Abstract
Expression of G protein alpha subunits of the Gq family with various G protein-coupled receptors induces activation of an inositol 1,4, 5-trisphosphate (IP3)/Ca2+-mediated Cl- conductance in Xenopus oocytes. Our present data show that two members of this family, the human Galpha16 subunit and the murine homologue Galpha15, can induce both activation and inhibition of these agonist-induced currents. Although extremely low amounts (10-50 pg) of injected Galpha16 subunit cRNA cause modest ( approximately 2-fold) enhancement of ligand-induced Cl- currents in oocytes co-injected with thyrotropin-releasing hormone (TRH) receptor cRNA 48 h postinjection, larger Galpha16 and Galpha15 cRNA injections cause >10-fold inhibition of TRH or 5HT2c receptor responses. The inhibition is analyzed in this study. The inhibited currents are recovered if various Gbetagamma subunit combinations are also expressed with the Galpha subunits. The constitutively active mutant, Galpha16Q212L, also causes a strong attenuation of the ligand-induced Cl- currents, but this inhibition is not recovered by co-expression of Gbetagamma subunits. These results indicate that the free Galpha subunit is responsible for the inhibitory signal. Although expression of TRH receptor alone produces maximum responses approximately 48 h after injection, co-expression of TRH receptor with Galpha16 results in enhanced responses 6-12 h postinjection, followed by complete attenuation at 36 h. Furthermore, injection of Galpha16 cRNA alone at comparable levels gives rise to spontaneous Cl- currents within 6-12 h postinjection, suggesting that the early spontaneous activation underlies the later suppression. Expression of other G protein alpha subunits of the Gq family, at cRNA levels considerably higher than effective for Galpha16, produces both analogous spontaneous Cl- currents and, later, inhibition of ligand-induced Cl- currents. Experiments with direct injection of IP3 and of Ca2+ suggest that this inhibition is consistent with the down-regulation of IP3 receptors. These data indicate that both enhancement and inhibition of signaling through G protein-coupled receptors can be mediated by the expression level and/or activity of an individual G protein.
Highlights
Many hormones, neurotransmitters, and growth factors act via G protein-coupled receptors (GPCRs)1 in a wide range of transduction processes
We suggest that this effect is part of a desensitization process controlling the stimulation of the phospholipase C (PLC) pathway and that both enhancement and inhibition of signaling through GPCRs can be mediated by the level of G protein expression and/or activity
Injection of 50 pmol of CaCl2 produced comparable responses in control oocytes and in oocytes injected with G␣16 (Fig. 5B). These results indicate that the ClϪ channels themselves were not affected by the prolonged stimulation of the PLC  pathway and suggest that the reduction of the peak currents observed in oocytes expressing G␣16, G␣15, or other G␣ subunits in excess is a consequence of physiological alterations in IP3 receptor responses
Summary
In Vitro Synthesis of RNA—In vitro transcription of sense RNA was carried out as described previously [25] with a few modifications. The transcription of linearized templates was performed in 7.6 mM Tris-HCl, pH 7.6, 6 mM MgCl2, 0.6 mM NaCl, and 10 mM dithiothreitol containing 0.5 mM each ATP, CTP, and UTP, 0.1 mM GTP, 0.5 mM 5Ј-(7-methyl)-GTP, and 180 units of the respective polymerase in a total volume of 250 l. The DNA template was subsequently removed by treatment with 5 units of RNase-free DNase I for 15 min at 37 °C. Oocytes were defolliculated and maintained at 18 °C in incubation medium containing ND96 (96 mM NaCl, 2 mM KCl, 1 mM MgCl2, 5 mM HEPES, pH 7.4), 1.8 mM CaCl2, 50 g/ml gentamycin, and 5% horse serum [27]. The oocytes were clamped at Ϫ80 mV and superfused continuously in ND96 medium; all drugs were applied in this solution.
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