Site-directed mutagenesis of the peripheral benzodiazepine receptor: identification of amino acids implicated in the binding site of Ro5- 4864

R Farges, E Joseph-Liauzun, D Shire, D Caput, G Le Fur and P Ferrara

Sanofi Recherche, Labege, France.

The peripheral benzodiazepine receptor (PBR) is an 18-kDa protein present in the outer mitochondrial membrane. The human PBR can be labeled with the benzodiazepine Ro5-4864 and with the isoquinoline carboxamide PK11195. The two ligands compete with each other in binding experiments, with previous results suggesting overlapping but not identical binding sites. To define the regions of the receptor interacting with PK11195 and Ro5-4864 and to address the question of the topology of the molecule in the membrane, we generated mutant human PBRs with amino- and carboxyl-terminal deletions and with point mutations in potentially accessible cytoplasmic regions. The mutant genes were expressed in yeast and analyzed in binding experiments using radiolabeled PK11195 and Ro5-4864. The results showed that, whereas deletions in the amino-terminal sequence had marked consequences for the binding affinity of both ligands, the final 13 amino acids at the carboxyl terminus could be deleted with no effect on the binding of either Ro5-4864 or PK11195. The site-directed mutagenesis experiments pinpointed four amino acids as participating in the binding site of Ro5- 4864. Three of these, Glu-29, Arg-32, and Lys-39, which are located in the first putative cytoplasmic loop, are conserved in human, bovine, rat, and mouse PBRs. The remaining residue, Val-154, which is found at the interface between the putative fifth transmembrane region and the cytoplasm, is present in the human, rat, and mouse sequences but is replaced by methionine in the bovine sequence. The exchange of Met-154 for valine in the bovine PBR introduced a binding site for Ro5-4864, which is absent in the native PBR. These four amino acids played a minor role, if any, in the binding site of PK11195. We also showed that the histidines previously suggested to be part of the binding site of PK11195 are not directly involved in the interaction of the human receptor with either PK11195 or Ro5-4864.

Volume 46, Issue 6, pp. 1160-1167, 12/01/1994
Copyright © 1994 by American Society for Pharmacology and Experimental Therapeutics

This article has been cited by other articles:

				A. A. Yeliseev and S. Kaplan TspO of Rhodobacter sphaeroides. A STRUCTURAL AND FUNCTIONAL MODEL FOR THE MAMMALIAN PERIPHERAL BENZODIAZEPINE RECEPTOR J. Biol. Chem., February 25, 2000; 275(8): 5657 - 5667. [Abstract] [Full Text] [PDF]

				M. Gavish, I. Bachman, R. Shoukrun, Y. Katz, L. Veenman, G. Weisinger, and A. Weizman Enigma of the Peripheral Benzodiazepine Receptor Pharmacol. Rev., December 1, 1999; 51(4): 629 - 650. [Abstract] [Full Text] [PDF]

				H. Li and V. Papadopoulos Peripheral-Type Benzodiazepine Receptor Function in Cholesterol Transport. Identification of a Putative Cholesterol Recognition/Interaction Amino Acid Sequence and Consensus Pattern Endocrinology, December 1, 1998; 139(12): 4991 - 4997. [Abstract] [Full Text] [PDF]

				E. Joseph-Liauzun, P. Delmas, D. Shire, and P. Ferrara Topological Analysis of the Peripheral Benzodiazepine Receptor in Yeast Mitochondrial Membranes Supports a Five-transmembrane Structure J. Biol. Chem., January 23, 1998; 273(4): 2146 - 2152. [Abstract] [Full Text] [PDF]

				E. Joseph-Liauzun, R. Farges, P. Delmas, P. Ferrara, and G. Loison The Mr 18,000 Subunit of the Peripheral-type Benzodiazepine Receptor Exhibits Both Benzodiazepine and Isoquinoline Carboxamide Binding Sites in the Absence of the Voltage-dependent Anion Channel or of the Adenine Nucleotide Carrier J. Biol. Chem., October 31, 1997; 272(44): 28102 - 28106. [Abstract] [Full Text] [PDF]