Purification, molecular cloning, and expression of the mammalian sigma(1)-binding site

Sigma-ligands comprise several chemically unrelated drugs such as haloperidol, pentazocine, and ditolylguanidine, which bind to a family of low molecular mass proteins in the endoplasmic reticulum. These so-railed sigma-receptors are believed to mediate various pharmacological effects of sigma-ligands by as yet unknown mechanisms. Based on their opposite enantioselectivity for benzomorphans and different molecular masses, two subtypes are differentiated. We purified the sigma(1)-binding site as a single 30-kDa protein from guinea pig liver employing the benzomorphan (+) [H-3]pentazocine and the arylazide (-) [H-3]azidopamil as specific probes. The purified (+) [H-3]pentazocine-binding protein retained its high affinity for haloperidol, pentazocine, and ditolylguanidine. Partial amino acid sequence obtained after trypsinolysis revealed no homolog to known proteins, Radiation inactivation of the pentazocine-labeled sigma(1)-binding site yielded a molecular mass of 24 +/- 2 kDa, The corresponding cDNA was cloned using degenerate oligonucleotides 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)-binding 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 also in agreement with the known ability of sigma(1)-binding sites to interact with steroids, such as progesterone.