QUICK SEARCH:   [advanced] Author: Keyword(s): Year:  Vol:  Page:

This Article Abstract Full Text (PDF) All Versions of this Article: jpet.104.067306v1 310/3/865    most recent Submit a response Alert me when this article is cited Alert me when eLetters are posted Alert me if a correction is posted Citation Map Services Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Schlag, B. D. Articles by Dunlop, J. Search for Related Content PubMed PubMed Citation Articles by Schlag, B. D. Articles by Dunlop, J.

CELLULAR AND MOLECULAR

Ligand Dependency of 5-Hydroxytryptamine 2C Receptor Internalization

Neuroscience Discovery Research, Wyeth Research, Princeton, New Jersey

Received for publication February 19, 2004
Accepted April 26, 2004.

	Abstract

Top Abstract Materials and Methods Results Discussion References

 
Agonist-induced internalization of G protein-coupled receptors (GPCRs) is a well characterized phenomenon believed to contribute to receptor desensitization. The 5-hydroxytryptamine (5-HT)_2C subtype of serotonin receptor is a GPCR that we have shown to internalize upon agonist incubation. In this study, we have examined the effects of 5-HT_2C receptor agonists serotonin, Ro 60-0175 [(S)-2-(6-chloro-5-fluoroindol-1-yl)-1-methylethylamine], and WAY-161503 [(4aR)-8,9-dichloro-2,3,4,4a-tetrahydro-1H-pyrazino[1,2-a]quinoxalin-5(6H)-one]; partial agonists mCPP [1-(m-chlorophenyl)piperazine] and DOI [(+)-1-(2,5-dimethoxy-4-iodophenyl)-2-amino-propane]; inverse agonists SB-206553 [N-3-pyridinyl-3,5-dihydro-5-methylbenzo(1,2-b:4,5-b')dipyrrole-1(2H)carboxamide] and mianserin; and neutral antagonists SB-242084 [6-chloro-5-methyl-1-[[2-[(2-methyl-3-pyridyl)oxy]-5-pyridyl]carbamoyl]-indoline] and 5-methoxygramine on the internalization of a C-terminal green fluorescent protein (GFP)-tagged 5-HT_2C receptor (VSV isoform) expressed in transiently transfected human embryonic kidney cells. We detected internalization with an automated, cell-based fluorescence-imaging system (Arrayscan) and monitored function with intracellular Ca²⁺ measurements (flourometric imaging plate reader). The 5-HT_2C-GFP construct exhibited appropriate pharmacology, and we observed that although all three agonists resulted in similar magnitudes of dose-dependent internalization, the partial agonists resulted in 50% less internalization, and the inverse agonists and neutral antagonists failed to induce internalization. These results were confirmed by confocal microscopy. They demonstrate that the 5-HT_2C receptor is internalized by incubation with agonists and partial agonists but not with inverse agonists or neutral antagonists.

Although agonist- and antagonist-induced internalization has been studied for the 5-HT_2A serotonin receptor, it has not been examined for the closely related 5-HT_2C receptor subtype. The 5-HT_2C receptor functions through the same mechanism as the other members of the 5-HT₂ family, activating phospholipase C, promoting the hydrolysis of membrane phospholipids, leading to increases in the intracellular levels of inositol phosphates and intracellular calcium (for review, see Boess and Martin, 1994). Both the 5-HT_2A and 5-HT_2C receptor subtypes have received considerable attention in pharmaceutical drug discovery. In the case of the 5-HT_2A receptor subtype, it has been recognized that drugs that are used to treat both schizophrenia and depression, in addition to hallucinogenic agents, display affinity for this target (Canton et al., 1990). The 5-HT_2C receptor has been implicated in a variety of conditions, including obesity, anxiety, depression, obsessive compulsive disorder, schizophrenia, migraine, nociception, and erectile dysfunction (Fozard and Gray, 1989; Kennett and Curzon, 1991; Sanders-Bush and Breeding, 1991; Gibson et al., 1994; Bos et al., 1997; Millan et al., 1997; Sasaki et al., 2003). Consequently, a number of pharmacological agents targeting both activation and antagonism of the 5-HT_2C receptor have been described previously (Bos et al., 1997; Kennett et al., 1996, 1997; Bishop and Nilsson, 2003).

To gain insight into the regulation of the 5-HT_2C receptor subtype by ligands with both positive and negative intrinsic activity, we examined the ligand dependence of 5-HT_2C receptor internalization by evaluating agonist and antagonist effects on internalization and desensitization. The 5-HT_2C receptor undergoes RNA editing to generate multiple isoforms, and the predominant human VSV isoform (Niswender et al., 2001) was chosen for study because this isoform will be a significant determinant of drug action in the human central nervous system. We tagged the C terminus of the 5-HT_2C receptor (VSV isoform) with GFP and confirmed appropriate functional pharmacology by intracellular calcium measurements using a fluorometric imaging plate reader (FLIPR; Molecular Devices, Sunnyvale, CA). Internalization was then monitored by measuring the formation of clusters of tagged receptors trafficked to endocytic recycling compartments (ERCs) using an automated, cell-based fluorescence imaging system (Arrayscan II; Cellomics, Pittsburgh, PA). Our results demonstrate that 5-HT_2C receptor agonists and partial agonists promote receptor internalization, whereas both inverse agonists and neutral antagonists do not. In addition, agonist intrinsic activity was similar for promoting internalization compared with a more conventional assay of functional receptor coupling, calcium mobilization.

	Materials and Methods

Top Abstract Materials and Methods Results Discussion References

 
Cloning of 5-HT_2C-GFP. 5-HT_2C-GFP was cloned by polymerase chain reaction amplification. The segment between a native, unique AvaI site and the C terminus of the gene was amplified by polymerase chain reaction using primers to remove the stop codon and add a ClaI site. The N-terminal portion of the receptor was obtained by cutting template with NotI and AvaI. These two pieces were ligated and cloned into pEGFP-N1 (BD Biosciences Clontech, Palo Alto, CA) using the Rapid DNA ligation kit (Roche Diagnostics, Indianapolis, IN).

Transfection. HEK-293 cells were transiently transfected using the LipofectAMINE Plus transfection reagent (Invitrogen, Carlsbad, CA) with 5 µg of DNA for approximately 2 x 10⁶ cells. The next day, they were replated for assay.

Intracellular Calcium Flux Measurements. Cells were plated 24 h before the experiment in poly-D-lysine-coated 96-well plates at a density of approximately 60,000 cells/well. In preparation of the assay, the confluent monolayer of cells was washed twice with Hanks' balanced salt solution supplemented with 20 mM Hepes and 2.5 mM probenecid (FLIPR buffer), and then the cells were loaded by adding 4 µM Fluo-4 AM (Molecular Probes, Eugene, OR) in FLIPR buffer for 1 h at 37°C. After loading, the cells were then rinsed twice with FLIPR buffer, and intracellular calcium increases were detected by measuring increases in fluorescence with FLIPR after ligand addition. For ligand preincubation experiments, ligands were included during the dye loading step and 10 µM 5-HT was added to detect the level of desensitized responses. In the experiments examining the effect of agonist preincubation, cells were washed before stimulation with 5-HT to evaluate desensitization without the confounding competition for agonist binding sites.

Internalization Measurements. Cells were plated 24 h before ligand addition in a 96-well plate at a density of approximately 25,000 cells/well. The next day, the cells were rinsed with OptiMem I (Invitrogen) and treated with up to 10 µM of ligand in OptiMem I overnight at 37°C. The cells were then rinsed once with Hanks' balanced salt solution and fixed with 3% paraformaldehyde for 15 min. After rinsing the cells with Tris-buffered saline with 0.1% Triton X-100 the cells were then incubated for 15 min with 10 µg/ml Hoechst 33342 in Tris-buffered saline with 0.1% Triton X-100. The cells were then rinsed with Tris-buffered saline and stored at 4°C. Images were then acquired at 20x with the Arrayscan II (Cellomics) and analyzed using an imaging processing algorithm (Receptor Internalization) designed to identify ERCs per field of cells.

Confocal Microscopy. Cells were plated at approximately 50,000 cells/well on eight-well culture slides coated with poly-D-lysine (BD Biosciences, Bedford, MA) and treated as described above. After fixation, coverslips were mounted with the use of the ProLong Antifade kit (Molecular Probes). Images were acquired at 63x with a TCS SP confocal system (Leica Microsystems, Inc., Deerfield, IL).

	Results

Top Abstract Materials and Methods Results Discussion References

 
Ligand Effects on Calcium Flux in HEK-293 Cells Expressing 5-HT_2C-GFP. To ensure appropriate pharmacology of the tagged 5-HT_2C-GFP construct, functional validation was performed by measuring intracellular calcium flux using the FLIPR. Compounds previously described to function as agonists (5-HT, Ro 60-0175, WAY-161503, mCPP, and DOI), inverse agonists (SB-242084 and 5-methoxygramine), and antagonists (SB-206553 and mianserin) on the 5-HT_2C receptor were selected for measuring their effects on calcium flux. We monitored the time course of calcium flux in response to 10 µM compound (Fig. 1A) and observed that the highest fluorescence counts (directly equated to the magnitude of calcium flux) were obtained with the agonists 5-HT and Ro 60-0175, whereas mCPP and DOI, compounds described previously as partial agonists for the 5-HT_2C receptor, had smaller maximal levels of calcium flux (Table 1). The EC₅₀ values obtained for the agonists studied, estimated from the log concentration-response curves shown (Fig. 1B) showed that 5-HT was the most potent at 1.8 ± 0.7 nM, with the least potent being mCPP at 49 ± 18 nM (Table 1). With the exception of Ro 60-0175 we observed a good correlation between the potencies of agonists to acutely stimulate Ca²⁺-signaling with their potencies to induce desensitization. All of the antagonists and inverse agonists tested showed no detectable calcium flux (Fig. 1B; Table 1). In control experiments, none of the compounds used in this study stimulated a calcium response in nontransfected HEK cells (data not shown).

View larger version (33K): [in this window] [in a new window]   Fig. 1. A, agonist stimulation (10 µM) of calcium signaling in HEK-293 cells transfected with 5-HT2C-GFP receptor. B, log concentration-response curves for ligand-stimulated calcium signaling in HEK-293 cells expressing 5-HT2C-GFP receptor. Data are expressed as percentage of 5-HT response from a representative experiment and represent the mean ± S.E.M. from quadruplicate wells.

Desensitization and Antagonism Effects on Calcium Flux in HEK-293 Cells Expressing 5HT_2C-GFP. Pharmacological characterization was also evaluated for measuring agonist effects on desensitization as well as inverse agonist and antagonist effects on 5-HT-stimulated responses. After 30-min preincubation with agonists, inverse agonists, or antagonists, cells were subsequently challenged with 10 nM 5-HT. All of the agonists induced desensitization with EC₅₀ values in the range of 1.6 ± 0.3 nM for 5-HT, the most potent, to 22 ± 6 nM for mCPP, the least potent (Fig. 2; Table 2). The inverse agonists and neutral antagonists exhibited antagonism under the same paradigm (Fig. 3). The ranges of IC₅₀ values were from 3150 ± 750 nM for 5-methoxygramine to less than 0.00015 ± 0.00005 nM for SB-242084 (Table 3).

View larger version (15K): [in this window] [in a new window]   Fig. 2. Desensitization. Effects of agonist pretreatment on 5-HT2C-GFP receptor coupled calcium signaling in HEK-293 cells. 5-HT (10 nM)-stimulated calcium signaling in HEK-293 cells expressing 5-HT2C-GFP was measured after a 30-min preincubation with agonists. Data are expressed as percentage of 10 nM 5-HT response measured in cells not pretreated with ligand and are from a representative experiment and represent the mean ± S.E.M. from triplicate wells.

View larger version (13K): [in this window] [in a new window]   Fig. 3. Antagonism. Effects of inverse agonist or neutral antagonist pretreatment on 5-HT2C-GFP receptor coupled calcium signaling in HEK-293 cells. 5-HT (10 nM)-stimulated calcium signaling in HEK-293 cells expressing 5-HT2C-GFP was measured after a 30-min preincubation with ligands. Data are expressed as percentage of 10 nM 5-HT response measured in cells not pretreated with ligand and are from a representative experiment and represent the mean ± S.E.M. from triplicate wells.

Detection of 5HT_2C-GFP Internalization upon Agonist and Antagonist Incubation. The pharmacology of receptor internalization was determined by monitoring the accumulation of green fluorescence within centralized intracellular locations, which are consistent with ERCs. To quantitate these events, we used the Arrayscan II (Cellomics) automated cell-based imaging and analysis system, using their receptor internalization algorithm. Figure 4, A–C, demonstrate the acquired Arrayscan images for 5-HT incubation with the applied algorithm shown as red overlay in A and C. Although under basal conditions the 5-HT_2C-GFP signal was predominantly associated with the cell surface (Fig. 4A) application of the algorithm results in the detection of a small number of intense bright spots (red overlay). This highlights an important limitation of any automated imaging procedure intended to measure cell surface removal (or incorporation), the inability to be able to define the plasma membrane and to eliminate this background signal. Nevertheless, we observed that incubation with the natural ligand, 5-HT, produced an increase in the number of intense bright spots within the field (Fig. 4A, B), which are also detectable by the algorithm and highlighted by the red overlay (Fig. 4A, C, same image with overlay). Ligand-induced internalization was monitored for 30 min, 1, 2, 4 h, and overnight (data not shown). Internalized receptors began to be detectable starting after approximately 1 h of ligand incubation, with overnight incubation, yielding results best detected with the Arrayscan. All of the results included in this study are from overnight incubation.

View larger version (61K): [in this window] [in a new window]   Fig. 4. Ligand-induced 5-HT2C-GFP receptor internalization is dependent upon intrinsic activity of ligand. A, A–C, example of Arrayscan II images (20x) of receptor localization under control conditions (A), 10 µM 5-HT (B), and with the Arrayscan overlay of detected ERCs (C). B, D–M, confocal images (63x; scale bar, 10 µm) of receptor localization (green) and nuclei (blue) under control conditions (D) and with agonists 5-HT (E), Ro 60-0175 (F), WAY-161503 (G), mCPP (H), and DOI (I); inverse agonists SB-206553 (J) and mianserin (K) and neutral antagonists SB-242084 (L) and 5-methoxygramine (M). Images shown were acquired after treatment with 10 µM drug.

Confocal microscopy was also used to verify effects of 5-HT_2C receptor ligands on 5-HT_2C-GFP internalization. Only the agonists 5-HT, Ro 60-0175, and WAY-161503 were able to fully induce ERC formation (Fig. 4B, D–G). The partial agonists mCPP and DOI induced formation of ERCs, albeit with significant remaining cell surface labeling (Fig. 4B, H and I). The inverse agonists and antagonists did not induce internalization (Fig. 4B, J–M).

Log concentration-response curves for the formation of ERCs, quantified automatically using the Arrayscan, after overnight incubation with the various ligands are shown (Fig. 5). The agonists tested yielded EC₅₀ values that were greater than those obtained with FLIPR, ranging from 1.3-fold (Ro 60-0175) to 45-fold (5-HT) (compare Table 1 and Table 4). The antagonists and inverse agonists tested resulted in no internalization at any of the concentrations of ligand tested (Fig. 5B).

View larger version (24K): [in this window] [in a new window]   Fig. 5. Log concentration-response curves for ligand-stimulated formation of ERCs in HEK-293 cells expressing 5-HT2C-GFP receptor. Data are from a representative experiment and are expressed as number of ERCs from triplicate wells of approximately five fields (mean ± S.E.M.).

	Discussion

Top Abstract Materials and Methods Results Discussion References

 
Although agonist-induced internalization is a well described phenomenon for many GPCRs, internalization by antagonists or inverse agonists has been documented for a limited number of GPCRs, suggesting this may be a receptor-specific property. In this study, we focused on determining the effects of a range of 5-HT_2C receptor ligands, including agonists, inverse agonists, and antagonists on receptor internalization. Our goal was to construct a 5HT_2C receptor-GFP fusion protein, validate pharmacological function, and then to monitor and quantitate any ligand-induced internalization using the Arrayscan II high content screening system (Cellomics) with their receptor internalization algorithm. This approach has been previously used for another member of the GPCR class of receptors, the parathyroid hormone receptor (Conway et al., 1999).

All of the 5HT_2C receptor ligands tested with our 5HT_2C receptor-GFP fusion protein resulted in functional pharmacology for calcium flux within anticipated ranges, validating the use of the construct for our subsequent internalization studies. Serotonin, Ro 60-0175, DOI, and mCPP have been reported to have EC₅₀ values from approximately 1 to 169 nM (Porter et al., 1999). The values obtained in this study were between 2 and 50 nM, with the same rank order potency as the published values: 5-HT > DOI > Ro 60-0175 > mCPP. Similarly, WAY-161503, another compound shown to be a 5HT_2C receptor agonist (Cryan and Lucki, 2000), also falls within this range at approximately 47 nM.

The inverse agonists and antagonists were also within ranges anticipated from previous studies. SB-206553, mianserin, and SB-242084 have been shown to have the following IC₅₀ values, respectively: 12, 8.6, and 0.5 nM (Kennett et al., 1996, 1997; Rauser et al., 2001). Although those values were from a different functional assay, phosphoinositide hydrolysis, the values were similar to the IC₅₀ values we obtained with Ca²⁺ mobilization studies performed using the FLIPR (Table 3). The only surprising difference is that we observed a more potent effect with SB-242084, 0.00015 nM compared with the published value of 0.5 nM for phosphoinositide hydrolysis (Kennett et al., 1997). Although it is unclear as to why this discrepancy exists, it is clear that SB-242084 functioned as an extremely potent antagonist (n = 4), and we have also confirmed this potent antagonism, with IC₅₀ in the subnanomolar range using a stable Chinese hamster ovary cell line expressing the human 5-HT_2C receptor (our unpublished observations). 5-Methoxygramine was the least potent antagonist tested, with an IC₅₀ of approximately 3 µM. Such potency was also anticipated because the binding constant for 5-methoxygramine has been shown to be 1.45 µM (Stollak and Furchgott, 1983).

Short-term pretreatment with all of the agonists resulted in functional desensitization of the 5-HT_2C-GFP receptor construct, as determined by measurement of 5-HT stimulated Ca²⁺ mobilization after the agonist pretreatment(s). All of the agonists tested resulted in EC₅₀ values for desensitization of between 1.8 and 22 nM supporting the idea that upon activation, the receptor becomes desensitized. All agonists resulted in full desensitization at the maximal concentrations tested. Although this experiment demonstrates a decrease in receptor function, it does not address the means by which the receptor function decreases. Receptor localization studies were next performed to explore the possibility that receptors were being internalized, a mechanism contributing to desensitization. We used the Arrayscan II technology (Cellomics) to monitor the pharmacology of internalization of 5-HT_2C-GFP. Agonist incubation resulted in internalization of 5-HT_2C-GFP from the membrane to a single intracellular compartment. Each of the agonists tested was able to increase the basal level of ERCs detected by approximately 4-fold. It is also noteworthy that measuring internalization seems to be less sensitive than the calcium flux method for determining EC₅₀ values. The potential for the C-terminal GFP tag to interfere with appropriate receptor translocation is an important confounding variable likely to have a larger impact on internalization compared with functional coupling measured by calcium flux. In contrast, agonist intrinsic activities determined using the internalization assay were consistent with those determined in the Ca²⁺ assay, as evidenced by the ability to distinguish full and partial agonists.

Although all of the agonists were able to induce detectable levels of internalization, the amount of time required for internalization was greater for 5-HT_2C-GFP than other GPCRs, such as parathyroid hormone receptor and the -adrenergic receptor, which have been demonstrated to show full internalization in under 1 h (Awaji et al., 1998; Conway et al., 2001). To get consistent images with internalization of 5-HT_2C-GFP, it was necessary for us to permit the incubation with agonists to proceed overnight. We hypothesize that the reason for the longer internalization time is that the GFP tag on the c-terminal tail of the receptor could be inhibiting proper trafficking of the receptor. This hypothesis is supported by evidence that other neuronal GPCRs, such as metabotropic glutamate receptors, use their cytoplasmic carboxy-terminal domains for appropriate targeting in neurons (Stowell and Craig, 1999). Also, a study has been performed comparing ligand regulation of GFP-tagged forms of -adrenoceptors with unmodified receptors (McLean and Milligan, 2000). This study specifically notes that c-terminal addition of GFP markedly decreased the rate of internalization of -adrenoceptors in response to agonist and that sustained exposure to agonist (24 h) was able to completely internalize receptor from the cell surface.

In addition to the slower time course of internalization, it was also noteworthy that incubation with inverse agonists and neutral antagonists do not result in any detectable internalization of 5-HT_2C-GFP. This is specifically interesting because the 5-HT_2A receptor has been shown to be internalized by both agonists and antagonists (Willins et al., 1998). The 5-HT_2C subtype of serotonin receptor seems to be different from 5-HT_2A in that antagonists do not induce internalization, at least when examined at the level of detecting internalized ERCs of a defined shape and size. This may prove to be a significant observation in helping to understand the differences in regulation of these two subtypes of serotonin receptor. During the course of submission of our study, Marion et al. (2004) reported differences in 5-HT_2C receptor internalization as a function of the edited isoform under evaluation. In the case of the VSV isoform tagged with GFP, they observed internalization after 5-HT treatment but not with SB-206553, an identical observation to ours. However, they also had sufficient resolution to detect a small proportion of constitutively internalized 5-HT_2C-VSV-GFP receptor, beyond the resolution of our analysis, which was externalized after treatment with SB206553. Furthermore, they demonstrated that the highly constitutively active INI isoform was predominantly intracellular, or constitutively internalized. These observations suggest that agonist-dependent internalization for the 5-HT_2C receptor will be critically dependent upon receptor isoform and be a more important property for investigation with fully and partially edited receptors such as VGV and VSV.

	Footnotes

 
These results have been presented in poster format (abstract 643.7) at the Experimental Biology Meeting (San Diego, CA), April 11–15, 2003.

Article, publication date, and citation information can be found at http://jpet.aspetjournals.org.

doi:10.1124/jpet.104.067306.

ABBREVIATIONS: GPCR, G protein-coupled receptor; 5-HT, 5-hydroxytryptamine (serotonin); GFP, green fluorescent protein; FLIPR, fluorometric imaging plate reader; ERC, endocytic recycling compartment; HEK, human embryonic kidney; Ro 60-0175, (S)-2-(6-chloro-5-fluoroindol-1-yl)-1-methylethylamine; WAY-161503, (4aR)-8,9-dichloro-2,3,4,4a-tetrahydro-1H-pyrazino[1,2-a]quinoxalin-5(6H)-one; mCPP, 1-(m-chlorophenyl)piperazine; DOI, (+)-1-(2,5-dimethoxy-4-iodophenyl)-2-amino-propane; SB-206553, N-3-pyridinyl-3,5-dihydro-5-methyl-benzo(1,2-b:4,5-b')dipyrrole-1(2H)carboxamide; SB-242084, 6-chloro-5-methyl-1-[[2-[(2-methyl-3-pyridyl)oxy]-5-pyridyl]carbamoyl]-indoline.

Address correspondence to: Dr. John Dunlop, Neuroscience Discovery Research, Wyeth Research, CN 8000, Princeton, NJ 08543. E-mail dunlopj{at}wyeth.com

	References

Top Abstract Materials and Methods Results Discussion References

 

Awaji T, Hirasawa A, Kataoka M, Shinoura H, Nakayama Y, Sugawara T, Izumi S, and Tsujimoto G (1998) Real-time optical monitoring of ligand-mediated internalization of 1b-adrenoceptor with green fluorescent protein. Mol Endocrinol 12: 1099-1111.[Abstract/Free Full Text]
Bishop MJ and Nilsson BM (2003) New 5-HT_2C receptor agonists. Exp Opin Ther Patents 13: 1691-1705.[CrossRef]
Boess FG and Martin IL (1994) Molecular biology of 5-HT receptors. Neuropharmacology 33: 275-317.[CrossRef][Medline]
Bos M, Jenck F, Martin JR, Moreau JL, Sleight AJ, Wichmann J, and Widmer U (1997) Novel agonists of 5-HT_2C receptors. Synthesis and biological evaluation of substituted 2-(indol-1-yl)-1-methylethylamines and 2-(indeno[1,2-b]pyrrol-1-yl)-1-methylethylamines. Improved therapeutics for obsessive compulsive disorder. J Med Chem 40: 2762-2769.[CrossRef][Medline]
Canton H, Verriele L, and Colpaert FC (1990) Binding of typical and atypical antipsychotics to 5-HT_1C and 5-HT₂ sites: clozapine potently interacts with 5-HT_1C sites. Eur J Pharmacol 191: 93-96.[CrossRef][Medline]
Conway BR, Minor LK, Xu JZ, D'Andrea MR, Ghosh RN, and Demarest KT (2001) Quantitative analysis of agonist-dependent parathyroid hormone receptor trafficking in whole cells using a functional green fluorescent protein conjugate. J Cell Physiol 189: 341-355.[CrossRef][Medline]
Conway BR, Minor LK, Xu JZ, Gunnet JW, DeBiasio R, D'Andrea MR, Rubin R, DeBiasio R, Giuliano K, Zhou L, et al. (1999) Quantification of G-protein coupled receptor internalization using G-protein coupled receptor-green fluorescent protein conjugates with the Arrayscan high-content screening system. J Biomol Screen 4: 75-86.[Abstract/Free Full Text]
Cryan JF and Lucki I (2000) Antidepressant-like behavioral effects mediated by 5-hydroxytryptamine_2C receptors. J Pharmacol Exp Ther 295: 1120-1126.[Abstract/Free Full Text]
Ferguson SS (2001) Evolving concepts in G protein-coupled receptor endocytosis: the role in receptor desensitization and signaling. Pharm Rev 53: 1-24.[Abstract/Free Full Text]
Fozard JR and Gray JA (1989) 5-HT_1C receptor activation: a key step in the initiation of migraine? Trends Pharmacol Sci 10: 307-309.[CrossRef][Medline]
Gibson EL, Barnfield AM, and Curzon G (1994) Evidence that mCPP-induced anxiety in the plus maze is mediated by postsynaptic 5-HT_2C receptors but not by sympathomimetic effects. Neuropharmacology 33: 457-465.[CrossRef][Medline]
Kennett GA and Curzon G (1991) Potencies of 5-HT_1C antagonists indicate that receptors mediated mCPP-induced hypophagia. Br J Pharmacol 103: 2016-2020.[Medline]
Kennett GA, Wood MD, Bright F, Cilia J, Piper DC, Gager T, Thomas D, Baxter GS, Forbes IT, Ham P, et al. (1996) In vitro and in vivo profile of SB 206553, a potent 5HT_2C/5HT_2B receptor antagonist with anxiolytic-like properties. Br J Pharmacol 117: 427-434.[Medline]
Kennett GA, Wood MD, Bright F, Trail B, Riley G, Holland V, Avenell KY, Stean T, Upton N, Bromidge S, et al. (1997) SB 242084, a selective and brain penetrant 5-HT_2C receptor antagonist. Neuropharmacology 36: 609-620.[CrossRef][Medline]
Marion S, Weiner DM, and Caron MG (2004) RNA editing induces variation in desensitization and trafficking of 5-hydroxytrptamine 2C receptor isoforms. J Biol Chem 279: 2945-2954.[Abstract/Free Full Text]
McLean AJ and Milligan G (2000) Ligand regulation of green fluorescent protein-tagged forms of the human ₁- and ₂-adrenoceptors: comparisons with the unmodified receptors. Br J Pharmacol 130: 1825-1832.[CrossRef][Medline]
Millan MJ, Peglion JL, Lavielle G, and Perrin-Monneyron S (1997) 5-HT_2C receptors mediate penile erections in rats: actions of novel and selective agonists and antagonists. Eur J Pharmacol 325: 9-12.[CrossRef][Medline]
Navarro A, Zapata R, Canela EI, Mallol J, Lluis C, and Franco R (1999) Epidermal growth factor (EGF)-induced up-regulation and agonist- and antagonist-induced desensitization and internalization of A1 adenosine receptors in a pituitary-derived cell line. Brain Res 816: 47-57.[CrossRef][Medline]
Niswender CM, Herrick-Davis K, Dilley GE, Meltzer HY, Overholser JC, Stckmeier CA, Emeson RB, and Sanders-Bush E (2001) RNA editing of the human serotonin 5-HT_2C receptor: alterations in suicide and implications for serotonergic pharmacotherapy. Neuropsychopharmacology 24: 478-491.[CrossRef][Medline]
Pfeiffer R, Kirsch J, and Fahrenholz F (1998) Agonist and antagonist-dependent internalization of the human vasopressin V2 receptor. Exp Cell Res 244: 327-339.[CrossRef][Medline]
Porter RH, Benwell KR, Lamb H, Malcolm CS, Allen NH, Revell DF, Adams DR, and Sheardown MJ (1999) Functional characterization of agonists at recombinant human 5-HT_2A, 5-HT_2B and 5HT_2C receptors in CHO-K1 cells. Br J Pharmacol 128: 13-20.[CrossRef][Medline]
Rauser L, Savage JE, Meltzer HY, and Roth BL (2001) Inverse agonist actions of typical and atypical antipsychotic drugs at the human 5-hydroxtryptamine_2C receptor. J Pharmacol Exp Ther 299: 83-89.[Abstract/Free Full Text]
Roettger BF, Ghanekar D, Rao R, Toledo C, Yingling J, Pinon D, and Miller LJ (1997) Antagonist-stimulated internalization of the G protein coupled cholecystokinin receptor. Mol Pharm 51: 357-362.[Abstract/Free Full Text]
Sanders-Bush E and Breeding M (1991) Choroid plexus epithelial cells in primary culture: a model of 5-HT_1C receptor activation by hallucinogenic drugs. Psychopharmacology 105: 340-346.[CrossRef][Medline]
Sasaki M, Obata H, Saito S, and Goto F (2003) Antinociception with intrathecal -methyl-5-hydroxytryptamine, a 5-hydroxytryptamine_2A/2C receptor agonist, in two rat models of sustained pain. Anesth Analg 96: 1072-1078.[Abstract/Free Full Text]
Stollak JS and Furchgott RF (1983) Use of selective antagonists for determining the types of receptors mediating the actions of 5-hydroxytryptamine and tryptamine in the isolated rabbit aorta. J Pharmacol Exp Ther 224: 215-221.[Abstract/Free Full Text]
Stowell JN and Craig AM (1999) Axon/Dendrite targeting of metabotropic glutamate receptors by their cytoplasmic carboxy-terminal domains. Neuron 22: 525-536.[CrossRef][Medline]
Willins DL, Alsayegh L, Berry SA, Bachstrom JR, Sanders-Bush E, Friedman L, Khan N, and Roth BL (1998) Serotonergic antagonist effects on trafficking of serotonin 5-HT2A receptors in vitro and in vivo. Ann NY Acad Sci 861: 121-127.[CrossRef][Medline]

This article has been cited by other articles:

				J.-X. Li, A. Unzeitig, M. A. Javors, K. C. Rice, W. Koek, and C. P. France Discriminative Stimulus Effects of 1-(2,5-Dimethoxy-4-methylphenyl)-2-aminopropane (DOM), Ketanserin, and (R)-(+)-{alpha}-(2,3-Dimethoxyphenyl)-1-[2-(4-fluorophenyl)ethyl]-4-pipidinemethanol (MDL100907) in Rats J. Pharmacol. Exp. Ther., November 1, 2009; 331(2): 671 - 679. [Abstract] [Full Text] [PDF]

				R. Lu, Y. Li, Y. Zhang, Y. Chen, A. D. Shields, D. G. Winder, T. Angelotti, K. Jiao, L. E. Limbird, Y. Zhou, et al. Epitope-tagged Receptor Knock-in Mice Reveal That Differential Desensitization of {alpha}2-Adrenergic Responses Is because of Ligand-selective Internalization J. Biol. Chem., May 8, 2009; 284(19): 13233 - 13243. [Abstract] [Full Text] [PDF]

				B. Chanrion, C. M. la Cour, S. Gavarini, M. Seimandi, L. Vincent, J.-F. Pujol, J. Bockaert, P. Marin, and M. J. Millan Inverse Agonist and Neutral Antagonist Actions of Antidepressants at Recombinant and Native 5-Hydroxytryptamine2C Receptors: Differential Modulation of Cell Surface Expression and Signal Transduction Mol. Pharmacol., March 1, 2008; 73(3): 748 - 757. [Abstract] [Full Text] [PDF]

				S. Gavarini, C. Becamel, C. Altier, P. Lory, J. Poncet, J. Wijnholds, J. Bockaert, and P. Marin Opposite Effects of PSD-95 and MPP3 PDZ Proteins on Serotonin 5-Hydroxytryptamine2C Receptor Desensitization and Membrane Stability Mol. Biol. Cell, November 1, 2006; 17(11): 4619 - 4631. [Abstract] [Full Text] [PDF]

				K. A. Berg, S. Navailles, T. A. Sanchez, Y. M. Silva, M. D. Wood, U. Spampinato, and W. P. Clarke J. Pharmacol. Exp. Ther., October 1, 2006; 319(1): 260 - 268. [Abstract] [Full Text] [PDF]

				C. R. Guthrie, A. T. Murray, A. A. Franklin, and M. W. Hamblin Differential Agonist-Mediated Internalization of the Human 5-Hydroxytryptamine 7 Receptor Isoforms J. Pharmacol. Exp. Ther., June 1, 2005; 313(3): 1003 - 1010. [Abstract] [Full Text] [PDF]

This Article Abstract Full Text (PDF) All Versions of this Article: jpet.104.067306v1 310/3/865    most recent Submit a response Alert me when this article is cited Alert me when eLetters are posted Alert me if a correction is posted Citation Map Services Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Schlag, B. D. Articles by Dunlop, J. Search for Related Content PubMed PubMed Citation Articles by Schlag, B. D. Articles by Dunlop, J.