Cook L A, Schey K L, Wilcox M D, Dingus J, Hildebrandt J D
Department of Cell and Molecular Pharmacology and Experimental Therapeutics, Medical University of South Carolina, Charleston 29425, USA.
Biochemistry. 1998 Sep 1;37(35):12280-6. doi: 10.1021/bi980230e.
The G protein gamma5 subunit is selectively associated with specific G protein alpha subunits [Wilcox, M. D., et al. (1995) J. Biol. Chem. 270, 4189] and is localized preferentially in focal adhesion plaques [Hansen, C. A., et al. (1996) J. Cell Biol. 126, 811]. What determines the differential association of G proteins and their subunits with specific cellular structures or compartments is not clear, but one factor could be variation in the pattern of processing of the proteins. To study gamma5 subunit diversity and modifications, G protein subunits were fractionated on an HPLC phenyl column and analyzed with a gamma5-specific antiserum. The gamma5 eluted from the column as two peaks of immunoreactivity. Analysis by matrix-assisted laser desorption ionization (MALDI) mass spectrometry and electrospray ionization tandem mass spectrometry revealed that the first immunoreactive peak corresponded to the predicted gamma5 isoform (N-terminally acetylated after removal of methionine, C-terminally geranylgeranylated and carboxymethylated with removal of the last three amino acids), while the second peak of immunoreactivity contained a gamma5 isoform isoprenylated at the C-terminus but retaining its three terminal amino acids. This alternatively processed protein is the predominant gamma5 subunit isoform associated with Go and Gi proteins purified from bovine brain. These results describe a new C-terminal processing pattern for G protein gamma subunits and establish the principle that G protein gamma subunits can be heterogeneously modified at their C-termini. This is a site on the gamma subunit critical for membrane and protein-protein interactions of G proteins. These results open the possibility that one determinant of the localization of G proteins in cells could be the pattern of processing of their gamma subunit constituents.
G蛋白γ5亚基与特定的G蛋白α亚基选择性相关[威尔科克斯，M.D.等人（1995年）《生物化学杂志》270卷，4189页]，并且优先定位于粘着斑[汉森，C.A.等人（1996年）《细胞生物学杂志》126卷，811页]。尚不清楚是什么决定了G蛋白及其亚基与特定细胞结构或区室的差异关联，但一个因素可能是蛋白质加工模式的变化。为了研究γ5亚基的多样性和修饰，将G蛋白亚基在HPLC苯基柱上进行分离，并用γ5特异性抗血清进行分析。γ5从柱上洗脱时呈现出两个免疫反应峰。通过基质辅助激光解吸电离（MALDI）质谱和电喷雾电离串联质谱分析表明，第一个免疫反应峰对应于预测的γ5同工型（甲硫氨酸去除后N端乙酰化，C端香叶基香叶基化并去除最后三个氨基酸后羧甲基化），而第二个免疫反应峰包含一个C端异戊二烯化但保留其三个末端氨基酸的γ5同工型。这种经过不同加工的蛋白质是与从牛脑纯化的Go和Gi蛋白相关的主要γ5亚基同工型。这些结果描述了G蛋白γ亚基一种新的C端加工模式，并确立了G蛋白γ亚基可在其C端进行异质性修饰的原则。这是γ亚基上对G蛋白的膜和蛋白质-蛋白质相互作用至关重要的位点。这些结果开启了一种可能性，即G蛋白在细胞内定位的一个决定因素可能是其γ亚基成分的加工模式。
