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NEUROPHARMACOLOGY

-1 Receptors (₁ Binding Sites) Form Raft-Like Microdomains and Target Lipid Droplets on the Endoplasmic Reticulum: Roles in Endoplasmic Reticulum Lipid Compartmentalization and Export

Cellular Pathobiology Unit, Cellular Neurobiology Research Branch, Intramural Research Program, National Institute on Drug Abuse, National Institutes of Health. Department of Health and Human Services, Baltimore, Maryland

Received for publication March 5, 2003
Accepted April 29, 2003.

	Abstract

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The brain -1 receptors can bind neurosteroids and psychotropic drugs, including neuroleptics and cocaine and are implicated in schizophrenia, depression, and drug dependence. In this study, we found that -1 receptors specifically target lipid storage sites (lipid droplets) on the endoplasmic reticulum by forming a distinct class of lipid microdomains. Both endogenously expressing -1 receptors and transfected C-terminally enhanced yellow fluorescent protein (EYFP)-tagged -1 receptors (Sig-1R-EYFP) target unique "ring-like" structures associated with endoplasmic reticulum reticular networks in NG108-15 cells. The ring-like structures contain neutral lipids and are enlarged by the oleate treatment, indicating that they are endoplasmic reticulum-associated lipid droplets (ER-LDs). -1 receptors colocalize with caveolin-2, a cholesterol-binding protein in lipid rafts on the ER-LDs, but not with adipocyte differentiation-related protein (ADRP), a cytosolic lipid droplet (c-LD)-specific protein. When the double-arginine ER retention signal on the N terminus of -1 receptors is truncated, -1 receptors no longer exist on ER-LDs, but predominantly target c-LDs, which contain ADRP. -1 receptors on ER-LDs form detergent-resistant raft-like lipid microdomains, the buoyancy of which is different from that of plasma membrane lipid rafts. (+)-Pentazocine causes -1 receptors to disappear from the microdomains. N-Terminally EYFP-tagged -1 receptors (EYFP-Sig-1R) failed to target ER-LDs. EYFP-Sig-1R-transfected cells showed an unrestricted distribution of neutral lipids all over the endoplasmic reticulum network, decreases in c-LDs and cholesterol in plasma membranes, and the bulbous aggregation of endoplasmic reticulum. Thus, -1 receptors are unique endoplasmic reticulum proteins that regulate the compartmentalization of lipids on the endoplasmic reticulum and their export from the endoplasmic reticulum to plasma membrane and c-LDs.

-1 receptors have one putative transmembrane domain and two hydrophobic stretches, and possess a double arginine endoplasmic reticulum retention signal at the N terminus (Hanner et al., 1996; Seth et al., 1997). A study recently proposed a two-transmembrane domain model for -1 receptors (Aydar et al., 2002). The sequence of -1 receptors exhibits no homology to any other mammalian protein; however, it has a 30.3% identity to the fungal sterol C8-C7 isomerase (Hanner et al., 1996). More strikingly, about 75% of amino acids on the hydrophobic domain in the center of -1 receptors are identical to the sterol binding pocket in fungal sterol C8-C7 isomerase (Keon et al., 1994). These suggest that -1 receptors might bear certain relation to sterols. However, -1 receptors lack the enzymatic activity as a sterol isomerase (Labit-Le Bouteiller et al., 1998), and the cloned mammalian C8-C7 sterol isomerase (Braverman et al., 1999) is totally different from -1 receptors. The biological action of -1 receptors on sterols, if any, has never been reported.

We reported recently that -1 receptors localize on smooth endoplasmic reticulum and form a dimeric complex with a cytoskeletal adaptor protein ankyrin-B (Hayashi and Su, 2001). Interestingly, upon stimulation by psychoactive drugs, -1 receptors translocate to plasmalemmal and nuclear membranes (Morin-Surun et al., 1999; Hayashi et al., 2000; Hayashi and Su, 2001). Here, in a series of investigations using immunocytochemistry and transfection of green fluorescent protein-tagged -1 receptors, we found that -1 receptors specifically target lipid droplets on smooth endoplasmic reticulum (ER-LDs) from whence cytosolic lipid droplets (c-LDs) are known to bud into cytosol (Murphy and Vance, 1999; Taguchi-Sato et al., 2002). Our findings in this report strongly suggest that -1 receptors are unique ER-LD targeting proteins that are regulated by psychoactive drugs and are intimately related to the compartmentalization of neutral lipids on the ER-LD and their transport thereof.

	Materials and Methods

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Cell Culture, Antibodies, and Peptides. Procedures for cell culture were described previously (Hayashi et al., 2000). Antibody and sources are as follows: polyclonal caveolin (Cav), monoclonal caveolin-1, caveolin-2, caveolin-3, Bip/GRB78, SRP54, or calnexin (Transduction Laboratories, San Diego, CA); Src or presenilin-1 (Santa Cruz Biotechnology, Inc., Santa Cruz, CA); ankyrin B (Oncogene Science, Cambridge, MA); NADPH-cytochrome P450 reductase (CYP450R; StressGen, Victoria, BC, Canada); cholera toxin subunit-B (CTx-B) (Sigma-Aldrich, St. Louis, MO); GFP (BD Biosciences Clontech, Palo Alto, CA); and Alexa Fluor-conjugated secondary antibodies and CTx-B (Molecular Probes, Eugene, OR). CTx-B was from Sigma-Aldrich and Alexa Fluor 590-conjugated CTx-B was from Molecular Probes. Polyclonal rabbit anti-guinea pig -1 receptors-A and -B were raised against guinea pig -1 receptor amino acid sequence 144 to 165.

Immunostaining of -1 Receptors. Cells grown on 12-mm poly-D-lysine/laminin-coated coverslips were fixed by 4% paraformaldehyde for 30 min. Paraformaldehyde was quenched by 100 mM glycine in HBSS (pH 8.5). Cells were permeabilized (0.1% Triton X-100 for 10 min) and blocked (10% nonfat milk for 60 min). In immunocytochemistry for -1 receptors and caveolins, fixed cells were treated with 0.02% SDS for 10 min for antigen retrieval (Brown et al., 1996). Cells were incubated with appropriate primary (4% bovine serum albumin + 0.5% Nonidet P-40) and secondary antibodies. Coverslips were mounted in the ProLong Antifade solution (Molecular Probes).

Construction and Expression of Enhanced Yellow Fluorescent Protein (EYFP)-Tagged -1 Receptors. Polymerase chain reaction amplifications of the mouse -1 receptors cDNA (GenBank accession no. AF030198 [GenBank] ) from pSPORT1--1 receptors (Seth et al., 1997) were used. Products from primers (5'-GGAATTC-GCTAGAATGCCGTGGGCG-3' and 5'-CGGGATCC-CGGGAGTCCTGCCAAAGAG-3' for Sig-1R-EYFP or 5'-GGAATTC-TATGCCGTGGGCTGCGGGA-3' and 5'-CGGGATCC-TCAGGAGTCTTGTCCAAA-3' for EYFP-Sig-1R) were subcloned into the pcDNA3.1/His cloning vector (Invitrogen, Carlsbad, CA). Purified vectors were digested by EcoRI and BamHI (sites underlined) to yield -1 receptors cDNAs for ligation into pEYFP-N1 or pEYFP-C1 vector (BD Biosciences Clontech) for expression of Sig-1R-EYFP and EYFP-Sig-1R, respectively. Italics indicated mutated nucleotides to avoid dimerization. Vectors were transfected by using LipofectAMINE-2000 (Invitrogen).

Filipin and Nile Red Fluorescence Stainings. NG108 cells were fixed and incubated for 3 h in PBS containing 1% bovine serum albumin and 125 µg/ml filipin (Polysciences, Warrington, PA). Images were captured by a confocal microscopy with an UV laser (365 nm). For Nile red (Sigma-Aldrich) staining, fixed cells were mounted in 50% glycerol/PBS containing 0.001% Nile red. For dual capturing of both Nile red and EYFP images in fixed cells, Nile red image was captured first (no crossover of EYFP to a red channel) and then the EYFP image was captured after the Nile red photobleach.

Labeling GM1 Gangliosides with CTx-B. For detection of intracellular GM1 ganglioside (Fig. 6c), fixed and permeabilized (0.5% Triton X-100 at 4°C) cells were incubated with 5 µg/ml Alexa conjugated-CTx-B (4°C, 30 min). Cells were washed with PBS containing 0.2% Triton X-100 and mounted. For Western blotting of CTx-B directed against plasma membrane raft GM1 gangliosides (Fig. 6d), NG108 cells were incubated with 5 µg/ml CTx-B (4°C, 30 min) in HBSS, solubilized with 0.5% Triton X-100 (4°C, 30 min), and subjected to floatation sucrose gradient and SDS-polyacrylamide gel electrophoresis. Boiling of samples before application to SDS-polyacrylamide gel electrophoresis was omitted to maintain the pentameric structure of GM1 ganglioside-binding CTx-B. Protein bands were detected by anti-CTx-B antibodies.

View larger version (48K): [in this window] [in a new window]   Fig. 6. Detergent insolubility of -1 receptor-containing ER-LD substructures. a, Sig-1R-EYFP-expressing NG108 cells were treated with cytochalasin-D (10 µM, 30 min) followed by extraction with 0.5% Triton X-100 (at 4°C, 5 min) and fixed thereafter. b and c, Sig-1R-EYFP-containing ER-LD stained with filipin (in blue lined circles), but not with CTx-B (red in c). In c, Sig-1R-EYFP-expressed cells were fixed, extracted with 0.5% Triton X-100 at 4°C, and incubated with Alexa Fluor 590-conjugated CTx-B. Scale bar, 10 µm. N, nucleus, G; Golgi. Arrows in b and c indicate positions of plasma membranes. Scale bars, 10 µm. d, buoyancy of plasma membrane raft markers (src, CTx-B-bound GM1 ganglioside; under Materials and Methods) in sucrose gradients containing Triton X-100. Lipid rafts in NG108 cell lysates were separated by centrifugation (4°C) in the presence of Triton X-100. In the graph, distribution of cholesterol is presented in percentage. e, buoyancy differences of microdomains containing -1 receptors or Cav-2. ANK, ankyrin B (135 kDa). f, buoyancy changes of -1 receptor-containing membrane microdomains induced by a low cell density culture, cytocharacin-D (10 µM, 60 min), 0.5% digitonin, or (+)-pentazocine (100 nM, 10 min). Note disappearance of -1 receptors, but not src and Cav-2, in fractions 3 and 4 induced by (+)-pentazocine. g, buoyancy of immunoreactive -1 receptors in Sig-1R-EYFP-, 2-8-Sig-1R-EYFP-, and EYFP-Sig-1R-expressing cells in low cell density culture. Western blottings for EYFP-tagged -1 receptors are shown with accompanying endogenously expressed -1 receptors. Note that in EYFP-Sig-1R-transfected cells, no EYFP-Sig-1R or endogenous -1 receptors was present in fractions 2 to 5.

Purification of Detergent-Insoluble Lipid Microdomains. NG108 cells were extracted for 30 min in TNE buffer (10 mM Tris, 150 mM NaCl, 5 mM EDTA, 1 mM phenylmethylsulfonyl fluoride, 10 µg/ml aprotinin, 2% sucrose; pH 7.2) containing 0.5% Triton X-100 (4°C). Triton X-100 extracts were adjusted to 40% sucrose (10 ml), placed in an ultracentrifuge tube, overlaid with sucrose gradients (35%, 12.5 ml; 15%, 2.5 ml; 5%, 2.5 ml; and 0%, 2.5 ml), and centrifuged at 141,000g (4°C, 24 h) in a SW28 rotor. Thirteen fractions (2.5 ml each from top) were collected.

Enzymatic Assays for Cholesterol. After fractionation of NG108 cell membranes, lipids in each fraction were extracted according to methods described elsewhere (Corvera et al., 2000). Total protein contents in each fraction were measured by bicinchoninic acid kit (Pierce Chemical, Rockford, IL). Lipid extracts dissolved in isopropanol were assayed enzymatically for total cholesterol (Wako Bioproducts, Richmond, VA).

	Results

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Endogenously Expressed -1 Receptors Exhibit a Unique Pattern of Localization in NG108 Cells. Endogenously expressed -1 receptors exhibit a unique pattern of localization when examined by immunocytohistochemistry in combination with an antigen retrieval method (see Materials and Methods). -1 receptors localized in perinuclear areas as dense clusters (Fig. 1a). Higher magnification indicated that -1 receptors accumulated predominantly as ring structures and a few accompanying tubular elements (Fig. 1b, inset). The size of the ring structure varied, ranging between 1 and 2.5 µm in a diameter.

View larger version (82K): [in this window] [in a new window]   Fig. 1. Distribution of endogenously expressed -1 receptors in NG108 cells. a, endogenously expressed -1 receptors in perinuclear regions. NG108 cells were fixed and antigen-retrieved, followed by staining with polyclonal anti-guinea pig -1 receptors antibody-B. b, -1 receptors-positive ring-like structures and associated tubular elements. Scale bar, 10 µm.

-1 Receptors Target ER-LDs: An Examination Using C-Terminally EYFP-Tagged -1 Receptors. To further examine the intracellular localization of -1 receptors, four different constructs of EYFP-tagged -1 receptors were transfected in NG108 cells (Fig. 2). C-terminally EYFP-tagged -1 receptors (Sig-1R-EYFP) showed the same localization pattern as that of endogenously expressed -1 receptors (Fig. 3a). Sig-1R-EYFPs were highly concentrated on the ring structures and accompanying tubular elements (Fig. 3b, arrows). In highly overexpressed cells, a portion of Sig-1R-EYFP occurred on nuclear envelope and apparently increased the size of ring structures. Although Sig-1R-EYFP-containing ring structures were negative to CYP450R (a smooth endoplasmic reticulum resident protein) staining, they were always connected to CYP450R-containing endoplasmic reticulum reticular networks (Fig. 3c). Moreover, tubular elements associated with ring structures could be stained with CYP450R, indicating that they are integral endoplasmic reticulum structures (Fig. 3d, arrows). Together, these results suggest that the -1 receptor-containing ring structure is a part of the smooth endoplasmic reticulum. The -1 receptor-containing ring structures were negative to stainings by all tested organelle markers [EEA-1 (endosome), Mito Tracker and bcl-2 (mitochondria), Lyso Tracker (lysosome), synapsin II (synaptic vesicles), Fas and CTx-B (plasma membranes), and GM130 (Golgi)] and were negative as well to stainings by other endoplasmic reticulum-associated proteins, including Bip/GRP78, SRP54, presenilin-1, and calnexin. The Sig-1R-EYFP-containing ring structures, however, could be stained with Nile red, which is known to stain neutral lipid-enriched LDs (Fig. 3e). The Nile red result suggests that -1 receptor reside specifically at the ER-LD. To confirm this speculation, we used the oleic acid treatment to see whether -1 receptor-containing ring structures might be enlarged by this treatment. Oleic acid (300 µM for 6 h), which facilitates neutral lipid formation, caused a significant enlargement of the -1 receptor-containing ring structures and retention of neutral lipids therein (Fig. 3f). Thus, -1 receptors or Sig-1R-EYFPs localize specifically on the ER-LDs.

View larger version (13K): [in this window] [in a new window]   Fig. 2. Diagram of constructs of -1 receptor mutants. Bold bars on the N termini are double-arginine ER retention signals; shaded areas, hydrophobic stretches on N and C termini; and black boxes, putative transmembrane domains.

View larger version (76K): [in this window] [in a new window]   Fig. 3. Sig-1R-EYFP targeting endoplasmic reticulum lipid droplets. a and b, Sig-1R-EYFP target perinuclear ring structures and accompanying tubular elements (arrows in b) in NG108 cells. c and d, Sig-1R-EYFP (green)-containing ring structures connecting to endoplasmic reticulum reticular networks (c). Colocalization of Sig-1R-EYFP-containing tubular elements with CYP450R (arrows in d). Sig-1R-EYFP in green, CYP450R in red. e and f, Nile red staining (red) in normal (e) or oleic acid-treated (f: 300 µM,6h) cells. Sig-1R-EYFP in green. g, endogenously expressed ADRP were visualized in red. Inset, ADRP-containing c-LDs in proximity to -1 receptors-containing ER-LD. H, Western blotting detecting endogenously expressed caveolins in NG108 cells using monoclonal antibodies against Cav-1 (left), Cav-2 (middle), and Cav-3 (right panel). i and j, localization of endogenously expressed Cav-2 or Cav-1 (in red) and Sig-1R-EYFP (green). Arrows, colocalization of caveolin-2 and Sig-1R-EYFP in ER-LD. k, transiently expressed caveolin-2-GFP (in green) and endogenously expressed -1 receptors stained by anti-guinea pig -1 receptors antibody-B (in red). Scale bars, 10 µm. N, nucleus.

-1 Receptors Are Unique Proteins Specifically Targeting ER-LDs. So far, only a limited number of proteins are known to be associated with lipid droplets such as adipocyte differentiation-related protein (ADRP), perilipin (Brasaemle et al., 1997; Londos et al., 1999), and caveolins (Fujimoto et al., 2001; Ostermeyer et al., 2001; Pol et al., 2001). We set forth to examine whether any of those proteins may also be specifically localized, like -1 receptors, at the ER-LD. ADRP was found to localize on c-LDs in NG108 cells, but not on ER-LD (Fig. 3g). ADRP has been suggested as being translated on free ribosomes and then recruited to nascent c-LD from the cytosol (Brasaemle et al., 1997; Londos et al., 1999). Interestingly, some, usually one to two, ADRP-positive c-LDs were in proximity to a single -1 receptors-containing ER-LDs (Fig. 3g, inset). Thus, in NG108 cells, ADRP might be recruited to nascent c-LDs during the budding stage of c-LDs from ER-LDs. Isoforms and mutants of caveolin, a cholesterol-binding protein found in caveolae type of cholesterol-rich lipid rafts (Anderson, 1998), were reported to accumulate on LDs (Fujimoto et al., 2001; Ostermeyer et al., 2001; Pol et al., 2001). NG108 cells express Cav-1, Cav-2, but not caveolin-3 (Fig. 3h). By using monoclonal anti-caveolin-2 antibodies, we detected the caveolin-2-like immunoreactivity on Sig-1R-EYFP-containing ER-LDs (Fig. 3i). Caveolin-2-like immunoreactivity was also seen in cytosolic small vesicles. Most caveolin-1 localized at Golgi and the plasma membrane, but not on ER-LDs (Fig. 3j). Among the isomers of caveolin-2, isoform (Cav-2) was reported to target LDs. However, we transiently transfected GFP-tagged Cav-2 into NG108 cells and found that they targeted mostly c-LDs rather than ER-LDs (Fig. 3k). Thus, endogenously expressed caveolin-2 isomers in NG108 cells may not be caveolin-2. Together, these results indicated that the only proteins known to target ER-LDs are -1 receptors and an unknown isomer of caveolin-2.

-1 Receptors Lacking an Endoplasmic Reticulum Retention Signal Target c-LDs. Structural requirement of -1 receptors targeting the ER-LDs was examined. Similar to the KKXX motif, the double-arginine motif is known to direct the retrieval of membrane proteins to the endoplasmic reticulum via a retrograde transport pathway (Schutze et al., 1994). The truncation of N-terminal seven amino acids containing a double-arginine endoplasmic reticulum retention signal from Sig-1R-EYFP (2-8-Sig-1R-EYFP) caused -1 receptors to occur predominantly on c-LDs (Fig. 4a). 2-8-Sig-1R-EYFP and ADRP colocalized on some c-LDs. Some c-LDs, however, contained only ADRP or only 2-8-Sig-1R-EYFP (Fig. 4b, arrows). Removal of a longer stretch of N-terminal amino acids from Sig-1R-EYFP (2-29-Sig-1R-EYFP) caused -1 receptors to localize solely on c-LDs. Also, a coalescence of c-LDs was observed (Fig. 4c). Live-cell imaging revealed that small c-LDs fused to become a large c-LDs (data not shown). A few large c-LDs thus formed were congregated by small c-LDs containing ADRP and/or 2-28-Sig-1R-EYFP (Fig. 4d).

View larger version (37K): [in this window] [in a new window]   Fig. 4. Distribution of N-terminally truncated Sig-1R-EYFP. a and b, colocalization of 2-8-Sig-1R-EYFP (green), Nile red (red in a), and ADRP (red in b). Arrows indicate c-LDs containing only ADRP or only 2-8-Sig-1R-EYFP. c and d, c-LD coalescence induced by 2-29-Sig-1R-EYFP (green) expression. Nile red (c) and ADRP (d) in red. Scale bars, 10 µm. N, nucleus.

N-Terminally EYFP-Tagged -1 Receptors Fail to Target ER-LDs. In contrast to C-terminally EYFP-tagged -1 receptors (Sig-1R-EYFP), N-terminally EYFP-tagged -1 receptors (EYFP-Sig-1R) were found to exist at the endoplasmic reticulum reticular network, but failed to target ER-LDs (Fig. 5a). EYFP-Sig-1R-transfected cells showed not only significant decrease in number of all lipid droplets but also, importantly, the unrestricted distribution of neutral lipids all over the endoplasmic reticulum network, suggesting a lack of ER-LDs in EYFP-Sig-1R-overexpressing cells (a lower cell in Fig. 5b). In 38% of EYFP-Sig-1R-transfected cells, a bulbous aggregation of the CYP450R-positive endoplasmic reticulum network was seen (Fig. 5c). Neutral lipids and free cholesterol (filipin staining) were retained in these bulbous aggregations (Fig. 5, d and e). In EYFP-Sig-1R-transfected cells with these peculiar ER aggregations, free cholesterol levels were significantly reduced in Golgi and plasma membrane (a lower cell in Fig. 5e). Although, as mentioned above, C-terminally EYFP-tagged -1 receptors (Sig-1R-EYFP) specifically target ER-LDs, the Sig-1R-EYFP failed to target ER-LD in the presence of brefeldin-A (0.5 µg/ml for 14 h), a forward inhibitor of the endoplasmic reticulum-to-Golgi network (Fig. 5f). Interestingly, as a result of the brefeldin-A treatment, Sig-1R-EYFP showed the same distribution pattern as that of EYFP-Sig-1R. These results suggest that protein sorting through the endoplasmic reticulum-to-Golgi secretory pathway is required for targeting of endogenous -1 receptors to ER-LDs. On the contrary, EYFP-Sig-1R may fail to enter the pathway.

View larger version (57K): [in this window] [in a new window]   Fig. 5. Localization of EYFP-Sig-1R. a, colocalization of EYFP-Sig-1R (green) and CYP450R (red). b, relatively diffused Nile red staining on the endoplasmic reticulum of EYFP-Sig-1R-transfected cells (lower cell in both panels): Comparison with those seen in a nontransfected cell (upper cell in right panel). c, bulbous aggregation of endoplasmic reticulum in EYFP-Sig-1R-transfected cells. EYFP-Sig-1R in green, CYP450R in red. d and e, EYFP-Sig-1R-enriched endoplasmic reticulum bulbs stained with Nile red (red in d) and filipin (arrow in e). Note no filipin staining at Golgi (G) in the lower cell (e). f, effect of brefeldin-A (0.5 µg/ml for 14 h) on Sig-1R-EYFP localization. Scale bars, 10 µm. N, nucleus.

-1 Receptors Form Raft-Like Microdomains on LDs. Because caveolins are known to form detergent-resistant cholesterol-rich microdomains, at least on caveolae (Anderson, 1998; Simons and Toomre, 2000), and because our data show that -1 receptors colocalize with caveolin-2 on ER-LDs (Fig. 3i), we tested whether the ER-LD membrane may contain raft-like microdomains and whether -1 receptors might exist in those domains. Because lipid rafts are known to be resistant to Triton X-100 at low temperature (Simons and Toomre, 2000), Sig-1R-EYFP-expressing cells were, at first, in situ extracted at 4°C in HBSS containing 0.5% Triton X-100 and examined for the presence of detergent-insoluble -1 receptors-containing structures. ER-LD membranes, but not endoplasmic reticulum-tubular elements, were resistant to 0.5% Triton X-100 at 4°C (Fig. 6a). The renowned classic lipid rafts are known to be composed of cholesterol and glycosphingolipids such as GM1 ganglioside (Simons and Toomre, 2000). Although containing relatively high amount of free cholesterol (stained with filipin), the ER-LD membranes observed in the present study, however, contained no detectable GM1 ganglioside (stained with CTx-B) (Fig. 6, b and c).

-1 receptor-containing detergent-resistant microdomains were further characterized by the Triton X-100-floatation sucrose gradient centrifugation, which is conventionally used for preparation of lipid rafts. Detergent-resistant lipid rafts are known to float during centrifugation in the presence of Triton X-100 at low temperature and can be separated from detergent-soluble proteins that remain on the bottom of the centrifuge tube (Simons and Toomre, 2000). Plasma membrane raft markers Src and CTx-B-bound GM1 gangliosides (under Materials and Methods) existed in high-buoyancy fractions (2–4; Fig. 6d) with accompanying high levels of cholesterol. Interestingly, both -1 receptor- and Cav-2-containing microdomains were less buoyant than plasma membrane lipid-rafts (seen in fractions 4 and 5; Fig. 6e). -1 receptors were also present in denser fractions 6–9 (Fig. 6e). Ankyrin B (135 kDa), an actin cytoskeleton adaptor protein associated with -1 receptors in NG108 cells (Hayashi and Su, 2001), exhibited the same buoyancy pattern to that of -1 receptors (Fig. 6e). Ankyrin coimmunoprecipitated with -1 receptors in fraction 4 (data not shown). Cells either cultured at low density (50 x 10⁴ cells/10-cm dish) or treated with cytochalasin-D which disrupts actin polymerization, showed a decrease of -1 receptors in fractions 6–8 and an increase of -1 receptor-containing microdomains to higher buoyancy (fraction 3; Fig. 6f). This result suggests that buoyancy of -1 receptor-containing microdomains is rendered lower, at least in part, by tight interactions with actin cytoskeletons. Disappearance of -1 receptor-containing microdomains by the treatment of cells with digitonin, a cholesterol-bound detergent, suggests that cholesterol is an important constituent for -1 receptor-containing microdomains on ER-LD (Fig. 6f). (+)-Pentazocine (100 nM for 10 min) decreased -1 receptor-containing microdomains, but not that of Src- or caveolin-2 (Cav-2)-containing rafts (Fig. 6f). These results suggest either that caveolin-2 may not in the same microdomains as those containing -1 receptors or that (+)-pentazocine can only mobilize -1 receptors and not caveolin-2 from the same microdomains. Thus, -1 receptors form unique cytoskeleton-associated ER-LD microdomains that differ from classical well recognized GM1 ganglioside-containing lipid rafts on the plasma membrane (Simons and Toomre, 2000).

Like endogenous -1 receptors, Sig-1R-EYFP formed detergent-resistant microdomains (Fig. 6g). 4-8-Sig-1R-EYFP, which predominantly target c-LDs (Fig. 4, a and b), formed substantial amount of detergent-resistant microdomains. This result suggests that -1 receptor-deletion mutants can form rafts on the c-LD membrane that is known to consist of phospholipid monolayer (Fujimoto et al., 2001; Tauchi-Sato et al., 2002). EYFP-Sig-1R failed to form the microdomains (Fig. 6g). In EYFP-Sig-1R-transfected cells, no endogenous -1 receptor-containing rafts could be detected (Fig. 6g, bottom panels). EYFP-Sig-1R might thus act as functionally dominant negative proteins for -1 receptors.

	Discussion

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Most eukaryotic cells store neutral lipids such as triglycerides and cholesteryl esters in lipid droplets. Lipid droplets are formed by coalescence of neutral lipids into discs inside the bilayer of the endoplasmic reticulum (i.e., ER-LDs). c-LDs are known to bud from ER-LDs (Murphy and Vance, 1999). Lipid droplets play an important role in energy storage and/or transport of lipids. Malfunctions in neutral lipid storage are implicated in certain human diseases (Londos et al., 1999; Murphy and Vance, 1999). However, the regulations of lipid droplets and associated neutral lipids are not well understood. Specific proteins such as oleosin (in plant seeds), perilipin (in mammalian adipocytes), and ADRP (in most mammalian cells) can target and regulate biogenesis, storage capacity, and maturation of c-LDs (Brasaemle et al., 1997; Londos et al., 1999). Recently, caveolins, specifically caveolin-2 (Fujimoto et al., 2001), have been shown to accumulate on c-LDs under certain conditions. Although N-terminally truncated dominant negative mutant of caveolin-1, caveolin-2, or caveolin-3 can associate with c-LDs or ER-LDs, respectively (Ostermeyer et al., 2001; Pol et al., 2001), no naturally existing protein has been so far described to specifically target ER-LDs. Thus, our present study constitutes the first report demonstrating -1 receptors as the first protein known to target ER-LDs under the physiological condition.

Topology of -1 receptors on membrane is not fully understood, but a recent report (Aydar et al., 2002) and our data (unpublished results) indicate that both N and C termini of -1 receptors are directed toward the cytoplasm. Although the N-terminal hydrophobic domain was originally proposed as a transmembrane domain in the first cloning study of -1 receptors (Hanner et al., 1996), several recent reports confirm that the hydrophobic domain on the center of -1 receptors, at least, can serve as a membrane-spanning region (Seth et al., 1997; Yamamoto et al., 1999; Aydar et al., 2002). Interestingly, the topology of -1 receptors is similar to that of oleosin or caveolin: a central hydrophobic domain flanked by two hydrophilic cytoplasmic domains. For caveolins and oleosins, the central hydrophobic domain seems to embed into the phospholipid monolayer of lipid droplets, and possibly into the neutral lipid core (Murphy and Vance, 1999). Because lipid droplets are filled with lipids, transmembrane proteins that have hydrophilic domains on the lumen side cannot accumulate on lipid droplets. Therefore, it is very likely that -1 receptors, at least on lipid droplets, lack a luminal hydrophilic domain and thus form a hair-pin structure on surfaces of lipid droplet membranes in a manner similar to that of caveolin or oleosin. The central hydrophobic domain of -1 receptors has a high homology (>70% identity) to the sterol binding pocket of fungal C8-C7 sterol isomerase. Therefore, -1 receptors might be able to bind tightly to lipids on surface and/or in the core of lipid droplets using this central hydrophobic domain.

Ostermeyer et al. (2001) showed that both KKSL (endoplasmic reticulum retrieval motif)-tagged caveolin-1 (Cav-KKSL) and caveolin-1 mutant lacking the N-terminal hydrophobic domain (Cav-N2) are both present on ADRP-positive c-LDs. These two mutants should theoretically stay at the endoplasmic reticulum according to the well known endoplasmic reticulum-to-Golgi pathway for caveolins. These suggest a possibility that "ER-LD to c-LD" may represent a newly recognized pathway with different structural requirement for exporting caveolins, or by extension, neutral lipids, out of the endoplasmic reticulum. Thus, caveolins either with a KKXX signal on C terminus or with a truncated N-terminal stretch can still be exported via the ER-LD to c-LD pathway. Our results with deletion mutant of -1 receptors indicate that the double arginine endoplasmic reticulum retention signal on the N terminus of a protein can be an important determinant to keep that protein (e.g., -1 receptors) on ER-LDs. Deletion of the double arginine endoplasmic reticulum retention signal on -1 receptors causes export of almost all -1 receptors to c-LD, just like Cav-KKSL and Cav-N2 (Fig. 5, a and b). In addition, although the molecular mechanism is unclear at present, our results suggest that the N-terminal hydrophobic stretch of -1 receptors seems to be involved in the membrane fusion of lipid droplets (Fig. 4, c and d). Further investigation is required to understand what controls the protein secretion in this ER-LD to c-LD pathway.

In this study, we found that -1 receptors form detergent-resistant lipid microdomains on ER-LD. Interestingly, a recent study shows that -2 receptors can also form lipid rafts (Gebreselassie and Bowen, 2002). The -1 receptor-containing lipid microdomains, however, possess different features from those of plasma membrane lipid rafts: 1) -1 receptor-containing microdomains have lower buoyancy in Triton X-100 sucrose floatation; 2) the buoyancy of -1 receptor-containing rafts is significantly affected by cytochalasin-D and -1 receptor ligands; and 3) -1 receptor-containing microdomains contain cholesterol, but not GM1 ganglioside. Glycosphingolipids are the well known components in plasma membrane lipid rafts (Simons and Toomre, 2000). Furthermore, concentrations of glycosphingolipids are known to be extremely low at the endoplasmic reticulum compared with those at the Golgi or the plasma membrane (van Meer and Holthuis, 2000). Together, these suggest that other as yet unidentified types of sphingolipids must participate in the formation of -1 receptor-containing detergent-resistant microdomains on ER-LDs. It would warrant very much to investigate what sphingolipid, if any, corroborates with cholesterol to form -1 receptor-containing detergent-resistant microdomains on the ER-LDs and how exactly -1 receptor ligands may affect this unique lipid assembly.

Our results showing that tagging EYFP to the N terminus of -1 receptors (i.e., EYFP-Sig-1R) renders -1 receptors functionally negative suggests that the resultant mutant fails to enter ER-Golgi secretory pathway and/or fails to acquire a post-translational modification at the Golgi. In EYFP-Sig-1R-transfected cells, several abnormalities have been observed: 1) retention of neutral lipids over entire ER network; 2) no compartmentalization of neutral lipids into ER-LDs; 3) a significant decrease in c-LDs; 4) formation of bulbous aggregations of endoplasmic reticulum that contain substantial amounts of neutral lipids and free cholesterol; and 5) decreases in free cholesterol in Golgi and plasma membrane. Neutral lipids such as cholesteryl esters and triglycerides are known to be synthesized in the entire endoplasmic reticulum network and are thought to be concentrated thereafter to specific loci (e.g., ER-LDs) (Tauchi-Sato et al., 2002). Therefore, we propose that -1 receptors may demarcate ER-LD surface from the bulk ER membrane and thus compartmentalize lipids inside the ER-LDs. Neutral lipids are known to be exported from the endoplasmic reticulum to c-LDs, and free cholesterol from the endoplasmic reticulum to Golgi and plasma membrane. Therefore, results mentioned above in 3 to 5 strongly suggest that EYFP-Sig-1R severely disturbs exports of lipids from the endoplasmic reticulum to expected destinations. It is thus plausible that -1 receptors serving to compartmentalize lipids in specialized loci on the endoplasmic reticulum can regulate export of lipids from the endoplasmic reticulum to c-LDs or to plasma membrane.

-1 receptors are present in high levels in the brain. In addition to certain steroid hormones, diverse kinds of psychotropic compounds, including neuroleptics, antidepressants, and psychostimulants can bind to -1 receptors (Su, 1982; Sharkey et al., 1988; Su et al., 1988; Narita et al., 1996). Neuroleptics, especially haloperidol, are known to function as -1 receptor antagonists, whereas cocaine and antidepressants as agonists (Hayashi and Su, 2001; Matsumoto et al., 2001; Takebayashi et al., 2002). -1 receptors are implicated in several physiological and pharmacological functions in the brain such as neuronal firing (Monnet et al., 1990), neurotransmitter release (Nuwayhid and Werling, 2003), psychostimulant sensitization (Ujike et al., 1996), and learning and memory (Maurice and Lockhart, 1997). How -1 receptors can affect such diverse functions in the brain is unknown. The brain is a lipid-enriched organ and contains approximately one-fourth of total cholesterol in the body (Dietschy and Turley, 2001). Brain lipids are mostly supplied via de novo synthesis and are used for energy consumption (highest proportion among organs), synaptic vesicle recycling and synaptogenesis (Dietschy and Turley, 2001). Thus, -1 receptor's regulation of lipid compartmentalization at and lipid export from the endoplasmic reticulum, as proposed in this study, may represent a novel mechanism whereby energy source and/or lipid membrane materials are distributed to certain parts of neurons in the brain. -1 receptors may therefore be related to certain brain disorders with altered cholesterol metabolism and malfunctions in brain repair mechanisms. Apparently, psychoactive drugs such as cocaine may affect membrane compositions via -1 receptors.

	Acknowledgements

 
We thank Dr. V. Ganapathy for a generous gift of the pSPORT1--1 receptors and Dr. T. Fujimoto for the caveolin-2-GFP vector.

	Footnotes

 
This study was supported by the Intramural Research Program of National Institute on Drug Abuse, National Institutes of Health.

DOI: 10.1124/jpet.103.051284.

ABBREVIATIONS: ER-LD, endoplasmic reticulum-associated lipid droplet; c-LD, cytosolic lipid droplet; CYP450R, NADPH-cytochrome P450 reductase; CTx-B, cholera toxin subunit-B; GFP, green fluorescent protein; HBSS, Hanks' balanced salt solution; PBS, phosphate-buffered saline; EYFP, enhanced yellow fluorescent protein; EYFP-Sig-1R, N-terminally EYFP-tagged -1 receptor; Sig-1R-EYFP, C-terminally EYFP-tagged -1 receptor; ADRP, adipocyte differentiation-related protein; Cav, caveolin.

Address correspondence to: Dr. Tsung-Ping Su, Cellular Pathobiology Unit, Cellular Neurobiology Research Branch, Intramural Research Program, National Institute on Drug Abuse, National Institutes of Health, Department of Health and Human Services, 5500 Nathan Shock Dr., Baltimore, MD 21224. E-mail: tsu{at}intra.nida.nih.gov

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