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Disabling the Gβγ-SNARE interaction disrupts GPCR-mediated presynaptic inhibition, leading to physiological and behavioral phenotypes.
Zurawski Z, Thompson Gray AD, Brady LJ, Page B, Church E, Harris NA, Dohn MR, Yim YY, Hyde K, Mortlock DP, Jones CK, Winder DG, Alford S, Hamm HE
(2019) Sci Signal 12:
MeSH Terms: Animals, Calcium, Exocytosis, GTP-Binding Protein alpha Subunits, Gi-Go, GTP-Binding Protein beta Subunits, GTP-Binding Protein gamma Subunits, Mice, Inbred C57BL, Mice, Inbred DBA, Mice, Knockout, Neural Inhibition, Phenotype, Protein Binding, Receptors, G-Protein-Coupled, Synaptic Transmission, Synaptosomal-Associated Protein 25
Show Abstract · Added February 22, 2019
G protein-coupled receptors (GPCRs) that couple to G proteins modulate neurotransmission presynaptically by inhibiting exocytosis. Release of Gβγ subunits from activated G proteins decreases the activity of voltage-gated Ca channels (VGCCs), decreasing excitability. A less understood Gβγ-mediated mechanism downstream of Ca entry is the binding of Gβγ to SNARE complexes, which facilitate the fusion of vesicles with the cell plasma membrane in exocytosis. Here, we generated mice expressing a form of the SNARE protein SNAP25 with premature truncation of the C terminus and that were therefore partially deficient in this interaction. SNAP25Δ3 homozygote mice exhibited normal presynaptic inhibition by GABA receptors, which inhibit VGCCs, but defective presynaptic inhibition by receptors that work directly on the SNARE complex, such as 5-hydroxytryptamine (serotonin) 5-HT receptors and adrenergic α receptors. Simultaneously stimulating receptors that act through both mechanisms showed synergistic inhibitory effects. SNAP25Δ3 homozygote mice had various behavioral phenotypes, including increased stress-induced hyperthermia, defective spatial learning, impaired gait, and supraspinal nociception. These data suggest that the inhibition of exocytosis by G-coupled GPCRs through the Gβγ-SNARE interaction is a crucial component of numerous physiological and behavioral processes.
Copyright © 2019 The Authors, some rights reserved; exclusive licensee American Association for the Advancement of Science. No claim to original U.S. Government Works.
1 Communities
3 Members
0 Resources
15 MeSH Terms
GHSR-D2R heteromerization modulates dopamine signaling through an effect on G protein conformation.
Damian M, Pons V, Renault P, M'Kadmi C, Delort B, Hartmann L, Kaya AI, Louet M, Gagne D, Ben Haj Salah K, Denoyelle S, Ferry G, Boutin JA, Wagner R, Fehrentz JA, Martinez J, Marie J, Floquet N, Galès C, Mary S, Hamm HE, Banères JL
(2018) Proc Natl Acad Sci U S A 115: 4501-4506
MeSH Terms: Dopamine, GTP-Binding Protein alpha Subunits, Gi-Go, Humans, Protein Multimerization, Receptors, Dopamine D2, Receptors, Ghrelin, Signal Transduction
Show Abstract · Added March 24, 2020
The growth hormone secretagogue receptor (GHSR) and dopamine receptor (D2R) have been shown to oligomerize in hypothalamic neurons with a significant effect on dopamine signaling, but the molecular processes underlying this effect are still obscure. We used here the purified GHSR and D2R to establish that these two receptors assemble in a lipid environment as a tetrameric complex composed of two each of the receptors. This complex further recruits G proteins to give rise to an assembly with only two G protein trimers bound to a receptor tetramer. We further demonstrate that receptor heteromerization directly impacts on dopamine-mediated Gi protein activation by modulating the conformation of its α-subunit. Indeed, association to the purified GHSR:D2R heteromer triggers a different active conformation of Gαi that is linked to a higher rate of GTP binding and a faster dissociation from the heteromeric receptor. This is an additional mechanism to expand the repertoire of GPCR signaling modulation that could have implications for the control of dopamine signaling in normal and physiopathological conditions.
0 Communities
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MeSH Terms
Hepatic Gi signaling regulates whole-body glucose homeostasis.
Rossi M, Zhu L, McMillin SM, Pydi SP, Jain S, Wang L, Cui Y, Lee RJ, Cohen AH, Kaneto H, Birnbaum MJ, Ma Y, Rotman Y, Liu J, Cyphert TJ, Finkel T, McGuinness OP, Wess J
(2018) J Clin Invest 128: 746-759
MeSH Terms: Animals, Blood Glucose, Extracellular Signal-Regulated MAP Kinases, Female, GTP-Binding Protein alpha Subunits, Gi-Go, Gene Expression Profiling, Glucagon, Gluconeogenesis, Glucose, Hepatocytes, Homeostasis, Humans, Liver, Male, Mice, Mice, Inbred C57BL, Mice, Knockout, Oxygen, Phosphatidylinositol 3-Kinases, Phosphorylation, Reactive Oxygen Species, Receptors, Glucagon, Signal Transduction
Show Abstract · Added March 14, 2018
An increase in hepatic glucose production (HGP) is a key feature of type 2 diabetes. Excessive signaling through hepatic Gs-linked glucagon receptors critically contributes to pathologically elevated HGP. Here, we tested the hypothesis that this metabolic impairment can be counteracted by enhancing hepatic Gi signaling. Specifically, we used a chemogenetic approach to selectively activate Gi-type G proteins in mouse hepatocytes in vivo. Unexpectedly, activation of hepatic Gi signaling triggered a pronounced increase in HGP and severely impaired glucose homeostasis. Moreover, increased Gi signaling stimulated glucose release in human hepatocytes. A lack of functional Gi-type G proteins in hepatocytes reduced blood glucose levels and protected mice against the metabolic deficits caused by the consumption of a high-fat diet. Additionally, we delineated a signaling cascade that links hepatic Gi signaling to ROS production, JNK activation, and a subsequent increase in HGP. Taken together, our data support the concept that drugs able to block hepatic Gi-coupled GPCRs may prove beneficial as antidiabetic drugs.
0 Communities
1 Members
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23 MeSH Terms
A Conserved Hydrophobic Core in Gαi1 Regulates G Protein Activation and Release from Activated Receptor.
Kaya AI, Lokits AD, Gilbert JA, Iverson TM, Meiler J, Hamm HE
(2016) J Biol Chem 291: 19674-86
MeSH Terms: Enzyme Activation, GTP-Binding Protein alpha Subunits, Gi-Go, Guanosine Diphosphate, Guanosine Triphosphate, Humans, Hydrophobic and Hydrophilic Interactions, Protein Structure, Secondary
Show Abstract · Added April 8, 2017
G protein-coupled receptor-mediated heterotrimeric G protein activation is a major mode of signal transduction in the cell. Previously, we and other groups reported that the α5 helix of Gαi1, especially the hydrophobic interactions in this region, plays a key role during nucleotide release and G protein activation. To further investigate the effect of this hydrophobic core, we disrupted it in Gαi1 by inserting 4 alanine amino acids into the α5 helix between residues Gln(333) and Phe(334) (Ins4A). This extends the length of the α5 helix without disturbing the β6-α5 loop interactions. This mutant has high basal nucleotide exchange activity yet no receptor-mediated activation of nucleotide exchange. By using structural approaches, we show that this mutant loses critical hydrophobic interactions, leading to significant rearrangements of side chain residues His(57), Phe(189), Phe(191), and Phe(336); it also disturbs the rotation of the α5 helix and the π-π interaction between His(57) and Phe(189) In addition, the insertion mutant abolishes G protein release from the activated receptor after nucleotide binding. Our biochemical and computational data indicate that the interactions between α5, α1, and β2-β3 are not only vital for GDP release during G protein activation, but they are also necessary for proper GTP binding (or GDP rebinding). Thus, our studies suggest that this hydrophobic interface is critical for accurate rearrangement of the α5 helix for G protein release from the receptor after GTP binding.
© 2016 by The American Society for Biochemistry and Molecular Biology, Inc.
1 Communities
3 Members
0 Resources
7 MeSH Terms
Gβγ Binds to the Extreme C Terminus of SNAP25 to Mediate the Action of Gi/o-Coupled G Protein-Coupled Receptors.
Zurawski Z, Rodriguez S, Hyde K, Alford S, Hamm HE
(2016) Mol Pharmacol 89: 75-83
MeSH Terms: Animals, Cattle, GTP-Binding Protein alpha Subunits, Gi-Go, GTP-Binding Protein beta Subunits, GTP-Binding Protein gamma Subunits, Mice, Petromyzon, Protein Binding, Protein Structure, Secondary, Synaptosomal-Associated Protein 25
Show Abstract · Added March 24, 2020
Gi/o-coupled G protein-coupled receptors can exert an inhibitory effect on vesicle release through several G protein-driven mechanisms, more than one of which may be concurrently present in individual presynaptic terminals. The synaptosomal-associated protein of 25 kDa (SNAP25) is a key downstream effector of Gβγ subunits. It has previously been shown that proteolytic cleavage of SNAP25 by botulinum toxin A reduces the ability of Gβγ to compete with the calcium sensor synaptotagmin 1 (Syt1) for binding to SNAP25 in a calcium-dependent manner. These truncated SNAP25 proteins sustain a low level of exocytosis but are unable to support serotonin-mediated inhibition of exocytosis in lamprey spinal neurons. Here, we generate a SNAP25 extreme C-terminal mutant that is deficient in its ability to bind Gβγ while retaining normal calcium-dependent Syt1 binding to soluble N-ethylmaleimide attachment protein receptor (SNARE) and vesicle release. The SNAP25Δ3 mutant, in which residue G204 is replaced by a stop codon, features a partial reduction in Gβ1γ2 binding in vitro as well as a partial reduction in the ability of the lamprey 5-hydroxytryptamine1b-type serotonin receptor to reduce excitatory postsynaptic current amplitudes, an effect previously shown to be mediated through the interaction of Gβγ with SNAP25. Syt1 calcium-dependent binding to SNAP25Δ3 was reduced by a small extent compared with the wild type. We conclude that the extreme C terminus of SNAP25 is a critical region for the Gβγ-SNARE interaction.
Copyright © 2015 by The American Society for Pharmacology and Experimental Therapeutics.
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MeSH Terms
A transient interaction between the phosphate binding loop and switch I contributes to the allosteric network between receptor and nucleotide in Gαi1.
Thaker TM, Sarwar M, Preininger AM, Hamm HE, Iverson TM
(2014) J Biol Chem 289: 11331-41
MeSH Terms: Allosteric Regulation, Amino Acid Substitution, Animals, Crystallography, X-Ray, GTP-Binding Protein alpha Subunits, Gi-Go, Guanosine 5'-O-(3-Thiotriphosphate), Guanosine Diphosphate, Mutation, Missense, Protein Structure, Secondary, Rats
Show Abstract · Added March 24, 2020
Receptor-mediated activation of the Gα subunit of heterotrimeric G proteins requires allosteric communication between the receptor binding site and the guanine nucleotide binding site, which are separated by >30 Å. Structural changes in the allosteric network connecting these sites are predicted to be transient in the wild-type Gα subunit, making studies of these connections challenging. In the current work, site-directed mutants that alter the energy barriers between the activation states are used as tools to better understand the transient features of allosteric signaling in the Gα subunit. The observed differences in relative receptor affinity for intact Gαi1 subunits versus C-terminal Gαi1 peptides harboring the K345L mutation are consistent with this mutation modulating the allosteric network in the protein subunit. Measurement of nucleotide exchange rates, affinity for metarhodopsin II, and thermostability suggest that the K345L Gαi1 variant has reduced stability in both the GDP-bound and nucleotide-free states as compared with wild type but similar stability in the GTPγS-bound state. High resolution x-ray crystal structures reveal conformational changes accompanying the destabilization of the GDP-bound state. Of these, the conformation for Switch I was stabilized by an ionic interaction with the phosphate binding loop. Further site-directed mutagenesis suggests that this interaction between Switch I and the phosphate binding loop is important for receptor-mediated nucleotide exchange in the wild-type Gαi1 subunit.
0 Communities
1 Members
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MeSH Terms
Myristoylation exerts direct and allosteric effects on Gα conformation and dynamics in solution.
Preininger AM, Kaya AI, Gilbert JA, Busenlehner LS, Armstrong RN, Hamm HE
(2012) Biochemistry 51: 1911-24
MeSH Terms: Allosteric Regulation, Animals, Deuterium Exchange Measurement, GTP-Binding Protein alpha Subunits, Gi-Go, Models, Molecular, Myristic Acid, Protein Conformation, Rats, Signal Transduction, Solutions
Show Abstract · Added December 10, 2013
Coupling of heterotrimeric G proteins to activated G protein-coupled receptors results in nucleotide exchange on the Gα subunit, which in turn decreases its affinity for both Gβγ and activated receptors. N-Terminal myristoylation of Gα subunits aids in membrane localization of inactive G proteins. Despite the presence of the covalently attached myristoyl group, Gα proteins are highly soluble after GTP binding. This study investigated factors facilitating the solubility of the activated, myristoylated protein. In doing so, we also identified myristoylation-dependent differences in regions of Gα known to play important roles in interactions with receptors, effectors, and nucleotide binding. Amide hydrogen-deuterium exchange and site-directed fluorescence of activated proteins revealed a solvent-protected amino terminus that was enhanced by myristoylation. Furthermore, fluorescence quenching confirmed that the myristoylated amino terminus is in proximity to the Switch II region in the activated protein. Myristoylation also stabilized the interaction between the guanine ring and the base of the α5 helix that contacts the bound nucleotide. The allosteric effects of myristoylation on protein structure, function, and localization indicate that the myristoylated amino terminus of Gα(i) functions as a myristoyl switch, with implications for myristoylation in the stabilization of nucleotide binding and in the spatial regulation of G protein signaling.
0 Communities
2 Members
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10 MeSH Terms
Suboptimal activation of protease-activated receptors enhances alpha2beta1 integrin-mediated platelet adhesion to collagen.
Marjoram RJ, Voss B, Pan Y, Dickeson SK, Zutter MM, Hamm HE, Santoro SA
(2009) J Biol Chem 284: 34640-7
MeSH Terms: Amino Acid Motifs, Animals, Blood Platelets, Carrier Proteins, Collagen, GTP-Binding Protein alpha Subunits, Gi-Go, Humans, Integrin alpha2beta1, Mice, Mice, Inbred C57BL, Peptides, Platelet Adhesiveness, Platelet Aggregation, Platelet Membrane Glycoproteins, Rats, Receptor, PAR-1, Receptors, Collagen, Receptors, G-Protein-Coupled, Receptors, IgG, Receptors, Thrombin, Signal Transduction, Thrombin, Type C Phospholipases
Show Abstract · Added December 10, 2013
Thrombin and fibrillar collagen are potent activators of platelets at sites of vascular injury. Both agonists cause platelet shape change, granule secretion, and aggregation to form the primary hemostatic plug. Human platelets express two thrombin receptors, protease-activated receptors 1 and 4 (PAR1 and PAR4) and two collagen receptors, the alpha(2)beta(1) integrin (alpha(2)beta(1)) and the glycoprotein VI (GPVI)/FcRgamma chain complex. Although these receptors and their signaling mechanisms have been intensely studied, it is not known whether and how these receptors cooperate in the hemostatic function of platelets. This study examined cooperation between the thrombin and collagen receptors in platelet adhesion by utilizing a collagen-related peptide (alpha2-CRP) containing the alpha(2)beta(1)-specific binding motif, GFOGER, in conjunction with PAR-activating peptides. We demonstrate that platelet adhesion to alpha2-CRP is substantially enhanced by suboptimal PAR activation (agonist concentrations that do not stimulate platelet aggregation) using the PAR4 agonist peptide and thrombin. The enhanced adhesion induced by suboptimal PAR4 activation was alpha(2)beta(1)-dependent and GPVI/FcRgamma-independent as revealed in experiments with alpha(2)beta(1)- or FcRgamma-deficient mouse platelets. We further show that suboptimal activation of other platelet G(q)-linked G protein-coupled receptors (GPCRs) produces enhanced platelet adhesion to alpha2-CRP. The enhanced alpha(2)beta(1)-mediated platelet adhesion is controlled by phospholipase C (PLC), but is not dependent on granule secretion, activation of alpha(IIb)beta(3) integrin, or on phosphoinositol-3 kinase (PI3K) activity. In conclusion, we demonstrate a platelet priming mechanism initiated by suboptimal activation of PAR4 or other platelet G(q)-linked GPCRs through a PLC-dependent signaling cascade that promotes enhanced alpha(2)beta(1) binding to collagens containing GFOGER sites.
1 Communities
2 Members
0 Resources
23 MeSH Terms
Trp fluorescence reveals an activation-dependent cation-pi interaction in the Switch II region of Galphai proteins.
Hamm HE, Meier SM, Liao G, Preininger AM
(2009) Protein Sci 18: 2326-35
MeSH Terms: Arginine, GTP-Binding Protein alpha Subunits, Gi-Go, Guanosine 5'-O-(3-Thiotriphosphate), Guanosine Diphosphate, Kinetics, Models, Molecular, Myristic Acid, Protein Conformation, Protein Structure, Tertiary, Spectrometry, Fluorescence, Tryptophan
Show Abstract · Added December 10, 2013
Crystal structures of Galpha(i) (and closely related family member Galpha(t)) reveal much of what we currently know about G protein structure, including changes which occur in Switch regions. Galpha(t) exhibits a low rate of basal (uncatalyzed) nucleotide exchange and an ordered Switch II region in the GDP-bound state, unlike Galpha(i), which exhibits higher basal exchange and a disordered Switch II region in Galpha(i)GDP structures. Using purified Galpha(i) and Galpha(t), we examined the intrinsic tryptophan fluorescence of these proteins, which reports conformational changes associated with activation and deactivation of Galpha proteins. In addition to the expected enhancement in tryptophan fluorescence intensity, activation of GalphaGDP proteins was accompanied by a modest but notable red shift in tryptophan emission maxima. We identified a cation-pi interaction between tryptophan and arginine residues in the Switch II of Galpha(i) family proteins that mediates the observed red shift in emission maxima. Furthermore, amino-terminal myristoylation of Galpha(i) resulted in a less polar environment for tryptophan residues in the GTPase domain, consistent with an interaction between the myristoylated amino terminus and the GTPase domain of Galpha proteins. These results reveal unique insights into conformational changes which occur upon activation and deactivation of G proteins in solution.
0 Communities
1 Members
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11 MeSH Terms
FcgammaRI ligation leads to a complex with BLT1 in lipid rafts that enhances rat lung macrophage antimicrobial functions.
Serezani CH, Aronoff DM, Sitrin RG, Peters-Golden M
(2009) Blood 114: 3316-24
MeSH Terms: Animals, Cholesterol, GTP-Binding Protein alpha Subunits, Gi-Go, Immunoglobulin G, Immunologic Capping, Klebsiella Infections, Klebsiella pneumoniae, Leukotriene B4, Macrophages, Alveolar, Membrane Microdomains, Mice, Mice, Knockout, Phagocytosis, Phosphorylation, Rats, Rats, Wistar, Receptors, IgG, Receptors, Leukotriene B4, Signal Transduction, src-Family Kinases
Show Abstract · Added May 4, 2017
Leukotriene (LT) B(4) is generated in response to engagement of the Fc gamma receptor (Fc gamma R) and potently contributes to Fc gamma R-mediated antimicrobial functions in pulmonary alveolar macrophages. In this study, we report that the LTB(4) receptor leukotriene B(4) receptor 1 (BLT1) redistributes from nonlipid raft (LR) to LR membrane microdomains upon immunoglobulin G-red blood cell, but not LTB(4), challenge. Cholesterol depletion to disrupt LRs abolished LTB(4)-induced enhancement of phagocytosis, microbicidal activity, and signaling. The dependence on LR integrity for BLT1 signaling correlated with formation of a complex consisting of BLT1, its primary coupled G protein G alpha i3, Src kinase, and Fc gamma RI within LRs. This association was dependent on Src-mediated phosphorylation of BLT1. These data identify a novel form of regulation in which engagement of a macrophage immunoreceptor recruits a stimulatory G protein-coupled receptor into a LR microdomain with resultant enhanced antimicrobial signaling.
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20 MeSH Terms