Showing papers on "GTP-Binding Protein alpha Subunits published in 2008"

G protein βγ subunits: central mediators of G protein-coupled receptor signaling.

Abstract: G protein βγ subunits are central participants in G protein-coupled receptor signaling pathways. They interact with receptors, G protein α subunits and downstream targets to coordinate multiple, different GPCR functions. Much is known about the biology of Gβγ subunits but mysteries remain. Here, we will review what is known about general aspects of structure and function of Gβγ as well as discuss emerging mechanisms for regulation of Gβγ signaling. Recent data suggest that Gβγ is a potential therapeutic drug target. Thus, a thorough understanding of the molecular and physiological functions of Gβγ has significant implications.

Structural diversity in the RGS domain and its interaction with heterotrimeric G protein alpha-subunits.

Abstract: Regulator of G protein signaling (RGS) proteins accelerate GTP hydrolysis by Gα subunits and thus facilitate termination of signaling initiated by G protein-coupled receptors (GPCRs). RGS proteins hold great promise as disease intervention points, given their signature role as negative regulators of GPCRs—receptors to which the largest fraction of approved medications are currently directed. RGS proteins share a hallmark RGS domain that interacts most avidly with Gα when in its transition state for GTP hydrolysis; by binding and stabilizing switch regions I and II of Gα, RGS domain binding consequently accelerates Gα-mediated GTP hydrolysis. The human genome encodes more than three dozen RGS domain-containing proteins with varied Gα substrate specificities. To facilitate their exploitation as drug-discovery targets, we have taken a systematic structural biology approach toward cataloging the structural diversity present among RGS domains and identifying molecular determinants of their differential Gα selectivities. Here, we determined 14 structures derived from NMR and x-ray crystallography of members of the R4, R7, R12, and RZ subfamilies of RGS proteins, including 10 uncomplexed RGS domains and 4 RGS domain/Gα complexes. Heterogeneity observed in the structural architecture of the RGS domain, as well as in engagement of switch III and the all-helical domain of the Gα substrate, suggests that unique structural determinants specific to particular RGS protein/Gα pairings exist and could be used to achieve selective inhibition by small molecules.

Functional analyses of heterotrimeric G protein Gα and Gβ subunits in Gibberella zeae

Abstract: The homothallic ascomycete fungus Gibberella zeae (anamorph: Fusarium graminearum) is a major toxigenic plant pathogen that causes head blight disease on small-grain cereals. The fungus produces the mycotoxins deoxynivalenol (DON) and zearalenone (ZEA) in infected hosts, posing a threat to human and animal health. Despite its agricultural and toxicological importance, the molecular mechanisms underlying its growth, development and virulence remain largely unknown. To better understand such mechanisms, we studied the heterotrimeric G proteins of G. zeae, which are known to control crucial signalling pathways that regulate various cellular and developmental responses in fungi. Three putative Gα subunits, GzGPA1, GzGPA2 and GzGPA3, and one Gβ subunit, GzGPB1, were identified in the F. graminearum genome. Deletion of GzGPA1, a homologue of the Aspergillus nidulans Gα gene fadA, resulted in female sterility and enhanced DON and ZEA production, suggesting that GzGPA1 is required for normal sexual reproduction and repression of toxin biosynthesis. The production of DON and ZEA was also enhanced in the GzGPB1 mutant, suggesting that both Gα GzGPA1 and Gβ GzGPB1 negatively control mycotoxin production. Deletion of GzGPA2, which encodes a Gα protein similar to A. nidulans GanB, caused reduced pathogenicity and increased chitin accumulation in the cell wall, implying that GzGPA2 has multiple functions. Our study shows that G. zeae heterotrimeric G protein subunits can regulate vegetative growth, sexual development, toxin production and pathogenicity.

Molecular architecture of Gαo and the structural basis for RGS16-mediated deactivation

Abstract: Heterotrimeric G proteins relay extracellular cues from heptahelical transmembrane receptors to downstream effector molecules. Composed of an α subunit with intrinsic GTPase activity and a βγ heterodimer, the trimeric complex dissociates upon receptor-mediated nucleotide exchange on the α subunit, enabling each component to engage downstream effector targets for either activation or inhibition as dictated in a particular pathway. To mitigate excessive effector engagement and concomitant signal transmission, the Gα subunit's intrinsic activation timer (the rate of GTP hydrolysis) is regulated spatially and temporally by a class of GTPase accelerating proteins (GAPs) known as the regulator of G protein signaling (RGS) family. The array of G protein-coupled receptors, Gα subunits, RGS proteins and downstream effectors in mammalian systems is vast. Understanding the molecular determinants of specificity is critical for a comprehensive mapping of the G protein system. Here, we present the 2.9 A crystal structure of the enigmatic, neuronal G protein Gαo in the GTP hydrolytic transition state, complexed with RGS16. Comparison with the 1.89 A structure of apo-RGS16, also presented here, reveals plasticity upon Gαo binding, the determinants for GAP activity, and the structurally unique features of Gαo that likely distinguish it physiologically from other members of the larger Gαi family, affording insight to receptor, GAP and effector specificity.

Submembraneous microtubule cytoskeleton: regulation of microtubule assembly by heterotrimeric G proteins

Abstract: Heterotrimeric Gproteins participate in signal transduction by transferring signals from cell surface receptors to intracellular effector molecules Gproteins also interact with microtubules and participate in microtubule-dependent centrosome/chromosome movement during cell division, as well as neuronal differentiation In recent years, significant progress has been made in our understanding of the biochemical/functional interactions between Gprotein subunits (alpha and betagamma) and microtubules, and the molecular details emerging from these studies suggest that alpha and betagamma subunits of Gproteins interact with tubulin/microtubules to regulate the assembly/dynamics of microtubules, providing a novel mechanism for hormone- or neurotransmitter-induced rapid remodeling of cytoskeleton, regulation of the mitotic spindle for centrosome/chromosome movements in cell division, and neuronal differentiation in which structural plasticity mediated by microtubules is important for appropriate synaptic connections and signal transmission

Distinct Roles for Two Gα–Gβ Interfaces in Cell Polarity Control by a Yeast Heterotrimeric G Protein

Abstract: Saccharomyces cerevisiae mating pheromones trigger dissociation of a heterotrimeric G protein (Gαβγ) into Gα-guanosine triphosphate (GTP) and Gβγ. The Gβγ dimer regulates both mitogen-activated pro...

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.

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

Exploring allosteric coupling in the α-subunit of Heterotrimeric G proteins using evolutionary and ensemble-based approaches

Abstract: Allosteric coupling, which can be defined as propagation of a perturbation at one region of the protein molecule (such as ligand binding) to distant sites in the same molecule, constitutes the most general mechanism of regulation of protein function. However, unlike molecular details of ligand binding, structural elements involved in allosteric effects are difficult to diagnose. Here, we identified allosteric linkages in the α-subunits of heterotrimeric G proteins, which were evolved to transmit membrane receptor signals by allosteric mechanisms, by using two different approaches that utilize fundamentally different and independent information. We analyzed: 1) correlated mutations in the family of G protein α-subunits, and 2) cooperativity of the native state ensemble of the Gαi1 or transducin. The combination of these approaches not only recovered already-known details such as the switch regions that change conformation upon nucleotide exchange, and those regions that are involved in receptor, effector or Gβγ interactions (indicating that the predictions of the analyses can be viewed with a measure of confidence), but also predicted new sites that are potentially involved in allosteric communication in the Gα protein. A summary of the new sites found in the present analysis, which were not apparent in crystallographic data, is given along with known functional and structural information. Implications of the results are discussed. A set of residues and/or structural elements that are potentially involved in allosteric communication in Gα is presented. This information can be used as a guide to structural, spectroscopic, mutational, and theoretical studies on the allosteric network in Gα proteins, which will provide a better understanding of G protein-mediated signal transduction.

Interaction of alpha1-syntrophin with multiple isoforms of heterotrimeric G protein alpha subunits.

Abstract: Syntrophins are components of the dystrophin-glycoprotein complex of the plasma membrane in muscular and neuronal cells, and recruit signaling proteins such as neuronal nitric oxide synthase via their multiple protein-protein interaction motifs. In this study, we found that alpha1-syntrophin binds to various subtypes of guanine nucleotide-binding protein alpha subunits (Galpha). A pull-down analysis using full-length recombinant alpha1-syntrophin and MS analysis showed that alpha1-syntrophin was coprecipitated with several isoforms of Galpha proteins in addition to known binding partners such as dystrobrevin and neuronal nitric oxide synthase. Further analysis using recombinant Galpha isoforms showed that alpha1-syntrophin associates with at least Galphai, Galphao, Galphas and Galphaq subtypes. The region of alpha1-syntrophin required for its interaction with Galphas was determined as the N-terminal half of the first pleckstrin homology domain. In addition, the syntrophin unique domain of alpha1-syntrophin was suggested to contribute to this interaction. In COS-7 cells, downregulation of alpha1-syntrophin by RNAi resulted in enhanced cAMP production and cAMP response element-binding protein phosphorylation induced by isoproterenol treatment. These results suggest that alpha1-syntrophin provides a scaffold for the Galpha family of heterotrimeric G proteins in the brain to regulate the efficiency of signal transduction evoked by G-protein-coupled receptors.

Rit contributes to neurite outgrowth triggered by the alpha subunit of Go.

Abstract: Heterotrimeric GTP-binding protein transduce signals initiated by a variety of hormones and neurotransmitters. Go, a member of the Go/Gi family, is the most abundant heterotrimeric GTP-binding protein in nervous tissues and has been implicated in neuronal differentiation. The mechanism by which Go modulates neuronal differentiation has not been, however, fully elucidated. Here, we identified small GTPase Rit as an interacting partner of the alpha-subunit of Go (Goalpha). The biochemical characterizations of Goalpha::Rit interaction revealed that Rit is a candidate downstream effector for Goalpha. Furthermore, dominant negative Rit inhibited Goalpha-induced neurite outgrowth and Erk phosphorylation in Neuro2a cells. These results suggest that Rit may be involved in the signaling pathway for Goalpha-mediated neuronal differentiation.

Palmitoylation participates in G protein coupled signal transduction by affecting its oligomerization.

Abstract: Much in vivo and in vitro evidence has shown that the α subunits of heterotrimeric GTP-binding proteins (G proteins) exist as oligomers in their base state and disaggregate when being activated. In this article, the influence of palmitoylation modification of Gαo on its oligomerization was explored extensively. Gαo protein was expressed and purified from Escherichia coli strain JM109 cotransformed with pQE60(Gαo) and pBB131(N-myristoyltransferase). Non-denaturing gel electrophoresis analysis revealed that Gαo existed to a small extent as monomers but mostly as oligomers including dimers, trimers, tetramers and pentamers which could disaggregate completely into monomers by GTPγS stimulation. Palmitoylated Gαo, on the other hand, only present as oligomers that were difficult to disaggregate into monomers. The effect of palmitoylation on oligomerization of Gαo was further investigated by several other biochemical and biophysical methods including gel filtration chromatography, analytical ultracentrifugation ...

The same mutation in Gsα and transducin α reveals behavioral differences between these highly homologous G protein α-subunits

Abstract: Mutating Arg-238 to Glu (R238E) in the switch 3 region of a transducin α (*Tα) in which 27 aa of the GTPase domain have been replaced with those of the α-subunit of the inhibitory G protein 1 (Gi1α), was reported to create an α-subunit that is resistant to activation by GTPγS, is devoid of resident nucleotide, and has dominant negative (DN) properties. In an attempt to create a DN stimultory G protein α (Gsα) with a single mutation we created Gsα–R265E, equivalent to *Tα–R238E. Gsα–R265E has facilitated activation by GTPγS, a slightly facilitated activation by GTP but much reduced receptor plus GTP stimulated activation, and an apparently unaltered ability to interact with receptor as seen in ligand binding studies. Further, the activity profile of Gsα–R265E is that of an α-subunit with unaltered or increased GTPase activity. The only change in Gsα that is similar to that in *Tα is that the apparent affinity for guanine nucleotides is decreased in both proteins. The molecular basis of the changed properties are discussed based on the known crystal structure of Gsα and the changes introduced by the same mutation in a *Tα (Gtα*) with only 23 aa from Gi1α. Gtα*–R238E, with four fewer mutations in switch 3, was reported to show no evidence of DN properties, is activated by GTPγS, and has reduced GTPase activity. The data highlight a critical role for the switch 3 region in setting overall properties of signal-transducing GTPases.

Functional coupling of μ-receptor-Gαi-tethered proteins in AtT20 cells

Abstract: Opioid efficacy on mu-receptor may be influenced by various Gi/o-G-protein subunits interacting with intracellular face of receptor. Pertussis toxin-insensitive Galphai1 and Galphai2 subunits tethered with mu-receptor were stably transfected into AtT20 cells to (i) determine coupling of different alpha-subunits on opioid efficacy, and (ii) determine coupling to downstream effectors, for example, calcium and potassium channels. After pertussis toxin, stimulation of [35S]GTP-gamma-S incorporation persisted. Both constructs were able to couple to native calcium and potassium channels, with endomorphins 1 and 2 equally effective. However, pertussis toxin abolished opioid actions on calcium and potassium channels suggesting strong coupling to endogenous G-proteins, and that differences in coupling efficacy to Galphai1 and Galphai2 previously observed are restricted to initial step of signaling cascade.