Ueda N, Iñiguez-Lluhi J A, Lee E, Smrcka A V, Robishaw J D, Gilman A G
Department of Pharmacology, University of Texas Southwestern Medical Center, Dallas 75235.
J Biol Chem. 1994 Feb 11;269(6):4388-95.
The beta and gamma subunits of heterotrimeric guanine nucleotide-binding regulatory proteins (G proteins) form tightly associated complexes. To examine functional differences among the large number of possible combinations of unique beta and gamma subunits, we have synthesized and characterized beta gamma complexes containing gamma 5 and gamma 7, two widely distributed gamma subunits. When either gamma 5 or gamma 7 is expressed concurrently with beta 1 or beta 2 subunits in a baculovirus/Sf9 cell system, all four subunit complexes support pertussis toxin-catalyzed ADP-ribosylation of rGi alpha 1 (where "r" indicates recombinant), indicating formation of functional complexes. Each of the complexes was purified by subunit exchange chromatography, using the G203A mutant of rGi alpha 1 as the immobilized ligand. The purified preparations were compared with other recombinant beta gamma subunits, including beta 1 gamma 1 and beta 1 gamma 2, for their ability to modulate type I and II adenylyl cyclase activities; stimulate phosphoinositide-specific phospholipase C beta; support pertussis toxin-catalyzed ADP-ribosylation of rGi alpha 1 and Go alpha; and inhibit steady-state GTP hydrolysis catalyzed by Gs alpha, Go alpha, and myristoylated rGi alpha 2. The results emphasize the unique properties of beta 1 gamma 1. The properties of the complexes containing gamma 5 or gamma 7 were similar to each other and to those of beta 1 gamma 2.
异三聚体鸟嘌呤核苷酸结合调节蛋白（G蛋白）的β和γ亚基形成紧密相关的复合物。为了研究大量独特的β和γ亚基可能组合之间的功能差异，我们合成并表征了包含γ5和γ7这两种广泛分布的γ亚基的βγ复合物。当γ5或γ7与β1或β2亚基在杆状病毒/Sf9细胞系统中同时表达时，所有四种亚基复合物都支持百日咳毒素催化的rGiα1（其中“r”表示重组）的ADP核糖基化，表明形成了功能复合物。每种复合物都通过亚基交换色谱法进行纯化，使用rGiα1的G203A突变体作为固定配体。将纯化的制剂与其他重组βγ亚基，包括β1γ1和β1γ2，比较它们调节I型和II型腺苷酸环化酶活性的能力；刺激磷酸肌醇特异性磷脂酶Cβ；支持百日咳毒素催化的rGiα1和Goα的ADP核糖基化；以及抑制由Gsα、Goα和肉豆蔻酰化的rGiα2催化的稳态GTP水解。结果强调了β1γ1的独特性质。包含γ5或γ7的复合物的性质彼此相似，且与β1γ2的性质相似。
