Vol. 292, Issue 1, 209-214, January 2000

Coupling of Human -Opioid Receptor to Retinal Rod Transducin in Chinese Hamster Ovary Cells¹

Eva V. Varga, Dagmar Stropova, Tae Kim, Man Wang, William R. Roeske and Henry I. Yamamura

Departments of Pharmacology, Biochemistry, and Psychiatry, and The Program for Neuroscience, College of Medicine, The University of Arizona Health Sciences Center, Tucson, Arizona

Reverse transcription-polymerase chain reaction was used to identify the pertussis toxin (Ptx)-sensitive G protein -subunit pool in Chinese hamster ovary (CHO) and mouse fibroblast (B82) cells. We detected the presence of mRNA for G_i2, G_i3, and G_o in both cell lines. G_i1 and G_z mRNAs were not detected. We also found a homolog of the retinal rod transducin (G_t1) in CHO, and the mouse cone transducin (G_t2) in B82 cells. The presence of the transducin -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 -opioid receptor (hDOR/CHO). (+)-4-[(R)--((2S,2R)-4-allyl-2,5-dimethyl-1-piperazinyl)-3-methoxybenzyl]-N,N-diethylbenzamide, a selective -opioid receptor agonist, stimulated guanosine-5'-O-(3-[³⁵S]thio)triphosphate binding by 293 ± 36% after Ptx pretreatment in the mutant cell line with an EC₅₀ value of 54 ± 32 nM, showing that transducin can functionally couple to the human -opioid receptors in these cells.