Metabolism of exogenous cholesterol by rat adrenal mitochondria is stimulated equally by physiological levels of free Ca2+ and by GTP

Kowluru, Renu A.; Yamazaki, Takeshi; McNamara, Brian C.; Jefcoate, Colin R.

doi:10.1016/0303-7207(94)03441-u

Cited by 30 publications

(15 citation statements)

References 35 publications

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“…One interesting possibility is that StAR participates in a process involving GTP hydrolysis. GTP hydrolysis is known to be important for substrate delivery to cytochrome P450 scc (25), and GTPases participate in membrane trafficking and membrane fusion events (29,30). Remarkably, there is homology between StAR and members of the RhoGAP family of GTPase activating proteins, although the homology does not encompass the catalytic domain.…”

Section: Discussionmentioning

confidence: 99%

“…Are Biologically Active-Before producing recombinant protein for in vitro studies, we documented that the His-tag did not affect the activity of StAR in COS-1 cells transfected with an expression plasmid for the human cholesterol side-chain cleavage system (1,20,25). Both the NH 2 -and the C-His-tag N-62 StAR proteins stimulated pregnenolone secretion by COS-1 cells to the same extent as wildtype StAR (Fig.…”

Section: His-tag Star Proteinsmentioning

confidence: 91%

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The Mechanism of Action of Steroidogenic Acute Regulatory Protein (StAR)

Arakane

Kallen

Watari

et al. 1998

Journal of Biological Chemistry

177

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Steroidogenic acute regulatory protein (StAR) plays an essential role in steroidogenesis, facilitating delivery of cholesterol to cytochrome P450 scc on the inner mitochondrial membrane. StAR is synthesized in the cytoplasm and is subsequently imported by mitochondria and processed to a mature form by cleavage of the NH 2 -terminal mitochondrial targeting sequence. To explore the mechanism of StAR action, we produced 6-histidinetagged N-62 StAR (His-tag StAR) constructs lacking the NH 2 -terminal 62 amino acids that encode the mitochondrial targeting sequence and examined their steroidogenic activity in intact cells and on isolated mitochondria. His-tag StAR proteins stimulated pregnenolone synthesis to the same extent as wild-type StAR when expressed in COS-1 cells transfected with the cholesterol side-chain cleavage system. His-tag StAR was diffusely distributed in the cytoplasm of transfected COS-1 cells whereas wild-type StAR was localized to mitochondria. There was no evidence at the light or electron microscope levels for selective localization of His-tag StAR protein to mitochondrial membranes. In vitro import assays demonstrated that wild-type StAR preprotein was imported and processed to mature protein that was protected from subsequent trypsin treatment. In contrast, His-tag StAR was not imported and protein associated with mitochondria was sensitive to trypsin. Using metabolically labeled COS-1 cells transfected with wild-type or His-tag StAR constructs, we confirmed that wild-type StAR preprotein was imported and processed by mitochondria, whereas His-tag StAR remained largely cytosolic and unprocessed. To determine whether cytosolic factors are required for StAR action, we developed an assay system using washed mitochondria isolated from bovine corpora lutea and purified recombinant His-tag StAR proteins expressed in Escherichia coli. Recombinant His-tag StAR stimulated pregnenolone production in a dose-and time-dependent manner, functioning at nanomolar concentrations. A point mutant of StAR (A218V) that causes lipoid congenital adrenal hyperplasia was incorporated into the His-tag protein. This mutant was steroidogenically inactive in COS-1 cells and on isolated mitochondria. Our observations conclusively document that StAR acts on the outside of mitochondria, independent of mitochondrial import, and in the absence of cytosol. The ability to produce bioactive recombinant StAR protein paves the way for refined structural studies of StAR and StAR mutants.

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Section: Discussionmentioning

confidence: 99%

Section: His-tag Star Proteinsmentioning

confidence: 91%

The Mechanism of Action of Steroidogenic Acute Regulatory Protein (StAR)

Arakane

Kallen

Watari

et al. 1998

Journal of Biological Chemistry

177

View full text Add to dashboard Cite

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“…497,521). Ca 2ϩ also activates the transport of cholesterol into mitochondria and its subsequent side-chain cleavage (223,299). Elevation of [Ca 2ϩ ] c by either ANG II or via cell permeation results in increased cholesterol content in the inner mitochondrial membrane, and especially in the contact sites between the outer and inner mitochondrial membrane.…”

Section: A Cholesterol Transportmentioning

confidence: 99%

Control of Aldosterone Secretion: A Model for Convergence in Cellular Signaling Pathways

Spät

Hunyady

2004

Physiological Reviews

416

351

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Aldosterone secretion by glomerulosa cells is stimulated by angiotensin II (ANG II), extracellular K(+), corticotrophin, and several paracrine factors. Electrophysiological, fluorimetric, and molecular biological techniques have significantly clarified the molecular action of these stimuli. The steroidogenic effect of corticotrophin is mediated by adenylyl cyclase, whereas potassium activates voltage-operated Ca(2+) channels. ANG II, bound to AT(1) receptors, acts through the inositol 1,4,5-trisphosphate (IP(3))-Ca(2+)/calmodulin system. All three types of IP(3) receptors are coexpressed, rendering a complex control of Ca(2+) release possible. Ca(2+) release is followed by both capacitative and voltage-activated Ca(2+) influx. ANG II inhibits the background K(+) channel TASK and Na(+)-K(+)-ATPase, and the ensuing depolarization activates T-type (Ca(v)3.2) Ca(2+) channels. Activation of protein kinase C by diacylglycerol (DAG) inhibits aldosterone production, whereas the arachidonate released from DAG in ANG II-stimulated cells is converted by lipoxygenase to 12-hydroxyeicosatetraenoic acid, which may also induce Ca(2+) signaling. Feedback effects and cross-talk of signal-transducing pathways sensitize glomerulosa cells to low-intensity stimuli, such as physiological elevations of [K(+)] (< or =1 mM), ANG II, and ACTH. Ca(2+) signaling is also modified by cell swelling, as well as receptor desensitization, resensitization, and downregulation. Long-term regulation of glomerulosa cells involves cell growth and proliferation and induction of steroidogenic enzymes. Ca(2+), receptor, and nonreceptor tyrosine kinases and mitogen-activated kinases participate in these processes. Ca(2+)- and cAMP-dependent phosphorylation induce the transfer of the steroid precursor cholesterol from the cytoplasm to the inner mitochondrial membrane. Ca(2+) signaling, transferred into the mitochondria, stimulates the reduction of pyridine nucleotides.

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“…The IMM metalloproteases (MMP) that cleave the StAR NTD appear likely to interact with OMM VDAC1 each as participants in the mitochondrial permeability core complex (Shanmughapriya et al, 2015). This pore complex mediates the Ca inhibition of mitochondrial cholesterol metabolism, which is blocked by cyclosporine, a drug that binds to cyclophilin, a component of the pore complex (Kowluru et al, 1995). NTD-modulatory activity beyond the mitochondrial import function may involve the StAR 30–62 sequences, which are downstream of the first conserved MMP cleavage site.…”

Section: What Is the Role Of The Star Ntd?mentioning

confidence: 99%

“…GTP enhances mitochondrial uptake of cholesterol in partnership with Ca (Kowluru et al, 1995). GTPases such as mitochondrial fusion-associated protein 2 (MFN2) and opacity associated (OPA) proteins, which mediates the continuous dynamics of inter-mitochondrial fusion, may also play pivotal roles in the cAMP-stimulated cholesterol transfer (Duarte et al, 2012).…”

Section: What Is the Role Of The Star Ntd?mentioning

confidence: 99%

A single cell level measurement of StAR expression and activity in adrenal cells

Lee

Yamazaki

Dong

et al. 2017

Molecular and Cellular Endocrinology

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The Steroidogenic acute regulatory protein (StAR) directs mitochondrial cholesterol uptake through a C-terminal cholesterol binding domain (CBD) and a 62 amino acid N-terminal regulatory domain (NTD) that contains an import sequence and conserved sites for inner membrane metalloproteases. Deletion of the NTD prevents mitochondrial import while maintaining steroidogenesis but with compromised cholesterol homeostasis. The rapid StAR-mediated cholesterol transfer in adrenal cells depends on concerted mRNA translation, p37 StAR phosphorylation and controlled NTD cleavage. The NTD controls this process with two cAMP-inducible modulators of, respectively, transcription and translation SIK1 and TIS11b/Znf36l1. High-resolution fluorescence in situ hybridization (HR-FISH) of StAR RNA resolves slow RNA splicing at the gene loci in cAMP-induced Y-1 cells and transfer of individual 3.5 kb mRNA molecules to mitochondria. StAR transcription depends on the CREB coactivator CRTC2 and PKA inhibition of the highly inducible suppressor kinase SIK1 and a basal counterpart SIK2. PKA-inducible TIS11b/Znf36l1 binds specifically to highly conserved elements in exon 7 thereby suppressing formation of mRNA and subsequent translation. Co-expression of SIK1, Znf36l1 with 3.5 kb StAR mRNA may limit responses to pulsatile signaling by ACTH while regulating the transition to more prolonged stress

show abstract

Metabolism of exogenous cholesterol by rat adrenal mitochondria is stimulated equally by physiological levels of free Ca2+ and by GTP

Cited by 30 publications

References 35 publications

The Mechanism of Action of Steroidogenic Acute Regulatory Protein (StAR)

The Mechanism of Action of Steroidogenic Acute Regulatory Protein (StAR)

Control of Aldosterone Secretion: A Model for Convergence in Cellular Signaling Pathways

A single cell level measurement of StAR expression and activity in adrenal cells

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