Berditchevski Fedor, Odintsova Elena, Sawada Shigeaki, Gilbert Elizabeth
Cancer Research UK Institute for Cancer Studies, The University of Birmingham, Edgbaston, Birmingham B15 2TA, United Kingdom.
J Biol Chem. 2002 Oct 4;277(40):36991-7000. doi: 10.1074/jbc.M205265200. Epub 2002 Jul 10.
Transmembrane proteins of the tetraspanin superfamily are assembled in multimeric complexes on the cell surface. Spatial orientation of tetraspanins within these complexes may affect signaling functions of the associated transmembrane receptors (e.g. integrins, receptor-type tyrosine kinases). The structural determinants that control assembly of the tetraspanin complexes are unknown. We have found that various tetraspanins and the alpha(3) integrin subunit are palmitoylated. The stability and molecular composition of the palmitoylated alpha(3)beta(1)-tetraspanin complexes are not affected by adhesion. To assess the significance of palmitoylation in the function of the alpha(3)beta(1)-tetraspanin complexes we mapped the sites of palmitoylation for CD151. Mutation of six cysteines, Cys(11), Cys(15), Cys(79), Cys(80), Cys(242), and Cys(243) was necessary to completely abolish palmitoylation of CD151. The association of the palmitoylation-deficient mutant of CD151 (CD151Cys8) with CD81 and CD63 was markedly decreased, but the interaction of the alpha(3)beta(1)-CD151Cys8 complex with phosphatidylinositol 4-kinase was not affected. Ectopic expression of CD151Cys8 in Rat-1 cells impaired the interactions of the endogenous CD63 and CD81 with the alpha(3)beta(1) integrin. Although the expression of the palmitoylation-deficient CD151 does not change cell spreading on the extracellular matrix, the number of focal adhesions increased. Adhesion-induced phosphorylation of PKB/c-Akt is markedly increased in cells expressing a palmitoylation-deficient mutant, thereby providing direct evidence for the role of the tetraspanin microdomains in regulation of the integrin-dependent phosphatidylinositol 3-kinase signaling pathway. In contrast, activation of FAK and ERK1/2 were not affected by the expression of CD151Cys8. Our results demonstrate that palmitoylation of tetraspanins is critical not only for the organization of the integrin-tetraspanin microdomains but also has a specific role in modulation of adhesion-dependent signaling.
四跨膜蛋白超家族的跨膜蛋白在细胞表面组装成多聚体复合物。这些复合物中四跨膜蛋白的空间取向可能会影响相关跨膜受体（如整合素、受体型酪氨酸激酶）的信号传导功能。控制四跨膜蛋白复合物组装的结构决定因素尚不清楚。我们发现各种四跨膜蛋白和α(3)整合素亚基都被棕榈酰化。棕榈酰化的α(3)β(1)-四跨膜蛋白复合物的稳定性和分子组成不受黏附的影响。为了评估棕榈酰化在α(3)β(1)-四跨膜蛋白复合物功能中的重要性，我们绘制了CD151的棕榈酰化位点。六个半胱氨酸（Cys(11)、Cys(15)、Cys(79)、Cys(80)、Cys(242)和Cys(243)）的突变是完全消除CD151棕榈酰化所必需的。棕榈酰化缺陷型CD151（CD151Cys8）与CD81和CD63的结合明显减少，但α(3)β(1)-CD151Cys8复合物与磷脂酰肌醇4-激酶的相互作用不受影响。CD151Cys8在大鼠-1细胞中的异位表达损害了内源性CD63和CD81与α(3)β(1)整合素的相互作用。虽然棕榈酰化缺陷型CD151的表达不会改变细胞在细胞外基质上的铺展，但粘着斑的数量增加了。在表达棕榈酰化缺陷型突变体的细胞中，黏附诱导的蛋白激酶B/蛋白激酶B（PKB/c-Akt）磷酸化明显增加，从而为四跨膜蛋白微区在整合素依赖性磷脂酰肌醇3-激酶信号通路调节中的作用提供了直接证据。相比之下，黏着斑激酶（FAK）和细胞外信号调节激酶1/2（ERK1/2）的激活不受CD151Cys8表达的影响。我们的结果表明，四跨膜蛋白的棕榈酰化不仅对整合素-四跨膜蛋白微区的组织至关重要，而且在调节黏附依赖性信号传导中具有特定作用。
