Yu, Bo and Simon, Melvin I. (1998) Interaction of the Xanthine Nucleotide Binding Goα Mutant with G Protein-coupled Receptors. Journal of Biological Chemistry, 273 (46). pp. 30183-30188. ISSN 0021-9258. http://resolver.caltech.edu/CaltechAUTHORS:YUBjbc98
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We constructed a double mutant version of the α subunit of Go that was regulated by xanthine nucleotides instead of guanine nucleotides (GoαX). We investigated the interaction between GoαX and G protein-coupled receptors in vitro. First, we found that the activated m2 muscarinic cholinergic receptor (MAChR) could facilitate the exchange of XTPγS for XDP in the GoαXβγ heterotrimer. Second, the GoαXβγ complex was able to induce the high affinity ligand-binding state in the N-formyl peptide receptor (NFPR). These experiments demonstrated that GoαX was able to interact effectively with G protein-coupled receptors. Third, we found that the empty form of GoαX, lacking a bound nucleotide and βγ, formed a stable complex with the m2 muscarinic cholingeric receptor associated with the plasma membrane. Finally, we investigated the interaction of GoαX with receptor in COS-7 cells. The empty form of GoαX bound tightly to the receptor and was not activated because XTP was not available intracellularly. We tested the ability of GoαX to inhibit the activities of several different G protein-coupled receptors in transfected COS-7 cells and found that Goα X specifically inhibited Go-coupled receptors. Thus the modified G proteins may act as dominant-negative mutants to trap and inactivate specific subsets of receptors.
|Additional Information:||Copyright © 1998 by the American Society for Biochemistry and Molecular Biology. (Received for publication, July 6, 1998, and in revised form, August 20, 1998) The recombinant baculovirus encoding m2 MAChR was a generous gift from Dr. E. Ross. We thank members of Dr. Simon's lab for helpful discussions, and Dr. Tau-Mu Yi for comments on the manuscript. The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.|
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