Neer E J, Pulsifer L, Wolf L G
Department of Medicine, Brigham and Women's Hospital, Boston, Massachusetts 02115.
J Biol Chem. 1988 Jun 25;263(18):8996-70.
The guanine nucleotide-binding proteins (G proteins), which transduce hormonal and light signals across the plasma membrane, are heterotrimers composed of alpha, beta, and gamma subunits. Activation of G proteins by guanine nucleotides is accompanied by dissociation of the heterotrimer: G + alpha.beta.gamma in equilibrium alpha G + beta.gamma. Brain contains several G proteins of which the most abundant are alpha 39.beta.gamma and alpha 41.beta.gamma. We have used proteolysis by trypsin to study the functional domains of the alpha subunits. In the presence of guanosine 5'-(3-O-thio)triphosphate, trypsin removes a 2-kDa peptide from the amino terminus of these proteins (Hurley, J. B., Simon, M. I., Teplow, D. B., Robishaw, J. D., and Gilman, A. G. (1984) Science 226, 860-862; Winslow, J. W., Van Amsterdam, J. R., and Neer, E. J. (1986) J. Biol. Chem. 261, 7571-7579). Tryptic cleavage does not affect the GTPase activity of the truncated molecule nor the apparent Km for GTP. However, removal of the 2-kDa amino-terminal peptide prevents association of the alpha subunits with beta.gamma. Since the apparent substrate for pertussis toxin-catalyzed ADP-ribosylation is the alpha.beta.gamma heterotrimer, the trypsin-cleaved alpha subunit is not a substrate for the toxin. Digestion of the carboxyl terminus of alpha 39 with carboxypeptidase A prevents ADP-ribosylation by pertussis toxin but does not interfere with the formation of alpha 39.beta.gamma heterotrimers. We do not yet know whether the amino-terminal region of alpha 39 interacts with beta gamma directly or whether it is necessary to maintain a conformation of alpha 39 which is required for heterotrimer formation. Further studies are needed to define the nature of the contracts between alpha and beta gamma subunits since understanding the structural basis for their reversible interaction is fundamental to understanding their function.
鸟嘌呤核苷酸结合蛋白（G蛋白）可跨质膜传导激素和光信号，它是由α、β和γ亚基组成的异源三聚体。鸟嘌呤核苷酸对G蛋白的激活伴随着异源三聚体的解离：G + αβγ处于平衡状态⇌αG + βγ。脑中含有多种G蛋白，其中含量最丰富的是α39βγ和α41βγ。我们利用胰蛋白酶进行蛋白水解来研究α亚基的功能结构域。在5'-（3-O-硫代）三磷酸鸟苷存在的情况下，胰蛋白酶从这些蛋白质的氨基末端去除一个2 kDa的肽段（赫尔利，J. B.，西蒙，M. I.，特普洛，D. B.，罗比肖，J. D.，以及吉尔曼，A. G.（1984年）《科学》226卷，860 - 862页；温斯洛，J. W.，范阿姆斯特丹，J. R.，以及尼尔，E. J.（1986年）《生物化学杂志》261卷，7571 - 7579页）。胰蛋白酶切割不影响截短分子的GTP酶活性，也不影响GTP的表观米氏常数。然而，去除2 kDa的氨基末端肽段会阻止α亚基与βγ的结合。由于百日咳毒素催化的ADP核糖基化作用的明显底物是αβγ异源三聚体，所以经胰蛋白酶切割的α亚基不是该毒素的底物。用羧肽酶A消化α39的羧基末端可防止百日咳毒素催化的ADP核糖基化作用，但不干扰α39βγ异源三聚体的形成。我们尚不清楚α39的氨基末端区域是否直接与βγ相互作用，也不清楚它对于维持α39形成异源三聚体所需构象是否必要。需要进一步研究来确定α与βγ亚基之间相互作用的本质，因为理解它们可逆相互作用的结构基础对于理解其功能至关重要。
