Desensitization of Inositol 1,4,5-Trisphosphate/Ca2+-induced Cl− Currents by Prolonged Activation of G Proteins in Xenopus Oocytes

Quick, Michael W.; Lester, Henry A.; Davidson, Norman; Aragay, Anna M.

doi:10.1074/jbc.271.50.32021

Cited by 19 publications

(18 citation statements)

References 44 publications

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“…This result was similar to that previously jpet.aspetjournals.org observed in oocytes expressing the thyrotropin-releasing hormone receptor and ␣ subunits of the Gq family G 16 (Quick et al, 1996). They suggested that overexpressed free ␣ subunits activated phospholipase C and desensitized the pathway.…”

Section: Discussionsupporting

confidence: 79%

Site of Action of the General Anesthetic Propofol in Muscarinic M1 Receptor-Mediated Signal Transduction

Murasaki

Kaibara²,

Nagase³

et al. 2003

J Pharmacol Exp Ther

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Although a potential target site of general anesthetics is primarily the GABA A receptor, a chloride ion channel, a previous study suggested that the intravenous general anesthetic propofol attenuates the M1 muscarinic acetylcholine receptor (M1 receptor)-mediated signal transduction. In the present study, we examined the target site of propofol in M1 receptor-mediated signal transduction. Two-electrode voltage-clamp method was used in Xenopus oocytes expressing both M1 receptors and associated G protein ␣ subunits (Gq␣). Propofol inhibited M1 receptor-mediated signal transduction in a dose-dependent manner (IC 50 ϭ 50 nM). Injection of guanosine 5Ј-3-O-(thio)triphosphate (GTP␥S) into oocytes overexpressing Gq␣ was used to investigate direct effects of propofol on G protein coupled with the M1 receptor. Propofol did not affect activation of Gq␣-mediated signal transduction with the intracellular injection of GTP␥S. We also studied effects of propofol on l-[N- Effects of propofol on Gs-and Gi/o-coupled signal transduction were investigated, using oocytes expressing the ␤2 adrenoceptor (␤2 receptor)/cystic fibrosis transmembrane conductance regulator or oocytes expressing the M2 muscarinic acetylcholine receptor (M2 receptor)/Kir3.1 (a member of G protein-gated inwardly rectifying K ϩ channels). Neither ␤2 receptor-mediated nor M2 receptor-mediated signal transduction was inhibited by a relatively high concentration of propofol (50 M). These results indicate that propofol inhibits M1 receptor-mediated signal transduction by selectively disrupting interaction between the receptor and associated G protein.

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Section: Discussionsupporting

confidence: 79%

Site of Action of the General Anesthetic Propofol in Muscarinic M1 Receptor-Mediated Signal Transduction

Murasaki

Kaibara²,

Nagase³

et al. 2003

J Pharmacol Exp Ther

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“…3D). Concentrations of the three basic amino acids at 3 M elicited no response, and higher concentrations (300 M) led to decreased currents compared with 100 M (data not shown) presumably because of desensitization of the receptor and/or the signaling pathway proteins, as reported by others (Minakami et al, 1994;Quick et al, 1996). L-Arg, L-Lys, and L-Orn were thus the most potent agonists tested, showing that the receptor has a preference for basic amino acids.…”

Section: Deorphanization Of Gprc6a 593mentioning

confidence: 58%

Deorphanization of GPRC6A: A Promiscuous l-α-Amino Acid Receptor with Preference for Basic Amino Acids

Wellendorph

Hansen²,

Balsgaard³

et al. 2005

Molecular Pharmacology

188

205

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One of the most important tasks of molecular pharmacology is the deorphanization of the large number of G-protein-coupled receptors with unidentified endogenous agonists. We recently reported the cloning and analysis of expression of a novel human family C G-protein-coupled receptor, termed hGPRC6A. To identify agonists at this orphan receptor, we faced the challenges of achieving surface expression in mammalian cell lines and establishing an appropriate functional assay. Generating a chimeric receptor construct, h6A/5.24, containing the ligand binding amino-terminal domain (ATD) of hGPRC6A with the signal transducing transmembrane and C terminus of the homologous goldfish 5.24 receptor allowed us to overcome these obstacles. Homology modeling of the hGPRC6A ATD based on the crystal structure of the metabotropic glutamate receptor subtype 1 predicted interaction with ␣-amino acids and was employed to rationally select potential ligands. Measurement of Ca 2ϩ -dependent chloride currents in Xenopus laevis oocytes facilitated the deorphanization of h6A/ 5.24 and identification of L-␣-amino acids as agonists. The most active agonists were basic L-␣-amino acids, L-Arg, L-Lys, and L-ornithine, suggesting that these may function as endogenous signaling molecules. Measurement of intracellular calcium in tsA cells expressing h6A/5.24 allowed determination of EC 50 values, which confirmed the agonist preferences observed in oocytes. Cloning, cell surface expression and deorphanization of the mouse ortholog further reinforces the assignment of the agonist preferences of hGPRC6A. This study demonstrates the utility of a chimeric receptor approach in combination with molecular modeling, for elucidating agonist interaction with GPRC6A, a novel family C G-protein-coupled receptor.

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“…In our experiments, injections of the cRNAs coding G␣ q family members (0.5-40 ng) always inhibited ginsenoside-induced Cl Ϫ current; we never detected enhancement of the oocyte response even at very low doses of the cRNAs. The reason Quick et al (17) observed enhancement of the oocyte response after injections of smaller doses of G␣ q or G␣ 16 cRNA might be simply that they co-injected TRH receptor cRNA. In this case, the exogenously expressed G␣ q or G␣ 16 might couple to co-expressed TRH receptors, increasing the efficacy of TRH in inducing the Cl Ϫ current.…”

Section: Fig 4 Increase In Ginsenoside-induced CLmentioning

confidence: 99%

“…Recently, Quick et al (17) reported that in the Xenopus oocyte, expression of a G␣ q family member (G␣ q or G␣ 16 ) significantly altered the amplitude of thyrotropin-releasing hormone (TRH)-induced Cl Ϫ current. This report coupled with the present findings points to the possibility that ginsenosides and TRH share the same signal transduction pathways.…”

Section: Fig 4 Increase In Ginsenoside-induced CLmentioning

confidence: 99%

Gαq/11 Coupled to Mammalian Phospholipase C β3-like Enzyme Mediates the Ginsenoside Effect on Ca2+-activated Cl− Current in theXenopus Oocyte

Choi

Kim

et al. 2001

Journal of Biological Chemistry

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Recently we demonstrated that ginsenosides, the active ingredients of Panax ginseng, enhanced Ca 2؉-activated Cl ؊ current in the Xenopus oocyte through a signal transduction mechanism involving the activation of pertussis toxin-insensitive G protein and phospholipase C (PLC). However, it has not yet been determined precisely which G protein subunit(s) and which PLC isoform(s) participate in the ginsenoside signaling. To provide answers to these questions, we investigated the changes in ginsenoside effect on the Cl ؊ current after intraoocyte injections of the cRNAs coding various G protein subunits, a regulator of G protein signaling (RGS2), and G␤␥-binding proteins. In addition, we examined which of mammalian PLC␤1-3 antibodies injected into the oocyte inhibited the action of ginsenosides on the Cl ؊ current. Injection of G␣ q or G␣ 11 cRNA increased the basal Cl ؊ current recorded 48 h after, and it further prevented ginsenosides from enhancing the Cl ؊ current, whereas G␣ i2 and G␣ oA cRNA injection had no significant effect. The changes following G␣ q cRNA injection were prevented when G␤ 1 ␥ 2 and G␣ q subunits were co-expressed by simultaneous injection of the cRNAs coding these subunits. Injection of cRNA coding G␣ q Q209L, a constitutively active mutant that does not bind to G␤␥, produced effects similar to those of G␣ q cRNA injection. The effects of G␣ q Q209L cRNA injection, however, were not prevented by co-injection of G␤ 1 ␥ 2 cRNA. Injection of the cRNA coding RGS2, which interacts most selectively with G␣ q/11 among various identified RGS isoforms and stimulates the hydrolysis of GTP to GDP in active GTP-bound G␣ subunit, resulted in a severe attenuation of ginsenoside effect on the Cl ؊ current. Finally, antibodies against PLC␤3, but not -␤1 and -␤2, markedly attenuated the ginsenoside effect examined at 3-h postinjection. These results suggest that G␣ q/11 coupled to mammalian PLC ␤3-like enzyme mediates ginsenoside effect on Ca 2؉

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Desensitization of Inositol 1,4,5-Trisphosphate/Ca2+-induced Cl− Currents by Prolonged Activation of G Proteins in Xenopus Oocytes

Cited by 19 publications

References 44 publications

Site of Action of the General Anesthetic Propofol in Muscarinic M1 Receptor-Mediated Signal Transduction

Site of Action of the General Anesthetic Propofol in Muscarinic M1 Receptor-Mediated Signal Transduction

Deorphanization of GPRC6A: A Promiscuous l-α-Amino Acid Receptor with Preference for Basic Amino Acids

Gαq/11 Coupled to Mammalian Phospholipase C β3-like Enzyme Mediates the Ginsenoside Effect on Ca2+-activated Cl− Current in theXenopus Oocyte

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