Coupling of GABA_B receptor GABA_B2 subunit to G proteins: evidence from Xenopus oocyte and baby hamster kidney cell expression system

Abstract

Coupling of functional GABAB receptors (GABABR) to G proteins was investigated with an expression system of baby hamster kidney (BHK) cells and Xenopus oocytes. Fluorescence resonance energy transfer (FRET) analysis of BHK cells coexpressing GABAB1a receptor (GB1aR) fused to Cerulean, a brighter variant of cyan fluorescent protein, and GABAB2 receptor (GB2R) fused to Venus, a brighter variant of yellow fluorescent protein, revealed that GB1aR-Cerulean and GB2R-Venus form a heterodimer. The GABABR agonists baclofen and 3-aminopropylphosphonic acid (3-APPA) elicited inward-rectifying K+ currents in a concentration-dependent manner in oocytes expressing GB1aR and GB2R, or GB1aR-Cerulean and GB2R-Venus, together with G protein-activated inward-rectifying K+ channels (GIRKs), but not in oocytes expressing GB1aR alone or GB2R alone together with GIRKs. Oocytes coexpressing GB1aR + Gαi2-fused GB2R (GB2R-Gαi2) caused faster K+ currents in response to baclofen. Furthermore, oocytes coexpressing GB1aR + GB2R fused to Gαqi5 (a chimeric Gαq protein that activates PLC pathways) caused PLC-mediated Ca2+-activated Cl− currents in response to baclofen. In contrast, these responses to baclofen were not observed in oocytes coexpressing GB1aR-Gαi2 or GB1aR-Gαqi5 together with GB2R. BHK cells and Xenopus oocytes coexpressing GB1aR-Cerulean + a triplet tandem of GB2R-Venus-Gαqi5 caused FRET and Ca2+-activated Cl− currents, respectively, with a similar potency in BHK cells coexpressing GB1aR-Cerulean + GB2R-Venus and in oocytes coexpressing GB1aR + GB2R-Gαqi5. Our results indicate that functional GABABR forms a heterodimer composed of GB1R and GB2R and that the signal transducing G proteins are directly coupled to GB2R but not to GB1R.

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

“…In our study as well as others, this protocol had almost no effect on Cerulean in the absence of Venus (11,16). According to this procedure, if FRET is occurring, then photobleaching of the acceptor (Venus) should yield a significant increase in fluorescence of the donor (Cerulean).…”

“…For transfection experiments, BHK cells were seeded at a density of 1 × 10 5 cells / 35-mm glass-bottomed culture dish (World Precision Instrument, Sarasota, FL, USA) for 24 h. For western blot assay, cells were seeded at a density of 1 × 10 6 cells / 35-mm dish. Transient transfection was then performed with Effectene transfection reagent (Qiagen, Tokyo) containing 0.2 μg each of cDNAs as described previously (11,15). Cells were used in confocal microscopy analysis, FRET analysis, and Western blotting assay 16 -24 h after transfection.…”

“…FRET was measured by imaging Cerulean before and after photo-bleaching Venus with the 100% intensity of 514-nm argon laser for 30 -60 s, a duration that efficiently bleached Venus with little effect on Cerulean (11,15). An increase of the donor fluorescence (Cerulean) was interpreted as evidence of FRET from Cerulean to Venus.…”

“…We also conducted coimmunoprecipitation with subsequent western blot assay in BHK cells expressing FLAG-tagged μOR and CB 1 R. Electrophysiological assay was also conducted to confirm the formation of functional heterodimers of μOR and CB 1 R. To this end, we developed an electrophysiological assay using the Xenopus oocyte expression system with μOR and CB 1 R fused to a chimeric Gα q protein, G qi5 . The chimeric G qi5 protein, whose last five amino acid residues in the C-terminus was replaced with the corresponding portion of G i protein, is useful for monitoring the responses mediated by stimulation of G i / o protein-coupled receptors in the Xenopus oocyte expression system, as reported previously by our laboratory (10,11). Chimeric G qi5 allows G i / o -coupled receptors to couple to the phospholipase C (PLC)-mediated signal pathway (12,13).…”

“…Chimeric G qi5 allows G i / o -coupled receptors to couple to the phospholipase C (PLC)-mediated signal pathway (12,13). Agonists for G i / o protein-coupled receptors can elicit Ca 2+ -activated Cl − currents in such oocytes only through the chimeric G qi5 , but not through G i / o endogenously expressed in oocytes (10,11). Based on the results, we proposed that our electrophysiological method for functional analyses could be useful for determination of signals mediated through heterodimerized G protein-coupled receptors.…”