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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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Identification of integrin palmitoylation sites. (A) Membrane-proximal regions containing candidate palmitoylation sites are shaded gray. The first lysine defines a putative transmembrane interface. A and B designate alternatively spliced forms of integrin cytoplasmic tails. (B) MDA-MB-435 cells stably expressing mutant or wild-type β4, or vector control, were [3H]palmitate labeled and lysed in 1% Brij 96. The α2 integrin was immunoprecipitated from vector control cells, and α6β4 was immunoprecipitated (using anti-α6 mAb GoH3) from β4-transfected cells. Shown are proteins labeled with [3H]palmitate (left) or blotted with anti-β4 antibody (right). (C) Murine B12 cells, stably expressing human integrin α subunits, were [3H]palmitate labeled and lysed in 1% RIPA. Integrins were immunoprecipitated using anti–human α2 (lane 1) and α3 (lanes 2–5) antibodies (A2-IIE10 and A3-X8) and resolved by SDS-PAGE, and [3H]palmitate was detected. The α3 subunits include α3-X3TC5 (α3 tail and transmembrane regions are replaced by those regions from α5; Yauch et al., 1998) and α3-C1067S (palmitoylation site point mutant).
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fig3: Identification of integrin palmitoylation sites. (A) Membrane-proximal regions containing candidate palmitoylation sites are shaded gray. The first lysine defines a putative transmembrane interface. A and B designate alternatively spliced forms of integrin cytoplasmic tails. (B) MDA-MB-435 cells stably expressing mutant or wild-type β4, or vector control, were [3H]palmitate labeled and lysed in 1% Brij 96. The α2 integrin was immunoprecipitated from vector control cells, and α6β4 was immunoprecipitated (using anti-α6 mAb GoH3) from β4-transfected cells. Shown are proteins labeled with [3H]palmitate (left) or blotted with anti-β4 antibody (right). (C) Murine B12 cells, stably expressing human integrin α subunits, were [3H]palmitate labeled and lysed in 1% RIPA. Integrins were immunoprecipitated using anti–human α2 (lane 1) and α3 (lanes 2–5) antibodies (A2-IIE10 and A3-X8) and resolved by SDS-PAGE, and [3H]palmitate was detected. The α3 subunits include α3-X3TC5 (α3 tail and transmembrane regions are replaced by those regions from α5; Yauch et al., 1998) and α3-C1067S (palmitoylation site point mutant).

Mentions: The membrane-proximal region of the β4 cytoplasmic tail contains seven potential cysteine palmitoylation sites (Fig. 3 A). Because palmitoylation often occurs on multiple clustered cysteines in the same molecule (Gundersen et al., 1994; Chapman et al., 1996; Berditchevski et al., 2002; Charrin et al., 2002; Yang et al., 2002), we prepared a “7C/S” β4 mutant, replacing all seven cysteines with serines. When stably expressed in MDA-MB-435 cells, wild-type β4, but not the 7C/S mutant, incorporated [3H]palmitate (Fig. 3 B, left). Although β4 palmitoylation was lost, the 7C/S mutant retained association with palmitoylated α6 (Fig. 3 B, left). Wild-type and mutant β4 were present at similar levels, as detected by anti-β4 immunoblotting (Fig. 3 B, right; and Fig. 4 B, middle), flow cytometry (Fig. 4 A), cell surface biotinylation (Fig. 4 B, top), and by [35S]methionine labeling (not depicted). Mutant and wild-type β4 associated similarly with their core partner, CD151 (Fig. 4 B, bottom), and brought similarly elevated amounts of CD151 and α6 to the cell surface (Fig. 4 A). Levels of cell surface α3 (Fig. 4 A) and β1 (not depicted) also remained very similar when either wild-type or mutant β4 was expressed.


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)

Identification of integrin palmitoylation sites. (A) Membrane-proximal regions containing candidate palmitoylation sites are shaded gray. The first lysine defines a putative transmembrane interface. A and B designate alternatively spliced forms of integrin cytoplasmic tails. (B) MDA-MB-435 cells stably expressing mutant or wild-type β4, or vector control, were [3H]palmitate labeled and lysed in 1% Brij 96. The α2 integrin was immunoprecipitated from vector control cells, and α6β4 was immunoprecipitated (using anti-α6 mAb GoH3) from β4-transfected cells. Shown are proteins labeled with [3H]palmitate (left) or blotted with anti-β4 antibody (right). (C) Murine B12 cells, stably expressing human integrin α subunits, were [3H]palmitate labeled and lysed in 1% RIPA. Integrins were immunoprecipitated using anti–human α2 (lane 1) and α3 (lanes 2–5) antibodies (A2-IIE10 and A3-X8) and resolved by SDS-PAGE, and [3H]palmitate was detected. The α3 subunits include α3-X3TC5 (α3 tail and transmembrane regions are replaced by those regions from α5; Yauch et al., 1998) and α3-C1067S (palmitoylation site point mutant).
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Related In: Results  -  Collection

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fig3: Identification of integrin palmitoylation sites. (A) Membrane-proximal regions containing candidate palmitoylation sites are shaded gray. The first lysine defines a putative transmembrane interface. A and B designate alternatively spliced forms of integrin cytoplasmic tails. (B) MDA-MB-435 cells stably expressing mutant or wild-type β4, or vector control, were [3H]palmitate labeled and lysed in 1% Brij 96. The α2 integrin was immunoprecipitated from vector control cells, and α6β4 was immunoprecipitated (using anti-α6 mAb GoH3) from β4-transfected cells. Shown are proteins labeled with [3H]palmitate (left) or blotted with anti-β4 antibody (right). (C) Murine B12 cells, stably expressing human integrin α subunits, were [3H]palmitate labeled and lysed in 1% RIPA. Integrins were immunoprecipitated using anti–human α2 (lane 1) and α3 (lanes 2–5) antibodies (A2-IIE10 and A3-X8) and resolved by SDS-PAGE, and [3H]palmitate was detected. The α3 subunits include α3-X3TC5 (α3 tail and transmembrane regions are replaced by those regions from α5; Yauch et al., 1998) and α3-C1067S (palmitoylation site point mutant).
Mentions: The membrane-proximal region of the β4 cytoplasmic tail contains seven potential cysteine palmitoylation sites (Fig. 3 A). Because palmitoylation often occurs on multiple clustered cysteines in the same molecule (Gundersen et al., 1994; Chapman et al., 1996; Berditchevski et al., 2002; Charrin et al., 2002; Yang et al., 2002), we prepared a “7C/S” β4 mutant, replacing all seven cysteines with serines. When stably expressed in MDA-MB-435 cells, wild-type β4, but not the 7C/S mutant, incorporated [3H]palmitate (Fig. 3 B, left). Although β4 palmitoylation was lost, the 7C/S mutant retained association with palmitoylated α6 (Fig. 3 B, left). Wild-type and mutant β4 were present at similar levels, as detected by anti-β4 immunoblotting (Fig. 3 B, right; and Fig. 4 B, middle), flow cytometry (Fig. 4 A), cell surface biotinylation (Fig. 4 B, top), and by [35S]methionine labeling (not depicted). Mutant and wild-type β4 associated similarly with their core partner, CD151 (Fig. 4 B, bottom), and brought similarly elevated amounts of CD151 and α6 to the cell surface (Fig. 4 A). Levels of cell surface α3 (Fig. 4 A) and β1 (not depicted) also remained very similar when either wild-type or mutant β4 was expressed.

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