SITE-DIRECTED MUTAGENESIS OF THE PERIPHERAL BENZODIAZEPINE RECEPTOR -IDENTIFICATION OF AMINO-ACIDS IMPLICATED IN THE BINDING-SITE OF RO5-4864

The peripheral benzodiazepine receptor (PER) is an 18-kDa protein pres ent in the outer mitochondrial membrane. The human PER can be labeled with the benzodiazepine Ro5-4864 and with the isoquinoline carboxamide PK11195. The two ligands compete with each other in binding experimen ts, with previous results suggesting overlapping but not identical bin ding sites. To define the regions of the receptor interacting with PK1 1195 and Ro5-4864 and to address the question of the topology of the m olecule in the membrane, we generated mutant human PBRs with amino- an d carboxyl-terminal deletions and with point mutations in potentially accessible cytoplasmic regions. The mutant genes were expressed in yea st and analyzed in binding experiments using radiolabeled PK11195 and Ro5-4864. The results showed that, whereas deletions in the amino-term inal sequence had marked consequences for the binding affinity of both ligands, the final 13 amino acids at the carboxyl terminus could be d eleted with no effect on the binding of either Ro5-4864 or PK11195. Th e 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 cytoplas mic loop, are conserved in human, bovine, rat, and mouse PBRs. The rem aining residue, Val-154, which is found at the interface between the p utative fifth transmembrane region and the cytoplasm, is present in th e human, rat, and mouse sequences but is replaced by methionine in the bovine sequence. The exchange of Met-154 for valine in the bovine PER introduced a binding site for Ro5-4864, which is absent in the native PER. These four amino acids played a minor role, if any, in the bindi ng site of PK11195. We also showed that the histidines previously sugg ested to be part of the binding site of PK11195 are not directly invol ved in the interaction of the human receptor with either PK11195 or Ro 5-4864.