Abstract
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.
Highlights
We recently reported that the xanthine nucleotide binding Go␣ mutant, Go␣X can interact with appropriate receptors on the membrane [5, 6]
We previously reported that Go␣X, the xanthine nucleotidebinding mutant protein of Go␣, formed stable complexes with their appropriate receptors and inhibited the activation of cognate receptors because of competitive binding [6]
After immunoprecipitating the mutant proteins incubated with radioactive nucleotides in COS-7 cell lysates, we found that G11␣X and G16␣X bound XTP␥S instead of GTP␥S, whereas wild-type G11␣ and G16␣ preferred GTP␥S
Summary
Materials—Purified bovine retinal transducin ␥ were generous gifts from Dr O. Mutagenesis of G11␣ and G16␣—The D277N mutation was introduced in both wild-type G11␣ and the activated mutant G11␣Q209L. The D280N mutation was introduced in both wild-type G16␣ and the activated G16␣Q213L. Expression and Purification of His6-tagged Go␣—Both wild-type Go␣ and mutant Go␣X were subcloned into the Escherichia coli expression vector pET-15b (Novagen) with a His tag at the N terminus [5]. The recombinant proteins were expressed and purified as described previously. Purified proteins were stored in TED buffer (20 mM Tris-HCl, pH 8.0, 1 mM EDTA, and 1 mM dithiothreitol) with 0.1 mM MgCl2. Membranes of the infected cells were prepared as described. Infected cells were centrifuged and resuspended at Ͻ107 cells/ml in HME/PI buffer (20 mM NaHepes, pH 8.0, 2 mM MgCl2, 1 mM EDTA, 0.1 mM phenylmethylsulfonyl fluoride, 2 g/ml aprotinin, and 10 g/ml leupeptin).
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