Coupling of GABAB receptor GABAB2 subunit to G proteins: evidence from Xenopus oocyte and baby hamster kidney cell expression system

• Published in: American Journal of Physiology: Cell Physiology 2006-01, Vol.290 (1), p.C200-C207
• Main Authors: Uezono, Yasuhito, Kanaide, Masato, Kaibara, Muneshige, Barzilai, Rachel, Dascal, Nathan, Sumikawa, Koji, Taniyama, Kohtaro
• Format: Article
• Language: English
• Subjects: Animals; Baclofen - pharmacology; Calcium - metabolism; Cell Line; Chlorides - metabolism; Cricetinae; Fluorescence Resonance Energy Transfer; Fluorescent Dyes; G Protein-Coupled Inwardly-Rectifying Potassium Channels - genetics; G Protein-Coupled Inwardly-Rectifying Potassium Channels - metabolism; GABA Agonists - pharmacology; GABA-B Receptor Agonists; Gene Expression; GTP-Binding Protein alpha Subunit, Gi2 - genetics; GTP-Binding Protein alpha Subunit, Gi2 - metabolism; GTP-Binding Proteins - metabolism; Humans; Kidney - cytology; Mice; Mutant Chimeric Proteins - genetics; Mutant Chimeric Proteins - metabolism; Protein Subunits - genetics; Protein Subunits - metabolism; Rats; Receptors, GABA-B - genetics; Receptors, GABA-B - metabolism; Xenopus laevis
• ISSN: 0363-6143; 1522-1563
• DOI: 10.1152/ajpcell.00269.2005

Departments of 1 Pharmacology and 2 Anesthesiology, Nagasaki University Graduate School of Biomedical Sciences, Nagasaki, Japan; and 3 Department of Physiology and Pharmacology, Sackler School of Medicine, Tel Aviv University, Ramat Aviv, Israel Submitted 8 June 2005 ; accepted in final form 17 August 2005 Coupling of functional GABA B receptors (GABA B R) 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 GABA B1a receptor (GB 1a R) fused to Cerulean, a brighter variant of cyan fluorescent protein, and GABA B2 receptor (GB 2 R) fused to Venus, a brighter variant of yellow fluorescent protein, revealed that GB 1a R-Cerulean and GB 2 R-Venus form a heterodimer. The GABA B R agonists baclofen and 3-aminopropylphosphonic acid (3-APPA) elicited inward-rectifying K + currents in a concentration-dependent manner in oocytes expressing GB 1a R and GB 2 R, or GB 1a R-Cerulean and GB 2 R-Venus, together with G protein-activated inward-rectifying K + channels (GIRKs), but not in oocytes expressing GB 1a R alone or GB 2 R alone together with GIRKs. Oocytes coexpressing GB 1a R + G i2 -fused GB 2 R (GB 2 R-G i2 ) caused faster K + currents in response to baclofen. Furthermore, oocytes coexpressing GB 1a R + GB 2 R fused to G qi5 (a chimeric G q protein that activates PLC pathways) caused PLC-mediated Ca 2+ -activated Cl – currents in response to baclofen. In contrast, these responses to baclofen were not observed in oocytes coexpressing GB 1a R-G i2 or GB 1a R-G qi5 together with GB 2 R. BHK cells and Xenopus oocytes coexpressing GB 1a R-Cerulean + a triplet tandem of GB 2 R-Venus-G qi5 caused FRET and Ca 2+ -activated Cl – currents, respectively, with a similar potency in BHK cells coexpressing GB 1a R-Cerulean + GB 2 R-Venus and in oocytes coexpressing GB 1a R + GB 2 R-G qi5 . Our results indicate that functional GABA B R forms a heterodimer composed of GB 1 R and GB 2 R and that the signal transducing G proteins are directly coupled to GB 2 R but not to GB 1 R. fluorescence resonance energy transfer Address for reprint requests and other correspondence: Y. Uezono, Dept. of Pharmacology, Nagasaki Univ. Graduate School of Biomedical Sciences, Nagasaki 852-8523, Japan (e-mail: uezonoy{at}alpha.med.nagasaki-u.ac.jp )