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 microM, 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.
Li
H
H
Division of Hormone Research, Departments of Cell Biology and Pharmacology and Neuroscience, Georgetown University School of Medicine, Washington, DC. 20007, USA.
Yao
Z
Z
Degenhardt
B
B
Teper
G
G
Papadopoulos
V
V
D000595:Amino Acid Sequence; D000818:Animals; D001665:Binding Sites; D002460:Cell Line; D002784:Cholesterol / Q000378:metabolism*; D003001:Cloning, Molecular; D016384:Consensus Sequence; D015696:Gene Products, tat / Q000737:chemistry* / Q000378:metabolism*; D006678:HIV / Q000378:metabolism; D007700:Kinetics; D007985:Leydig Cells / Q000502:physiology*; D008297:Male; D051379:Mice; D008928:Mitochondria / Q000378:metabolism; D008969:Molecular Sequence Data; D011374:Progesterone / Q000378:metabolism*; D011397:Promegestone / Q000493:pharmacokinetics; D011963:Receptors, GABA-A / Q000737:chemistry* / Q000378:metabolism*; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D014316:Tritium; D054322:tat Gene Products, Human Immunodeficiency Virus
D015696:Gene Products, tat; D011963:Receptors, GABA-A; D011994:Recombinant Proteins; D054322:tat Gene Products, Human Immunodeficiency Virus; D014316:Tritium; D011374:Progesterone; D002784:Cholesterol; D011397:Promegestone