Cloning and expression of a pharmacologically unique bovine peripheral-type benzodiazepine receptor isoquinoline binding protein.

Department of Pharmacology and Toxicology, College of Pharmacy, University of Arizona, Tucson 85721.
Journal of Biological Chemistry (Impact Factor: 4.65). 08/1991; 266(21):14082-7.
Source: PubMed

ABSTRACT High affinity binding of isoquinolines, such as PK 11195, is a conserved feature of peripheral-type benzodiazepine receptors (PBR) across species. However, species differences in PBR ligand binding have been described based on the affinity for N1-alkyl-1,4-benzodiazepines, such as Ro5-4864. Ro5-4864 binds with high affinity to the rat receptor but has low affinity for the bovine PBR. Photolabeling with an isoquinoline ligand, [3H]PK 14105, identifies a 17-kDa protein, the PBR isoquinoline binding protein (PBR/IBP), in both species. To further elucidate the role of the PBR/IBP in determining PBR benzodiazepine and isoquinoline binding characteristics, the bovine PBR/IBP was cloned and expressed. Using a cDNA encoding a rat PBR/IBP to screen a fetal bovine adrenal cDNA library, a bovine cDNA encoding a polypeptide of 169 residues was cloned. The bovine and rat PBR/IBPs had similar hydropathy profiles exhibiting five potential transmembrane domains. Transfecting the cloned bovine PBR/IBP cDNA into COS-7 cells resulted in an 11-fold increase in the density of high affinity [3H]PK 11195 binding sites which had only low affinity for Ro5-4864. Expression of the bovine PBR/IBP yields a receptor which is pharmacologically distinct from both endogenous COS-7 PBR and the rat PBR based on the affinity for several N1-alkyl-1,4-benzodiazepine ligands. These results suggest the PBR/IBP is the minimal functional component required for PBR ligand binding characteristics and the different protein sequences account for the species differences in PBR benzodiazepine ligand binding.

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    ABSTRACT: The translocator protein (18 kDa; TSPO), previously known as peripheral-type benzodiazepine receptor, is a high-affinity cholesterol- and drug-binding mitochondrial protein involved in various cell functions including steroidogenesis, apoptosis, and proliferation. TSPO is highly expressed in secretory and glandular tissues, especially in steroidogenic cells, and its expression is altered in certain pathological conditions such as cancer and neurological diseases. In this study, we characterized the regulatory elements present in the region of the TPSO promoter extending from 515 to 805 bp upstream of the transcription start site, an area previously identified as being important for transcription. Promoter fragments extending 2.7 kb and 805 bp upstream of the transcription start site were able to direct enhanced green fluorescent protein expression to Leydig cells of the testis, theca cells of the ovary, and cells of the adrenal cortex in transgenic animals. This expression pattern perfectly mimicked endogenous TSPO expression. Functional characterization of the 515-805 bp region revealed the presence of one specificity protein 1/specificity protein 3 (Sp1/Sp3) and two v-ets erythroblastosis virus E26 oncogene homologue (Ets) binding sites that are important for transcriptional activity in both MA-10 mouse Leydig tumor cells and NIH/3T3 whole mouse embryo fibroblasts. GA-binding protein alpha (GABPalpha), a member of the Ets family of transcription factors, was found to be associated with the endogenous TSPO promoter. We conclude that Sp1/Sp3 and members of the Ets family of transcription factors bind to specific binding sites in the TSPO promoter to drive basal TSPO gene transcription.
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