Reverse transcription-polymerase chain reaction was used to identify the pertussis toxin (Ptx)-sensitive G protein alpha-subunit pool in Chinese hamster ovary (CHO) and mouse fibroblast (B82) cells. We detected the presence of mRNA for G(ialpha2), G(ialpha3), and G(oalpha) in both cell lines. G(ialpha1) and G(alphaz) mRNAs were not detected. We also found a homolog of the retinal rod transducin (G(talpha1)) in CHO, and the mouse cone transducin (G(talpha2)) in B82 cells. The presence of the transducin alpha-subunit proteins in CHO and B82 cells was confirmed by immunoprecipitation with specific antibodies. To test the interaction of heterologously expressed receptors with transducin in CHO cells, a Ptx-insensitive (C347S) rod transducin mutant was transfected into a CHO cell line stably expressing the human delta-opioid receptor (hDOR/CHO). (+)-4-[(alphaR)-alpha-((2S,2R)-4-allyl-2, 5-dimethyl-1-piperazinyl)-3-methoxybenzyl]-N,N-diethylbenzamide, a selective delta-opioid receptor agonist, stimulated guanosine-5'-O-(3-[(35)S]thio)triphosphate binding by 293 +/- 36% after Ptx pretreatment in the mutant cell line with an EC(50) value of 54 +/- 32 nM, showing that transducin can functionally couple to the human delta-opioid receptors in these cells.
Authors
Varga, E V; Stropova, D; Kim, T; Wang, M; Roeske, W R; Yamamura, H I