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NEUROPHARMACOLOGY
11 Protein Reduces Coupling to 5-HT2A ReceptorsDepartment of Pharmacology and Experimental Therapeutics, Loyola University Chicago, Stritch School of Medicine, Maywood, Illinois (J.S., K.J.D., G.A.C., F.G., A.J.G., M.L., N.R.S., G.B.); and Department of Pharmacology and Toxicology, School of Pharmacy, Malott Hall, University of Kansas, Lawrence, Kansas (R.K.S., N.A.M.)
Received for publication
March 19, 2007
Accepted
July 20, 2007.
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Abstract |
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11 protein at serine 154 and that this phosphorylation causes desensitization of serotonin (5-HT) 2A receptor signaling in A1A1v cells (Shi et al., 2007
). We now report that treatment of A1A1v cells with DOI for 24 h produces a greater reduction in the Bmax of [125I](±)-DOI-labeled high-affinity binding sites (46%) than the reduction of [3H]ketanserin binding sites (25%). Although the KD values are not altered, there is a smaller amount of GTP
S [guanosine 5'-3-O-(thio)triphosphate]-sensitive [125I](±)-DOI binding in DOI-treated cells. These results suggest that DOI treatment causes down-regulation of 5-HT2A receptors and reductions in G protein-coupled 5-HT2A receptors. In contrast, in cells transfected with the phosphorylation state mimic G11S154D, GTPS-sensitive [125I](±)-DOI binding was decreased by 48%; however, there was no significant difference in the KD and Bmax values of [125I](±)-DOI-labeled receptors. The receptor binding experiments suggest that phosphorylation of G11 on serine 154 reduces coupling of 5-HT2A receptors, whereas DOI causes down-regulation of 5-HT2A receptors in addition to the phosphorylation-induced uncoupling of G11 to 5-HT2A receptors. To determine whether DOI increases phosphorylation of Gq/11 protein in vivo, rats were treated with 1 mg/kg/day DOI or saline for 1 to 7 days. Seven days of DOI treatment significantly decreased phospholipase C activity stimulated by an Emax concentration of 5-HT by 40% and increased phosphorylation of Gq/11 proteins by 51% in the frontal cortex. These data suggest that DOI causes phosphorylation of Gq/11 in vivo and could thereby contribute to the desensitization of 5-HT2A receptors.
; Hoyer et al., 1994). Desensitization of 5-HT2A/2C receptor signaling may underlie the mechanism of action of several drug treatments for neuropsychiatric disorders (Dean and Hayes, 1996). For example, several antipsychotic drugs, such as olanzapine, desensitize both 5-HT2A and 5-HT2C receptors (Kuoppamäki et al., 1995; Leysen et al., 1993; Roth and Ciaranello, 1991; Schmidt et al., 1995). Mechanisms leading to and resulting from the desensitization of 5-HT2A receptors may explain the 2-week delay in full symptomatic improvement seen with these antipsychotic drugs. However, the molecular mechanisms that underlie the desensitization of 5-HT2A receptor signaling are not well understood.
Previous studies suggested that three possible mechanisms might be involved in the desensitization of 5-HT2A receptors: receptor uncoupling from G proteins, internalization (sequestration of the receptor away from the cell surface) (Bhattacharyya et al., 2002; Hanley and Hensler, 2002), or down-regulation (reduced ligand-bound receptor). For example, some in vivo and in vitro studies have demonstrated that agonist exposure resulted in the desensitization and down-regulation 5-HT2A receptors (Anji et al., 2000; Buckholtz et al., 1988; McKenna et al., 1989; Valdez et al., 2002). However, other studies showed that agonists may cause desensitization without down-regulation or desensitization in the presence of increased densities of 5-HT2A receptors (Akiyoshi et al., 1993; Grotewiel and Sanders-Bush, 1994; Roth et al., 1995). The reduction of G-protein coupling to 5-HT2A receptors has been suggested by a greater reduction in the Bmax of agonist DOI [(–)-1-(2,5-dimethoxy-4-iodophenyl)-2-aminopropane HCl]-labeled high-affinity receptors than the reduction in Bmax of antagonist ketanserin-labeled 5-HT2A receptors after chronic agonist treatment (McKenna et al., 1989). These studies suggest that multiple mechanisms may be responsible for the desensitization of 5-HT2A receptors, but with the specific mechanism(s) involved, dependent on the specific cellular milieu in which the receptors are expressed (Roth et al., 1998). These findings further suggest that, although studies using cell culture models are informative, it is important to determine whether molecular mechanisms revealed in cell culture models reflect the nature of mammalian brain using complementary in vivo studies.
Our previous data in an embryonic rat cortical cell line, A1A1v cells, demonstrated that phosphorylation of G11 protein on serine 154 by protein kinase C and calcium-calmodulin dependent kinase II contributes to agonist-induced desensitization of 5-HT2A receptor signaling (Shi et al., 2007). Exposure of A1A1v cells to DOI for 24 h increased phosphorylation of Gq/11 protein. Using site-directed mutagenesis, we found that mutation of serine 154 to alanine on G11 protein reduced the desensitization of 5-HT2A receptor signaling and prevented the increase in phosphorylation of Gq/11 protein caused by DOI. Mutation of G11 protein serine 154 to aspartic acid, a phosphorylation mimic, directly reduced 5-HT2A receptor signaling. In contrast, mutation of Gq to alanine had no effect on agonist-induced desensitization of 5-HT2A receptor signaling.
As described above, it is important to determine whether the mechanisms revealed in cell culture models reflect the nature of the response in vivo; therefore, we examined the phosphorylation of Gq/11 protein in rat brain. Now we report the outcome of a time-course study to determine the impact of DOI on phosphorylation of Gq/11 protein and desensitization of 5-HT2A receptor signaling in rat frontal cortex. In rat frontal cortex, G11 protein mRNA is more abundant than Gq protein mRNA (Tanaka et al., 2000), suggesting that increased phosphorylation of G11 protein would affect 5-HT2A receptor signaling in this brain region. Furthermore, we examined the effects of another drug that alters 5-HT2A receptor signaling on phosphorylation of Gq/11 protein, a serotonin reuptake inhibitor, fluoxetine, which increases 5-HT2A receptor signaling.
We further hypothesize that phosphorylation of G11 protein reduces 5-HT2A receptor signaling by altering the interactions of G11 protein with other proteins such as the 5-HT2A receptors. This hypothesis is consistent with our previous study, which measured phospholipase C (PLC) activity stimulated by a EC80 dose of 5-HT or GTPS [guanosine 5'-3-O-(thio)triphosphate], and suggested that an alteration at the receptor or an alteration in coupling of 5-HT2A receptors to Gq/11 protein occurs with agonist treatment (Damjanoska et al., 2004). To determine the impact of phosphorylation of G11 protein and agonist treatment on 5-HT2A receptor coupling, we examined the density (Bmax) and receptor affinity (KD) of 5-HT2A receptors by using radioligand binding assays with [125I](±)-DOI, which labels high-affinity state of the 5-HT2A receptor, and [3H]ketanserin, which labels total 5-HT2A receptor density. Most importantly, we examined coupling of G proteins to 5-HT2A receptors by measuring the inhibitory effects of GTPS (tetrasodium salt solution) on [125I](±)-DOI binding.
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Materials and Methods |
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S were purchased from Sigma-Aldrich (St. Louis, MO). [3H]Phosphatidylinositol, [3H]ketanserin (specific activity, 76.5 Ci/mmol), and [125I](±)-DOI (specific activity, 2200 Ci/mmol) were purchased from PerkinElmer Life and Analytical Sciences (Boston, MA).
Cell Culture and Transfections. A1A1v neuronal cells endogenously express the 5-HT2A receptor signaling system and were kindly provided by Dr. William Clarke and Kelly Berg (University of Texas Health Science Center, San Antonio, TX). The A1A1v cells were grown in poly-L-ornithine-coated plates in Dulbecco's modified Eagle's medium supplemented with 10% fetal bovine serum in a humidified atmosphere containing 5% CO2. Serum was heat-inactivated and charcoal-treated to remove monoamines. Cells were treated with either 100 nM DOI or vehicle (Hanks' balanced buffer) for 24 h before harvesting for the receptor-binding assay. Cells were transiently transfected with either wild-type G11 or mutant G11S154D using Lipofectamine Plus (Invitrogen) according to the manufacturer's recommendations. A total of 4 µg/plate DNA was used in each transfection. After transfection (48 h), cells were harvested for radioligand binding assays.
Cell Homogenates. To harvest cells, culture plates were washed twice with ice-cold phosphate-buffered saline. We then added 50 mM Tris, pH 7.4, containing protease inhibitor cocktail (containing 104 µM 4-(2-aminoethyl)benzenesulfonyl fluoride, 0.08 µM aprotinin, 2 µM leupeptin, 4 µM bestatin, 1.5 µM pepstatin A, and 1.4 µM E-64, pH 7.4) from Sigma-Aldrich. Cells were harvested by scraping cells off of the surface of the plate. Cells were then centrifuged at 30,000g for 20 min at 4°C. The pellet was resuspended in 50 mM Tris buffer and stored at –80°C. The homogenates were thawed on the day of the binding assay and homogenized by hand with five up-and-down strokes with a glass/glass homogenizer and then centrifuged at 30,000g for 20 min. After centrifugation, the pellet was resuspended in 50 mM Tris buffer and incubated at 37°C for 15 min and centrifuged at 30,000g for 20 min. The resultant pellet was washed once and resuspended in binding assay buffer (50 mM Tris pH 7.4, 0.5 mM EDTA, and 10 mM MgSO4). The final protein concentration was determined by using a bicinchoninic acid protein assay kit (Pierce Chemical, Rockford, IL).
[125I](±)-DOI Saturation Analysis. To determine changes in the density and affinity of high-affinity 5-HT2A receptors, increasing concentrations of [125I](±)-DOI (0.5–4.0 nM) were incubated with the cell homogenates (
100 µg/tube protein) for 1.5 h at room temperature in the same assay buffer as described above. The concentration range of [125I](±)-DOI was based on the KD in our previous study (Li et al., 1997). Nonspecific binding was defined in the presence of 100 nM MDL 100,907, a selective 5-HT2A receptor antagonist. GTPS binding experiments were performed using a concentration (0.4 nM) of [125I](±)-DOI below its KD. The reaction was terminated by rapid filtration over Whatman GF/C glass fiber filters that had been presoaked with 1.0% polyethylenimine. The filters were then washed with 15 ml of ice-cold 50 mM Tris buffer, pH 7.4. The amount of [125I](±)-DOI on the filters was determined by using a Micromedic 4/200 Plus -counter. All radioligand receptor binding assays were performed in triplicate in a final volume of 0.5 ml. Specific binding was defined as the total binding minus nonspecific binding. Determination of Bmax and KD values by either computer-assisted analyses of saturation data (Prism; GraphPad Software, Inc., San Diego, CA) or linear transformation of saturation data via Scatchard plots gave comparable results.
[3H]Ketanserin Saturation Analysis. To measure the total 5-HT2A receptor density (Bmax) and receptor affinity (KD), [3H]ketanserin radioligand binding assays were performed. In saturation experiments, a range of [3H]ketanserin concentrations (1.0–30.0 nM) was incubated with cell homogenates containing 100 µg/tube protein for 1 h at room temperature in assay buffer. Prazosin (30 nM) was included in all assays to preclude binding to 1 receptors. Nonspecific binding was defined in the presence of 1 µM spiperone. The reactions were terminated by filtration and washing as described above. The amount of [3H]ketanserin on the filters was counted using a Beckman LS 6500 scintillation counter. All radioligand receptor binding assays were performed in triplicate in a final volume of 0.5 ml. Specific binding was defined as the total binding minus nonspecific binding. Determination of Bmax and KD values by either computer-assisted analyses of saturation data (Prism; GraphPad Software, Inc.) or by linear transformation of saturation data via Scatchard plots gave comparable results.
Animals. Male Sprague-Dawley rats (225–275 g; Harlan Laboratories, Indianapolis, IN) were housed two per cage in an environment controlled for temperature, humidity, and lighting (7:00 AM-7:00 PM). Food and water were provided ad libitum. Seven to 11 rats were used per experimental group. All procedures were conducted in accordance with the National Institutes of Health (Institute of Laboratory Animal Resources, 1996) as approved by the Loyola University Institutional Animal Care and Use Committee.
Animal Treatment Procedures. The rats were handled during the treatment periods to acclimate them to human contact and to minimize stress. Rats were randomly assigned to the various experimental groups, cage mates in the same experimental groups. The body weight of each rat was recorded every other day.
Sustained Agonist (DOI) Treatment. Rats received daily injections of DOI dissolved in 0.9% saline (1 mg/kg i.p.) for 1, 4, or 7 days or 0.9% saline (1 ml/kg i.p.) for 7 days. Rats receiving DOI injections for 1 or 4 days were given injections of 0.9% saline (1 ml/kg i.p.) on the days prior to the commencement of DOI treatment. Thus, every group received injections for a total of 7 days, which allowed us to control for any potential injection effects. Twenty-four hours after the last DOI treatment, the rats were decapitated. Brains were quickly removed, frozen on dry ice, and stored at –80°C for biochemical and molecular analyses.
Chronic Fluoxetine Treatment. Rats were injected with fluoxetine (10 mg/kg/day i.p.), dissolved in 0.9% saline or 0.9% saline (2 ml/kg i.p.) daily for 21 days. Eighteen hours after the last injection, the rats were decapitated. The brains were quickly removed, frozen on dry ice, and stored at –80°C.
PLC Assay. 5-HT- and GTPS-stimulated PLC activity in frontal cortex was conducted as described previously (Damjanoska et al., 2003, 2004; Wolf and Schutz, 1997). In brief, membrane fractions were prepared from rat frontal cortex. Protein concentrations were determined using a bicinchoninic acid protein assay kit (Pierce Chemical). The membrane protein was diluted to a concentration of 30 µg/100 µl with a buffer containing 25 mM HEPES-Tris, 3 mM EGTA, 10 mM LiCl, 12 mM MgCl2, 1.44 mM sodium deoxycholate with 1 µM GTPS (a nonhydrolyzable form of GTP), 300 nM free Ca2+, 1 mM unlabeled phosphatidylinositol, and 100 µM[3H]phosphatidylinositol (PerkinElmer Life and Analytical Sciences). Two concentrations of 5-HT were used to stimulate PLC activity: 0.3 µM (EC50) and 10.0 µM(Emax) (Wolf and Schutz, 1997). This extends our previous study, which used only a single EC80 dose of 5-HT to measure the desensitization response to repeated DOI treatments (Damjanoska et al., 2004). 5-HT-stimulated PLC activity in the frontal cortex is a selective measure of 5-HT2A receptor function as previously demonstrated using selective antagonists (Wolf and Schutz, 1997).
Analysis of Phosphorylation of Gq/11 Proteins. Frontal cortex tissues from rats treated with DOI and saline were homogenized in a 50 mM Tris buffer, pH 7.7, containing 150 mM NaCl, 10% sucrose, and 0.5 mM phenylmethanesulfonyl fluoride, 50 mM NaF, 2 mM activated sodium orthovanadate, and 1:1000 protease inhibitor cocktail (Sigma-Aldrich). The homogenate was then centrifuged for 60 min at 20,000g. The pellet was resuspended by sonication in a 20 mM Tris buffer, pH 8, containing 1 mM EDTA, 100 mM NaCl, and 1% sodium cholate. The resuspended pellet was agitated at 4°C for 60 min and was then centrifuged for 60 min at 100,000g. The supernatant was saved from each sample and stored at –80°C before use in the immunoprecipitation assay. Protein concentrations were measured using a bicinchoninic acid protein assay kit (Pierce Chemical).
Immunoprecipitation of Gq/11 proteins was performed as described previously (Shi et al., 2007) using a Gq/11 polyclonal antibody (Santa Cruz Biotechnology Inc., Santa Cruz, CA). The immunoprecipitated Gq/11 proteins were resolved by loading 7 µl of supernatant from each sample onto a SDS-polyacrylamide gel containing 0.1% SDS, 8% acrylamide/bisacrylamide (30:0.2), 4.6 M urea, and 375 mM Tris, pH 8.7. Gels loaded with samples from the DOI experiment were designed to contain three samples from the saline-treated control group and three samples from each DOI treatment group. Gels loaded with tissue from the fluoxetine experiment contained six samples from the 21-day fluoxetine treatment group and six samples of the 21-day saline treatment group. The proteins were electrophoretically transferred from the gels onto nitrocellulose or polyvinylidene difluoride membranes, and phosphorylated proteins were detected with a phospho-Ser/Thr/Tyr antibody (1:200; Spring Bioscience, Fremont, CA) as described previously (Shi et al., 2007) or with the Pro-Q Diamond Phosphoprotein Stain as per the manufacturer's directions (Invitrogen). Levels of Gq/11 proteins were examined to verify equal loading of protein in each lane (using Gq/11 antibody, 1:500; and a horseradish peroxidase-labeled, anti-rabbit antibody, 1:100,000; Santa Cruz Biotechnology Inc.).
Films were analyzed densitometrically using the Scion Image program (Scion Corp., Frederick, MD). For normalization, the integrated optical density (IOD) of phosphorylated Gq/11 protein bands on each blot were divided by the mean IOD of saline-treated animals and by the IOD of the respective Gq/11 protein bands (independent of their phosphorylation state).
A total of eight samples per group were analyzed for the DOI experiment. For each animal, immunoprecipitations were performed in triplicate, and the Western blot analysis of each immunoprecipitated sample was performed at least twice. The data presented for the DOI treatment are the means of all three immunoprecipitations. For the fluoxetine experiment, a total of six samples per group were analyzed. Immunoprecipitations for the fluoxetine treatment paradigm were performed twice, and the Western blot analysis of each immunoprecipitated sample was performed at least twice. The data are the means of six samples in one representative assay.
Statistical Analyses. All data are presented as group means ± S.E.M. The KD and Bmax data were analyzed using Student t tests. The GTPS-sensitive binding data, 5-HT-stimulated PLC activity, and 5-HT to GTPS ratio data were analyzed using a two-way ANOVA followed by a Newman-Keuls post hoc analysis. GTPS-stimulated PLC activity was analyzed using one-way ANOVA. Because of nonhomogeneity of variance assessed using Bartlett's Chi Square (p < 0.05), data from the immunoprecipitation assays for the DOI-treatment paradigm were analyzed using nonparametric Kruskal-Wallis one-way ANOVA. This was followed by a Newman-Keuls post hoc analysis. The immunoprecipitation assays of the fluoxetine-treatment paradigm, comparing fluoxetine with saline treatment, were analyzed using a Student's t test. GB-STAT software (Dynamic Microsystems, Inc., Silver Spring, MD) was used for all statistical analyses. A probability level of p < 0.05 was considered to be statistically significant for all statistical tests.
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To further determine whether 24-h DOI-induced desensitization of 5-HT2A receptors is a result of impaired capacity of the 5-HT2A receptor to couple to G proteins, GTPS-sensitive [125I](±)-DOI binding was performed at a single concentration (0.4 nM). As shown in Fig. 2, treatment of cells with DOI for 24 h reduced the amount of [125I](±)-DOI binding by 85.8% compared with vehicle-treated cells [F(1,11) = 18.11, p < 0.01]. GTPS (20 µM) significantly reduced the specific binding of [125I](±)-DOI in vehicle-treated cells by 73.6% [F(1,11) = 16.25, p < 0.01]. In cells treated with DOI for 24 h, GTPS did not significantly reduce [125I](±)-DOI binding. These data suggest that 24-h DOI treatment reduced the number of 5-HT2A receptors coupled to G proteins rather than altering binding to a high-affinity non-G protein-coupled conformational state of the receptor.
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Effect of Expression of G11S154D on [125I](±)-DOI-Labeled High-Affinity State of the 5-HT2A Receptor in A1A1v Cells. The results from our previous study (Shi et al., 2007) suggested that phosphorylation at G11 serine 154 contributes to DOI-induced desensitization of 5-HT2A receptor signaling. Here we sought to determine whether phosphorylation at G11 serine 154 residue decreased the coupling of G proteins with 5-HT2A receptor. [125I](±)-DOI-labeled density of 5-HT2A receptors was examined in A1A1v cells transfected with either the phosphorylation state mimic G11S154D or wild-type G11. The transfection efficiency was approximately 40 to 60%. As shown in Table 2, no significant difference in KD or the density of 5-HT2A receptors was observed between cells transfected with wild-type G11 or the phosphorylation state mimic. GTPS-sensitive [125I](±)-DOI binding was then determined at the KD concentration (0.4 nM). No significant difference in the density of 5-HT2A receptors was observed between cells transfected with wild-type G11 or phosphorylation state mimic at the KD concentration. However, in cells transfected with the phosphorylation state mimic G11S154D, GTPS-sensitive binding was reduced to 47.6 ± 9.6% GTPS-sensitive binding in cells transfected with wild-type G11 (Fig. 3). These data suggest less 5-HT2A-G protein coupling with the G11S154D phosphorylation mimic.
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Effect of in Vivo DOI Treatment on 5-HT-Stimulated PLC Activity. As shown in Fig. 4A, 5-HT stimulated PLC activity in the frontal cortex in a concentration-dependent manner. Pretreatment with daily injections of DOI (1 mg/kg) significantly decreased 10 µM 5-HT-stimulated PLC activity in the frontal cortex of rats [F(2,42) = 11.1, p = 0.0001]. With the 10 µM concentration, 5-HT-stimulated PLC activity was decreased by 32% after 4 days of DOI treatment (p < 0.01) and by 40% after 7 days (p < 0.01) of DOI treatment (Fig. 4A). DOI treatment reduced the PLC response to 0.3 µM 5-HT by 26% at 4 days and 39% at 7 days, but this difference did not reach statistical significance (Fig. 4A). A two-way ANOVA revealed a significant effect of 5-HT [F(1,42) = 19.3, p < 0.0001] and a significant effect of DOI [F(2,41) = 4.46, p < 0.05], but there was no significant interaction between 5-HT and DOI treatment [F(2,42) = 1.1, p > 0.05]. As shown in Fig. 4B, DOI treatment for either 4 or 7 days did not significantly affect GTPS-stimulated PLC activity in the frontal cortex [F(2,21) = 0.6, p = 0.56]. Although the GTPS-stimulated PLC activity was not significantly altered, we examined the ratio of 5-HT- to GTPS-stimulated PLC activity to determine whether variations in the GTPS-stimulated PLC activity for individual animals would influence 5-HT-stimulated effects. As shown in Fig. 4C, DOI treatment decreased the ratio of 10 µM 5-HT- to GTPS-stimulated PLC activity [F(2,41) = 4.46, p = 0.02] by 30% after 4 days (p < 0.05) and by 39% after 7 days (p < 0.05), similar to that seen by 10 µM 5-HT stimulation directly (Fig. 4A). With 0.3 µM 5-HT stimulation, the DOI-induced reductions in the ratio on 5-HT to GTPS-stimulated PLC activity were not statistically significant (Fig. 4C).
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Effect of in Vivo DOI Treatment on Levels of Phosphorylated Gq/11 Proteins. Using immunoprecipitation and Western blots, we demonstrated that Gq/11 proteins in the frontal cortex are phosphorylated as shown in Fig. 5. DOI treatment increased the levels of phosphorylated Gq/11 proteins in the frontal cortex in a time-dependent manner (Fig. 5, A and B). The levels of phosphorylation of Gq/11 proteins were 2, 31, and 51% above control levels after 1, 4, and 7 days of sustained DOI treatment, respectively. A nonparametric Kruskal-Wallis one-way ANOVA revealed a significant (p < 0.05) overall effect of DOI treatment. Newman-Keuls post hoc analysis indicated that phosphorylation of Gq/11 proteins after 7 days of DOI treatment was significantly increased (p < 0.05) over saline controls and 1 day of DOI treatment (Fig. 5A). There was no significant effect of 4 days of DOI treatment on phosphorylation of Gq/11 proteins. The specificity of the immunoprecipitation of Gq/11 proteins is shown in Fig. 5C. A second method was used to detect the phosphorylated proteins on blots prepared with immunoprecipitated Gq/11 proteins. As shown in Fig. 5D, using the Pro-Q Diamond Phosphoprotein Stain, the levels of phosphorylated Gq/11 proteins were higher in frontal cortex from rats treated for 7 days with DOI compared with saline-treated controls.
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Effect of in Vivo Chronic Fluoxetine Treatment on Phosphorylated Levels of Gq/11 Proteins. To determine whether the regulation of G protein phosphorylation is specific for agonist-induced desensitization or is a more general mechanism regulating 5-HT2A receptor signaling, we examined G protein phosphorylation in a model of indirect and nonselective 5-HT receptor stimulation using treatment with the serotonin reuptake inhibitor fluoxetine. We examined Gq/11 protein phosphorylation in the frontal cortex of rats treated with chronic fluoxetine, based on our previous findings that 21 days of daily fluoxetine increases 5-HT2A receptor-mediated PLC activity in rat frontal cortex (Damjanoska et al., 2003). As shown in Fig. 6, A and B, chronic fluoxetine treatment did not change the levels of phosphorylated Gq/11 proteins in the frontal cortex (Student's t test, p = 0.89).
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11 protein from 5-HT2A receptors in A1A1v cells. Our results also suggest that the increased phosphorylation of G11 protein induced by DOI treatment results in an uncoupling of G proteins from 5-HT2A receptors in A1A1v cells based on the use of a phosphorylation state mimic mutation, S154D, in G11 protein. However, the phosphorylation state mimic did not reduce agonist binding to 5-HT2A receptors. The additional effects of DOI treatment on receptor binding are consistent with our previous data suggesting that phosphorylation of G11 protein contributes approximately half of the total desensitization response caused by DOI treatment (Shi et al., 2007).
Previous studies found that chronic administration of the 5-HT2A receptor agonist DOI resulted in reductions in the density of 5-HT2A receptors in the cortex (Anji et al., 2000; Hensler and Truett, 1998; McKenna et al., 1989). In our current study, we found that DOI treatment significantly lowered the Bmax of [3H]ketanserin-labeled total 5-HT2A receptor binding. These data suggest that down-regulation of 5-HT2A receptors was involved in DOI-induced desensitization in A1A1v cells. DOI treatment did not result in changes in the KD value of either [125I](±)-DOI or [3H]ketanserin binding, suggesting that the affinity of the 5-HT2A receptors was not altered by DOI treatment. Our current data show significant reductions in [125I](±)-DOI binding to 5-HT2A receptors following 24-h DOI treatment in A1A1v cells and that the 46.5% reduction in agonist binding is much greater than the 24.8% reduction in antagonist binding. These relative differences in agonist and antagonist binding are consistent with previous studies on rat frontal cortex in which agonist binding was reduced 86% and antagonist binding was reduced 51% (McKenna et al., 1989). Based on the ternary complex model, these results suggest that, in addition to overall reductions in 5-HT2A receptor density, DOI treatment causes a decreased coupling of 5-HT2A receptors with G proteins. The data from GTPS binding experiments demonstrate that GTPS-sensitive binding is decreased in cells treated with DOI in comparison with vehicle-treated cells, which further confirms the decreased coupling of 5-HT2A receptors with G proteins.
The decreased coupling of 5-HT2A receptors with G proteins may be due to the phosphorylation of 5-HT2A receptors, phosphorylation of G proteins, or a reduction in G protein levels. Results from previous studies (Damjanoska et al., 2004; Roth et al., 1995) suggest that the agonist-induced desensitization of 5-HT2A receptor signaling is not likely due to alterations in the levels of G proteins. Although phosphorylation of 5-HT2A receptors could result in the uncoupling of 5-HT2A receptors with G proteins and 5-HT2A receptors containing several consensus sites for effector kinases, protein kinase C and calcium-calmodulin dependent kinase II, an increase in the phosphorylation of 5-HT2A receptors by these enzymes during agonist-induced desensitization has yet to be shown (Gray et al., 2003).
To examine the hypothesis that DOI-induced phosphorylation of G11 results in the uncoupling of G protein with 5-HT2A receptors, cells were transfected with either the phosphorylation state mimic G11S154D or wild-type G11. Forty-eight hours after transfection, the [125I](±)-DOI-labeled high-affinity state 5-HT2A receptors were determined. If phosphorylation of G11 results in uncoupling of G protein from 5-HT2A receptors, according to the ternary complex model, we would expect to see a lower Bmax of [125I](±)-DOI binding in cells transfected with G11S154D compared with cells transfected with wild-type G11. We were unable to detect the difference in Bmax and KD value between cells transfected with wild-type G11 and G11S154D. However, GTPS-sensitive binding was decreased in cells transfected with phosphorylation state mimic G11S154D (p < 0.05). One possible explanation of this result is that endogenous Gq/11 prevented detection of the difference, because the percentage of cells transiently transfected is approximately 40 to 60%. Another possibility is that, although 5-HT2A receptors exist in a high-affinity-G protein coupled state (Battaglia et al., 1984), some receptors can exist in a high-affinity state that is not coupled to G as suggested by a revised ternary model for 5-HT2A receptors (Egan et al., 2000; Roth et al., 1997). Based on the revised ternary model, GTPS-sensitive binding would be the better index of receptor-G protein coupling. In cells transfected with either wild-type or mutant G11 proteins, we found some agonist binding remaining after adding GTPS, suggesting that there is a population of 5-HT2A receptor proteins in the membrane that is able to bind agonist but is not G protein-coupled, consistent with the revised ternary model for 5-HT2A receptors (Egan et al., 2000; Roth et al., 1997).
Phosphorylation of G11 protein could hinder the coupling to 5-HT2A receptors by either directly interrupting of the interaction of G11 protein with 5-HT2A receptors or by directly interrupting the interaction of G
with G11 protein and thereby preventing the formation of the heterotrimeric G protein complex, which binds to 5-HT2A receptors. Phosphorylation of tyrosine residues in Gq/11 proteins disrupts the interaction of Gq/11 proteins with M1 muscarinic receptors (Umemori et al., 1997). Protein kinase C-mediated phosphorylation of Gz prevents association with G and conversely association of Gz with G prevents phosphorylation in vitro (Fields and Casey, 1995). However, Gz is primarily phosphorylated at Ser27 and is also phosphorylated at Ser16. Likewise, protein kinase C-mediated phosphorylation of G12 in cells and in vitro is prevented by its interaction with G (Kozasa and Gilman, 1996). However, G12 is also phosphorylated in the amino terminus of the protein, possibly within the first 50 amino acids based on digestion experiments. Previous studies have also shown that the amino terminus of Gq is important for binding to G, especially isoleucine 25 (Evanko et al., 2005). These results make decreased binding of G to Ser154-phosphorylated Gq/11 a less likely possibility. Previous studies have also found that phosphorylation of G proteins can reduce binding to other proteins. For example, phosphorylation of Gi2 inhibits interaction with the effector enzyme (Strassheim and Malbon, 1994), and phosphorylation of Gz inhibits interaction with regulator of G protein signaling (RGS) proteins (Glick et al., 1998).
In vivo, sustained treatment with DOI decreases the maximal response (Emax) of 5-HT-stimulated PLC activity and increases the phosphorylation of Gq/11 proteins. Maximal 5-HT-stimulated PLC activity was reduced in frontal cortex by 32% after 4 days and 40% after 7 days of DOI treatment. In the frontal cortex, phosphorylated Gq/11 proteins increase gradually and are 31% above control levels after 4 days and 51% above control levels after 7 days of DOI treatment. The time course and magnitude of the increase in phosphorylation of Gq/11 proteins correlates with the time course and magnitude of the desensitization 5-HT2A receptor-mediated PLC activity; although at 4 days of DOI, the increase in phosphorylation was not statistically significant. The lack of a statistically significant increase in phosphorylation of Gq/11 proteins after 4 days of DOI is probably due to experimental variability in the immunoprecipitation and Western blot analysis. These correlational results, together with the cell culture data, suggest that the increase in phosphorylated G11 protein contributes to desensitization of 5-HT2A receptors in the frontal cortex. Although for the rat brain experiments, we cannot distinguish between phosphorylation of Gq and G11 protein because the immunoprecipitation assay can only be performed with an antibody that cannot distinguish between the proteins, the cell culture data suggest that phosphorylation of only G11 is likely contributing to the desensitization response in frontal cortex. Sustained DOI treatment for 4 and 7 days induces desensitization of PLC activity stimulated by an Emax concentration of 5-HT. This treatment does not, however, induce a desensitization of GTPS-mediated PLC activity in the frontal cortex. These differential effects suggest that the desensitization of 5-HT2A receptor signaling is due to a disruption between the receptor and the G protein and not between the G protein and the effector.
This regulation of phosphorylated Gq/11 proteins is treatment-specific because it occurs with agonist-induced desensitization of 5-HT2A receptors but not with chronic fluoxetine treatment. Chronic stimulation of 5-HT2A receptors as found with fluoxetine treatment, resulted in an increase in 5-HT-stimulated PLC activity in the frontal cortex (Damjanoska et al., 2003), rather than a decrease in 5-HT-stimulated PLC activity as seen with sustained DOI treatment. The increase in PLC activity induced by chronic fluoxetine was not accompanied by a change in phosphorylation of Gq/11 proteins. Chronic activation of other 5-HT receptors by fluoxetine treatment, notably 5-HT1A receptors, probably has an impact on the regulation of 5-HT2A receptor signaling (Darmani et al., 1990; Krebs-Thomson and Geyer, 1998; Zhang et al., 2001) and possibly the phosphorylation of Gq/11 protein. It is clinically relevant to determine whether stimulation of other serotonin receptors, such as 5-HT1A receptors, inhibits phosphorylation of G11 protein in future studies. Alternately, desensitization of 5-HT1A receptors, as found with chronic fluoxetine treatment, could prevent G11 protein phosphorylation.
In conclusion, the present data support the hypothesis that phosphorylation of G11 proteins induced by DOI treatment causes uncoupling of 5-HT2A receptors with G11 protein in A1A1v cells and thereby contributes to desensitization of 5-HT2A receptor signaling. The in vivo data demonstrate that increased phosphorylation of Gq/11 proteins correlates with desensitization of 5-HT2A receptor signaling in rat frontal cortex, suggesting that similar mechanisms are involved in desensitization in vivo as in A1A1v cells.
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Article, publication date, and citation information can be found at http://jpet.aspetjournals.org.
ABBREVIATIONS: 5-HT, serotonin; DOI, (–)-1-(2,5-dimethoxy-4-iodophenyl)-2-aminopropane HCl; IOD, integrated optical density; PLC, phospholipase C; ANOVA, analysis of variance; E-64, N-(trans-epoxysuccinyl)-L-leucine 4-guanidinobutylamide; GTPS, guanosine 5'-3-O-(thio)triphosphate; MDL 100,907, 4-piperidinemethanol, 1-[2-[4-fluorophenyl]ethyl]--(2,3-dimethoxyphenyl)-, (R).
Address correspondence to: Dr. Nancy A. Muma, Department of Pharmacology and Toxicology, University of Kansas, School of Pharmacy, Lawrence, KS 66045. E-mail: nmuma{at}ku.edu
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