Cholesterol binding at the cholesterol recognition/ interaction amino acid consensus (CRAC) of the peripheral-type benzodiazepine receptor and inhibition of steroidogenesis by an HIV TAT-CRAC peptide

We previously defined a cholesterol recognition/interaction amino acid consensus (CRAC; ATVLNYYVWRDNS) in the carboxyl terminus of the peripheral-type benzodiazepine receptor (PBR), an outer mitochondrial membrane protein involved in the regulation of cholesterol transport into the mitochondria, the rate-determining step in steroid biosynthesis. We examined (i) the PBR–cholesterol interaction by UV crosslinking of the C17 side-chain containing progestin, promegestone, and (ii) the role of the CRAC domain of PBR in Leydig cell steroidogenesis by using a transducible peptide composed of the TAT domain of HIV and the CRAC domain of PBR. [(3)H]Promegestone photoincorporated into recombinant PBR, and this labeling was displaced by cholesterol. [(3)H]Promegestone also photoincorporated into the TAT-CRAC peptide. [(3)H]Promegestone crosslinking to TAT-CRAC could be displaced by cholesterol and promegestone, with IC50 values of 1 and 200 μM, respectively. TAT-CRAC efficiently transduced into MA-10 Leydig cells and inhibited the hCG- and cAMP-stimulated steroid production in a dose-dependent manner. TAT-CRAC did not affect the hCG-induced cAMP synthesis and the 22R-hydroxycholesterol-supported steroidogenesis. Mutated TAT-CRAC lost its ability to bind [(3)H]promegestone and to inhibit the hCG-stimulated steroidogenesis. These results show that TAT-CRAC binds cholesterol and competes for cholesterol interaction with endogenous PBR, suggesting that the cytosolic carboxyl-terminal domain of PBR is responsible for taking up and bringing steroidogenic cholesterol into the mitochondria.