Interaction of the Xanthine Nucleotide Binding Goα Mutant with G Protein-coupled Receptors

Bo Yu,Melvin I Simon

doi:10.1074/jbc.273.46.30183

Abstract

We constructed a double mutant version of the alpha subunit of Go that was regulated by xanthine nucleotides instead of guanine nucleotides (GoalphaX). We investigated the interaction between GoalphaX and G protein-coupled receptors in vitro. First, we found that the activated m2 muscarinic cholinergic receptor (MAChR) could facilitate the exchange of XTPgammaS for XDP in the GoalphaXbetagamma heterotrimer. Second, the GoalphaXbetagamma complex was able to induce the high affinity ligand-binding state in the N-formyl peptide receptor (NFPR). These experiments demonstrated that GoalphaX was able to interact effectively with G protein-coupled receptors. Third, we found that the empty form of GoalphaX, lacking a bound nucleotide and betagamma, formed a stable complex with the m2 muscarinic cholingeric receptor associated with the plasma membrane. Finally, we investigated the interaction of GoalphaX with receptor in COS-7 cells. The empty form of GoalphaX bound tightly to the receptor and was not activated because XTP was not available intracellularly. We tested the ability of GoalphaX to inhibit the activities of several different G protein-coupled receptors in transfected COS-7 cells and found that GoalphaX specifically inhibited Go-coupled receptors. Thus the modified G proteins may act as dominant-negative mutants to trap and inactivate specific subsets of receptors.

Highlights

We recently reported that a mutant version of Go␣, Go␣X (Go␣D273N/Q205L), was regulated by xanthine nucleotides, not by guanine nucleotides [6]
We found that Go␣X mutant proteins retained the receptor binding specificity of the wildtype Go␣ and were able to interact with Go-coupled receptors, such as the m2 muscarinic cholinergic receptor (MAChR), Nformyl peptide receptor (NFPR), and thrombin receptor, but not with m1 MAChR or thyrotropin-releasing hormone (TRH) receptor which do not couple to wild-type Go
Stimulation of XTP␥S Binding of Go␣X by Activated M2 Muscarinic Receptor—To test if Go␣X could interact with G protein-coupled receptors, we investigated the receptor-stimulated nucleotide binding of Go␣X

Summary

Introduction

We recently reported that a mutant version of Go␣, Go␣X (Go␣D273N/Q205L), was regulated by xanthine nucleotides, not by guanine nucleotides [6]. We reconstituted purified Go␣X with Sf9 cell membranes containing m2 MAChR in the presence of XDP and G protein ␤␥ subunits purified from bovine retina, and followed agonist-dependent stimulation of XTP␥S binding to Go␣X. In control experiments using wild-type Sf9 cell membranes, both atropine and carbachol had no effect on the XTP␥S binding of Go␣X (Fig. 1a).

Results

Conclusion