Coupling of human delta-opioid receptor to retinal rod transducin in Chinese hamster ovary cells

Reverse transcription-polymerase chain reaction was used to identify the pe rtussis toxin (Ptx)-sensitive G protein alpha-subunit pool in Chinese hamst er ovary (CHO) and mouse fibroblast (B82) cells. We detected the presence o f mRNA for G(i alpha 2),G(i alpha 3), and G(o alpha) in both cell lines. G( i alpha 1) and G(alpha z) mRNAs were not detected. We also found a homolog of the retinal rod transducin (G(t alpha 1)) in CHO, and the mouse cone tra nsducin (G(t alpha 2))in B82 cells. The presence of the transducin alpha-su bunit proteins in CHO and B82 cells was confirmed by immunoprecipitation wi th specific antibodies. To test the interaction of heterologously expressed receptors with transducin in CHO cells, a Ptx-insensitive (C347S) rod tran sducin mutant was transfected into a CHO cell line stably expressing the hu man delta-opioid receptor (hDOR/CHO). (+)-4-[(alpha R)-alpha- ((2S,2R)-4-al lyl-2,5-dimethyl-1-piperazinyl)- 3-methoxybenzyl]-N,N-diethylbenzamide, a s elective delta-opioid receptor agonist, stimulated guanosine-5'-O-(3-[ S-35 ] thio) triphosphate binding by 293 +/- 36% after Ptx pretreatment in the m utant cell line with an EC50 value of 54 +/- 32 nM, showing that transducin can functionally couple to the human delta-opioid receptors in these cells .