PURIFICATION, MOLECULAR-CLONING, AND EXPRESSION OF THE MAMMALIAN SIGMA(1)-BINDING SITE

Sigma-ligands comprise several chemically unrelated drugs such as halo peridol, pentazocine, and ditolylguanidine, which bind to a family of low molecular mass proteins in the endoplasmic reticulum. These so-rai led sigma-receptors are believed to mediate various pharmacological ef fects of sigma-ligands by as yet unknown mechanisms. Based on their op posite enantioselectivity for benzomorphans and different molecular ma sses, two subtypes are differentiated. We purified the sigma(1)-bindin g site as a single 30-kDa protein from guinea pig liver employing the benzomorphan (+) [H-3]pentazocine and the arylazide (-) [H-3]azidopami l as specific probes. The purified (+) [H-3]pentazocine-binding protei n retained its high affinity for haloperidol, pentazocine, and ditolyl guanidine. Partial amino acid sequence obtained after trypsinolysis re vealed no homolog to known proteins, Radiation inactivation of the pen tazocine-labeled sigma(1)-binding site yielded a molecular mass of 24 +/- 2 kDa, The corresponding cDNA was cloned using degenerate oligonuc leotides and cDNA library screening, Its open reading frame encoded a 25.3-kDa protein with at least one putative transmembrane segment. The protein expressed in yeast cells transformed with the cDNA showed the pharmacological characteristics of the brain and liver sigma(1)-bindi ng site. The deduced amino acid sequence was structurally unrelated to known mammalian proteins but it shared homology with fungal proteins involved in sterol synthesis, Northern blots showed high densities of the sigma(1)-binding site mRNA in sterol-producing tissues, This is al so in agreement with the known ability of sigma(1)-binding sites to in teract with steroids, such as progesterone.