MDA-MB-435 cells (Shaw et al., 1997). Expression levels for α3β1 and α6β1 were essentially unchanged, regardless of whether mutant or wild-type β4 was present, as seen by flow cytometry (Fig. 4 A) and immunoblotting (not depicted).


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)

β1-dependent cell adhesion is unaffected by β4 mutation. (A) To assess cell adhesion, MDA-MB-435 cells were plated on specific substrates for 30 min, and then nonadherent cells were removed by washing and adherent cells were stained using Wright-Giemsa and then quantitated at OD 590 using an ELISA 96-well plate reader. Starting with 70,000 cells/well, ∼70% cell adhesion corresponds to OD 590 = 0.3. (B) Cells were incubated with ∼10 μg/ml of the indicated mAbs at RT for 15 min before being plated on laminin-5. Adhesion was determined as in A. (C) Effects of indicated antibodies on adhesion of wild-type (Wt) β4 cells was determined as in A and B. Results shown are the means of triplicate determinations ± SD.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2172609&req=5

fig6: β1-dependent cell adhesion is unaffected by β4 mutation. (A) To assess cell adhesion, MDA-MB-435 cells were plated on specific substrates for 30 min, and then nonadherent cells were removed by washing and adherent cells were stained using Wright-Giemsa and then quantitated at OD 590 using an ELISA 96-well plate reader. Starting with 70,000 cells/well, ∼70% cell adhesion corresponds to OD 590 = 0.3. (B) Cells were incubated with ∼10 μg/ml of the indicated mAbs at RT for 15 min before being plated on laminin-5. Adhesion was determined as in A. (C) Effects of indicated antibodies on adhesion of wild-type (Wt) β4 cells was determined as in A and B. Results shown are the means of triplicate determinations ± SD.
Mentions: The spreading defect does not arise because of diminished cell adhesion. In a 30-min static cell adhesion assay, all cells adhered similarly to laminin-1, laminin-5, and vitronectin (Fig. 6 A). Adhesion to laminin-5 (Fig. 6 B) or laminin-1 (not depicted) was dependent on β1 rather than β4 integrins, as indicated by nearly complete blocking with an anti-β1 monoclonal antibody. On laminin-1, adhesion was almost entirely α6β1 dependent, whereas on laminin-5, α3β1 also contributed (Fig. 6 C). On vitronectin, antibodies to laminin receptors had minimal inhibitory effect. The use of α6β1, but not α6β4, for adhesion is in accord with a previous study of untransfected and β4-transfected MDA-MB-435 cells (Shaw et al., 1997). Expression levels for α3β1 and α6β1 were essentially unchanged, regardless of whether mutant or wild-type β4 was present, as seen by flow cytometry (Fig. 4 A) and immunoblotting (not depicted).

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
Related in: MedlinePlus