Cloning and Functional Characterization of a σ Receptor from Rat Brain

Abstract: We have cloned a σ receptor from rat brain and established its functional identity using a heterologous expression system. The cloned cDNA (1,582 bp long) codes for a protein of 223 amino acids that possesses a single putative transmembrane domain. The amino acid sequence of the rat brain σ receptor is highly homologous to that of the σ receptor recently cloned from guinea pig liver and a human placental cell line but is not related to any other known mammalian receptors. When expressed in HeLa cells, the rat brain σ receptor cDNA leads to a two‐ to threefold increase in haloperidol binding, and this cDNA‐induced binding is sensitive to inhibition by several σ receptor‐specific ligands. Kinetic analysis using the heterologous expression system has revealed that the rat brain σ receptor interacts with haloperidol with an apparent dissociation constant (KD) of 3 nM. Functional expression of the cloned rat brain σ receptor in HeLa cells also leads to an increase in the binding of two other σ ligands, namely, (+)‐pentazocine and (+)‐3‐(3‐hydroxyphenyl)‐N‐(1‐propyl)piperidine (PPP). Pharmacological characterization of the cloned rat brain σ receptor reveals that it exhibits severalfold higher affinity for clorgyline than for 1,3‐di(2‐tolyl)guanidine, it interacts with progesterone and testosterone, and its interaction with PPP is markedly enhanced by phenytoin. In addition, transfection of MCF‐7 cells, which do not express type 1 σ receptor mRNA or activity, with the cloned rat brain cDNA leads to the appearance of haloperidol‐sensitive binding of (+)‐pentazocine, a selective type 1 σ receptor ligand. These data show that the cloned rat brain cDNA codes for a functional type 1 σ receptor. Northern blot analysis with poly(A)+ RNA isolated from various rat tissues has indicated that the σ receptor‐specific transcript, 1.6 kb in size, is expressed abundantly in liver and moderately in intestine, kidney, brain, and lung.