期刊名称：Proceedings of the National Academy of Sciences
印刷版ISSN：0027-8424
电子版ISSN：1091-6490
出版年度：2015
卷号：112
期号：11
页码：E1181-E1190
DOI：10.1073/pnas.1417573112
语种：English
出版社：The National Academy of Sciences of the United States of America
摘要：SignificanceG-protein-coupled receptors (GPCRs) are the target of 30% of all drugs on the market. Nevertheless, the lack of detailed knowledge of GPCR signaling impedes the development of highly specific drugs. In this respect, additional structures of GPCR/G-protein complexes could greatly enhance our knowledge on how to design specific drugs. Unfortunately, the nature of the GPCR/G-protein complex is characterized by a transient interaction and an intrinsic instability, thereby hampering structure determination. In our study, we show--besides new insights into the combinatorial diversity of heterotrimeric G proteins--that the combination of evolved, stable GPCR mutants and G-protein combinations selected from the natural pool of G proteins yields promisingly stable GPCR/G-protein complexes. Agonist binding to G-protein-coupled receptors (GPCRs) triggers signal transduction cascades involving heterotrimeric G proteins as key players. A major obstacle for drug design is the limited knowledge of conformational changes upon agonist binding, the details of interaction with the different G proteins, and the transmission to movements within the G protein. Although a variety of different GPCR/G protein complex structures would be needed, the transient nature of this complex and the intrinsic instability against dissociation make this endeavor very challenging. We have previously evolved GPCR mutants that display higher stability and retain their interaction with G proteins. We aimed at finding all G-protein combinations that preferentially interact with neurotensin receptor 1 (NTR1) and our stabilized mutants. We first systematically analyzed by coimmunoprecipitation the capability of 120 different G-protein combinations consisting of i1 or sL and all possible {beta}{gamma}-dimers to form a heterotrimeric complex. This analysis revealed a surprisingly unrestricted ability of the G-protein subunits to form heterotrimeric complexes, including {beta}{gamma}-dimers previously thought to be nonexistent, except for combinations containing {beta}5. A second screen on coupling preference of all G-protein heterotrimers to NTR1 wild type and a stabilized mutant indicated a preference for those Gi1{beta}{gamma} combinations containing {gamma}1 and {gamma}11. Heterotrimeric G proteins, including combinations believed to be nonexistent, were purified, and complexes with the GPCR were prepared. Our results shed new light on the combinatorial diversity of G proteins and their coupling to GPCRs and open new approaches to improve the stability of GPCR/G-protein complexes.
关键词：heterotrimeric G proteins ; G-protein–coupled receptor ; membrane protein ; protein complex ; protein–protein interaction
