Abstract
The ς binding site present in the Jurkat human T lymphocyte cell line was investigated. Jurkat cell membranes were found to have a single saturable binding site for [3H]haloperidol, a ς ligand (dissociation constant, 3.9 ± 0.3 nM). The binding of [3H]haloperidol was inhibited by several ς ligands. Northern analysis and reverse transcription-polymerase chain reaction provided evidence for the expression of the recently cloned type 1 ς-receptor (ς-R1) in Jurkat cells. The ς-R1 cDNA cloned from these cells was functional in heterologous expression systems. When expressed in mammalian cells, the cDNA-induced binding was saturable with dissociation constants of 1.9 ± 0.3 nM for [3H]haloperidol and 12 ± 2 nM for (+)-pentazocine. The binding of [3H]progesterone, a putative endogenous ligand to ς-R1, to the Jurkat cell ς-receptor could be directly demonstrated by using heterologously expressed ς-R1 cDNA. The binding of [3H]progesterone was saturable, with a dissociation constant of 88 ± 7 nM. Progesterone and haloperidol interacted with the receptor competitively. Reverse transcription-polymerase chain reaction also produced evidence for the existence of an alternatively spliced ς-R1 variant in Jurkat cells. This splice variant was found to be nonfunctional in ligand binding assays. This constitutes the first report on the molecular characterization of the ς-receptor in immune cells.
Footnotes
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Send reprint requests to: Dr. Vadivel Ganapathy, Department of Biochemistry and Molecular Biology, Medical College of Georgia, Augusta, Georgia. E-mail: vganapat{at}mail.mcg.edu
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↵1 This work was supported by National Institutes of Health Grants DA 10045 (V.G.) and GM 54122 (M.E.G.).
- Abbreviations:
- (+)-PPP
- (+)-1-propyl-3-(3-hydroxyphenyl)piperidine
- DTG
- 1,3-di(2-tolyl)guanidine
- (−)-PPAP
- (−)-N-(3-phenyl-1-propyl)-1-phenyl-2-aminopropane
- ς-R1
- type 1 ς-receptor
- RT-PCR
- reverse transcription-polymerase chain reaction
- ς-R1A
- alternatively spliced ς-R1
- hς-R1
- human ς-R1
- Received May 1, 1998.
- Accepted November 23, 1998.
- The American Society for Pharmacology and Experimental Therapeutics
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