PURIFICATION AND CHARACTERIZATION OF THE HUMAN SR 31747A-BINDING PROTEIN - A NUCLEAR-MEMBRANE PROTEIN RELATED TO YEAST STEROL ISOMERASE

SR 31747A, defined as a sigma ligand, is a novel immunosuppressive age nt that blocks proliferation of human and mouse lymphocytes. Using a r adiolabeled chemical probe, we here purified a target of SR 31747A and called it SR 31747A-binding protein (SR-BP). Purified SR-BP retained its binding properties and migrated on SDS-polyacrylamide gel as a M-r 28,000 protein. Cloning of the cDNA encoding human SR-BP shows an ope n reading frame for a 223-amino acid protein, which is homologous to t he recently cloned sigma 1 receptor. Interestingly, the deduced amino acid sequence was found to be related to fungal C8-C7 sterol isomerase , encoded by the ERG2 gene. The ERG2 gene product has been identified recently as the molecular target of SR 31747A that mediates antiprolif erative effects of the drug in yeast. Northern blot analysis of SR-BP gene expression revealed a single transcript of 2 kilobases which was widely expressed among organs, with the highest abundance in liver and the lowest abundance in brain. Subcellular localization analysis in v arious cells, using a specific monoclonal antibody raised against SR-B P, demonstrated that this protein was associated with the nuclear enve lope. When studying the binding of SR 31747A on membranes from yeast e xpressing SR-BP, we found a pharmacological profile of sigma 1 recepto rs; binding was displaced by (+)-pentazocine, haloperidol, and (+)-SKF 10,047, with (+)-SKF 10,047 being a more potent competitor than (-)-S KF 10,047. Scatchard plot analysis revealed K-d values of 7.1 nM and 0 .15 nM for (+)-pentazocine and SR 31747A, respectively, indicating an affinity of SR-BP 50-fold higher for SR 31747A than for pentazocine. A dditionally, we showed that pentazocine, a competitive inhibitor of SR 31747A binding, also prevents the immunosuppressive effect of SR 3174 7A. Taken together, these findings strongly suggest that SR-BP represe nts the molecular target for SR 31747A in mammalian tissues, which cou ld be critical for T cell proliferation.