Involvement of G protein βγ-subunits in diverse signaling induced by G_i/o-coupled receptors: study using the Xenopus oocyte expression system

Abstract

.-We studied the functions of ␤␥-subunits of Gi/o protein using the Xenopus oocyte expression system. Isoproterenol (ISO) elicited cAMP production and slowly activating Cl Ϫ currents in oocytes expressing ␤2-adrenoceptor and the protein kinase A-dependent Cl , and baclofen enhanced ISO-induced cAMP levels and CFTR currents in oocytes expressing ␤2-adrenoceptor-CFTR and 5-HT1A receptor (5-HT1AR), ␦-opioid receptor, or GABAB receptor, respectively. 5-HT also enhanced pituitary adenylate cyclase activating peptide (PACAP) 38-induced cAMP levels and CFTR currents in oocytes expressing PACAP receptor, CFTR and 5-HT1AR. The 5-HT-induced enhancement of Gs-coupled receptormediated currents was abrogated by pretreatment with pertussis toxin (PTX) and coexpression of G transducin ␣ (Gt␣). The 5-HT-induced enhancement was further augmented by coexpression of the G␤␥-activated form of adenylate cyclase (AC) type II but not AC type III. Thus ␤␥-subunits of Gi/o protein contribute to the enhancement of Gs-coupled receptor-mediated responses. 5-HT and DADLE did not elicit any currents in oocytes expressing 5-HT1AR or ␦-opioid receptor alone. They elicited Ca 2ϩ -activated Cl Ϫ currents in oocytes coexpressing these receptors with the G␤␥-activated form of phospholipase C (PLC)-␤2 but not with PLC-␤1. These currents were inhibited by pretreatment with PTX and coexpression of Gt␣, suggesting that ␤␥-subunits of Gi/o protein activate PLC-␤2 and then cause intracellular Ca 2ϩ mobilization. Our results indicate that ␤␥-subunits of Gi/o protein participate in diverse intracellular signals, enhancement of Gs-coupled receptor-mediated responses, and intracellular Ca 2ϩ mobilization.

μ-Opioid Receptor Forms a Functional Heterodimer With Cannabinoid CB1 Receptor: Electrophysiological and FRET Assay Analysis

“…In the electrophysiological study, Xenopus oocytes coexpressing G i / o -coupled μOR or CB 1 R together with GIRK1/ 2 caused inward rectifying K + currents in response to the selective μOR agonist DAMGO and CB 1 R agonist CP55,940, respectively, as reported previously (19,22). The signals generated by stimulation of each receptor are transmitted to GIRK via Gβγ subunits liberated from G i / o endogenously expressed in oocytes (10,19).…”

“…tions and durations of PTX treatment used in the present study were reported to produce almost complete inactivation of the G i / o proteins in oocytes (11,19). Under these conditions, oocytes coexpressing μOR + CB 1 R-G qi5 showed no responses to DAMGO or CP55,940, whereas both agonists caused Cl − currents in oocytes coexpressing μOR + CB 1 R-G qi5(m) (Fig.…”

“…Immature V and VI oocytes from Xenopus were enzymatically dissociated as described previously (18,19). Isolated oocytes were incubated at 18°C in ND-96 medium (96 mM NaCl, 2 mM KCl, 1.8 mM CaCl 2 , 1 mM MgCl 2 , and 5 mM 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid [HEPES], pH 7.4) containing 2.5 mM sodium pyruvate and 50 μg / ml gentamicin.…”

“…The final volume of the injectate was less than 50 nl in all cases. Oocytes were incubated in ND-96 and used 3 -8 days after injection as reported previously (11,18,19).…”

“…Voltage recording microelectrodes were filled with 3 M KCl and their tip resistance was 1.0 -2.5 MΩ. Currents were continuously recorded and stored with a MacLab (AD Instruments, Castle Hill, NSW, Australia) and a Macintosh computer, as described previously (18,19). All test compounds applied to oocytes were dissolved into ND-96 or HK (49 mM K + ) medium.…”