GTP-Binding Protein alpha Subunits

About: GTP-Binding Protein alpha Subunits is a research topic. Over the lifetime, 304 publications have been published within this topic receiving 19915 citations.

Assay for G protein-dependent activation of phospholipase C beta using purified protein components.

Abstract: The activity of mammalian phosphoinositide-specific phospholipase C beta (PLC beta) is regulated by the alpha q family of G protein alpha subunits and by beta gamma subunits thought to be released from Gi. Interactions between G protein subunits and PLC beta can be assayed by measuring the stimulation of PLC beta enzymatic activity on reconstituting the purified G protein subunits with purified PLC beta on artificial phospholipid vesicles containing the substrate, phosphatidylinositol-4,5-bisphosphate (PIP2). These vesicles are doped with [3H]-inositol PIP2 and the rate of hydrolysis is determined by quantitating the amount of [3H]-inositol triphosphate (IP3) released from the vesicle into the aqueous phase. This assay provides a relatively simple method for assessing the activity PLC activity and its ability to be regulated by beta gamma and alpha(q) subunits. It can also be used to assess the functionality of the components after modification by mutagenesis, chemical modification, or in the presence of competing molecules.

Activated Alleles of the Schizosaccharomyces pombe gpa2+ Gα Gene Identify Residues Involved in GDP-GTP Exchange

Abstract: The Schizosaccharomyces pombe glucose/cyclic AMP (cAMP) signaling pathway includes the Gpa2-Git5-Git11 heterotrimeric G protein, whose Gpa2 Gα subunit directly binds to and activates adenylate cyclase in response to signaling from the Git3 G protein-coupled receptor. To study intrinsic and extrinsic regulation of Gpa2, we developed a plasmid-based screen to identify mutationally activated gpa2 alleles that bypass the loss of the Git5-Git11 Gβγ dimer to repress transcription of the glucose-regulated fbp1+ gene. Fifteen independently isolated mutations alter 11 different Gpa2 residues, with all but one conferring a receptor-independent activated phenotype upon integration into the gpa2+ chromosomal locus. Biochemical characterization of three activated Gpa2 proteins demonstrated an increased GDP-GTP exchange rate that would explain the mechanism of activation. Interestingly, the amino acid altered in the Gpa2(V90A) exchange rate mutant protein is in a region of Gpa2 with no obvious role in Gα function, thus extending our understanding of Gα protein structure-function relationships.

Expression of GTP-binding protein alpha subunits in human thymocytes.

Abstract: In this report, we investigate G protein α subunit diversity in human thymocytes, utilizing common properties shared by these genes and reverse transcription-polymerase chain reaction (RT-PCR). Sequence analysis of PCR amplified gene portions, indicate the presence of members from all four G-protein families that have been described thus far. The α subunit genes identified are: Gαi1-3 and Gαz but not Gαo from the Gi family, Gαs from the Gs family, Gα11, Gαq, and Gα16 from the Gq family, and Gα12 and Gα13 from the G12 family. Also in this report we present the nucleotide and predicted amino acid sequences of the human Gα13 cloned from a thymocyte cDNA library. The sequence of the human Gα13 has not been previously reported. Comparison of this sequence with the reported murine Gα13 shows >90% identity at the deduced amino acid sequence level. We conclude that thymocytes represent a useful experimental system fo the study of G protein involvement in immune responses and lymphocyte development.

The β Subunit of the Heterotrimeric G Protein Triggers the Kluyveromyces lactis Pheromone Response Pathway in the Absence of the γ Subunit

Abstract: The Gγ subunit of the heterotrimeric G protein is not required to trigger the mating pathway in K. lactis. When the Gγ subunit is missing, the Gα subunit carry Gβ to the plasma membrane and the phe...

The GTP hydrolysis defect of the Saccharomyces cerevisiae mutant G-protein Gpa1(G50V).

Abstract: The Saccharomyces cerevisiae haploid cell response to pheromone involves two seven-transmembrane-domain pheromone receptors that couple to a heterotrimeric G protein. The G50V mutation in the G protein alpha subunit (G(alpha)), Gpa1p, is analogous to the p21(ras) transforming mutation Gly-->Val 12, and has been extensively examined for the phenotypes it produces in yeast cells. Here we have characterized the Gpa1(G50V) mutant protein in vitro by examining GTPgammaS binding, GDP exchange, GTP occupancy and guanosine triphosphatase (GTPase) activity. Compared to wild-type (WT) Gpa1p, Gpa1(G50V)p was found to have a moderately reduced GTPase activity and increased GTP occupancy, while GTPgammaS binding and GDP exchange were not significantly altered. The yeast regulator of G protein Signalling (RGS) protein, Sst2p, was also expressed and purified, and found to have a significantly reduced ability to stimulate the initial rate of GTP hydrolysis of Gpa1(G50V)p compared to its effect on WT Gpa1p. Probing conformational transitions by a protease sensitivity assay suggested that Gpa1(G50V)p did not bind the transition state mimetic GDP/AlF(4)(-) as efficiently as the WT Gpa1p. These biochemical results can explain many of the known gpa1(G50V) yeast cell phenotypes.