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

Integrin α3, α6, and β4 subunits undergo palmitoylation. (A) MDA-MB-231 human breast carcinoma cells were pulsed with [3H]palmitate and incubated at 4°C with mAb anti-CD82 (mAb M104) or anti-CD151 (mAb 5C11). Unbound antibody was removed, cells were lysed in 1% Brij 96, and immune complexes were collected using protein G–agarose. Remaining lysate was then used for further immunoprecipitation. In, intracellular fraction; Sur, surface fraction. (B) A431 cells were pulsed with [3H]palmitate, surface labeled with biotin, and lysed in RIPA buffer, and then human α2, α3, and α6 integrins were immunoprecipitated using mAbs A2-IIE10, A3-X8, and GoH3, respectively. Proteins labeled with biotin (lanes 1–3) and [3H]palmitate (lanes 4–6) are shown. Kidney epithelial B12 cells expressing human integrin α2 and α6 were also labeled with [3H]palmitate, and then α2 and α6 were immunoprecipitated (lanes 7 and 8). Comparable cell surface α2 and α6 levels were seen by flow cytometry (not depicted). Note that, under RIPA conditions, human α6 remains associated with human (lane 6), but not mouse (lane 8) β4. White lines indicate that intervening lanes have been spliced out. (C) A431 cells were pulsed with [3H]palmitate and then chased in the presence of 100 μM unlabeled palmitic acid for up to 6 h, as indicated. Anti-α6 and anti-CD151 immunoprecipitates were analyzed for [3H] labeling of β4 and CD151 (top) or by immunoblotting (loading controls, bottom) using anti-α6 cyto tail antibody (to evaluate α6β4) and anti-α3 cyto tail antibody (to evaluate α3β1–CD151 complexes). Question mark indicates unknown protein.
© Copyright Policy
Related In: Results  -  Collection


getmorefigures.php?uid=PMC2172609&req=5

fig1: Integrin α3, α6, and β4 subunits undergo palmitoylation. (A) MDA-MB-231 human breast carcinoma cells were pulsed with [3H]palmitate and incubated at 4°C with mAb anti-CD82 (mAb M104) or anti-CD151 (mAb 5C11). Unbound antibody was removed, cells were lysed in 1% Brij 96, and immune complexes were collected using protein G–agarose. Remaining lysate was then used for further immunoprecipitation. In, intracellular fraction; Sur, surface fraction. (B) A431 cells were pulsed with [3H]palmitate, surface labeled with biotin, and lysed in RIPA buffer, and then human α2, α3, and α6 integrins were immunoprecipitated using mAbs A2-IIE10, A3-X8, and GoH3, respectively. Proteins labeled with biotin (lanes 1–3) and [3H]palmitate (lanes 4–6) are shown. Kidney epithelial B12 cells expressing human integrin α2 and α6 were also labeled with [3H]palmitate, and then α2 and α6 were immunoprecipitated (lanes 7 and 8). Comparable cell surface α2 and α6 levels were seen by flow cytometry (not depicted). Note that, under RIPA conditions, human α6 remains associated with human (lane 6), but not mouse (lane 8) β4. White lines indicate that intervening lanes have been spliced out. (C) A431 cells were pulsed with [3H]palmitate and then chased in the presence of 100 μM unlabeled palmitic acid for up to 6 h, as indicated. Anti-α6 and anti-CD151 immunoprecipitates were analyzed for [3H] labeling of β4 and CD151 (top) or by immunoblotting (loading controls, bottom) using anti-α6 cyto tail antibody (to evaluate α6β4) and anti-α3 cyto tail antibody (to evaluate α3β1–CD151 complexes). Question mark indicates unknown protein.

Mentions: While studying palmitoylated CD151 (Yang et al., 2002), we noticed that it associates with palmitoylated proteins resembling the β4, α3, and α6 integrin subunits (Fig. 1 A, lanes 3 and 4). [3H]palmitate-labeled integrin subunits from [3H]palmitate-labeled MDA-MB-231 breast carcinoma cells were more abundant on the cell surface (Fig. 1 A, lane 3) than intracellularly (Fig. 1 A, lane 4), and were not present in tetraspanin CD82 immunoprecipitations (Fig. 1 A, lanes 1 and 2). Integrin palmitoylation was confirmed by recovery of [3H]palmitate-labeled α3 (Fig. 1 B, lane 5), α6 (Fig. 1 B, lanes 6 and 8), and β4 (Fig. 1 B, lane 6), but not α2 (Fig. 1 B, lanes 4 and 7), from stringent detergent (RIPA) lysates of A431 cells or B12 kidney epithelial cells. In A431 cells, α2, α3, and α6 integrin subunits were present at comparable levels, as indicated by cell surface labeling (Fig. 1 B, lanes 1–3), and in B12 cells, α2 and α6 were again at comparable levels (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)

Integrin α3, α6, and β4 subunits undergo palmitoylation. (A) MDA-MB-231 human breast carcinoma cells were pulsed with [3H]palmitate and incubated at 4°C with mAb anti-CD82 (mAb M104) or anti-CD151 (mAb 5C11). Unbound antibody was removed, cells were lysed in 1% Brij 96, and immune complexes were collected using protein G–agarose. Remaining lysate was then used for further immunoprecipitation. In, intracellular fraction; Sur, surface fraction. (B) A431 cells were pulsed with [3H]palmitate, surface labeled with biotin, and lysed in RIPA buffer, and then human α2, α3, and α6 integrins were immunoprecipitated using mAbs A2-IIE10, A3-X8, and GoH3, respectively. Proteins labeled with biotin (lanes 1–3) and [3H]palmitate (lanes 4–6) are shown. Kidney epithelial B12 cells expressing human integrin α2 and α6 were also labeled with [3H]palmitate, and then α2 and α6 were immunoprecipitated (lanes 7 and 8). Comparable cell surface α2 and α6 levels were seen by flow cytometry (not depicted). Note that, under RIPA conditions, human α6 remains associated with human (lane 6), but not mouse (lane 8) β4. White lines indicate that intervening lanes have been spliced out. (C) A431 cells were pulsed with [3H]palmitate and then chased in the presence of 100 μM unlabeled palmitic acid for up to 6 h, as indicated. Anti-α6 and anti-CD151 immunoprecipitates were analyzed for [3H] labeling of β4 and CD151 (top) or by immunoblotting (loading controls, bottom) using anti-α6 cyto tail antibody (to evaluate α6β4) and anti-α3 cyto tail antibody (to evaluate α3β1–CD151 complexes). Question mark indicates unknown protein.
© Copyright Policy
Related In: Results  -  Collection

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

fig1: Integrin α3, α6, and β4 subunits undergo palmitoylation. (A) MDA-MB-231 human breast carcinoma cells were pulsed with [3H]palmitate and incubated at 4°C with mAb anti-CD82 (mAb M104) or anti-CD151 (mAb 5C11). Unbound antibody was removed, cells were lysed in 1% Brij 96, and immune complexes were collected using protein G–agarose. Remaining lysate was then used for further immunoprecipitation. In, intracellular fraction; Sur, surface fraction. (B) A431 cells were pulsed with [3H]palmitate, surface labeled with biotin, and lysed in RIPA buffer, and then human α2, α3, and α6 integrins were immunoprecipitated using mAbs A2-IIE10, A3-X8, and GoH3, respectively. Proteins labeled with biotin (lanes 1–3) and [3H]palmitate (lanes 4–6) are shown. Kidney epithelial B12 cells expressing human integrin α2 and α6 were also labeled with [3H]palmitate, and then α2 and α6 were immunoprecipitated (lanes 7 and 8). Comparable cell surface α2 and α6 levels were seen by flow cytometry (not depicted). Note that, under RIPA conditions, human α6 remains associated with human (lane 6), but not mouse (lane 8) β4. White lines indicate that intervening lanes have been spliced out. (C) A431 cells were pulsed with [3H]palmitate and then chased in the presence of 100 μM unlabeled palmitic acid for up to 6 h, as indicated. Anti-α6 and anti-CD151 immunoprecipitates were analyzed for [3H] labeling of β4 and CD151 (top) or by immunoblotting (loading controls, bottom) using anti-α6 cyto tail antibody (to evaluate α6β4) and anti-α3 cyto tail antibody (to evaluate α3β1–CD151 complexes). Question mark indicates unknown protein.
Mentions: While studying palmitoylated CD151 (Yang et al., 2002), we noticed that it associates with palmitoylated proteins resembling the β4, α3, and α6 integrin subunits (Fig. 1 A, lanes 3 and 4). [3H]palmitate-labeled integrin subunits from [3H]palmitate-labeled MDA-MB-231 breast carcinoma cells were more abundant on the cell surface (Fig. 1 A, lane 3) than intracellularly (Fig. 1 A, lane 4), and were not present in tetraspanin CD82 immunoprecipitations (Fig. 1 A, lanes 1 and 2). Integrin palmitoylation was confirmed by recovery of [3H]palmitate-labeled α3 (Fig. 1 B, lane 5), α6 (Fig. 1 B, lanes 6 and 8), and β4 (Fig. 1 B, lane 6), but not α2 (Fig. 1 B, lanes 4 and 7), from stringent detergent (RIPA) lysates of A431 cells or B12 kidney epithelial cells. In A431 cells, α2, α3, and α6 integrin subunits were present at comparable levels, as indicated by cell surface labeling (Fig. 1 B, lanes 1–3), and in B12 cells, α2 and α6 were again at comparable levels (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