Spiegel A M, Simonds W F, Jones T L, Goldsmith P K, Unson C G
Molecular Pathophysiology Branch, National Institute of Diabetes, Digestive and Kidney Diseases, National Institutes of Health, Bethesda, MD 20892.
Biochem Soc Symp. 1990;56:61-9.
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.
针对与G蛋白α亚基C末端相对应的合成十肽产生的抗血清的特异性得到了严格界定。针对α亚基C末端十肽产生的抗血清被证明能够免疫沉淀其同源G蛋白，以及识别天然细胞膜中的这些蛋白。因此，αs特异性抗血清能够阻断天然膜中激动剂刺激的腺苷酸环化酶活性，还能免疫沉淀活化的αs-腺苷酸环化酶复合物。αi2特异性抗血清（而非αi3和αz特异性抗血清）能够阻断人血小板膜中激动剂介导的腺苷酸环化酶抑制作用。这些结果表明，C末端十肽参与G蛋白与受体的偶联，但不参与与效应器的偶联。这些抗血清在免疫沉淀COS细胞中的内源性和转染的α亚基方面也被证明是有用的。使用这种方法，我们能够证明αs和αi都与膜相关，但只有后者被豆蔻酰化。突变的αi1（第二个残基甘氨酸变为丙氨酸）无法掺入豆蔻酸，并且定位于可溶部分。因此，豆蔻酰化对于αi而非αs的膜附着至关重要。
