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

• Published in: The Journal of biological chemistry Vol. 266; no. 21; pp. 14082 - 14087
• Main Authors: A L Parola, D G Stump, D J Pepperl, K E Krueger, J W Regan, H E Laird, 2nd
• Format: Journal Article
• Language: English
• Published: Bethesda, MD American Society for Biochemistry and Molecular Biology 25.07.1991
• Subjects: Affinity Labels; Amino Acid Sequence; Animals; Base Sequence; Biological and medical sciences; Blotting, Northern; Blotting, Southern; Carrier Proteins - chemistry; Carrier Proteins - genetics; Carrier Proteins - metabolism; Cattle; Chlorocebus aethiops; Cloning, Molecular; DNA - genetics; Gabaergic and benzodiazepinic system; Gene Expression; In Vitro Techniques; Isoquinolines - metabolism; Medical sciences; Molecular Sequence Data; Neuropharmacology; Neurotransmitters. Neurotransmission. Receptors; Pharmacology. Drug treatments; Receptors, GABA-A - chemistry; Receptors, GABA-A - genetics; Receptors, GABA-A - metabolism; RNA, Messenger - genetics; Solubility; Transfection; Ligand binding; Bovine; Rat; Nucleotide sequence; Rodentia; Benzodiazepine receptor; Hydrophobicity; Photoaffinity labelling; Vertebrata; Mammalia; Transfection; Isoquinoline; Artiodactyla; Molecular cloning; Ungulata; Aminoacid sequence
• ISSN: 0021-9258; 1083-351X
• DOI: 10.1016/S0021-9258(18)92812-7

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.