Showing papers on "GTP-Binding Protein alpha Subunits published in 1997"

Crystal structure of the adenylyl cyclase activator Gsalpha

Abstract: The crystal structure of Gsalpha, the heterotrimeric G protein alpha subunit that stimulates adenylyl cyclase, was determined at 2.5 A in a complex with guanosine 5'-O-(3-thiotriphosphate) (GTPgammaS). Gsalpha is the prototypic member of a family of GTP-binding proteins that regulate the activities of effectors in a hormone-dependent manner. Comparison of the structure of Gsalpha.GTPgammaS with that of Gialpha.GTPgammaS suggests that their effector specificity is primarily dictated by the shape of the binding surface formed by the switch II helix and the alpha3-beta5 loop, despite the high sequence homology of these elements. In contrast, sequence divergence explains the inability of regulators of G protein signaling to stimulate the GTPase activity of Gsalpha. The betagamma binding surface of Gsalpha is largely conserved in sequence and structure to that of Gialpha, whereas differences in the surface formed by the carboxyl-terminal helix and the alpha4-beta6 loop may mediate receptor specificity.

G proteins in Ustilago maydis: transmission of multiple signals?

Abstract: In the phytopathogenic fungus Ustilago maydis, cell fusion is governed by a pheromone signalling system. The pheromone receptors belong to the seven transmembrane class that are coupled to heterotrimeric G proteins. We have isolated four genes (gpa1 to gpa4) encoding alpha subunits of G proteins. Gpa1, Gpa2 and Gpa3 have homologues in other fungal species, while Gpa4 is novel. Null mutants in individual genes were viable and only disruption of gpa3 caused a discernible phenotype. gpa3 mutant strains were unable to respond to pheromone and thus were mating-deficient. A constitutively active allele of gpa3 (gpa3(Q206L)) was generated by site-directed mutagenesis. Haploid strains harbouring gpa3(Q206L) were able to mate without pheromone stimulation, indicating that Gpa3 plays an active role in transmission of the pheromone signal. Surprisingly, Gpa3 is also required for pathogenic development, although pheromone signalling is not essential for this process.

The small GTP-binding protein Rho links G protein-coupled receptors and Galpha12 to the serum response element and to cellular transformation.

Abstract: Receptors coupled to heterotrimeric G proteins can effectively stimulate growth promoting pathways in a large variety of cell types, and if persistently activated, these receptors can also behave as dominant-acting oncoproteins. Consistently, activating mutations for G proteins of the Galphas and Galphai2 families were found in human tumors; and members of the Galphaq and Galpha12 families are fully transforming when expressed in murine fibroblasts. In an effort aimed to elucidate the molecular events involved in proliferative signaling through heterotrimeric G proteins we have focused recently on gene expression regulation. Using NIH 3T3 fibroblasts expressing m1 muscarinic acetylcholine receptors as a model system, we have observed that activation of this transforming G protein-coupled receptors induces the rapid expression of a variety of early responsive genes, including the c-fos protooncogene. One of the c-fos promoter elements, the serum response element (SRE), plays a central regulatory role, and activation of SRE-dependent transcription has been found to be regulated by several proteins, including the serum response factor and the ternary complex factor. With the aid of reporter plasmids for gene expression, we observed here that stimulation of m1 muscarinic acetylcholine receptors potently induced SRE-driven reporter gene activity in NIH 3T3 cells. In these cells, only the Galpha12 family of heterotrimeric G protein alpha subunits strongly induced the SRE, while Gbeta1gamma2 dimers activated SRE to a more limited extent. Furthermore, our study provides strong evidence that m1, Galpha12 and the small GTP-binding protein RhoA are components of a novel signal transduction pathway that leads to the ternary complex factor-independent transcriptional activation of the SRE and to cellular transformation.

The stoichiometry of G alpha(s) palmitoylation in its basal and activated states.

Abstract: Palmitoylation is the dynamic modification of proteins by the addition of palmitate to cysteine residues. The alpha subunits of heterotrimeric G proteins undergo palmitoylation on their amino terminus, and activation of alpha(s) accelerates its palmitate turnover. In previous studies, palmitoylation was assessed by incorporation or turnover of [3H]palmitate. These studies did not determine the fraction of alpha(s) that is palmitoylated because the specific activity of [3H]palmitoyl-CoA within cells is indeterminate. We developed an HPLC method to determine the fraction of alpha(s) that was palmitoylated in the basal and activated states. COS and S49 cells were radiolabeled with [35S]methionine, and alpha(s) was immunoprecipitated from the particulate fraction. The immunoprecipitated proteins were separated by reverse phase HPLC into two peaks that were determined to contain the modified and unmodified forms of alpha(s). Approximately 77% of the endogenous alpha(s) in COS cells and 70% in S49 lymphoma cells were palmitoylated. The fraction of alpha(s) that was modified did not change after treatment with isoproterenol, a beta-adrenergic receptor agonist that causes turnover of palmitate on alpha(s). These results suggest that receptor activation of alpha(s) caused a rapid turnover of palmitate to maintain most of alpha(s) in its palmitoylated form.

Analysis of the receptor binding domain of Gpa1p, the G(alpha) subunit involved in the yeast pheromone response pathway.

Abstract: The Saccharomyces cerevisiae G protein alpha subunit Gpa1p is involved in the response of both MATa and MAT alpha cells to pheromone. We mutagenized the GPA1 C terminus to characterize the receptor-interacting domain and to investigate the specificity of the interactions with the a- and alpha-factor receptors. The results are discussed with respect to a structural model of the Gpa1p C terminus that was based on the crystal structure of bovine transducin. Some mutants showed phenotypes different than the pheromone response and mating defects expected for mutations that affect receptor interactions, and therefore the mutations may affect other aspects of Gpa1p function. Most of the mutations that resulted in pheromone response and mating defects had similar effects in MATa and MAT alpha cells, suggesting that they affect the interactions with both receptors. Overexpression of the pheromone receptors increased the mating of some of the mutants tested but not the wild-type strain, consistent with defects in mutant Gpa1p-receptor interactions. The regions identified by the mating-defective mutants correlated well with the regions of mammalian G(alpha) subunits implicated in receptor interactions. The strongest mating type-specific effects were seen for mutations to proline and a mutation of a glycine residue predicted to form a C-terminal beta turn. The analogous beta turn in mammalian G(alpha) subunits undergoes a conformational change upon receptor interaction. We propose that the conformation of this region of Gpa1p differs during the interactions with the a- and alpha-factor receptors and that these mating type-specific mutations preclude the orientation necessary for interaction with one of the two receptors.

TNF translationally modulates the expression of G1 protein alpha(i2) subunits in human polymorphonuclear leukocytes.

Abstract: Priming of polymorphonuclear leukocyte responses to chemoattractants by TNF plays an important role in host defenses and inflammatory responses. TNF-induced priming is associated with an 80% increase in the membrane density of G alpha(i2) protein that is coupled to chemoattractant receptors. The present study examines the hypothesis that TNF stimulates increased synthesis of alpha(i2). Within 10 min of addition, TNF stimulated a significant increase in total cellular G alpha(i2), as determined by pertussis toxin-catalyzed ADP ribosylation, which was blocked by the translation inhibitor cycloheximide. Immunoprecipitation of biosynthetically labeled alpha(i2) showed that TNF increased alpha(i2) synthesis by about 20% at 10 min. Nuclear run-ons showed no change in alpha(i2) mRNA synthesis in TNF-treated cells; however, steady state alpha(i2) mRNA levels were reduced following a 10-min exposure to TNF. Pretreatment with cycloheximide prevented the TNF-induced reduction in steady state alpha(i2) mRNA levels. Therefore, TNF stimulates alpha(i2) protein synthesis and mRNA degradation in the same time frame as priming. The increased alpha(i2) synthesis results from increased translation, not transcription, of alpha(i2) mRNA. Simultaneous G alpha(i2) protein synthesis and mRNA degradation provide a mechanism by which TNF priming is associated with a rapid, self-limiting increase in G protein expression.

Inactivation of the G alpha i2 and G alpha o genes by homologous recombination.

Abstract: G proteins couple receptors to effectors and thus regulate multiple biological processes. Here we report on the phenotypes of G alpha i2-deficient and G alpha o-deficient mice. G alpha i2-deficient mice display a blunted inhibitory regulation of adenylyl cyclase, alterations in T cell maturation and function, a growth retardation and also develop a lethal diffuse colitis with clinical and histopathological features closely resembling ulcerative colitis in humans, including the development of adenocarcinoma of the colon. G alpha o-deficient mice are also viable, but significantly smaller than wild-type controls.