Antibodies as probes of G-protein receptor-effector coupling and of G-protein membrane attachment.
Antibodies as probes of G-protein receptor-effector coupling and of G-protein membrane attachment.
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抗体作为 G 蛋白受体效应物偶联和 G 蛋白膜附着的探针。
DOI：
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发表时间：
1990
期刊：
影响因子：
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通讯作者：
C. Unson
中科院分区：
文献类型：
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作者：
A. Spiegel;W. Simonds;T. L. Jones;P. Goldsmith;C. Unson
严格定义了针对对应于G蛋白α-亚基C-末端的合成十肽产生的抗血清的特异性。针对α-亚基C-末端十肽产生的抗血清证明能够免疫沉淀它们的同源G-蛋白，以及识别天然细胞膜中的这些蛋白。因此，α s-特异性抗血清可以阻断激动剂刺激的腺苷酸环化酶活性在天然膜，也免疫沉淀活化的α s-腺苷酸环化酶复合物。α i 2-特异性抗血清，而不是α i 3-和α z-特异性抗血清可以阻断激动剂介导的对人血小板膜中腺苷酸环化酶的抑制。这些结果表明，C-末端十肽参与G-蛋白受体，但不效应，耦合。这些抗血清也被证明是有用的免疫沉淀的内源性和转染的α-亚基在COS细胞。使用这种方法，我们能够证明α s和α i都是膜相关的，但只有后者是肉豆蔻化的。突变体α 11（第二个残基Gly变为Ala）不能掺入肉豆蔻酸酯，并且定位于可溶性级分中。因此，肉豆蔻酰化对于α i而不是α s的膜附着是必需的。
The specificity of antisera raised against synthetic decapeptides corresponding to the C-terminus of G-protein alpha-subunits was rigorously defined. Antisera raised against alpha-subunit C-terminal decapeptides proved capable of immuno-precipitating their cognate G-proteins, as well as recognizing these proteins in native cell membranes. Thus the alpha s-specific antiserum could block agonist-stimulated adenylyl cyclase activity in native membranes and also immuno-precipitate an activated alpha s-adenylyl cyclase complex. The alpha i 2-, but not alpha i 3- and alpha z-specific antiserum could block agonist-mediated inhibition of adenylyl cyclase in human platelet membranes. These results indicate that the C-terminal decapeptide is involved in G-protein receptor, but not effector, coupling. These antisera also proved useful in immunoprecipitation of endogenous and transfected alpha-subunits in COS cells. Using this approach, we were able to show that both alpha s and alpha i are membrane associated, but only the latter is myristylated. A mutant alpha i 1 (second residue Gly changed to Ala) fails to incorporate myristate and is localized in the soluble fraction. Myristylation is thus essential for membrane attachment of alpha i, but not alpha s.