Abstract
σ-1 receptors (Sig-1Rs) that bind diverse synthetic and endogenous compounds have been implicated in the pathophysiology of several human diseases such as drug addiction, depression, neurodegenerative disorders, pain-related disorders, and cancer. Sig-1Rs were identified recently as novel ligand-operated molecular chaperones. Although Sig-1Rs are predominantly expressed at endoplasmic reticulum (ER) subdomains apposing mitochondria [i.e., the mitochondria-associated ER membrane (MAM)], they dynamically change the cellular distribution, thus regulating both MAM-specific and plasma membrane proteins. However, what determines the location of Sig-1R at the MAM and how the receptor translocation is initiated is unknown. Here we report that the detergent-resistant membranes (DRMs) play an important role in anchoring Sig-1Rs to the MAM. The MAM, which is highly capable of accumulating ceramides, is enriched with both cholesterol and simple sphingolipids, thus forming Triton X-114-resistant DRMs. Sig-1Rs associate with MAM-derived DRMs but not with those from microsomes. A lipid overlay assay found that solubilized Sig-1Rs preferentially associate with simple sphingolipids such as ceramides. Disrupting DRMs by lowering cholesterol or inhibiting de novo synthesis of ceramides at the ER largely decreases Sig-1R at DRMs and causes translocation of Sig-1R from the MAM to ER cisternae. These findings suggest that the MAM, bearing cholesterol and ceramide-enriched microdomains at the ER, may use the microdomains to anchor Sig-1Rs to the location; thus, it serves to stage Sig-1R at ER-mitochondria junctions.
Footnotes
↵ The online version of this article (available at http://molpharm.aspetjournals.org) contains supplemental material.
This study was supported by the Intramural Research Program, National Institute on Drug Abuse, National Institutes of Health, Department of Health and Human Services.
Article, publication date, and citation information can be found at http://molpharm.aspetjournals.org.
doi:10.1124/mol.109.062539.
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ABBREVIATIONS:
- Sig-1R
- σ-1 receptor
- ANS
- 8-anilinonaphthalene-1-sulfonate
- BODIPY-Cer TR
- N-((4-(4,4-difluoro-5-(2-thienyl)-4-bora-3a,4a-diaza-s-indacene-3-yl)phenoxy)acetyl)sphingosine
- BSA
- bovine serum albumin
- CHO
- Chinese hamster ovary
- CHAPS
- 3-[(3-cholamidopropyl) dimethylammonio]-1-propanesulfonate
- CYP450R
- cytochrome P450 reductase
- DRM
- detergent-resistant membrane
- ER
- endoplasmic reticulum
- EYFP
- enhanced yellow fluorescent protein
- FB1
- fumonisin B1
- GFP
- green fluorescent protein
- GlcCer
- glucosylceramide
- HPTLC
- high-performance thin-layer chromatography
- IP3R3
- type-3 inositol 1,2,5-trisphosphate receptor
- MAM
- mitochondria-associated endoplasmic reticulum membrane
- MβC
- methyl-β-cyclodextrin
- NBD-Cer
- 6-((N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)amino)hexanoyl)sphingosine
- PBS
- phosphate-buffered saline
- PAGE
- polyacrylamide gel electrophoresis
- TNE
- Tris-NaCl-EDTA buffer
- TNC
- Tris-NaCl-CHAPS buffer
- Tx
- Triton X-100
- PDMP
- (±)-threo-1-phenyl-2-decanoyl-amino-3-morpholino-1-propanol hydrochloride
- MβC-Chol
- methyl-β-cyclodextrin presaturated with cholesterol.
- Received November 17, 2009.
- Accepted January 6, 2010.
- U.S. Government work not protected by U.S. copyright
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