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 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.
Varga
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EV
Departments of Pharmacology, Biochemistry, and Psychiatry, and The Program for Neuroscience, College of Medicine, The University of Arizona Health Sciences Center, Tucson, AZ 85724, USA.
Stropova
D
D
Kim
T
T
Wang
M
M
Roeske
W R
WR
Yamamura
H I
HI
D000818:Animals; D000906:Antibodies / Q000276:immunology; D001483:Base Sequence; D016466:CHO Cells; D006224:Cricetinae; D004305:Dose-Response Relationship, Drug; D005347:Fibroblasts / Q000378:metabolism*; D019204:GTP-Binding Proteins / Q000378:metabolism*; D016244:Guanosine 5'-O-(3-Thiotriphosphate) / Q000378:metabolism; D006801:Humans; D051379:Mice; D008969:Molecular Sequence Data; D009154:Mutation; D009292:Narcotic Antagonists / Q000494:pharmacology; D037342:Pertussis Toxin; D011233:Precipitin Tests; D012333:RNA, Messenger / Q000145:classification / Q000302:isolation & purification; D017465:Receptors, Opioid, delta / Q000378:metabolism*; D017948:Retinal Rod Photoreceptor Cells / Q000378:metabolism*; D020133:Reverse Transcriptase Polymerase Chain Reaction; D015293:Transducin / Q000378:metabolism*; D014161:Transduction, Genetic; D014162:Transfection; D010566:Virulence Factors, Bordetella / Q000494:pharmacology*
D000906:Antibodies; D009292:Narcotic Antagonists; D012333:RNA, Messenger; D017465:Receptors, Opioid, delta; D010566:Virulence Factors, Bordetella; D016244:Guanosine 5'-O-(3-Thiotriphosphate); D037342:Pertussis Toxin; D019204:GTP-Binding Proteins; D015293:Transducin