EVIDENCE FOR A REGULATED INTERACTION BETWEEN HETEROTRIMERIC G-PROTEINS AND CAVEOLIN
EVIDENCE FOR A REGULATED INTERACTION BETWEEN HETEROTRIMERIC G-PROTEINS AND CAVEOLIN
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DOI：
10.1074/jbc.270.26.15693
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发表时间：
1995-06-30
影响因子：
4.8
通讯作者：
LISANTI, MP
中科院分区：
文献类型：
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作者：
LI, SW;OKAMOTO, T;LISANTI, MP
小凹是闪光形状的质膜特化，22-kDa 蛋白，小窝蛋白，是体内小凹膜的主要成分，最近的证据表明小凹可能参与 G 蛋白偶联信号传导事件，我们研究了小窝蛋白与异三聚体 G 蛋白的潜在相互作用，使用细胞分级分离技术，我们发现 G(s α) 的突变或药理学激活可防止其与小凹蛋白共分级，在第二种独立方法中，我们直接检查了相互作用为此，我们将 Caveolin 重组表达为谷胱甘肽 S-转移酶融合蛋白。使用体外结合测定，我们发现 Caveolin 与 G 蛋白 α 亚基（G(s)、G(o) 和 G(i)）相互作用。 G(α) 亚基的突变或药理激活（用鸟苷 5'-O-（硫代三磷酸））可阻止这种相互作用，表明 G(α) 亚基的非活性 GDP 结合形式优先与小窝蛋白相互作用。这种 G 蛋白结合活性位于小窝蛋白细胞质 N 末端的 41 个氨基酸区域内。结构域（残基 61-101），进一步的功能分析表明，源自 Caveolin 该区域（残基 82-101）的多肽可有效抑制纯化 G 蛋白的基础活性，显然是通过抑制 GDP/GTP 交换。该 Caveolin 序列与 Rab GDP 解离抑制剂（一种已知的 Rab 蛋白 GDP/GTP 交换抑制剂）的区域同源。这些数据表明，caveolin 可以发挥负调节异源三聚体G蛋白。
Caveolae are flash-shaped plasma membrane specializations, A 22-kDa protein, caveolin, is a principal component of caveolar membranes in vivo, As recent evidence suggests that caveolae may participate in G protein-coupled signaling events, we have investigated the potential interaction of caveolin with heterotrimeric G proteins, Using cell fractionation techniques, we found that mutational or pharmacologic activation of G(s alpha) prevents its co-fractionation with caveolin, In a second independent approach, we directly examined the interaction of G proteins with caveolin, For this purpose, we recombinantly expressed caveolin as a glutathione S-transferase fusion protein, Using an in vitro binding assay, we found that caveolin interacts with G protein alpha subunits (G(s), G(o), and G(i)). Mutational or pharmacologic activation (with guanosine 5'-O-(thiotriphosphate)) of G(alpha) subunits prevents this interaction, indicating that the inactive GDP-bound form of G(alpha) subunits preferentially interacts with caveolin, This G protein binding activity is located within a 41-amino acid region of caveolin's cytoplasmic N-terminal. domain (residues 61-101), Further functional analysis shows that a polypeptide derived from this region of caveolin (residues 82-101) effectively suppresses the basal activity of purified G proteins, apparently by inhibiting GDP/GTP exchange, This caveolin sequence is homologous to a region of the Rab GDP dissociation inhibitor, a known inhibitor of GDP/GTP exchange for Rab proteins, These data suggest that caveolin could function to negatively regulate the activation state of heterotrimeric G proteins.