Detergent-Resistant Microdomains Determine the Localization of -1 Receptors to the Endoplasmic Reticulum-Mitochondria Junction

-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. The receptor is believed to serve as a novel target for therapeutic drugs. Although originally proposed as a subtype of opioid receptors, a series of recent studies has confirmed that the receptor is a nonopioid intracellular protein involved in a variety of functions of the brain and other organs (Snyder and Largent, 1989; Hayashi and Su, 2008). There are two known subtypes: -1 and -2 (Bowen, 2000). The -1 receptors (Sig1Rs) were cloned, and the structure and molecular biological functions have just begun to be unveiled (Hanner et al., 1996; Hayashi and Su, 2008). Sig-1Rs have been implicated in the pathophysiology of certain human diseases such as psychiatric disorders, neurodegenerative diseases, pain-related disorders, and cancer (Maurice et al., 2002; Matsumoto et al., 2003; Mei and Pasternak, 2007; Palmer et al., 2007). Recent evidence indicates potential neuroprotective and antidepressant-like actions for -1 agonists (Maurice et al., 2002). In contrast, -1 antagonists have been demonstrated to possess analgetic, anticancer, and anti–drug-abuse actions (Matsumoto et al., 2003; Mei and Pasternak, 2007; Palmer et al., 2007). The Sig-1Rs are the integral membrane proteins with two transmembrane domains and a long C terminus in the lumen 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. □S The online version of this article (available at http://molpharm. aspetjournals.org) contains supplemental material. ABBREVIATIONS: Sig-1R, -1 receptor; ANS, 8-anilinonaphthalene-1-sulfonate; BODIPY-Cer TR, N-((4-(4,4-difluoro-5-(2-thienyl)-4-bora-3a,4adiaza-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; M C, methyl-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; M C-Chol, methyl-cyclodextrin presaturated with cholesterol. 0026-895X/10/7704-517–528 MOLECULAR PHARMACOLOGY Vol. 77, No. 4 U.S. Government work not protected by U.S. copyright 62539/3569035 Mol Pharmacol 77:517–528, 2010 Printed in U.S.A. 517 http://molpharm.aspetjournals.org/content/suppl/2010/01/06/mol.109.062539.DC1 Supplemental material to this article can be found at: at A PE T Jornals on Jne 5, 2017 m oharm .aspeurnals.org D ow nladed from of the ER (Hayashi and Su, 2007). A characteristic of Sig-1Rs is their moderate to high affinity for diverse synthetic and endogenous compounds including benzomorphans, sterols, sphingosine, and trace amines (Su et al., 1988; Bowen, 2000; Fontanilla et al., 2009; Ramachandran et al., 2009). Sig-1Rs were recently identified as novel ligand-operated molecular chaperones expressed predominantly at the ER subdomainapposing mitochondria (the mitochondria-associated ER membrane, MAM) (Hayashi and Su, 2007). Thus, Sig-1Rs are positioned to regulate the cross-talk of signals between ER and mitochondria. For example, Sig-1R chaperones stabilize the conformation of MAM-residing type-3 inositol 1,4,5trisphosphate receptors (IP3R3), thus regulating the Ca influx from the ER to mitochondria (Hayashi et al., 2009). On the other hand, a number of studies demonstrate that Sig1Rs also regulate cellular events at plasma membranes, such as neurotransmitter release, neurotrophic factor signaling, and opening of voltage-gated ion channels (Aydar et al., 2002; Nuwayhid and Werling, 2003; Hayashi and Su, 2008; Herrera et al., 2008). The tonic inhibition of the potassium Kv1.4 channel by Sig-1Rs involves the physical association between Sig-1R and Kv1.4 channels (Aydar et al., 2002). Although Sig-1Rs are predominantly expressed at the MAM, the proteins are highly dynamic under certain conditions, such as glucose deprivation and ligand treatment, which promote translocation of Sig-1R from the MAM to loci close to plasma membranes (e.g., plasmalemma) (Hayashi and Su, 2003a, 2007). The phenomenon is particularly important and relevant for understanding the Sig-1R’s regulatory role on both ER and plasma membrane proteins. The dynamic shift of the receptor distribution may be to switch the action site of Sig-1R from the MAM to the plasma membrane/plasmalemma (Hayashi and Su, 2008). What determines the location of Sig-1R at the MAM and how the receptor translocation is initiated/restricted, however, remain largely unknown. Lipid rafts composed of cholesterol and sphingolipids are small-membrane domains that facilitate specificity and efficacy of signaling events by positioning involved molecules to the specific loci of the membrane (Simons and Toomre, 2000; Jacobson et al., 2007). Lipid rafts are resistant to extraction by nonionic detergents such as Triton X-100 (Tx) at 4°C; therefore, lipid rafts can be purified as detergent-resistant membranes (DRMs) by centrifugation (Simons and Toomre, 2000). We reported previously that Sig-1Rs are associated with DRM at cholesterol-rich ER subdomains (now identified as the MAM) in different types of cells (Hayashi and Su, 2003b, 2004) and that Sig-1R ligands, which can cause translocation of Sig-1R from MAM to the bulk of ER membranes, also down-regulate Sig-1R at DRMs (Hayashi and Su, 2003a,b). Because of DRMs serving as platforms to cluster signaling molecules, these findings raise a potential hypothesis that ER-specific DRMs may play a role in the targeting of Sig-1Rs at the ER-mitochondria junction. Thus, the objective of this study was to examine whether DRMs are involved in the localization of Sig-1Rs at the MAM and in the receptor translocation. Materials and Methods Materials and Methods. Reagents for cell culture were purchased from Invitrogen (Carlsbad, CA). Sources of antibodies included the following: anti-N-CAM was from BD Biosciences Pharmingen (San Diego, CA); anti-IP3R3 and anti-BiP were from BD Biosciences (San Jose, CA); anti-Src, anti-cytochrome P450 reductase (CYP450R), and anti-calreticulin were from Santa Cruz Biotechnology (Santa Cruz, CA); anti-ceramide was from Sigma-Aldrich (St. Louis, MO), anti-mitofusin-2 and anti-ERp57 were from Abcam Inc. (Cambridge, MA); anti-ATP synthase inhibitor and anti-cytochrome c oxidase subunit I were from Invitrogen; and anti-green fluorescent protein (GFP) was from Clontech (Mountain View, CA). Anti-Sig-1R antibodies were developed as described previously (Hayashi and Su, 2007). 6-((N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)amino)hexanoyl) sphingosine (NBD-Cer) and N-((4-(4,4-difluoro-5-(2-thienyl)-4-bora3a,4a-diaza-s-indacene-3-yl)phenoxy)acetyl)sphingosine (BODIPYCer TR) were from Invitrogen. Chemicals and lipids were from Sigma-Aldrich or Avanti Polar Lipids (Alabaster, AL). Expression vectors for Mito-DsRed, Mito-GFP, and KDEL-DsRed were purchased from Clontech. The structures of lipids are provided in the supplemental figure. Cell Culture. Chinese hamster ovary (CHO; American Type Culture Collection, Manassas, VA) cells were maintained in minimum essential mediumcontaining 2 mM GlutaMax (Invitrogen) and 10% heat-inactivated fetal calf serum at 37°C with 5% CO2. The vector encoding the mouse Sig-1R with an enhanced yellow fluorescent protein (EYFP)-tag on the C terminus was constructed in the pEYFP-N1 vector (Clontech) as described previously (Hayashi and Su, 2003b). CHO cell lines stably expressing EYFP or Sig-1R-EYFP were established by transiently transfecting pEYFP-N1 or pEYFPSig-1R vectors followed by colony selections with G418 (Geneticin; Invitrogen). Lipofectamine 2000 (Invitrogen) was used for gene transfection. CHO cells were treated with fumonisin B1 (FB1), methyl-cyclodextrin (M C), or cholesterol-conjugated M C in culture medium without serum. FB1 was dissolved in ethanol and used at 5 g/ml. M C was dissolved in phosphate-buffered saline (PBS) and used at 5 mM with 5 M lovastatin. Cholesterol-conjugated M C was prepared by rotating 1 ml of M C solution (100 mM) with N2-dried cholesterol (4 mg) overnight at room temperature. DRM Preparation. For the sucrose gradient fractionation of DRMs, CHO cells were cultured in a 10-cm dish at 80 to 90% confluence. CHO cells (2 10 cells) were lysed at 4°C in 0.5 ml of TNE-Tx buffer (10 mM Tris at pH 7.4, 150 mM NaCl, 1 mM phenylmethylsulfonyl fluoride, 5 mM EDTA, 48 Kallikrein inactivation units/ml aprotinin, and 0.5% Tx). After being mixed with an equal volume of 80% sucrose, the cell lysate was subjected to sucrose gradient centrifugation at 100,000g, and a total of 13 fractions was collected from the top, as described previously (Hayashi and Su, 2003b). The same volume from each fraction was applied to SDS-PAGE. To prepare the Tx-insoluble pellet enriched with DRMs and the Tx-soluble supernatant by differential centrifugations, cell membranes were incubated in TNE-Tx for 30 min at 4°C. After sonication (10 s, three times), samples were centrifuged at 12,000g for 20 min. The supernatant was further centrifuged at 100,000g for 1 h in a Ti rotor. The pellet and the supernatant were collected as Tx-insoluble (pellet) and Tx-soluble (supernatant) fractions, respectively. MAM Fractionation. The MAM fraction was prepared as described previously (Rusiñol et al., 1994). In brief, CHO cells were grown on two 15-cm dishes at 90 to 100% confluence. Harvested cells were homogenized by a glass Dounce homogenizer in a homogenization buffer (0.25 M sucrose and 10 mM HEPES/KOH, pH 7.4). The homogenate was centrifuged twice at 500g for 5 min to yield the P1 nuclear fraction. The supernatant was spun down at 10,300g for 20 min to yield crude mitochondria membranes as a pellet (P2 fraction). The supernatant was centrifuged at 100,000g for 1 h in a 50Ti rotor to obtain the P3 microsomal and cytosolic fractions. Crude mitochondrial membranes were suspended in 0.5 ml of isolation medium (250 mM mannitol, 5 mM HEPES/KOH pH 7.4, and 0.5 mM EGTA/KOH), layered on Percoll solution [225 mM mannitol, 25 mM HEPES/KOH, pH 7.4, 1 mM EGTA/KOH, and 30% (v/v) Percoll (GE Healthcare, 518 Hayashi and Fujimoto at A PE T Jornals on Jne 5, 2017 m oharm .aspeurnals.org D ow nladed from Chalfont St. Giles, Buckinghamshire, UK)], and centrifuged at 95,000g for 30 min in an SW 55Ti rotor. Purified mitochondrial and MAM fractions were collected and washed twice with an isolation medium by centrifugation. Immunocytochemistry and Confocal Microscopy. Immunocytochemistry and confocal microscopy were performed as described earlier (Hayashi and Su, 2003b). Primary antibodies were used at 1:100 for anti-Sig-1R or 1:50 for anti-IP3R3. The filipin staining was performed according to a previous report (Hayashi and Su, 2003b). Lipid Overlay Assay. CHO cells were grown in a 15-cm dish at 90% confluence. Cells were harvested in ice-cold PBS and centrifuged at 2000g for 10 min. The cell pellet was suspended in 1 ml of 50 mM Tris, pH 7.4, containing 0.2% CHAPS. The cell lysate was rotated at 4°C for 2 h, followed by a centrifugation at 100,000g in a 50Ti rotor for 1 h. The supernatant was filtered by a Millipore 0.22-mm filter unit (Millipore, Billerica, MA). The total protein concentration was measured with a BCA protein assay kit (Thermo Fisher Scientific, Waltham, MA). The lysate was stored at 80°C