Coupling of GABAB receptor GABAB2 subunit to G proteins: evidence from Xenopus oocyte and baby hamster kidney cell expression system.
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 + Galphai2-fused GB2R (GB2R-Galphai2) caused faster K+ currents in response to baclofen. Furthermore, oocytes coexpressing GB1aR + GB2R fused to Galphaqi5 (a chimeric Galphaq 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-Galphai2 or GB1aR-Galphaqi5 together with GB2R. BHK cells and Xenopus oocytes coexpressing GB1aR-Cerulean + a triplet tandem of GB2R-Venus-Galphaqi5 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-Galphaqi5. 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.
Uezono
Yasuhito
Y
Dept. of Pharmacology, Nagasaki Univ. Graduate School of Biomedical Sciences, Nagasaki 852-8523, Japan. uezonoy@alpha.med.nagasaki-u.ac.jp
Kanaide
Masato
M
Kaibara
Muneshige
M
Barzilai
Rachel
R
Dascal
Nathan
N
Sumikawa
Koji
K
Taniyama
Kohtaro
K
D000818:Animals; D001418:Baclofen / Q000494:pharmacology; D002118:Calcium / Q000378:metabolism; D002460:Cell Line; D002712:Chlorides / Q000378:metabolism; D006224:Cricetinae; D031541:Fluorescence Resonance Energy Transfer; D005456:Fluorescent Dyes; D051676:G Protein-Coupled Inwardly-Rectifying Potassium Channels / Q000235:genetics / Q000378:metabolism; D018755:GABA Agonists / Q000494:pharmacology; D058786:GABA-B Receptor Agonists; D051960:GTP-Binding Protein alpha Subunit, Gi2 / Q000235:genetics / Q000378:metabolism; D019204:GTP-Binding Proteins / Q000378:metabolism*; D015870:Gene Expression; D006801:Humans; D007668:Kidney / Q000166:cytology; D051379:Mice; D050506:Mutant Chimeric Proteins / Q000235:genetics / Q000378:metabolism; D021122:Protein Subunits / Q000235:genetics / Q000378:metabolism; D051381:Rats; D018080:Receptors, GABA-B / Q000235:genetics* / Q000378:metabolism*; D014982:Xenopus laevis
D002712:Chlorides; D005456:Fluorescent Dyes; D051676:G Protein-Coupled Inwardly-Rectifying Potassium Channels; D018755:GABA Agonists; D058786:GABA-B Receptor Agonists; D050506:Mutant Chimeric Proteins; D021122:Protein Subunits; D018080:Receptors, GABA-B; D019204:GTP-Binding Proteins; D051960:GTP-Binding Protein alpha Subunit, Gi2; D001418:Baclofen; D002118:Calcium