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NEUROPHARMACOLOGY

Intracellular Dynamics of -1 Receptors (₁ Binding Sites) in NG108-15 Cells

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 March 5, 2003; accepted April 29, 2003.

	Abstract

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The -1 receptors bind diverse kinds of psychoactive compounds, including cocaine, and translocate upon stimulation by these compounds. However, the exact intracellular localization and dynamics of -1 receptors have been unclear. We recently found that -1 receptors specifically localize on cholesterol-enriched loci on the endoplasmic reticulum (ER) membrane that function as neutral lipid storage sites (i.e., ER lipid droplets or ER-LDs) from which neutral lipids bud out to form cytosolic lipid droplets. By combining immunocytochemistry and real-time monitoring of enhanced yellow fluorescent protein (EYFP)-tagged -1 receptors (Sig-1R-EYFP) in living cells, we characterized the -1 receptor translocation in this study. (+)-Pentazocine, a selective -1 receptor agonist, causes a significant decrease of -1 receptors in ER-LDs and a diffused distribution of -1 receptors over the entire endoplasmic reticulum reticular network in NG108-15 cells. In the presence of -1 receptor agonists, Sig-1R-EYFP move out from ER-LDs and slide along the endoplasmic reticulum network toward nuclear envelope and the tip of neurites. Fluorescence recovery after photobleaching analysis demonstrates that Sig-1R-EYFP on endoplasmic reticulum reticular network are highly mobile compared with those in ER-LDs. A sucrose gradient fractionation study shows that (+)-pentazocine shifts -1 receptors from ER-LD membranes to higher density membranes. These results indicate that -1 receptors localize on ER-LDs and upon stimulation translocate on continuous endoplasmic reticulum reticular network toward peripheries of cells. Because -1 receptors specifically target ER lipid storage sites and compartmentalize neutral lipids therein, these results suggest that -1 receptors' dynamic translocation might affect lipid transport and distribution in neuronal cells.

-1 receptors have been cloned (Hanner et al., 1996; Seth et al., 1997). The sequence of -1 receptors exhibits no homology to any of other mammalian proteins but has a 30.3% identity to the sequence of a fungal sterol C8-C7 isomerase (Hanner et al., 1996). -1 receptors, however, lack the sterol isomerase activity (Labit-Le Bouteiller et al., 1998). The exact biological action of -1 receptors is still not totally clarified at present.

-1 receptors bind diverse classes of compounds, including psychotherapeutics agents (Su et al., 1982; Narita et al., 1996), cocaine (Sharkey et al., 1988; Matsumoto et al., 2001), and steroid hormones such as progesterone (Su et al., 1988). Haloperidol, a clinically used neuroleptic, functions as a -1 receptor antagonist (Okuyama and Nakazato, 1996). Certain antidepressants, in addition to cocaine, however, act as agonists (Hayashi and Su, 2001; Matsumoto et al., 2001; Takebayashi et al., 2002). -1 receptors and their ligands show modulatory actions in vivo and in vitro. For example, -1 receptors modulate Kv 1.4 potassium channels in nerve terminals (Aydar et al., 2002), inositol 1,3,5-trisphosphate (IP₃) receptor-mediated Ca²⁺ signaling at the ER (Hayashi et al., 2000; Hayashi and Su, 2001), and the N-methyl-D-aspartate-induced neuronal firing or dopamine release in the brain (Monnet et al., 1990; Nuwayhid and Werling, 2003). Notably, in most studies, -1 receptor agonists showed no effect by themselves, but exerted modulatory actions on signal transductions related to ion channels or neurotransmitters.

Morin-Surun et al. (1999) and we (Hayashi et al., 2000; Hayashi and Su, 2001) reported that -1 receptors translocate inside cells. -1 receptor agonists can cause translocation of -1 receptors from light-density microsomal fractions to other subcellular fractions in a period of 10 min (Hayashi et al., 2000). Translocation of -1 receptors might ensue important biological functions afforded -1 receptors. In fact, we demonstrated that in NG108 cells, a portion of -1 receptors are coupled to IP₃ receptors on the endoplasmic reticulum membrane and that -1 receptors amplify IP₃ receptor-medicated Ca²⁺ signaling at the endoplasmic reticulum vis-à-vis their translocation away from the endoplasmic reticulum (Hayashi et al., 2000; Hayashi and Su, 2001). Furthermore, we recently found that Sig-1R specifically target neutral lipid-enriched subdomains on the endoplasmic reticulum membrane [i.e., lipid droplets on the ER (ER-LDs); Hayashi and Su, 2003]. Specifically, studies with functionally negative -1 receptors in that report strongly suggested that -1 receptors at ER-LDs are crucial in regulating lipid compartmentalization at the endoplasmic reticulum (Hayashi and Su, 2003). However, the temporal and spatial characteristics of -1 receptor translocation in cells are still unclear. In this study, we explored the intracellular dynamics of -1 receptors and the associated effects exerted by Sig-1R ligands by using immunocytochemistry, real-time monitoring of C-terminally enhanced yellow fluorescent protein-tagged Sig-1R (Sig-1R-EYFP), as well as sucrose gradient subcellular fractionation in NG108-15 cells.

	Materials and Methods

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Cell Culture, Antibodies, and Chemicals. Procedures for cell culture were described previously (Hayashi et al., 2000). Antibodies and their sources are as follows: caveolin-2, VLA-2, GM130, Lamp-1, or EEA-1 (Transduction Laboratories, San Diego, CA); Src (Santa Cruz Biotechnology, Inc., Santa Cruz, CA); NADPH-cytochrome P450 reductase (CYP450R), bcl-2, Lamp-1, or synapsin II (StressGen, Victoria, BC, Canada); and Alexa Fluor-conjugated secondary antibodies (Molecular Probes, Eugene, OR). Polyclonal rabbit anti-guinea pig -1 receptor-A and -B were raised against guinea pig -1 receptor amino acid sequence 144 to 165. Chemicals not specified here are all from Sigma-Aldrich (St. Louis, MO).

Immunostaining and the Semiquantification of -1 Receptor Translocation. 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, fixed cells were treated with 0.05% 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 Probe). For the counting of the population of -1 receptor-translocated cells, images of NG108 cells stained with anti-guinea pig -1 receptor antibody-B were captured randomly as a field that contains at least four cells. In nontranslocated cells, -1 receptor-containing ring structures were found to surround the nucleus (usually >40 ring structures/cell). For the semiquantification of the -1 receptor translocation, cells displaying ring structures fully surrounding or covering at least 25% of the nucleus were counted as nontranslocated; otherwise as translocated. In other words, -1 receptors "translocated" cells, as defined in this study, have less than a quarter of the perinuclear area surrounded by -1 receptor-containing ring structures. The performance of the semiquantification was done by a person blind to experimental conditions.

Construction and Expression of EYFP-Tagged -1 Receptors. Procedures were described in elsewhere (Hayashi and Su, 2003). Briefly, polymerase chain reaction amplifications of the mouse -1 receptor cDNA (GenBank accession no. AF030198 [GenBank] ) from pSPORT1-Sig-1R (Seth et al., 1997) were subcloned into the pcDNA3.1/His cloning vector (Invitrogen, Carlsbad, CA). -1 receptor cDNA was digested by EcoRI and BamHI and ligated in pEYFP-N1 vector (BD Biosciences Clontech, Palo Alto, CA) for expression of C-terminally EYFP-tagged -1 receptors (Sig-1R-EYFP). Vectors were transfected by using LipofectAMINE-2000 (Invitrogen).

Nile Red Fluorescence Stainings. For Nile red staining, fixed cells were mounted in 50% glycerol/phosphate-buffered saline 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.

Sucrose Gradient Fractionation for ER-LDs and Cystolic Lipid Droplets (c-LDs). NG108 cells from two confluent 15-cm dishes were incubated at 4°C for 10 min in the hypotonic TME buffer (10 mM Tris, 5 mM MgCl₂, 0.5 mM EDTA, 1 mM phenylmethylsulfonyl fluoride, 10 µg/ml aprotinin; pH 7.2). TME buffer containing 68.4% sucrose was then added to obtain a final sucrose concentration at 8.6%. Cells in suspension were homogenized by a Dounce homogenizer (20 strokes). Homogenates were centrifuged at 900g and resultant supernatants collected. Pellets were homogenized again (10 strokes) and centrifuged (900g). The supernatants were overlaid (2 ml) on the top of a sucrose gradient [22 ml; consisting 11 layers from 68.4% (bottom) to 15.0% (top) sucrose]. Finally, the TME buffer with 0% sucrose (2 ml) was placed as the top layer and samples were centrifuged at 120,000g for 16 h. Under this condition, c-LDs float to the top layer (0% sucrose), cytosolic soluble proteins and synaptic vesicle remain in the original layer (8.6%), and other membranes move to lower layers according to their densities (see Fig. 5 under Results). Thirteen fractions were collected from the top. Differential centrifugation for P3H and P3L were described elsewhere (Hayashi and Su, 2001).

View larger version (39K): [in this window] [in a new window]   Fig. 5. Separation of -1 receptor-containing ER-LDs and the c-LDs from NG108 cell homogenates by sucrose gradients. a, low-density vesicles enriched in -1 receptors exist in 15 to 25% sucrose fractions. Note the enrichment of caveolin-2 (Cav-2), but not ADRP, in the same fractions. -1 receptors were detected by anti-guinea pig -1 receptor antibody-A. b, enrichment of organelle markers in each respective fraction. PM, plasma membrane. Each protein was assessed by Western blottings using specific antibodies followed by densitometric analysis. c, moderate enrichment of neutral lipids and free cholesterol (FC) in -1 receptor-containing fractions. Lipids in each fraction were extracted and separated by TLC. TLC spots visualized by H2SO4 spray (and baking) were densitometrically analyzed. TG, triglycerides; CE, cholesterylesters. d, vesicles in the 20.5% sucrose fraction from Sig-1R-EYFP-transfected NG108 cells. Left, Nomarski image; right, overlaid image (Sig-1R-EYFP in green). e, vesicles from 0% sucrose fraction. Inset, higher magnification. Scale bar, 5 µm.

Western Blotting. NG108 cell lysates were prepared in sodium dodecyl sulfate sample buffer and separated by 12% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (%C = 1.5). Proteins were electrophoretically transferred on PVDF membranes at 30 V overnight (4°C) in Towbin buffer without methanol. We found that methanol severely disturbs -1 receptor transfer onto PVDF membranes. PVDF membranes were blocked with 10% nonfat milk for 6 h at 4°C and incubated over night at 4°C with anti-guinea pig -1 receptor antibody-A (1:1500) in Tris-buffered saline/Tween 20 containing 1.0% Nonidet P-40. Protein bands were visualized by an enhanced chemiluminescence kit (Amersham Biosciences Inc., Piscataway, NJ). For Western blotting of extracellular -1 receptors, NG108 cells on 10-cm dishes were washed with prewarmed HBSS twice and incubated in HBSS at 37°C in the presence of cocaine. Cell supernatants were collected and centrifuged at 3000g for 5 min to pellet cell debris. Proteins in supernatants were subjected to SDS-polyacrylamide gel electrophoresis. Nuclei were purified by the detergent-based nucleus purification system (Sigma-Aldrich). Proteins in cell supernatants and sucrose fractions were concentrated by trichloroacetic acid precipitation.

Time-Lapse Fluorescence Microscopy on Living Cells. NG108 cells expressing EYFP-tagged -1 receptors were grown on 15-mm poly-D-lysine/laminin-coated glass coverslips. A coverslip was placed on a closed-bath imaging chamber (chamber volume, 36 µl) and heater platform (Warner Instrument, Hamden, CT). Cells were perfused by HBSS at 32°C (0.3 ml/min) and examined using an inverted Axiovert 135 microscopy (Carl Zeiss, Oberkochen, Germany). Images were collected digitally every 10 or 15 s with the Image series software (Carl Zeiss). A water immersion 63x C-Apochromat objective lens (numerical aperture, 1.2; working distance, 0.24 mm; coverslip thickness, 0.14–0.18 mm; Carl Zeiss) was used. Fluorescence recovery after photobleaching (FRAP) analysis was performed as described elsewhere (Nehls et al., 2000; Reits and Neefjes, 2001). Briefly, living cells on a coverslip were continuously perfused as mentioned above. Sig-1R-EYFP in the defined region (2.0 x 2.0 µm²) was photobleached at a full laser power (2 s), and 1 s after photobleaching, the recovery of fluorescence was monitored by scanning the whole cell at a minimal power output. Operation of the confocal microscope and the data collection were carried out by using FRAP software (Carl Zeiss). Mobility fraction (Mf) and diffusion time (_D) were calculated according to the method described elsewhere (Reits and Neefjes, 2001).

TLC for Lipid Analysis. After fractionation of NG108 cell membranes, lipids in each obtained fraction were extracted according to methods described elsewhere (Hayashi and Su, 2003). Total protein content in each fraction was measured by bicinchoninic acid kit (Pierce Chemical, Rockford, IL). Lipid extracts were dissolved in chloroform/methanol (2:1) and separated on Silica Gel TLC plates (Merck, Billerica, MA) with hexane/ether/acetic acid (80:20:1). Plates were sprayed with the H₂SO₄ solution followed by charring at 110°C for 40 min. Spots were analyzed by NIH Image software.

Statistical Analyses. One-way analysis of variance followed by Fisher's protected least significant difference post hoc test was used (significance level at p < 0.05).

	Results

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Translocation of Endogenously Expressed -1 Receptors by (+)-Pentazocine in NG108 Cells. Endogenously expressed -1 receptors and Sig-1R-EYFP both localized in perinuclear areas as dense clusters (Fig. 1, a–d). Higher magnification indicated that -1 receptors and Sig-1R-EYFP were on "ring-like" ER-LD structures and accompanying endoplasmic reticulum tubular elements (Fig. 1b, inset) (Hayashi and Su, 2003). Sig-1R-EYFP-positive ER-LDs contained neutral lipids such as cholesteryl esters and triglycerides that are stained with Nile red (Fig. 1c). Enlargement of ER-LDs was seen in some Sig-1R-EYFP-overexpressing cells (Fig. 1d). (+)-Pentazocine decreased densities of -1 receptors in the perinuclear ER-LDs and concomitantly caused an even distribution of -1 receptors over the endoplasmic reticulum reticular structure (Fig. 1, e and f). We attempted to semiquantify the -1 receptor translocation caused by (+)-pentazocine (see Materials and Methods). Results show that -1 receptor translocation was seen in about 40% of cells even without any exogenous stimulation. This suggests a possibility that certain endogenous -1 receptor ligand(s) mediate the -1 receptor translocation. In the presence of (+)-pentazocine, -1 receptor translocation was seen in 70 to 90% of cells (Fig. 1g).

View larger version (71K): [in this window] [in a new window]   Fig. 1. Translocation of endogenously expressed -1 receptors in NG108 cells. NG108 cells were fixed, and antigen-retrieved, followed by staining with polyclonal anti-guinea pig -1 receptor antibody-B. a and b, perinuclear distribution of -1 receptors in cells without treatment (inset in b, -1 receptor-positive ring structures with tubular elements at a higher magnification). c and d, cellular localization of Sig-1R-EYFP similar to that of endogenous -1 receptors in NG108 cells. A single Sig-1R-EYFP-positive ER-LD stained with Nile red in c. Note the enlargement of ER-LDs by overexpression of Sig-1R-EYFP in d. e and f, (+)-pentazocine [(+)-PTZ; 100 nM, 10 min)-treated cells. Arrows in e indicate cells after -1 receptor translocation from ring structures (reticular diffused pattern instead of clustered bright spots). Scale bar, 10 µm. g, percentages of cells showing translocation of -1 receptors from the ring structures. (+)-Pentazocine was present in the culture medium (37°C) over the indicated period of experiment. Columns represent mean ± S.E. (N = 10–22). Translocation of -1 receptors (percentage) are significantly higher in all (+)-pentazocine-treated groups (at all concentrations and time points) compared with nontreated groups (p < 0.01).

Intracellular Dynamics of -1 Receptors in Living NG108 Cells. Movements of EYFP-tagged Sig-1R in living NG108 cells were monitored. Not all Sig-1R-EYFP moved. Some Sig-1R-EYFP, especially those clustered in ER-LDs, did not move. However, a portion of Sig-1R-EYFP apparently moved. They moved out from ER-LDs and slid on the endoplasmic reticulum reticular network even in the absence of -1 receptor ligands. Under confocal microscopy, only the movement from ER-LDs to endoplasmic reticulum tubular elements was observed (Fig. 2a). The reverse movement, if any, from tubular elements to ER-LDs was not seen, at least under the microscopic observation. -1 receptor agonists (+)-pentazocine and cocaine increased the mobility of Sig-1R-EYFP in the following manner: 1) clustered Sig-1R-EYFP moved anterogradely on a neurite toward the tip or varicosities (Fig. 2b); and 2) Sig-1R-EYFP moved along endoplasmic reticulum tubular elements toward the nuclear envelope (Fig. 2c). Interestingly, Sig-1R-EYFP reached at varicosities did not stay and accumulate at these loci (Fig. 2b, arrow 1). Because in NG108 cells vesicles (e.g., synaptic vesicles) are known to be exocytosed from varicosities and tips of neurites (Fried and Han, 1995), this result suggested a possibility that -1 receptors at these loci could be released to extracellular space and/or transported back toward cell body. To test this possibility, we examined the content of -1 receptors in the extracellular space after -1 receptor agonist stimulations (see Materials and Methods). Cocaine dose and time dependently caused increases in -1 receptors in the extracellular space as well as in the nucleus (Fig. 2d). These results are in agreement with the dynamic patterns of -1 receptors shown in Fig. 2, a and b, and are supportive of a possibility that at least a portion of -1 receptors could pass the plasma membrane and be exocytosed.

View larger version (82K): [in this window] [in a new window]   Fig. 2. Intracellular dynamics of Sig-1R-EYFP in living NG108 cells. a, Sig-1R-EYFP movement from a single ER-LD to an endoplasmic reticulum tubular element (arrow). b and c, movement of Sig-1R-EYFP in the presence of (+)-pentazocine (b; 100 nM) or cocaine (c; 10 µM). N, nucleus. Scale bars, 10 µm. Rectangular areas were monitored for time-lapse images in b and c. d, increases of -1 receptor protein levels by cocaine (10 min) in the extracellular space and nuclei (see Materials and Methods). Western blotting for -1 receptors was visualized by anti-guinea pig -1 receptor antibody-A.

FRAP Analysis. To further characterize dynamics of -1 receptors, FRAP analysis was performed. When fluorescent molecules are irreversibly photobleached in a small area of the cell by a high-powered focused laser beam, subsequent diffusion of surrounding nonbleached fluorescent molecules into the bleached area leads to a recovery of fluorescence. Thus, FRAP enables one to measure the mobility of fluorescent molecules on continuous membranes by monitoring fluorescence recovery in a photobleached area. When Sig-1R-EYFP on a single ER-LD were photobleached, no significant recovery of fluorescence was seen until at least 30 min after photobleaching (Mf = 9.0 ± 0.7% at 32°C; n = 4) (Fig. 3, a and c). Thus, the movement of -1 receptors from endoplasmic reticulum tubular elements into ER-LDs is highly restricted, consistent with the result in time-lapse monitoring that shows the movement of -1 receptors only from ER-LDs to endoplasmic reticulum tubular elements. Sig-1R-EYFP on endoplasmic reticulum tubular elements were however highly mobile (Mf = 76.3 ± 5.2%, diffusion time _D = 15.5 ± 1.4 s at 32°C; n = 4) (Fig. 3, b and c). These results suggest that mobility of -1 receptors in the ER-LDs is different from that at the endoplasmic reticulum tubular element and that certain mechanism may exist in regulating the lateral diffusion of proteins between these two endoplasmic reticulum subcompartments.

View larger version (69K): [in this window] [in a new window]   Fig. 3. FPAP analysis of Sig-1R-EYFP mobility in living NG108 cells. a and b, rectangular areas (a; single ER-LD, b; endoplasmic reticulum tubular element) were first photobleached. Images were obtained before photobleaching and at the indicated time points after photobleaching. Cells were perfused continuously with HBSS at 33°C. Fluorescence recovery was monitored every 10 s. Scale bars, 10 µm. c, degree and time course of fluorescence recovery for Sig-1R-EYFP in either ER-LDs or endoplasmic reticulum tubular elements (normalized to prebleach values).

-1 Receptor Translocation Assessed by Sucrose Gradient Fractionation. In a subcellular fractionation study using differential centrifugation, we found that endogenously expressed -1 receptors in NG108 cells are enriched in the light-density microsomal fraction (P3L) (Hayashi and Su, 2001). (+)-Pentazocine (100 nM for 10 min) caused a reduction of -1 receptors in P3L, but an increase in P1, P2, and heavy-density microsomal (P3H) fractions (Fig. 4). Here, we examined -1 receptor translocation more extensively by fractionating NG108 cell membranes in sucrose gradients. Furthermore, because -1 receptors localize specifically on ER-LDs (Hayashi and Su, 2003), but not on c-LDs, which are formed by neutral lipids budding from ER-LDs into cytosol (Murphy and Vance, 1999; Brown, 2001; van Meer, 2001), we wanted to successfully separate ER-LDs, c-LDs, and other ER membrane into different fractions. Therefore, sucrose gradients consisting of 13 fractions (0–53.4% sucrose) were used (see Materials and Methods). Results show that -1 receptors and caveolin-2, both shown to localize on ER-LDs in our previous study (Hayashi and Su, 2003), were enriched in 15 to 25% sucrose fractions (Fig. 5a). Adipocyte differentiation-related protein (ADRP), a c-LD protein (Brasaemle et al., 1997; Murphy and Vance, 1999), existed in the 0% sucrose fraction (Fig. 5a, third panel). The -1 receptor-enriched fractions did not contain any organelle marker proteins except a very low amount of CYP450R (Fig. 5b). Most of CYP450R (90%) were in 46.2 to 53.4% sucrose fractions, indicating that most endoplasmic reticulum tubular elements were in these heavy fractions (Fig. 5b). -1 receptor-enriched fractions (15–25% sucrose) contained moderate levels of neutral lipids and free cholesterol (Fig. 5c). The top fraction (ADRP-positive c-LD fraction) contained high levels of neutral lipids and free cholesterol (Fig. 5c). Sig-1R-EYFP-transfected NG108 cells were accordingly fractionated, and the resultant fractions were observed under fluorescence confocal microscopy. In the 20.5% sucrose fraction, vesicular particles varied in size and shape, but most of them contained Sig-1R-EYFP (Fig. 5d). No Sig-1R-EYFP was seen in the c-LD fraction. On the other hand, the top fraction (0% sucrose) contained round lipid droplets that are uniformed in size and shape (Fig. 5e). Together, these results confirmed a successful separation of ER-LDs, c-LDs, and other suborganelles using this 13-sucrose layer fractionation method.

View larger version (75K): [in this window] [in a new window]   Fig. 4. Effect of (+)-pentazocine on subcellular distribution of -1 receptors in NG108 cell membranes separated by differential centrifugation. Enrichment of -1 receptors in each fraction was analyzed by Western blotting using anti-guinea pig -1 receptor antibody-A. Twenty micrograms of total protein lysates was loaded in each lane. P3L, light-density microsome; P3H, heavy-density microsome; (+)-PTZ, treatment with 100 nM (+)-pentazocine for 10 min at 37°C.

With this method successfully established as shown above, we examined effects of (+)-pentazocine on the -1 receptor translocation. The treatment of NG108 cells with (+)-pentazocine (1 µM, for 30 min at 37°C) caused a significant decrease of -1 receptors in the ER-LD fraction, but an increase of -1 receptors in heavier fractions containing endoplasmic reticulum tubular elements (Fig. 6). Importantly, (+)-pentazocine did not cause any significant change of -1 receptors in the c-LD-containing fractions, suggesting that -1 receptors on ER-LDs translocate to endoplasmic reticulum tubular network but not to c-LDs.

View larger version (10K): [in this window] [in a new window]   Fig. 6. Translocation of -1 receptors from ER-LDs to high-density fractions caused by (+)-pentazocine. (+)-Pentazocine [(+)-PTZ; 1 µM, 30 min at 37°C] caused a decrease of -1 receptors in ER-LD-enriched fractions (3–5) as shown by sucrose density gradients (see Fig. 5). Note a concomitant increase of -1 receptors in fractions 11 and 12. -1 receptors were detected by Western blottings using anti-guinea pig -1 receptor antibody-A.

	Discussion

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We reported previously (Hayashi and Su, 2003) and now that endogenous -1 receptors as well as transfected Sig-1R-EYFP localize mainly on the endoplasmic reticulum. Although several other studies suggest the existence of -1 receptors on both endoplasmic reticulum and plasma membrane (McCann and Su, 1990; Ramamoorthy et al., 1995; Alonso et al., 2000), we could not detect significant levels of -1 receptors on the plasma membrane. It is also reported that -1 receptors localize on the plasma membrane when expressed by gene transfection in oocytes (Aydar et al., 2002). A plausible explanation may be that the subcellular localization is different between cell types and/or stages of cell differentiation. Alternatively, -1 receptors might move between endoplasmic reticulum and plasma membrane, but cannot stay and accumulate on the plasma membrane in NG108 cells used in this study. Our results showing that -1 receptors could be detected in the extracellular space after an agonist-stimulation (Fig. 2d) suggest that at least a portion of -1 receptors can reach plasma membranes.

-1 receptors localized on both ER-LDs and endoplasmic reticulum tubular network, but are predominantly abundant on ER-LDs. The real-time monitoring of Sig-1R-EYFP in living cells indicates that -1 receptors translocate from ER-LDs to the endoplasmic reticulum tubular network. Because -1 receptors are significantly decreased in low-density microsomes by the treatment with (+)-pentazocine, and because -1 receptors are membrane proteins, we previously speculated that a vesicle transport might be involved in the -1 receptor translocation (Hayashi and Su, 2001). However, our present data show that -1 receptor translocate on continuous endoplasmic reticulum structures through lateral movements and not via vesicular translocation after budding processes. But, on the other hand, we also observed an accumulation of Sig-1R-EYFP caused by (+)-pentazocine on the plasmalemmal cortices, which consist of cytoskeleton lattice (data not shown). Furthermore, Sig-1R-EYFP disappeared from the plasmalemmal corticies when fixed cells were permeabilized with a detergent (0.5–0.2% Triton X-100) in immunocytochemical studies. Interestingly, the unique cytosolic transport vesicle "argosomes" that are exocytosed, presumably for cell-to-cell communication, have also been shown to disappear after a similar membrane permeabilization procedure (Greco et al., 2001). Therefore, it remains possible that because of their proximity to the plasma membrane, -1 receptors may be separated from endoplasmic reticulum membrane as vesicles that could be exocytosed (Fig. 2d).

The translocation of -1 receptors on the endoplasmic reticulum structure may not be simply due to a lateral diffusion of -1 receptors on phospholipid bilayers. Translocation of -1 receptors is regulated by specific ligands and temperature (data not shown), and the translocation directions are vectorial (i.e., one-way toward plasma membrane and nuclear membrane). Because -1 receptors are associated with ankyrin (Hayashi and Su, 2001), a cytoskeleton adaptor protein, it is tempting to speculate that cytoskeletal filaments might be involved in directing the intracellular translocation of -1 receptors.

Dynamics of -1 receptors at the ER-LDs and at the endoplasmic reticulum reticular network are apparently different. The FRAP data suggest that mobility of -1 receptors from the endoplasmic reticulum reticular network to ER-LDs is highly restricted. After a complete photobleaching of Sig-1R-EYFP fluorescence in a single ER-LD, no significant recovery of Sig-1R-EYFP fluorescence could be seen until at least 30 min thereafter, indicating that -1 receptors on endoplasmic reticulum reticular network cannot move back into ER-LDs, or very slowly, if any. This result is consistent with our previous observation that once -1 receptor translocate, it takes more than 1 h, after removal of -1 receptor agonist, for -1 receptors to return to normal levels at the original loci (Su and Hayashi, 2001). At present, we do not know what regulates protein movements between ER-LDs and other endoplasmic reticulum structures. However, in a recent study, we found that -1 receptors in ER-LDs form raft-like microdomains enriched in cholesterol (Hayashi and Su, 2003). Lipid membrane fluidity on ER-LDs should be lower than other ER membranes due to an enrichment of cholesterol on the ER-LDs (Barenholz, 2002). Therefore, the mobility of -1 receptors might be highly restricted in the so-called "liquid-ordered" phase of lipid raft membranes (Simons and Toomre, 2000), whereas -1 receptor mobility in other areas of the endoplasmic reticulum membrane is not.

-1 receptors have been shown in several reports to be present in microsomes, suggesting that they are on the endoplasmic reticulum (McCann and Su, 1990; Hayashi and Su, 2001). However, McCann and Su (1990) demonstrated that when brain membranes are further fractionated by a sucrose density gradient, -1 receptors are present in a unique fraction different from those containing either plasma membrane marker or endoplasmic reticulum marker. Our present results from immunocytochemistry and sucrose fractionation studies confirmed that -1 receptors locate on the specialized area of endoplasmic reticulum membranes (also see Hayashi and Su, 2003). It is very likely that -1 receptor-containing membranes have a lower density than that of other endoplasmic reticulum membranes due to the enrichment of neutral lipids and cholesterol. Therefore, -1 receptor-containing membranes can be separated from other endoplasmic reticulum membranes in the sucrose gradient centrifugation.

Newly synthesized neutral lipids (e.g., cholesteryl esters and triglycerides) are stored at ER-LDs and eventually bud out to form c-LDs (Murphy and Vance, 1999; van Meer, 2001). It is known that in adipocytes and steroidogenic cells the neutral lipid mass in matured c-LDs is regulated by specific c-LD proteins and other receptor-mediated signal transductions (Londos et al., 1999). The activation of adrenaline or insulin receptors causes the protein kinase A activation and a subsequent phosphorylation of perilipin and hormone-sensitive lipase (Londos et al., 1999). The phosphorylation causes translocation of perilipin and hormone-sensitive lipase, resulting in the facilitation of lipolytic reaction and changes in the neutral lipid mass in c-LDs. However, mechanisms that regulate the export of neutral lipids from ER-LDs to c-LDs and the formation of c-LDs are totally unknown. In this study, we found that a selective -1 receptor agonist (+)-pentazocine apparently causes translocation of -1 receptors from ER-LDs. It is plausible that the level of -1 receptors on ER-LDs might affect the ER-LD membrane environment that in turn affects compartmentalization of neutral lipids and their export at the endoplasmic reticulum (Hayashi and Su, 2003). Translocation of -1 receptors thus may have a significant impact on biological functions at the endoplasmic reticulum. In fact, in our recent study, we found that transfection of a functional negative -1 receptors alters the homeostasis of lipids and the morphology of the endoplasmic reticulum in NG108 cells (Hayashi and Su, 2003).

In summary, our present study demonstrates that -1 receptors target unique lipid-enriched sites on the endoplasmic reticulum (ER-LDs) and translocate to the endoplasmic reticulum-associated reticular network upon stimulation by psychoactive drugs. Furthermore, this translocation of -1 receptors uses lateral movement on the continuous endoplasmic reticulum network to reach peripheries of cells, apparently not involving vesicle buddings from ER-LDs. Our data, on the other hand, raise some questions that should be addressed in the future: 1) why do -1 receptors translocate even in the absence of exogenous -1 receptor ligands (endogenous activators?); and 2) what are the translocated -1 receptors doing at the periphery of cells? Because -1 receptors have a high homology to a fungal sterol C8-C7 isomerase (Hanner et al., 1996), some reports speculate that -1 receptors act as an enzyme in cholesterol metabolism (Moebius et al., 1997; Labit-Le Bouteiller et al., 1998). Indeed, a number of enzymes involved in lipid metabolisms are located on the smooth endoplasmic reticulum similar to -1 receptors (Koning et al., 1996; Khelef et al., 1998). However, most well characterized cholesterol-metabolizing enzymes are on the endoplasmic reticulum reticular network (Koning et al., 1996; Khelef et al., 1998), suggesting that -1 receptors might not be cholesterol-metabolizing enzymes. Biological roles of ER-LDs may be intimately related to the molecular function of -1 receptors. Because -1 receptors specifically target lipid storage sites on the endoplasmic reticulum, our results with the dynamic translocation of -1 receptors indicate that in neuronal cells -1 receptors might affect lipid transport on the endoplasmic reticulum as well as on the Ca²⁺ signaling as previously reported by us (Hayashi and Su, 2001). Sig-1R ligands such as neurosteroids and cocaine may thus modulate plasma membrane remodeling, including promotion of raft formation and synaptogenesis by affecting lipid mobilization at the endoplasmic reticulum. Thus, a demonstration of -1 receptor involvement in the formulation of cholesterol/sphingolipid-associated rafts, in other neuronal systems such as primary cultures, would be critical in advancing understanding on the role of -1 receptors in the central nervous system and perhaps other central nervous system-related behaviors or diseases such as addiction and cholesterol/lipid-related psychiatric or neurological disorders.

	Acknowledgements

 
We thank Dr. V. Ganapathy for a generous gift of the pSPORT1--1 receptor vector.

	Footnotes

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

DOI: 10.1124/jpet.103.051292.

ABBREVIATIONS: IP₃, inositol 1,3,5-trisphosphate; ER, endoplasmic reticulum; Sig-1R-EYFP, C-terminally enhanced fluorescent yellow protein-tagged -1 receptors; ER-LD, endoplasmic reticulum-associated lipid droplet; CYP450R, NADPH-cytochrome P450 reductase; HBSS, Hanks' balanced salt solution; c-LD, cytosolic lipid droplet; PVDF, polyvinylidene difluoride; FRAP, fluorescence recovery after photobleaching; Mf, mobility fraction; TLC, thin layer chromatography; ADRP, adipocyte differentiation-related protein.

Address correspondence to: Dr. Tsung-Ping Su, Cellular Pathobiology Unit, Cellular Neurobiology Research Branch, TRIAD Bldg., 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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