GI2 MEDIATES ALPHA-2-ADRENERGIC INHIBITION OF ADENYLYL CYCLASE IN PLATELET MEMBRANES - INSITU IDENTIFICATION WITH G-ALPHA C-TERMINAL ANTIBODIES
GI2 MEDIATES ALPHA-2-ADRENERGIC INHIBITION OF ADENYLYL CYCLASE IN PLATELET MEMBRANES - INSITU IDENTIFICATION WITH G-ALPHA C-TERMINAL ANTIBODIES
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DOI：
10.1073/pnas.86.20.7809
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发表时间：
1989-10-01
影响因子：
11.1
通讯作者：
SPIEGEL, AM
中科院分区：
文献类型：
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作者：
SIMONDS, WF;GOLDSMITH, PK;SPIEGEL, AM
对应于鸟嘌呤核苷酸结合调节蛋白（G蛋白）α C末端的合成十肽的一组抗体。在兔子体内产生了亚基。每种抗体的特异性通过ELISA的肽结合和通过免疫印迹的与确定的重组G α的结合来评估。在大肠杆菌中表达的亚基。用这些抗体对人血小板膜进行免疫印迹鉴定了多种内源性G蛋白，包括Gs（刺激性）、Gi 2（抑制性）、Gi 3和Gx（z）（功能未知）。用与Gi 2反应但不与Gi 3或Gx（z）抗体反应的C-末端抗体预处理血小板膜，阻断腺苷酸环化酶的α 2-肾上腺素能抑制。这鉴定了Gi 2为该途径中环化酶抑制的主要介质。这种方法可以提供一个通用的手段，确定相关的功能相互作用的G蛋白与受体和效应器在原位。
A panel of antibodies to synthetic decapeptides corresponding to the C termini of guanine nucleotide-binding regulatory protein (G protein) .alpha. subunits has been generated in rabbits. The specificity of each antibody was assessed by ELISA for peptide binding and by immunoblotting for binding to defined, recombinant G.alpha. subunits expressed in Escherichia coli. Immunoblotting of human platelet membranes with these antibodies identified a variety of endogenous G proteins including Gs (stimulatory), Gi2 (inhibitory), Gi3, and Gx(z) (unknown function). Pretreatment of platelet membranes with C-terminal antibodies reactive with Gi2, but not with antibodies to Gi3 or Gx(z), blocked .alpha.2-adrenergic inhibition of adenylyl cyclase. This identifies Gi2 as the dominant mediator of cyclase inhibition in this pathway. This approach may provide a general means of identifying relevant functional interactions of G proteins with receptors and effectors in situ.