Iñiguez-Lluhi J A, Simon M I, Robishaw J D, Gilman A G
Department of Pharmacology, University of Texas Southwestern Medical Center, Dallas 75235.
J Biol Chem. 1992 Nov 15;267(32):23409-17.
Heterotrimeric guanine nucleotide-binding regulatory proteins (G proteins) consist of a nucleotide-binding alpha subunit and a high-affinity complex of beta and gamma subunits. There is molecular heterogeneity of beta and gamma, but the significance of this diversity is poorly understood. Different G protein beta and gamma subunits have been expressed both singly and in combinations in Sf9 cells. Although expression of individual subunits is achieved in all cases, beta gamma subunit activity (support of pertussis toxin-catalyzed ADP-ribosylation of rGi alpha 1) is detected only when beta and gamma are expressed concurrently. Of the six combinations of beta gamma tested (beta 1 or beta 2 with gamma 1, gamma 2, or gamma 3), only one, beta 2 gamma 1, failed to generate a functional complex. Each of the other five complexes has been purified by subunit exchange chromatography using Go alpha-agarose as the chromatographic matrix. We have detected differences in the abilities of the purified proteins to support ADP-ribosylation of Gi alpha 1; these differences are attributable to the gamma component of the complex. When assayed for their ability to inhibit calmodulin-stimulated type-I adenylylcyclase activity or to potentiate Gs alpha-stimulated type-II adenylylcyclase, recombinant beta 1 gamma 1 and transducin beta gamma are approximately 10 and 20 times less potent, respectively, than the other complexes examined. Prenylation and/or further carboxyl-terminal processing of gamma are not required for assembly of the beta gamma subunit complex but are indispensable for high affinity interactions of beta gamma with either G protein alpha subunits or adenylylcyclases.
异源三聚体鸟嘌呤核苷酸结合调节蛋白（G蛋白）由一个核苷酸结合α亚基和一个β亚基与γ亚基的高亲和力复合物组成。β亚基和γ亚基存在分子异质性，但这种多样性的意义尚不清楚。不同的G蛋白β亚基和γ亚基已在Sf9细胞中单独或组合表达。虽然在所有情况下都能实现单个亚基的表达，但只有当β亚基和γ亚基同时表达时，才能检测到βγ亚基活性（支持百日咳毒素催化的rGiα1的ADP核糖基化）。在所测试的六种βγ组合（β1或β2与γ1、γ2或γ3）中，只有一种，即β2γ1，未能形成功能性复合物。其他五种复合物中的每一种都已通过使用Goα-琼脂糖作为色谱基质的亚基交换色谱法进行了纯化。我们检测到纯化后的蛋白质在支持Giα1的ADP核糖基化能力方面存在差异；这些差异归因于复合物的γ组分。当检测它们抑制钙调蛋白刺激的I型腺苷酸环化酶活性或增强Gsα刺激的II型腺苷酸环化酶活性的能力时，重组β1γ1和转导素βγ的效力分别比所检测的其他复合物低约10倍和20倍。γ亚基的异戊二烯化和/或进一步的羧基末端加工对于βγ亚基复合物的组装不是必需的，但对于βγ与G蛋白α亚基或腺苷酸环化酶的高亲和力相互作用是不可或缺的。
