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.

Proteolytic processing of chromogranins.

Abstract: A number of peptides derived from the large chromogranin precursor proteins have been identified in neuroendocrine tissues and several of them have been attributed with functional data. In addition, various fragments were shown to be liberated by the action of prohormone convertases. This is in accordance with the concept that these peptides represent the mediators of at least part of the yet unidentified physiological functions of the chromogranins.

Heterotrimeric Galphaq11 co-immunoprecipitates with surface-anchored GRP78 from plasma membranes of alpha2M*-stimulated macrophages.

Abstract: We have previously shown that a fraction of newly expressed GRP78 is translocated to the cell surface in association with the co-chaperone MTJ-1. Proteinase and methylamine-activated alpha(2)M (alpha(2)M*) bind to cell surface-associated GRP78 activating phosphoinositide-specific phospholipase C coupled to a pertussis toxin-insensitive heterotrimeric G protein, generating IP(3)/calcium signaling. We have now studied the association of pertussis toxin-insensitive Galphaq11, with GRP78/MTJ-1 complexes in the plasma membranes of alpha(2)M*-stimulated macrophages. When GRP78 was immunoprecipitated from plasma membranes of macrophages stimulated with alpha(2)M*, Galphaq11, and MTJ-1 were co-precipitated. Likewise Galphaq11 and GRP78 co-immunoprecipitated with MTJ-1 while GRP78 and MTJ-1 co-immunoprecipitated with Galphaq11. Silencing GRP78 expression with GRP78 dsRNA or MTJ-1 with MTJ-1 dsRNA greatly reduced the levels of Galphaq11 co-precipitated with GRP78 or MTJ-1. In conclusion, we show here that plasma membrane-associated GRP78 is coupled to pertussis toxin-insensitive Galphaq11 and forms a ternary signaling complex with MTJ-1.

Functional reconstitution of the human chemokine receptor CXCR4 with G(i)/G (o)-proteins in Sf9 insect cells.

Abstract: The chemokine stromal cell-derived factor-1α (SDF-1α) binds to the chemokine receptor CXCR4 that couples to pertussis toxin-sensitive G-proteins of the Gi/Go-family. CXCR4 plays a role in the pathogenesis of autoimmune diseases, human immunodeficiency virus infection and various tumors, fetal development as well as endothelial progenitor and T-cell recruitment. To this end, most CXCR4 studies have focused on the cellular level. The aim of this study was to establish a reconstitution system for the human CXCR4 that allows for the analysis of receptor/G-protein coupling at the membrane level. We wished to study specifically constitutive CXCR4 activity and the G-protein-specificity of CXCR4. We co-expressed N- and C-terminally epitope-tagged human CXCR4 with various Gi/Go-proteins and regulator of G-protein signaling (RGS)-proteins in Sf9 insect cells. Expression of CXCR4, G-proteins, and RGS-proteins was verified by immunoblotting. CXCR4 coupled more effectively to Gαi1 and Gαi2 than to Gαi3 and Gαo and insect cell G-proteins as assessed by SDF-1α-stimulated high-affinity steady-state GTP hydrolysis. The RGS-proteins RGS4 and GAIP enhanced SDF-1α-stimulated GTP hydrolysis. SDF-1α stimulated [35S]guanosine 5′-[γ-thio]triphosphate (GTPγS) binding to Gαi2. RGS4 did not enhance GTPγS binding. Na+ salts of halides did not reduce basal GTPase activity. The bicyclam, 1-[[1,4,8,11-tetrazacyclotetradec-1-ylmethyl)phenyl]methyl]-1,4,8,11-tetrazacyclotetradecane (AMD3100), acted as CXCR4 antagonist but was devoid of inverse agonistic activity. Halides reduced the maximum SDF-1α-stimulated GTP hydrolysis in the order of efficacy I− > Br− > Cl−. In addition, salts reduced the potency of SDF-1α at activating GTP hydrolysis. From our data, we conclude the following: (1) Sf9 cells are a suitable system for expression of functionally intact human CXCR4; (2) Human CXCR4 couples effectively to Gαi1 and Gαi2; (3) There is no evidence for constitutive activity of CXCR4; (4) RGS-proteins enhance agonist-stimulated GTP hydrolysis, showing that GTP hydrolysis becomes rate-limiting in the presence of SDF-1α; (5) By analogy to previous observations made for the β2-adrenoceptor coupled to Gs, the inhibitory effects of halides on agonist-stimulated GTP hydrolysis may be due to increased GDP-affinity of Gi-proteins, reducing the efficacy of CXCR4 at stimulating nucleotide exchange.

Modulation of a GEF switch: autoinhibition of the intrinsic guanine nucleotide exchange activity of p115-RhoGEF.

Abstract: p115-RhoGEF (p115) belongs to the family of RGS-containing guanine nucleotide exchange factors for Rho GTPases (RGS-RhoGEFs) that are activated by G12 class heterotrimeric G protein α subunits. All RGS-RhoGEFs possess tandemly linked Dbl-homology (DH) and plekstrin-homology (PH) domains, which bind and catalyze the exchange of GDP for GTP on RhoA. We have identified that the linker region connecting the N-terminal RGS-homology (RH) domain and the DH domain inhibits the intrinsic guanine nucleotide exchange (GEF) activity of p115, and determined the crystal structures of the DH/PH domains in the presence or absence of the inhibitory linker region. An N-terminal extension of the canonical DH domain (the GEF switch), which is critical to GEF activity, is well folded in the crystal structure of DH/PH alone, but becomes disordered in the presence of the linker region. The linker region is completely disordered in the crystal structure and partially disordered in the molecular envelope calculated from measurements of small angle x-ray scattering (SAXS). It is possible that Gα subunits activate p115 in part by relieving autoinhibition imposed by the linker region.

Heterotrimeric G-Protein Subunit Function in Candida albicans: both the α and β Subunits of the Pheromone Response G Protein Are Required for Mating

Abstract: Received 29 February 2008/Accepted 15 July 2008 A pheromone-mediated signaling pathway that couples seven-transmembrane-domain (7-TMD) receptors to a mitogen-activated protein kinase module controls Candida albicans mating. 7-TMD receptors are typically connected to heterotrimeric G proteins whose activation regulates downstream effectors. Two G subunits in C. albicans have been identified previously, both of which have been implicated in aspects of pheromone response. Cag1p was found to complement the mating pathway function of the pheromone receptor-coupled G subunit in Saccharomyces cerevisiae, and Gpa2p was shown to have a role in the regulation of cyclic AMP signaling in C. albicans and to repress pheromone-mediated arrest. Here, we show that the disruption of CAG1 prevented mating, inactivated pheromone-mediated arrest and morphological changes, and blocked pheromone-mediated gene expression changes in opaque cells of C. albicans and that the overproduction of CAG1 suppressed the hyperactive cell cycle arrest exhibited by sst2 mutant cells. Because the disruption of the STE4 homolog constituting the only C. albicans gene for a heterotrimeric G subunit also blocked mating and pheromone response, it appears that in this fungal pathogen the G and G subunits do not act antagonistically but, instead, are both required for the transmission of the mating signal. Many fungi have well-defined mating systems. Currently, the most thoroughly studied is that of the baker’s or brewer’s yeast Saccharomyces cerevisiae (2, 12). In this yeast, a signaling pathway