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 + 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.
Subject(s)
GTP-Binding Proteins/metabolism , Receptors, GABA-B/genetics , Receptors, GABA-B/metabolism , 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 , GTP-Binding Protein alpha Subunit, Gi2/genetics , GTP-Binding Protein alpha Subunit, Gi2/metabolism , Gene Expression , Humans , Kidney/cytology , Mice , Mutant Chimeric Proteins/genetics , Mutant Chimeric Proteins/metabolism , Protein Subunits/genetics , Protein Subunits/metabolism , Rats , Xenopus laevisABSTRACT
Human L-type voltage-dependent Ca(2+) channels (alpha(1C), or Ca(v)1.2) are up-regulated by protein kinase C (PKC) in native tissues, but in heterologous systems this modulation is absent. In rat and rabbit, alpha(1C) has two N-terminal (NT) isoforms, long and short, with variable initial segments of 46 and 16 amino acids, respectively. The initial 46 amino acids of the long-NT alpha(1C) are crucial for PKC regulation. However, only a short-NT human alpha(1C) is known. We assumed that a long-NT isoform of human alpha(1C) may exist. By homology screening of human genomic DNA, we identified a stretch (termed exon 1a) highly homologous to rabbit long-NT, separated from the next known exon of alpha(1C) (exon 1b, which encodes the alternative, short-NT) by an approximately 80 kb-long intron. The predicted 46-amino acid protein sequence is highly homologous to rabbit long-NT. Reverse transcriptase PCR showed the presence of exon 1a transcript in human cardiac RNA. Expression of human long-NT alpha(1C) in Xenopus oocytes produced Ca(2+) channel enhanced by a PKC activator, whereas the short-NT alpha(1C) was inhibited. The long-NT isoform may be the Ca(2+) channel enhanced by PKC-activating transmitters in human tissues.
