Inhibition of subsets of G protein-coupled receptors by empty mutants of G protein α subunits in G0, G11, and G16

We previously reported that the xanthine nucleotide binding G(o)alpha mutant, G(o)alpha X, 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)alpha X and G(16)alpha X expressed in COS-7 cells bound xanthine 5'-O-(thiotriphosphate) instead of guanosine 5'-O-(thiotriphosphate). Second, we found that G(11)alpha X and G(16)alpha X interacted with beta gamma subunits in the presence of xanthine diphosphate. These experiments demonstrated that G(11)alpha X and G(16)alpha X were xanthine nucleotide-binding proteins, similar to G(o)alpha X. Third, in COS-7 cells, both G(11)alpha X and G(16)alpha X inhibited the activation of G(q)-coupled receptors, whereas only G(16)alpha X inhibited the activation of G(i)-coupled receptors. Therefore, when in the nucleotide-free state, empty G(11)alpha X and G(16)alpha X appeared to retain the same receptor binding specificity as their wild-type counterparts. Finally, we found that G(o)alpha X, G(11)alpha X, and G(16)alpha X all inhibited the endogenous thrombin receptors and lysophosphatidic acid receptors in NIH3T3 cells, whereas G(11)alpha X and G(16)alpha X, but not G(o)alpha X, 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.