Inhibition of subsets of G protein-coupled receptors by empty mutants of G protein alpha subunits in g(o), G(11), and G(16).
We previously reported that the xanthine nucleotide binding G(o)alpha mutant, G(o)alphaX, inhibited the activation of G(i)-coupled receptors. We constructed similar mutations in G(11)alpha and G(16)alpha and characterized their nucleotide binding and receptor interaction. First, we found that G(11)alphaX and G(16)alphaX expressed in COS-7 cells bound xanthine 5'-O-(thiotriphosphate) instead of guanosine 5'-O-(thiotriphosphate). Second, we found that G(11)alphaX and G(16)alphaX interacted with betagamma subunits in the presence of xanthine diphosphate. These experiments demonstrated that G(11)alphaX and G(16)alphaX were xanthine nucleotide-binding proteins, similar to G(o)alphaX. Third, in COS-7 cells, both G(11)alphaX and G(16)alphaX inhibited the activation of G(q)-coupled receptors, whereas only G(16)alphaX inhibited the activation of G(i)-coupled receptors. Therefore, when in the nucleotide-free state, empty G(11)alphaX and G(16)alphaX appeared to retain the same receptor binding specificity as their wild-type counterparts. Finally, we found that G(o)alphaX, G(11)alphaX, and G(16)alphaX all inhibited the endogenous thrombin receptors and lysophosphatidic acid receptors in NIH3T3 cells, whereas G(11)alphaX and G(16)alphaX, but not G(o)alphaX, inhibited the activation of transfected m1 muscarinic receptor in these cells. We conclude that these empty G protein mutants of G(o)alpha, G(11)alpha, and G(16)alpha can act as dominant negative inhibitors against specific subsets of G protein-coupled receptors.
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Division of Biology, 147-75, California Institute of Technology, Pasadena, California 91125, USA.
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Simon
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D016475:3T3 Cells; D000818:Animals; D019206:GTP-Binding Protein alpha Subunits, Gi-Go / Q000378:metabolism; D016244:Guanosine 5'-O-(3-Thiotriphosphate) / Q000378:metabolism; D020962:Heterotrimeric GTP-Binding Proteins / Q000235:genetics* / Q000378:metabolism*; D051379:Mice; D018727:Muscarinic Antagonists / Q000378:metabolism; D009154:Mutation; D043585:Receptor, Muscarinic M2; D011956:Receptors, Cell Surface / Q000037:antagonists & inhibitors*; D043562:Receptors, G-Protein-Coupled; D049368:Receptors, Lysophosphatidic Acid; D011976:Receptors, Muscarinic / Q000378:metabolism; D018179:Receptors, Thrombin / Q000037:antagonists & inhibitors; D012265:Ribonucleotides / Q000378:metabolism; D015398:Signal Transduction; D013873:Thionucleotides / Q000378:metabolism; D014970:Xanthines / Q000378:metabolism
D018727:Muscarinic Antagonists; D043585:Receptor, Muscarinic M2; D011956:Receptors, Cell Surface; D043562:Receptors, G-Protein-Coupled; D049368:Receptors, Lysophosphatidic Acid; D011976:Receptors, Muscarinic; D018179:Receptors, Thrombin; D012265:Ribonucleotides; D013873:Thionucleotides; D014970:Xanthines; C115713:xanthine 5'-O-(3-thiotriphosphate); D016244:Guanosine 5'-O-(3-Thiotriphosphate); C065326:xanthosine 5'-triphosphate; D019206:GTP-Binding Protein alpha Subunits, Gi-Go; D020962:Heterotrimeric GTP-Binding Proteins