Interaction of the xanthine nucleotide binding Goalpha mutant with G protein-coupled receptors.
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.
Yu
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Division of Biology, 147-75, California Institute of Technology, Pasadena, California 91125, USA.
Simon
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MI
D000818:Animals; D019556:COS Cells; D002417:Cattle; D019206:GTP-Binding Protein alpha Subunits, Gi-Go; D019204:GTP-Binding Proteins / Q000378:metabolism*; D009240:N-Formylmethionine Leucyl-Phenylalanine / Q000378:metabolism; D009711:Nucleotides / Q000378:metabolism*; D043585:Receptor, Muscarinic M2; D011956:Receptors, Cell Surface / Q000378:metabolism*; D044042:Receptors, Formyl Peptide; D011971:Receptors, Immunologic / Q000378:metabolism; D011976:Receptors, Muscarinic / Q000378:metabolism; D018000:Receptors, Peptide / Q000378:metabolism; D012265:Ribonucleotides / Q000378:metabolism; D018411:Spodoptera; D013873:Thionucleotides / Q000378:metabolism; D019820:Xanthine / Q000378:metabolism*; D014970:Xanthines / Q000378:metabolism
D009711:Nucleotides; D043585:Receptor, Muscarinic M2; D011956:Receptors, Cell Surface; D044042:Receptors, Formyl Peptide; D011971:Receptors, Immunologic; D011976:Receptors, Muscarinic; D018000:Receptors, Peptide; D012265:Ribonucleotides; D013873:Thionucleotides; D014970:Xanthines; C115713:xanthine 5'-O-(3-thiotriphosphate); D019820:Xanthine; D009240:N-Formylmethionine Leucyl-Phenylalanine; C065326:xanthosine 5'-triphosphate; D019204:GTP-Binding Proteins; D019206:GTP-Binding Protein alpha Subunits, Gi-Go