Molecular cloning and pharmacological characterization of the rat sigma(1)receptor

Citation
Jf. Mei et Gw. Pasternak, Molecular cloning and pharmacological characterization of the rat sigma(1)receptor, BIOCH PHARM, 62(3), 2001, pp. 349-355
Citations number
23
Language
INGLESE
art.tipo
Article
Categorie Soggetti
Pharmacology & Toxicology
Journal title
BIOCHEMICAL PHARMACOLOGY
ISSN journal
0006-2952 → ACNP
Volume
62
Issue
3
Year of publication
2001
Pages
349 - 355
Database
ISI
SICI code
0006-2952(20010801)62:3<349:MCAPCO>2.0.ZU;2-5
Abstract
In an effort to further understand the pharmacology of sigma receptors, we have cloned the rat homolog of the sigma, receptor. We isolated a cDNA clon e (rs2-2) from rat brain tissue using reverse transcriptase-polymerase chai n reaction (RT-PCR) and 5' and 3' rapid amplification of cDNA ends (RACE) t hat encoded a full-length sequence of 223 amino acids. The predicted protei n sequence of the clone has high homology with that of the murine (93.3%), guinea pig (93.7%), and human (96%) sigma, receptors. Northern analysis sho wed a major mRNA band of approximately 1.8 kb. RT-PCR revealed the presence of the mRNA in all the tissues tested, with high levels in the brain, spin al cord, liver, thymus, adrenal glands, and kidneys. When expressed in Chin ese hamster ovary (CHO) cells, the level of sigma, binding increased marked ly, and the binding profile was consistent with sigma, sites. However, meas urable levels of sigma, binding present in the cell lines before transfecti on made the interpretation of these results difficult. To ensure that the b inding reflected the transfected protein, we tagged the receptors with a he magglutinin (HA) epitope at the amino terminus and examined binding in immu noprecipitated receptors. Western analysis using an antisera against the HA epitope revealed a molecular weight of similar to 28 kDa, close to the pre dicted value. The receptor binding profile of the immunopurified receptor w as consistent with that seen with traditional sigma, binding sites. Thus, r s2-2.HA encodes a high-affinity [H-3](+)-pentazocine binding site with char acteristics of a rat sigma, receptor. (C) 2001 Elsevier Science Inc. All ri ghts reserved.