Abstract
Reverse transcription-polymerase chain reaction was used to identify the pertussis toxin (Ptx)-sensitive G protein α-subunit pool in Chinese hamster ovary (CHO) and mouse fibroblast (B82) cells. We detected the presence of mRNA for Giα2, Giα3, and Goα in both cell lines. Giα1 and Gαz mRNAs were not detected. We also found a homolog of the retinal rod transducin (Gtα1) in CHO, and the mouse cone transducin (Gtα2) in B82 cells. The presence of the transducin α-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 δ-opioid receptor (hDOR/CHO). (+)-4-[(αR)-α-((2S,2R)-4-allyl-2,5-dimethyl-1-piperazinyl)-3-methoxybenzyl]-N,N-diethylbenzamide, a selective δ-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 δ-opioid receptors in these cells.
Footnotes
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Send reprint requests to: Henry I. Yamamura, Ph.D., Department of Pharmacology, College of Medicine, The University of Arizona Health Sciences Center, Tucson, AZ 85724. E-mail:hiy{at}u.arizona.edu
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↵1 This study was supported by grants from the National Institute on Drug Abuse and the Arizona Disease Control Research Commission, and the Undergraduate Biology Research Program.
- Abbreviations:
- CHO
- Chinese hamster ovary
- B82 cells
- murine fibroblast cells
- [35S]GTPγS
- guanosine-5′-O-(3-[35S]thio)triphosphate
- Ptx
- pertussis toxin
- RT-PCR
- reverse transcription-polymerase chain reaction
- hDOR
- human δ-opioid receptor
- PAGE
- polyacrylamide gel electrophoresis
- SNC 80
- (+)-4-[(αR)-α-((2S,2R)-4-allyl-2,5-dimethyl-1-piperazinyl)-3-methoxybenzyl]-N,N-diethylbenzamide
- NTI
- naltrindole
- IMDM
- Iscove's modified Dulbecco's medium
- Received August 2, 1999.
- Accepted September 15, 1999.
- The American Society for Pharmacology and Experimental Therapeutics
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