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

• Published in: Proceedings of the National Academy of Sciences - PNAS Vol. 98; no. 3; pp. 1267 - 1272
• Main Authors: Li, Hua, Yao, Zhi-xing, Degenhardt, Babett, Teper, Gary, Papadopoulos, Vassilios
• Format: Journal Article
• Language: English
• Published: United States National Academy of Sciences 30.01.2001; National Acad Sciences; The National Academy of Sciences
• Subjects: Amino Acid Sequence; Animals; Binding Sites; Biological Sciences; Cell Line; Cholesterol - metabolism; Cloning, Molecular; Consensus Sequence; CRAC peptide; Gene Products, tat - chemistry; Gene Products, tat - metabolism; HIV - metabolism; Human immunodeficiency virus; Kinetics; Leydig Cells - physiology; Male; Mice; Mitochondria - metabolism; Molecular Sequence Data; PBR protein; Progesterone - metabolism; promegestone; Promegestone - pharmacokinetics; Receptors, GABA-A - chemistry; Receptors, GABA-A - metabolism; Recombinant Proteins - chemistry; Recombinant Proteins - metabolism; tat Gene Products, Human Immunodeficiency Virus; Tritium
• ISSN: 0027-8424; 1091-6490
• DOI: 10.1073/pnas.031461598

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. [3H]Promegestone photoincorporated into recombinant PBR, and this labeling was displaced by cholesterol. [3H]Promegestone also photoincorporated into the TAT-CRAC peptide. [3H]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 [3H]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.