Cloning and expression of 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: 4.57). 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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Available from: Donald G Stump, Sep 28, 2015
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    • "Rat, human, mouse and bovine TSPO were cloned and expressed in various cells that contained low TSPO levels (Garnier et al., 1994; Parola et al., 1991; Riond et al., 1991b; Sprengel et al., 1989). "
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    ABSTRACT: Steroidogenesis begins with cholesterol transfer into mitochondria through the transduceosome, a complex composed of cytosolic proteins that include steroidogenesis acute regulatory protein (STAR), 14-3-3 adaptor proteins, and the outer mitochondrial membrane proteins Translocator Protein (TSPO) and Voltage-Dependent Anion Channel (VDAC). TSPO is a drug- and cholesterol- binding protein found at particularly high levels in steroid synthesizing cells. Its aberrant expression has been linked to cancer, neurodegeneration, neuropsychiatric disorders and primary hypogonadism. Brain steroids serve as local regulators of neural development and excitability. Reduced levels of these steroids have been linked to depression, anxiety and neurodegeneration. Reduced serum testosterone is common among subfertile young men and aging men, and is associated with depression, metabolic syndrome and reduced sexual function. Although testosterone-replacement therapy is available, there are undesired side-effects. TSPO drug ligands have been proposed as therapeutic agents to regulate steroid levels in the brain and testis. Copyright © 2015 Elsevier Ireland Ltd. All rights reserved.
    Molecular and Cellular Endocrinology 03/2015; 408. DOI:10.1016/j.mce.2015.03.014 · 4.41 Impact Factor
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    • "It is highly hydrophobic and rich in tryptophan (Casellas et al., 2002). The cDNA encoding TSPO has been cloned from various species such as rodents, bovines and humans (Chang et al., 1992; Garnier et al., 1994; Parola et al., 1991; Riond et al., 1991; Sprengel et al., 1989) and there is an 80% sequence homology amongst these species (Casellas et al., 2002). The location of the TSPO gene is in the q13.3 region of the long arm of human chromosome 22 (Riond et al., 1991). "
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    ABSTRACT: For over 15 years, the peripheral benzodiazepine receptor (PBR), recently named translocator protein 18 kDa (TSPO) has been studied as a biomarker of reactive gliosis and inflammation associated with a variety of neuropathological conditions. Early studies documented that in the brain parenchyma, TSPO is exclusively localized in glial cells. Under normal physiological conditions, TSPO levels are low in the brain neuropil but they markedly increase at sites of brain injury and inflammation making it uniquely suited for assessing active gliosis. This research has generated significant efforts from multiple research groups throughout the world to apply TSPO as a marker of "active" brain pathology using in vivo imaging modalities such as Positron Emission Tomography (PET) in experimental animals and humans. Further, in the last few years, there has been an increased interest in understanding the molecular and cellular function(s) of TSPO in glial cells. The latest evidence suggests that TSPO may not only serve as a biomarker of active brain disease but also the use of TSPO-specific ligands may have therapeutic implications in brain injury and repair. This review presents an overview of the history and function of TSPO focusing on studies related to its use as a sensor of active brain disease in experimental animals and in human studies.
    Pharmacology [?] Therapeutics 05/2008; 118(1):1-17. DOI:10.1016/j.pharmthera.2007.12.004 · 9.72 Impact Factor
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    • "While it is highly hydrophobic and rich in tryptophan, it also has a positive charge with a pI of 9.6. The cDNA encoding the PBR has been cloned from various species, including rodents (Sprengel et al., 1989; Garnier et al., 1994), cows (Parola et al., 1991) and humans (Riond et al., 1991), and shows an 80% sequence homology. Three-dimensional modeling reveals a structure with five ␣-helices spanning the membrane lipid bilayer (Bernassau et al., 1993; Joseph-Liauzun et al., 1998). "
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    ABSTRACT: For over 20 years, numerous investigations have focused on elucidating the function of the peripheral benzodiazepine receptor (PBR). This relatively small protein (18kDa) arouses great interest because of its association with numerous biological functions, including the regulation of cellular proliferation, immunomodulation, porphyrin transport and heme biosynthesis, anion transport, regulation of steroidogenesis and apoptosis. Although the receptor was first identified as a binding site for the benzodiazepine, diazepam, in peripheral organ systems, the PBR was subsequently found to be distinct from the central benzodiazepine receptor (CBR) in terms of its pharmacological profile, structure, subcellular localization, tissue distribution and physiological functions. The PBR is widely expressed throughout the body, with high densities found in steroid-producing tissues. In contrast, its expression in the CNS is restricted to ependymal cells and glia. The benzodiazepine Ro5-4864 and the isoquinoline carboxamide PK11195 exhibit nanomolar affinity for the PBR, and are the archtypic pharmacological tools for characterizing the receptor and its function. Primary among these functions are its regulation of steroidogenesis and apoptosis, which reflect its mitochondrial localization and involvement in oxidative processes. This review will evaluate the basic pharmacology and molecular biology of the PBR, and highlight its role in regulating mitochondrial function, the mitochondrial transmembrane potential and its sensitivity to reactive oxygen species (ROS), and neurosteroid synthesis, processes relevant to the pathogenesis of a number of neurological and neuropsychiatric disorders.
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