Cholesterol Binding Does Not Predict Activity of the Steroidogenic Acute Regulatory Protein, StAR

Abstract

Steroidogenic acute regulatory protein (StAR) stimulates adrenal and gonadal steroidogenesis by increasing the influx of cholesterol into mitochondria, where it is converted to pregnenolone to initiate steroidogenesis. StAR acts on the outer mitochondrial membrane where each molecule stimulates the mitochondrial import of several hundred molecules of cholesterol, but the precise mechanism of the action of StAR remains uncertain. StAR has a sterol-binding pocket that can accommodate one molecule of cholesterol. Direct assays show that StAR can bind cholesterol with stoichiometry approaching 1:1, and several disease-causing mutants with decreased or absent activity have correspondingly decreased cholesterol binding. We show that the StAR mutant R182L, which causes severe disease and is devoid of measurable activity in transfected cells or with isolated steroidogenic mitochondria, nevertheless, can bind as much [ C]-or NBD-cholesterol as wild-type StAR under equilibrium conditions and can transfer cholesterol between liposomes in vitro.Similarly, the artificial mutant S195A had 46.5% of the activity of wild-type StAR but bound cholesterol indistinguishably from wild-type. Competition assays showed that the rate of binding (t1 ⁄ 2on ) for R182L was only 36% of the wild-type and the rate of dissociation (t1 ⁄ 2off ) was 57% of wild-type, whereas the t1 ⁄ 2on and t1 ⁄ 2off for S195A and S195D were essentially the same for wild-type. These data indicate that cholesterol binding and transfer activities are distinct from its activity to induce steroidogenesis. StAR appears to act by other mechanisms in addition to cholesterol binding.In the adrenal and gonad, the steroidogenic acute regulatory protein (StAR) 2 facilitates the flow of cholesterol from the outer mitochondrial membrane (OMM) to the inner mitochondrial membrane (IMM) where it is converted to pregnenolone by the cholesterol side chain cleavage enzyme, P450scc, thus initiating the synthesis of steroid hormones (1, 2). Loss of StAR activity ablates adrenal and gonadal steroidogenesis, causing lifethreatening congenital lipoid adrenal hyperplasia (3). Fulllength StAR is expressed as a 37-kDa, 285-residue phosphoprotein having a mitochondrial leader sequence that is cleaved to a 30-kDa intramitochondrial form (4 -7). Nevertheless, StAR acts on the OMM, and is inactive inside the mitochondrion: deletion of 62 NH 2 -terminal residues yields a protein (N-62 StAR) that remains in the cytoplasm, yet retains full biologic activity (8), and affixing StAR to the OMM yields constituitive activity, whereas localizing it to the intramembranous space or the matrix side of the IMM ablates activity (9). Only the COOH-terminal ␣-helix (C-helix) of StAR interacts with the OMM (10). Biophysical data show that StAR undergoes an acid-induced structural change to a molten globule state (11), and the association of StAR with the OMM is stronger under acidic conditions (10,(12)(13)(14)(15).The structure of StAR has not been determined experimentally, but the structures of three clos...

A sensitive assay for ABCA1-mediated cholesterol efflux using BODIPY-cholesterol

“…It partitions into the liquid-disordered and liquid-ordered phases of model bilayer membranes and cell membranes ( 28 ), with a preference for the liquid-ordered phase in giant unilamellar vesicles ( 29 ). BODIPY-cholesterol has been used for live-cell monitoring of cholesterol traffi cking ( 30 ) and for analysis of the kinetics of transfer of self-quenched BODIPY-cholesterol from donor to acceptor vesicles in the presence of StAR protein ( 31 ).…”

Steroidogenic Activity of StAR Requires Contact with Mitochondrial VDAC1 and Phosphate Carrier Protein

“…StAR acts exclusively on the OMM prior to import (3), must undergo a structural transition to exert activity (9,32), and phosphorylation of Ser 195 augments its steroidogenic activity (4). The ability of StAR to transfer cholesterol between membranes is distinct from its ability to induce steroidogenesis (33). The proposal that StAR acts through an OMM receptor was challenged by data showing it had activity with mitochondria subjected to proteolysis (34).…”

Early steps in steroidogenesis: intracellular cholesterol trafficking

“…StAR has been reported to interact with PAP7 and be phosphorylated by cAMP-dependent protein kinase regulatory subunit 1 ␣ (PKA-RI ␣ ) ( 227,235 ), but more recent data indicate that the PKA anchor protein AKAP121, one of four AKAP proteins arising from the alternately spliced AKAP1 gene ( 238 ), recruits the type II PKA regulatory subunit ␣ not needed for its action; however, the mutant R182L, which is biologically inactive ( 201,220 ), can also transfer cholesterol to membranes in vitro ( 221 ), and other proteins are known to interact with StAR. Thus, StAR's action to promote steroidogenesis is distinct from its cholesterolbinding and cholesterol-transfer activity; cholesterol binding is necessary but not suffi cient for StAR activity.…”