CLONING AND FUNCTIONAL-CHARACTERIZATION OF A SIGMA-RECEPTOR FROM RAT-BRAIN

We have cloned a a receptor from rat brain and established its functio nal 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 a receptor is highly homologous to that of the sigma recepto r recently cloned from guinea pig liver and a human placental cell lin e but is not related to any other known mammalian receptors. When expr essed in HeLa cells, the rat brain sigma receptor cDNA leads to a two- to threefold increase in haloperidol binding, and this cDNA-induced bi nding is sensitive to inhibition by several sigma receptor-specific li gands. Kinetic analysis using the heterologous expression system has r evealed that the rat brain a receptor interacts with haloperidol with an apparent dissociation constant (K-D) of 3 nM. Functional expression of the cloned rat brain sigma receptor in HeLa cells also leads to an increase in the binding of two other sigma ligands, namely, (+)-penta zocine and (+)-3-(3-hydroxyphenyl)-N-(1-propyl)piperidine (PPP). Pharm acological characterization of the cloned rat brain sigma receptor rev eals that it exhibits severalfold higher affinity for clorgyline than for 1,3-di(2-tolyl) guanidine, it interacts with progesterone and test osterone, and its interaction with PPP is markedly enhanced by phenyto in. In addition, transfection of MCF-7 cells, which do not express typ e 1 sigma receptor mRNA or activity, with the cloned rat brain cDNA le ads to the appearance of haloperidol-sensitive binding of (+)-pentazoc ine, a selective type 1 sigma receptor ligand. These data show that th e cloned rat brain cDNA codes for a functional type 1 sigma receptor. Northern blot analysis with poly(A)(+) RNA isolated from various rat t issues has indicated that the sigma receptor-specific transcript, 1.6 kb in size, is expressed abundantly in liver and moderately in intesti ne, kidney, brain, and lung.