ADP Ribosylation by Cholera Toxin Identifies Three G-Proteins That Are Activated by the Galanin Receptor: Studies With RINm5F Cell Membranes

Gillison, S. L.; Sharp, Geoffrey W. G.

doi:10.2337/diab.43.1.24

Cited by 24 publications

(17 citation statements)

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“…PTx-pretreatment did not significantly affect (not shown) uptake of [ 3 H]methionine and subsequent incorporation into SAM pools. Alternatively, PTx-catalyzed ribosylation of ␣ subunits was quantitated using total particulate fraction (17,38). For this purpose, PTx holoenzyme was activated at 30ЊC for 30 min in 10-mM Tris-HCl consisting of 10-mM DTT and 100-M ATP.…”

Section: Gtp-depletion and Ptx-pretreatment Studiesmentioning

confidence: 99%

Glucose activates the carboxyl methylation of gamma subunits of trimeric GTP-binding proteins in pancreatic beta cells. Modulation in vivo by calcium, GTP, and pertussis toxin.

Kowluru¹,

Li²,

Metz³

1997

J. Clin. Invest.

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The ␥ subunits of trimeric G-proteins ( ␥ 1 , ␥ 2 , ␥ 5 , and ␥ 7 isoforms) were found to be methylated at their carboxyl termini in normal rat islets, human islets and pure ␤ [HIT-T15] cells. Of these, GTP ␥ S significantly stimulated the carboxyl methylation selectively of ␥ 2 and ␥ 5 isoforms. Exposure of intact HIT cells to either of two receptor-independent agonists-a stimulatory concentration of glucose or a depolarizing concentration of K ϩ -resulted in a rapid (within 30 s) and sustained (at least up to 60 min) stimulation of ␥ subunit carboxyl methylation. Mastoparan, which directly activates G-proteins (and insulin secretion from ␤ cells), also stimulated the carboxyl methylation of ␥ subunits in intact HIT cells. Stimulatory effects of glucose or K ϩ were not demonstrable after removal of extracellular Ca 2 ϩ or depletion of intracellular GTP, implying regulatory roles for calcium fluxes and GTP; however, the methyl transferase itself was not directly activated by either. The stimulatory effects of mastoparan were resistant to removal of extracellular Ca 2 ϩ , implying a mechanism of action that is different from glucose or K ϩ but also suggesting that dissociation of the ␣␤␥ trimer is conducive to ␥ subunit carboxyl methylation. Indeed, pertussis toxin also markedly attenuated the stimulatory effects of glucose, K ϩ or mastoparan without altering the rise in intracellular calcium induced by glucose or K ϩ . Glucose-induced carboxyl methylation of ␥ 2 and ␥

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Section: Gtp-depletion and Ptx-pretreatment Studiesmentioning

confidence: 99%

Glucose activates the carboxyl methylation of gamma subunits of trimeric GTP-binding proteins in pancreatic beta cells. Modulation in vivo by calcium, GTP, and pertussis toxin.

Kowluru¹,

Li²,

Metz³

1997

J. Clin. Invest.

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“…Galanin exerts its actions via binding to specific membrane receptors (4). Biochemical and molecular studies, performed in brain and pancreas, indicate that the galanin receptor is a glycoprotein of 54 kDa (5)(6)(7) coupled to the inhibitory guanine nucleotide binding (G) protein Gi, identified as Gil, Gi2, and Gi3 in pancreatic 83 cells (8,9). Depending on the target tissue, different pathways for intracellular signaling by galanin are involved: inhibition of adenylate cyclase (10), blockage of voltage-dependent Ca2+ channels (11), and activation of ATP-sensitive K+ channels (12).…”

mentioning

confidence: 99%

Molecular cloning of a functional human galanin receptor.

Habert-Ortoli¹,

Amiranoff²,

Loquet³

et al. 1994

Proc. Natl. Acad. Sci. U.S.A.

322

225

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The ubiquitous neuropeptide galanin controls numerous functions such as endocrine secretions, intestinal motility, and behavioral activities. These regulatory effects of galanin are mediated through the interaction with specific membrane receptors and involve the pertussis toxin-sensitive guanine nucleotide binding proteins Gj/G0 as transducing elements. We report here the isolation of a cDNA coding for a human galanin receptor from a Bowes melanoma cell line cDNA expression library, by using a radioligand binding strategy. The nucleotide sequence ofthe cloned receptor reveals an open reading frame encoding a 349-amino acid protein with seven putative hydrophobic transmembrane domains and significant homology with members of the guanine nucleotide binding protein-coupled neuropeptide receptor family. The cloned receptor expressed in COS cells specifically binds human, porcine, and rat galanin with high affinity (Kd in the nanomolar range) and mediates the galanin inhibition of adenylate cyclase. A 2.8-kb galanin receptor transcript was identified in several human tissues. Cloning of this galanin receptor should enhance our knowledge of its distribution, structure, and function in human physiology and pathophysiology.Galanin, a 29-amino acid neuropeptide (30 amino acids in humans), was originally isolated from pig intestine (1) and later reported to be widely distributed in the central and peripheral nervous systems of numerous species (2). Galanin is unrelated to the other known families of regulatory peptides and, to date, remains the only known member of its own family. It exerts multiple regulatory functions such as (i) control of endocrine and exocrine pancreatic secretions, (ii) regulation of intestinal motility, and (iii) modulation of behavioral, cognitive, and sensory functions such as feeding, learning, memory, and nociception (for reviews, see refs. 2 and 3). Galanin exerts its actions via binding to specific membrane receptors (4). Biochemical and molecular studies, performed in brain and pancreas, indicate that the galanin receptor is a glycoprotein of 54 kDa (5-7) coupled to the inhibitory guanine nucleotide binding (G) protein Gi, identified as Gil, Gi2, and Gi3 in pancreatic 83 cells (8,9). Depending on the target tissue, different pathways for intracellular signaling by galanin are involved: inhibition of adenylate cyclase (10), blockage of voltage-dependent Ca2+ channels (11), and activation of ATP-sensitive K+ channels (12). Structure-activity studies, with galanin fragments (13-15) and chimeric peptides (16,17), generally emphasize the importance of the N-terminal fragment of galanin for the interaction with the peptide receptor. These studies also raise the possibility of the existence of galanin receptor subtypes, an issue that may be properly addressed with the molecular cloning of the galanin receptor.In this context, we describe here the expression cloning of a cDNA encoding a galanin receptor from the human Bowes melanoma cell line (18) (vol/vol) fetal calf serum, 6 mM glutami...

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“…The functionality of the receptors was measured by their ability to activate their specific G-proteins by means of a fluorescence spectroscopic assay. We used Gi1 for GalR 1 and Gi2 for 5-HT 1A activation, as it has been previously described that our target receptors are able to signal via these specific Gα subunits [27,28], when they are activated by their corresponding ligands. Here, adding 8-OH-DPAT for 5-HT 1A or galanin peptide for GalR 1 was not necessary because the receptors have been already purified bound to their ligands during the purification process.…”

Section: Resultsmentioning

confidence: 99%

Zinc Is Involved in Depression by Modulating G Protein-Coupled Receptor Heterodimerization

et al. 2015

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5-Hydroxytryptamine 1A receptor and galanin receptor 1 belong to the G-protein-coupled receptors superfamily and they have been described to heterodimerize triggering an anomalous physiological state that would underly depression. Zinc supplementation has been widely reported to improve treatment against major depressive disorder. Our work has focused on the study and characterization of these receptors and its relationships with zinc both under purified conditions and in cell culture. To this aim, we have designed a strategy to purify the receptors in a conformationally active state. We have used receptors tagged with the monoclonal Rho-1D4 antibody, and employed ligand assisted purification in order to successfully purify both receptors in a properly folded and active state. The interaction between both purified receptors has been analyzed by surface plasmon resonance in order to determine the kinetics of dimerization. Zinc effect on heteromer has also been tested using the same methodology, but exposing the 5-hydroxytryptamine 1A receptor to zinc before the binding experiment. These results, combined with FRET measurements, in the absence and presence of zinc, suggest that this ion is capable of disrupting this interaction. Moreover, molecular modelling suggests that there is a coincidence between zinc binding sites and heterodimerization interfaces for the serotonin receptor.Our results establish a rational explanation for the role of zinc in the molecular processes associated with receptor-receptor interactions and its relationship with depression, in agreement with previously reported evidence for the positive effects of zinc in depression treatment, and the involvement of our target dimer in the same disease.

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ADP Ribosylation by Cholera Toxin Identifies Three G-Proteins That Are Activated by the Galanin Receptor: Studies With RINm5F Cell Membranes

Cited by 24 publications

References 0 publications

Glucose activates the carboxyl methylation of gamma subunits of trimeric GTP-binding proteins in pancreatic beta cells. Modulation in vivo by calcium, GTP, and pertussis toxin.

Glucose activates the carboxyl methylation of gamma subunits of trimeric GTP-binding proteins in pancreatic beta cells. Modulation in vivo by calcium, GTP, and pertussis toxin.

Molecular cloning of a functional human galanin receptor.

Zinc Is Involved in Depression by Modulating G Protein-Coupled Receptor Heterodimerization

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