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Ernest Gallo Clinic and Research Center, University of California, San Francisco, California, USA
2Correspondence: Ernest Gallo Clinic and Research Center, 5858 Horton St., Ste. 200, Emeryville, CA 94608, USA. E-mail: jennifer.whistler{at}ucsf.edu
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Key Words: degradation • GPCR • postendocytic sorting • tolerance
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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. Cannabinoid receptors (CBR) belong to the most prevalent 7TM receptor family (i.e., the family A receptors) and have at least two deorphanized members, the CB1 and CB2 receptors. Although additional members have been proposed (2
, 3)
, little is known of their function. The CB1R is one of the most abundant 7TM receptors in the central nervous system (CNS) and is expressed at high levels in cerebral cortex, hippocampus, and striatum (4
, 5)
. It plays an important role in several pathways, including pain transmission, feeding, and the rewarding effects of drugs like alcohol, tobacco, and cannabis (6
7
8
9)
. CB1Rs couple primarily to Gi/o proteins, which in turn inhibit cAMP accumulation and selective types of calcium channels, and activate mitogen-activated protein (MAP) kinases and a subset of potassium channels (10)
. When expressed in cell lines or in neuronal tissue, CB1Rs display some constitutive activity (i.e., ligand-independent receptor activation) (reviewed in ref. 11
). CB1Rs are further activated by endocannabinoids such as anandamide and by exogenously administered compounds like the synthetic agonist WIN55,212–2 [(R)-(+)-[2,3-dihydro-5-methyl-3-(4-morpholinylmethyl)pyrrolo[1,2,3-de]-1,4-benzoxazin-6-yl]-1-naphthalenylmethanone] or the naturally occurring delta(9)-THC [delta(9)-tetrahydrocannabinol], the psychoactive ingredient of cannabis. The response of many 7TM receptors to drugs fades after prolonged exposure, which often is referred to as tolerance; as a result, increased doses of the drug are required to achieve the same physiological effect. At the molecular level at least three principal mechanisms can contribute to the development of tolerance to 7TM receptor ligands. These include 1) homologous receptor desensitization, 2) receptor down-regulation, and 3) heterologous desensitization. Homologous receptor desensitization is characterized by an uncoupling of the receptor from its G-proteins. Receptor down-regulation involves a loss of receptor number caused by either receptor degradation or decreases in receptor synthesis, which results in fewer available ligand binding sites. Heterologous desensitization is a disruption of signaling pathways in the cell that decreases the effectiveness of the agonist-occupied receptor to regulate the level of second messengers, even though receptor/G-protein coupling and receptor number may remain unchanged. Any or all of these mechanisms can contribute to tolerance to a specific drug-receptor pair.
After activation, most 7TM receptors are desensitized and endocytosed. Subsequently, the receptors are either recycled back to the membrane—thus ready for a new encounter with a ligand—or degraded in lysosomes. As a consequence, the postendocytic fate of a receptor after exposure to a certain drug can determine what role internalization plays in modulating receptor availability and thereby signal transduction. Thus, elucidating the trafficking properties of a receptor can, by extension, provide insight into the mechanism(s) responsible for drug tolerance. For receptors that are recycled, endocytosis would not contribute to receptor down-regulation and would, in fact, promote resensitization of receptor function, possibly preventing tolerance. On the other hand, for receptors that are degraded, endocytosis would serve as the first step toward receptor down-regulation and perhaps promote tolerance.
Numerous reports have described the development of tolerance after chronic treatment with cannabinoids, including WIN55,212–2, delta(9)-THC and CP55,940 (12
13
14
15
16)
. When administered in vivo, anandamide is metabolized within minutes (17)
, which might explain why long-term treatment with this agonist has not been shown to promote tolerance. While the specific mechanism mediating tolerance to cannabinoids remains unresolved, several studies have found CB1 receptor down-regulation after chronic administration (13
14
15
16
, 18
19
20
21
22
23)
. In vivo, it is unknown whether this down-regulation of receptor number is mediated at the level of gene expression or receptor degradation. However, Northern blot and in situ hybridization studies have shown that mRNA levels of CB1R either remained constant or increased in most brain regions (16
, 23
, 24)
.
It is well-established that a broad range of cannabinoids induce receptor internalization (25
, 26)
. If the CB1R were targeted for degradation after endocytosis, it is reasonable to hypothesize that down-regulation of the CB1R and, by extension, tolerance to cannabinoids, is mediated, at least in part, by selective postendocytic sorting of the receptor to the degradative pathway. Here we show that treatment of the CB1R with either anandamide or WIN55,212–2 results in receptor degradation and that the G-protein-coupled receptor-associated sorting protein (GASP1) (27)
plays a major role in the postendocytic sorting of CB1R to the degradative pathway.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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, 28)
. Mouse monoclonal LAMP1 and LAMP2 antibodies developed by J. T. August and J. E. K. Hildreth were from the Developmental Studies Hybridoma Bank developed under the auspices of the NICHD and maintained by the University of Iowa, Department of Biological Sciences, Iowa City, IA, USA). Anandamide, WIN55,212–2 mesylate, and AM251 were purchased from Tocris Bioscience (Ellisville, MO, USA) and dissolved in either ethanol or DMSO. Protein A Sepharose and 35S-methionine were purchased from Amersham Biosciences (GE Healthcare, NJ, USA).
Cell culture and generation of constructs
Human embryonic kidney (HEK293) cells (American Type Culture Collection, Rockville, MD, USA) were grown in Dulbecco’s modified Eagle medium (DMEM) (Gibco BRL, Carlsbad, CA, USA) supplemented with 10% FBS (HyClone, Logan, UT, USA). A signaling peptide sequence/FLAG epitope (MKTIIALSYIFCLVFA/DYKDDDDA) was fused N-terminally to the human cannabinoid 1 receptor (CB1R) and inserted in the pcDNA3.1 (+) vector. The CB1R and a GFP-cGASP construct (27)
were then either transiently or stably expressed in HEK293 cells after transfection with Fugene6 (Roche, Basel, Switzerland) according to the manufacturer’s instructions. To generate clonal stable HEK293 cell lines, single colonies were chosen and propagated in appropriate selection media (0.8 µg/ml Zeocin 228, 0.5 mg/ml Geneticin®).
Immunocytochemistry
Recycling experiments for CB1R
Cells stably expressing CB1R were grown on coverslips treated with poly-D-lysine (Sigma-Aldrich, St. Louis, MO, USA) to 
50% confluency and fed M1 antibody directed against the FLAG epitope (1:1000, 30 min). After incubation with ligand, residual surface receptors (those not internalized by agonist) were stripped of antibody by washing twice in 0.04 mM PBS-EDTA (the M1 interaction is calcium sensitive). Cells were then treated with the inverse agonist AM251 for 60 min (Fig. 1
A) or 120 min (Fig. 1B
) to assess receptor recycling and fixed in 4% formaldehyde in PBS for 20 min. The cells shown in Fig. 1A, B
(left panels) were then permeabilized in blotto for 30 min (3% milk, 0.1% Triton X-100, 50 mM Tris·HCl, pH 7.5) and stained with Alexa Fluor 488 goat anti-mouse IgG2b antibody (1:500, 20 min). Coverslips shown in Fig. 1B
(center and right panels) were immunostained in the absence of TritonX-100, then mounted using 4',6'-diam idino-2-phenylidole (DAPI) -containing mounting-media from Vectashield.
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Recycling experiments for CB1R and cGASP
As described above, except that CB1R was stained with Alexa Fluor 594 goat anti-mouse IgG2b antibody.
Colocalization of CBIR with transferrin
Cells stably expressing CB1R were grown on coverslips treated with poly-D-lysine (Sigma-Aldrich) to 50% confluency and serum-starved for 60 min prior to incubation with M1 antibody directed against the FLAG epitope (1:1000, 30 min). The cells were then treated with ligand for 30, 90, or 180 min and incubated with Texas Red-conjugated transferrin (1:500) for the last 30 min. This was followed by fixation in 4% formaldehyde in PBS, permeabilization in blotto, and staining with Alexa Fluor 488 goat anti-mouse IgG2b antibody (1:500, 20 min).
Colocalization of CB1R with LAMP
HEK293 cells stably expressing CB1R were grown on coverslips treated with poly-D-lysine (Sigma-Aldrich) to 50% confluency and incubated with M1 antibody (IgG2b) directed against the FLAG epitope (1:1000, 30 min). The cells were treated with ligand for 30, 90, or 180 min and fixed in 4% formaldehyde in PBS. The cells were then permeabilized in blotto and incubated with monoclonal antibodies directed against LAMP1 and LAMP2 (IgG11:500, 45 min), followed by staining with subtype selective antibodies: Alexa Fluor 488-conjugated IgG2b against CB1R and Alexa Fluor 594-conjugated IgG1 against LAMP1 and LAMP2 (1:500, 20 min).
Colocalization of CB1R and cGASP with LAMP
HEK293 cells stably expressing CB1R were transfected with GFP-cGASP using Fugene6 (Roche, Basel, Switzerland) according to the manufacturer’s instructions and plated on coverslips treated with poly-D-lysine (Sigma-Aldrich). The coverslips were treated essentially as described above, except using Alexa Fluor 594-conjugated IgG2b against CB1R and Alexa Fluor 633-conjugated IgG1 against LAMP1 and LAMP2 (1:500, 20 min) to allow visualization of GFP-cGASP in the green channel. Confocal microscopy images in the merged field were subsequently pseudo-colored green (receptor) and red (LAMP) in Adobe Photoshop CS.
Generation of primary striatal culture
Striatum was dissected from newborn rats (P1), placed in saline with papain for 45 min at 37°C, and rinsed in buffer containing 2.5 mg/ml trypsin inhibitor. Cells were then dissociated and plated at a density of 106 cells/ml on poly-D-lysine (Sigma-Aldrich) -treated coverslips and in culture slides (Becton Dickinson, MA, USA) in neurobasal media supplemented with B27 (Invitrogen). Neurons were fed with fresh neurobasal media on days 7 and 14.
Immunocytochemistry of primary striatal culture
Primary striatal neurons at 80% confluency were fixed in PBS containing 4% formaldehyde and 4% sucrose for 15 min. The cells were permeabilized in PBS with 0.1% Triton X-100 for 20 min, blocked in 5% normal donkey serum for 40 min, incubated with goat anti-CB1R antibody (1:100) and rabbit anti-GASP1 antibody (1:250) for 2 h, then with Alexa 594-conjugated donkey anti-goat (1:100) and Alexa 488-conjugated donkey anti-rabbit (1:250) antibodies for 1 h. The CB1R and GASP signals were blocked by preincubation of the primary antisera with their respective purified immunizing proteins. The coverslips incubated under these conditions were mounted using Vectashield mounting media containing DAPI to stain cell nuclei.
Immunocytochemistry of rat brain sections
Adult rats were deeply anesthetized with halothane and perfused with 4% paraformaldehyde in phosphate buffer; brains were sectioned into 40 µm-thick sagittal sections using a Vibratome (Leica Microsystems Inc.; Bannockburn, IL, USA). Single or double immunofluorescence of free-floating sections was performed using rabbit anti-GASP (1:1000) and/or goat anti-CB1R antibody (1:500, Santa Cruz Biotechnology). Secondary fluorescent antibodies were Alexa 594-conjugated donkey anti-goat (1:500) and Alexa 488-conjugated donkey anti-rabbit (1:500) antibodies (Invitrogen) or donkey anti-rabbit Cy-3 (1:250, Jackson ImmunoResearch Laboratories, Inc. West Grove, PA, USA). Sections were mounted on gelatinized slides with Vectashield mounting media (Vector labs, Burlingame, CA, USA) containing DAPI.
Microscopy
Mounted slides were examined using a 63x oil objective mounted on an LSM 510 laser confocal microscope (Zeiss, Thornwood, NY, USA).
Biotin protection degradation assay (BPA)
HEK293 cells stably expressing CB1R either alone or in combination with GFP-cGASP were grown to confluency in poly-D-lysine pretreated 10 cm plates. Before the experiment, a subset of plates was pretreated with 200 µM chloroquine (Sigma-Aldrich) dissolved in water at 37°C for 60 min. Next, all cells were treated with 3 µg/ml disulfide-cleavable biotin (Pierce, Rockford, IL, USA) at 4°C for 30 min, washed in TBS (137 mM NaCl, 25 mM Tris-base, 3 mM KCl, 1 mM CaCl2), placed in DMEM with or without the presence of 200 µM chloroquine, and treated with ligand (10 nM–1 µM WIN55,212–2) or vehicle (DMSO) for the periods specified. Fresh ligand was added every 60 min. Concurrent with ligand treatments, the 100% and strip plates remained at 4°C. All plates were washed in PBS, and except for the 100% plate, the remaining cell surface biotinylated receptors were removed in strip buffer (50 mM glutathione, 0.3 M NaCl, 75 mM NaOH, 1% FBS) at 4°C for 30 min. All plates were then quenched in buffer containing 9 mg/ml iodoacetamide and 10 mg/ml BSA at 4°C for 20 min, followed by cell lysis in buffer (150 mM NaCl, 25 mM KCl, 10 mM Tris·HCl, 0.1% Triton X-100, pH 7.4) with added protease inhibitors (Complete, Roche, Basel, Switzerland) and 1 mg/ml iodoacetamide. Cellular debris was removed by centrifugation at 10,000 x g at 4°C for 10 min, then lysates were immunoprecipitated with anti-FLAG M2 antibodies linked with rabbit anti-mouse linker antibodies to protein A Sepharose beads at 4°C, washed extensively, and treated with PNGaseF at 37°C for 1 h. Samples were denatured in SDS sample buffer with no reducing agent added, resolved by SDS/PAGE using 4–20% Tris-glycine precast gels (Invitrogen), transferred to nitrocellulose membrane, overlaid with streptavidin (Vectastain ABC immunoperoxidase reagent; Vector Laboratories, Burlingame, CA, USA), and finally developed with enhanced chemiluminescence (ECL) plus reagents (Amersham). For quantification, data generated on five independent blots were analyzed using Scion Image software (Scion Corp., Frederick, MD, USA), which was corrected for background (signal in strip-lane) and where treatment with WIN55,212–2 for 30 min was designated as 100% signal. For statistical analysis, unpaired t test, two-tailed P value was performed using Graphpad Prism version 4.00 (San Diego, CA, USA). Anandamide was not suited for analysis via BPA, most likely because the biotinylating reagent modifies all accessible lysine residues, including one recognized by the ligand (29)
.
Determination of cGASP to GASP ratio
To analyze levels of GFP-cGASP relative to GASP1, 20 µl of the 800 µl lysate from a biotinylated 100% plate (shown in Fig. 5B
) was diluted in lysis buffer, denatured in reducing sample buffer, resolved on 4–20% Tris-glycine precast gels (Invitrogen), and electroblotted onto nitrocellulose membrane. The blot was incubated with rabbit anti-GASP1 antibody (1:1000, 1 h), then with IRDye800-conjugated goat anti-rabbit antibody (1:10,000, 1 h), and visualized and quantified using the Licor Odyssey Infrared Imaging System (Lincoln, NE, USA).
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Glutathione S-transferase (GST) pull-down
The last 14 amino acids of the human CB1R and 21 amino acids of human delta opioid receptor (DOP-R) were subcloned into the pGEX-4T-1 expression vector (Amersham), expressed in Escherichia coli strain BL21 bacteria, and bound to glutathione-agarose. GASP1 was in vitro translated and labeled with 35S-methionine using the TNT T7-Coupled Reticulocyte Lysate System (Promega, Fitchburg, WI, USA). The GST fusion proteins were tested for binding to GASP1 for 60 min at room temperature, then the probes were eluted in sample buffer, resolved by SDS-PAGE (SDS/PAGE) in Tris-glycine buffer, and developed by autoradiography.
Coimmunoprecipitation from HEK293 cells
HEK293 cells or HEK293 cells stably expressing CB1R were grown to confluency, washed twice with PBS, and lysates were prepared in buffer (150 mM NaCl, 25 mM KCl, 10 mM Tris·HCl, 0.1% Triton X-100, pH 7.4) with protease inhibitors added (Complete, Roche Diagnostics). Cleared lysates were incubated with M2 anti-FLAG affinity resin at 4°C for 1 h, washed extensively, deglycosylated with PNGaseF, and eluted in reducing sample buffer. Precipitates were resolved on 4–20% Tris-glycine precast gels (Invitrogen) and electroblotted onto polyvinylidene fluoride membrane. The blots were cut below the 75 kDa marker band to separately immunoblot for either receptor or GASP1. GASP1 blots were first incubated with rabbit anti-GASP1 antibody (1:1000, 1 h), then with HRP-conjugated anti-rabbit antibody (1:4000, 1 h). Receptor blots were first incubated with BioM2 antibody (1:250, 1 h), then with streptavidin overlay (Vectastain ABC reagents, Vector Laboratories). Both blots were finally visualized with ECL plus reagent (Amersham).
Coimmunoprecipitation from rat brain
Rat brain synaptosomes were prepared from adult rats as described (30)
in lysis buffer (150 mM NaCl, 25 mM KCl, 10 mM Tris·HCl, pH 7.4, with added protease inhibitors (Sigma-Aldrich) and Complete (Roche, Basel, Switzerland) supplemented with 0.1% Triton X-100. Cleared lysates were incubated with protein G-agarose beads coated with either rabbit anti-rat CB1R antibody (5 µg/ml, Chemicon AB5636) or 1 mg/ml BSA (Sigma-Aldrich) at 4°C for 2 h, washed extensively with lysis buffer containing 1% Triton X-100, then deglycosylated with PNGaseF for 2 h. CB1R was eluted by addition of CB1R blocking peptide (Cayman Chemical Company, MI, USA); sample buffer was added and boiled at 95°C for 5 min, resolved on 4–20% Tris-glycine precast gels (Invitrogen), and transferred to nitrocellulose membrane. The blots were cut below the 75 kDa marker band and immunoblotted separately for either GASP1 or receptor. First, the GASP1 blots were incubated with rabbit anti-GASP1 antibody (1:1000, 1 h) and the receptor blot was incubated with anti-CB1R antibody (1:1500 1 h). This was followed by incubation with HRP-conjugated anti-rabbit antibody (1:4000, 1 h), and finally visualized with ECL plus reagent (Amersham).
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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, but also accumulates intracellularly in the absence of ligand. This has been attributed to the ability of the CB1R to constitutively internalize (31)
. We incubated human embryonic kidney (HEK293) cells stably expressing CB1R, modified with an N-terminal signal sequence and epitope tag, with M1-antibody for 30 min to specifically label surface receptors. This assay thereby eliminates any receptors that are retained in the secretory pathway from the analysis and therefore selectively follows the movement of receptors that have reached the plasma membrane. Utilizing this assay, we observed receptors both on the surface and intracellularly in the absence of agonist (Fig. 1A, no
treatment), which supports previous findings suggesting that the CB1 receptor is constitutively internalized. Incubation with the endogenous agonist anandamide (1 µM) resulted in prominent internalization of the receptor (Fig. 1A
, anandamide 90 min), whereas the inverse agonist AM251 efficiently prevented internalization (Fig. 1A
, anandamide+AM251 90 min). Receptors in cells first treated with anandamide, then with AM251, remained intracellular, suggesting that the CB1R did not recycle after internalization (Fig. 1A
, anandamide 90 min, strip, AM251 60 min). Incubation with the synthetic agonist WIN55,212–2 (1 µM) resulted in an intracellular distribution pattern of the CB1R similar to that observed with anandamide. The receptors internalized by WIN55, 212–2 were also resistant to recycling (Fig. 1A
, lower panels).
Since previous reports have described recycling of the CB1R both in the absence of agonist stimulation and after stimulation with WIN55,212–2 (25)
, we tested whether the failure of the CB1 receptor to efficiently recycle was agonist concentration- and/or time-dependent (Fig. 1B
and data not shown). In good agreement with previous findings, we observed efficient recycling of the CB1 receptor, as evidenced by increased cell surface staining, after exposure to low concentrations (0.1 µM) of WIN55,212–2 for a short duration (20 min), followed by 2 h recovery in AM251-containing media (Fig. 1B
, middle panels). Recycling was also observed in the absence of agonist at a 20 min point (Fig. 1B
, top panels). However, the CB1Rs failed to recycle after a short incubation (20 min), with a higher concentration of WIN55,212–2 (1 µM, Fig. 1B
, lower panels). Furthermore, CB1 receptors failed to efficiently recycle after agonist incubation for 90 min at all WIN55, 212–2 concentrations tested (0.1–1 µM) (data not shown, but see Fig. 2
C). Together, these data suggest that prolonged exposure of CB1 receptors, even to low doses of agonist, or short exposure to high doses of agonist target the CB1 receptors out of the recycling pathway.
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Consistent with this observation, CB1Rs treated with 1 µM WIN55,212–2 showed little colocalization with transferrin, a marker of recycling endosomes (Fig. 2A
). In contrast, the CB1R showed substantial time-dependent colocalization with the lysosomal markers LAMP (lysosomal-associated membrane protein)1 and LAMP2. These data suggest that the CB1R is targeted to lysosomes after agonist stimulation, where it is likely to be degraded.
To monitor the postendocytic fate of the CB1R, we utilized the biotin protection assay, which specifically follows the stability of internalized receptors. Cells stably expressing CB1Rs were initially treated with a cell-impermeable biotinylating agent to label surface-expressed receptors, then incubated with WIN55,212–2 for various times. The cells were then stripped of remaining surface-associated biotin and receptors were immunoprecipitated. Finally, internalized "protected" biotinylated receptors were detected using a streptavidin overlay. As was observed in Fig. 1A
, CB1Rs showed some constitutive internalization (Fig. 2B
, lane 3), but internalization was substantially enhanced after treatment with WIN55,212–2 (Fig. 2B
, lane 4). The pool of receptors that was internalized by the agonist was substantially degraded during prolonged agonist treatment (Fig. 2B
, lanes 5, 6). Furthermore, receptor proteolysis could be reduced by simultaneous treatment with the lysosomotropic agent chloroquine (Fig. 2B
, right panel). Postendocytic targeting of the CB1 receptor to the degradative pathway, though time dependent (see Fig. 1B
), was not dose dependent. Although lower doses of WIN55, 2212–2 promoted less receptor internalization (Fig. 2C
, compare 30' time points across doses), the pool of receptors internalized in the presence of 10, 100, or 1000 nM WIN55,212–2 were substantially degraded after 3 h of agonist exposure (Fig. 2C
, compare 30' to 180' at each dose).
GASP1 modulates CB1R trafficking
GASP1 has been shown to interact with the cytoplasmatic tail of many 7TM receptors (32
, 33)
. It shows preferential binding for degrading members of the opioid receptor (27)
and the dopamine receptor families (28)
, suggesting that this interaction could facilitate degradation of 7TM receptors. We thus examined whether degradation of the CB1R was modulated by GASP1. Indeed, GASP1 interacted with a GST fusion protein containing the CB1R tail (Fig. 3
A). In addition, full-length CB1R coimmunoprecipitated with endogenous GASP1 in HEK293 cells (Fig. 3B
).
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In previous studies (27
, 28)
, a C-terminal fragment of GASP1, cGASP, has been used as a dominant-negative to compete with full-length GASP1 for receptor binding, thus changing the cellular sorting of receptors. If indeed GASP1 mediated targeted degradation of the CB1R, we would expect cGASP to likewise alter its postendocytic sorting. To examine this possibility, CB1Rs in adjacent cells that either expressed only endogenous GASP1 or overexpressed cGASP were examined for lysosomal targeting and receptor recycling.
CB1Rs in cells overexpressing cGASP displayed reduced lysosomal colocalization compared with cells expressing the CB1 receptor alone (Fig. 4
A). To investigate whether reduced lysosomal targeting led to coincidental facilitated receptor recycling, cells were incubated with the inverse agonist AM251 after agonist treatment and recovery of receptors at the surface was monitored. Cells coexpressing CB1R and cGASP did indeed show enhanced surface recovery of the receptor compared with cells expressing only CB1R (Fig. 4B
), suggesting that overexpression of cGASP promoted the redirection of internalized receptors to the recycling pathway. In summary, these data suggest that the CB1R degrades after incubation with WIN55,212–2 and that GASP1 is a key cellular mediator in this postendocytic sorting process.
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We next quantified the effect of the dominant-negative cGASP on postendocytic CB1R degradation. Cells stably expressing CB1Rs and overexpressing cGASP in 100x excess to endogenous GASP1 (Fig. 5
A) were generated. Biotin protection assays performed on these cells demonstrated that the CB1Rs in cells overexpressing cGASP showed significantly delayed receptor degradation during WIN55,212–2-exposure compared with CB1Rs in cells expressing CB1R alone (Fig. 5B
). These effects of cGASP were observed in three different cell lines and are quantified in Fig. 5C
. These data further support the idea that trafficking of the CB1R after internalization is directed to the degradative pathway in a GASP1-dependent manner. Thus, we have identified a mechanism that can contribute to down-regulation of CB1Rs in vitro.
Development of tolerance to cannabinoids has been widely described in vivo with concomitant findings of receptor down-regulation. If CB1R and GASP1 were colocalized in vivo, this could provide a molecular mechanism to promote CB1R down-regulation. Both the CB1R (4
, 5
, 34)
and GASP1 (35)
are widely expressed in the CNS. The striatum is the major input nucleus of the basal ganglia, which forms an important pathway controlling locomotion. The CB1R is highly expressed within the striatum and its projection nuclei (5
, 36)
, and therefore may be a target for the inhibitory motoric actions of cannabinoids. Furthermore, the striatum forms a critical region in rewarding circuits, and previous findings have shown that the CB1R is down-regulated in striatal areas after chronic exposure to cannabinoids (22)
. CB1 receptors are also highly expressed in interneurons of the hippocampus. We found that the CB1R and GASP1 were localized to the same neurons both in primary striatal cultures (Fig. 6
A) and in hippocampal sections (Fig. 6B-D
). In addition, CB1R could be coimmunoprecipitated with GASP1 from a whole rat brain (Fig. 6G
), providing evidence of a specific protein-protein interaction in an in vivo setting.
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Collectively, these results imply that the CB1R and GASP1 interact both in vitro and in vivo. Thus, this protein-protein interaction between CB1R and GASP1 may promote postendocytic receptor trafficking toward degradation, and therefore may contribute to the development of tolerance to cannabinoids after long-term administration.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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, 19
, 20)
. Several mechanisms underlying tolerance to cannabinoids have been proposed, including changes in cell signaling (37)
, receptor desensitization (14
, 38)
, and receptor down-regulation (13
14
15
, 18
19
20
21
22
23
, 39)
. While receptor desensitization appears to be specific to selected brain areas, receptor down-regulation is a prominent cellular hallmark in tolerant animals and has been observed in multiple brain regions (14
, 22)
. However, the molecular mechanisms mediating this loss of receptor number have remained unclear. Sim-Selley and colleagues recently proposed that down-regulation of CB1 receptors was mediated at the level of the receptor protein rather than through changes in gene expression (16)
. Our findings, whereby we propose a molecular mechanism that modulates the postendocytic down-regulation of the CB1 receptor, are entirely consistent with this hypothesis.
In this study we have found that treatment of the CB1R with either anandamide or WIN55,212–2 resulted in receptor internalization, followed by pronounced colocalization with lysosomes (Fig. 2A
). Biotinylation protection assays further supported these findings and showed that internalized receptors were indeed degraded, a process that could be prevented by adding protease inhibitors (Fig. 2B
). In good agreement with these findings, Hsieh and co-workers previously demonstrated that recovery of CB1Rs to the cell surface after 90 min of agonist treatment depended on new protein synthesis in AtT20 cells (25)
. However, they also found that the CB1R was able to recycle after shorter agonist treatments (20 min), which could reflect that prolonged presence of agonist is required for receptors to proceed to the degradation pathway (Fig. 1B, C
).
It has become evident that 7TM receptor trafficking is regulated by interaction with multiple intracellular protein partners. Purified receptor fragments in in vitro assays and intact receptors in coimmunoprecipitation experiments have been widely applied to identify potential trafficking proteins. The G-protein-coupled receptor-associated sorting protein GASP1 has been shown to associate preferably with degrading receptors, but not with recycling members of either opioid or dopamine receptor families (27
, 28)
. Here we demonstrate that GASP1 interacts with the CB1R in cell lines (Fig. 3B
) and in the brain (Fig. 6G
). We believe that this receptor-GASP1 interaction directs CB1R trafficking, because the dominant negative cGASP delays receptor degradation and facilitates receptor recycling (Figs. 4
, 5)
. It is likely that this interaction is a direct protein-protein interaction and is mediated, at least in part, by the C-terminal tail of the receptor, which binds GASP1 in vitro (Fig. 3A
). Different 7TM receptors appear to bind GASP1 with varying affinities (33)
. In fact, we found that the CB1R bound GASP1 with lower affinity in vitro than did the delta opioid receptor DOP-R (Fig. 3A
). Thus it could be speculated that, given a less tight binding to GASP1, the CB1 receptor is capable of recycling after short-term agonist treatment, as described by Hseih et al. (25)
, whereas long-term stimulation would ultimately lead to receptor degradation.
Correct axonal targeting of neuronal CB1Rs depends on receptor recycling (40)
. This would be the expected fate of receptors in the drug-free state, when endogenous ligand is released in a pulsatile manner and has a short half-life. It is intriguing, then, to speculated that receptors could be mistargeted in the presence of prolonged agonist exposure—for example, during exogenous drug use.
Protein interactions such as those between GASP and the CB1 receptor have the ability to convey temporal and spatial specificity to membrane trafficking. In particular, regional differences in GASP expression could explain why CB1Rs are down-regulated more prevalently in some brain regions after chronic agonist treatment (14
, 18
, 20
21
22)
. The fact that GASP is an X-linked gene may also explain why there are gender-specific differences in responsiveness to ligands which activate receptors that have been shown to interact with GASP, including the kappa opioid peptide receptor (41)
and the CB1 receptor (42
, 43)
. In addition to mediating the specificity and regulation of 7TM receptor membrane trafficking after initial endocytosis, specific protein interactions, such as those between CB1R and GASP, that control postendocytic sorting of 7TM receptors, could be therapeutically important. Ligands at the CB1R are being developed as therapeutics for nicotine dependence as well as eating disorders. Although little is known about the trafficking properties of these receptor-ligand complexes, it is clear that CB1Rs are down-regulated both in vitro and in vivo in response to some CB1 ligands. Therefore, a compound that disrupted the interaction of CB1 with GASP might be expected to block down-regulation and promote functional resensitization to ligands that promote receptor endocytosis. As a result, the signaling capacity of CB1R in response to prolonged or repeated exposure to endogenously produced cannabinoids or exogenously administered drugs could be enhanced.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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Received for publication August 15, 2006. Accepted for publication September 25, 2006.
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REFERENCES |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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