Zhang J H, Simonds W F
Metabolic Diseases Branch, National Institute of Diabetes and Digestiveand Kidney Diseases, National Institutes of Health, Bethesda, Maryland 20892, USA.
J Neurosci. 2000 Feb 1;20(3):RC59. doi: 10.1523/JNEUROSCI.20-03-j0004.2000.
A structurally divergent G-protein beta subunit expressed in brain and retina, Gbeta5, exhibits functional specialization in its protein-protein interactions in vitro. In retina, Gbeta5 has been isolated in a soluble complex with regulator of G-protein signaling RGS7. The function and molecular associations of Gbeta5 in brain are unknown. To identify tightly bound proteins associated with Gbeta5 in the brain, it was immunoaffinity-purified from a nonionic detergent extract of washed mouse brain membranes using an antibody directed against its N terminus. Elution with cognate peptide revealed a broad band of 55 kDa that coeluted with Gbeta5 on SDS-PAGE. The copurifying 55 kDa band was identified as an approximately 1:1 mixture of RGS6 and RGS7 by matrix-assisted laser desorption ionization mass spectroscopic analysis of tryptic peptides. Gbeta5 and RGS7 could be reciprocally coimmunoprecipitated from unfractionated brain membrane extracts confirming the tight association of native proteins. In contrast, immunoblotting of the peptide eluate revealed no copurifying Galphaq/11, Galphai1/2, Ggamma2, Ggamma3, or Ggamma7. These findings implicate RGS6 and RGS7 in the function of Gbeta5 in the brain and suggest that a large fraction of membrane-targeted Gbeta5 has no associated G subunit and therefore functions outside the canonical framework of G(beta)(gamma) interactions.
一种在大脑和视网膜中表达的结构不同的G蛋白β亚基Gβ5，在体外其蛋白质-蛋白质相互作用中表现出功能特异性。在视网膜中，Gβ5已被分离出与G蛋白信号调节因子RGS7形成可溶性复合物。Gβ5在大脑中的功能和分子关联尚不清楚。为了鉴定与大脑中Gβ5紧密结合的蛋白质，使用针对其N端的抗体从洗涤过的小鼠脑膜的非离子去污剂提取物中进行免疫亲和纯化。用同源肽洗脱显示出一条55 kDa的宽带，在SDS-PAGE上与Gβ5共洗脱。通过对胰蛋白酶肽段的基质辅助激光解吸电离质谱分析，将共纯化的55 kDa条带鉴定为RGS6和RGS7的大约1:1混合物。Gβ5和RGS7可以从未分级的脑膜提取物中相互免疫共沉淀，证实了天然蛋白质的紧密结合。相比之下，肽洗脱液的免疫印迹显示没有共纯化的Gαq/11、Gαi1/2、Gγ2、Gγ3或Gγ7。这些发现表明RGS6和RGS7参与了Gβ5在大脑中的功能，并表明大部分靶向膜的Gβ5没有相关的G亚基，因此在G（β）（γ）相互作用的经典框架之外发挥作用。
