Taylor J M, Jacob-Mosier G G, Lawton R G, Remmers A E, Neubig R R
Department of Pharmacology, University of Michigan, Ann Arbor 48109.
J Biol Chem. 1994 Nov 4;269(44):27618-24.
The structural basis of receptor-G protein interactions was examined using a photoaffinity derivative of a G protein-activating receptor-derived peptide (Q peptide) from the carboxyl-terminal region of the third intracellular loop of alpha 2 adrenergic receptor. A diazopyruvoyl photoaffinity derivative of this peptide (DAP-Q) was cross-linked to purified bovine brain Go. Specific, competable cross-linking of 750 nM DAP-Q to sites on both the alpha o and beta subunits was observed. No specific cross-linking was seen with non-target proteins or heat-denatured G protein subunits. 125I-DAP-Q labeled the 2-kDa amino-terminal fragment of alpha o as determined by protease digestion of the cross-linked G protein followed by gel electrophoresis or h igh pressure liquid chromatography purification and mass spectroscopy of the radiolabeled proteolysis fragment. The functional significance of incorporation into beta gamma subunit is supported by the absolute requirement of beta gamma subunit for DAP-Q stimulation of Go/Gi GTPase. Thus, specific interactions of G protein-coupled receptors with the beta subunit of G protein, in addition to those with the alpha subunit, appear to be important for receptor-G protein coupling.
利用来自α2肾上腺素能受体第三细胞内环羧基末端区域的G蛋白激活受体衍生肽（Q肽）的光亲和衍生物，研究了受体 - G蛋白相互作用的结构基础。该肽的重氮丙酮酰光亲和衍生物（DAP - Q）与纯化的牛脑Go进行交联。观察到750 nM DAP - Q与αo和β亚基上的位点发生特异性、可竞争的交联。未观察到与非靶蛋白或热变性G蛋白亚基的特异性交联。通过对交联的G蛋白进行蛋白酶消化，然后进行凝胶电泳或高压液相色谱纯化以及对放射性标记的蛋白水解片段进行质谱分析，125I - DAP - Q标记了αo的2 kDa氨基末端片段。βγ亚基对DAP - Q刺激Go / Gi GTPase的绝对需求支持了其掺入βγ亚基的功能意义。因此，除了与α亚基的相互作用外，G蛋白偶联受体与G蛋白β亚基的特异性相互作用似乎对受体 - G蛋白偶联也很重要。
