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Phosphorylation of G₁₁ Protein Contributes to Agonist-Induced Desensitization of 5-HT_2A Receptor Signaling

Department of Pharmacology and Experimental Therapeutics, Loyola University Chicago, Stritch School of Medicine, Maywood, Illinois

Received for publication June 22, 2006.

Accepted for publication October 20, 2006.

	Abstract

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Agonist treatment causes desensitization of many G proteincoupled receptor systems. Recent advances have delineated changes in receptors in the desensitization response; however, the role of G proteins remains unclear. We investigated the role of phosphorylation of G_q/11 proteins in agonist-induced desensitization of serotonin 2A (5-HT_2A) receptors. In an embryonic rat cortical cell line (A1A1v), 24-h treatment with 100 nM (-)-1-(2,5-dimethoxy-4-iodophenyl)-2-aminopropane HCl (DOI), a 5-HT_2A/2C receptor agonist, decreased DOI-stimulated inositol phosphate accumulation and increased the phosphorylation of G_q/11 proteins, as demonstrated by immunoprecipitation of G_q/11 and both incorporation of ³²P phosphate and labeling with a S/T/Y phosphorylation-dependent antibody. Treatment with DOI for 30 min induced desensitization but did not increase phosphorylation of G_q/11 proteins, suggesting that different mechanisms are involved in desensitization after short- and long-term treatments. Mutation of S154A in a protein kinase C (PKC) and calcium/calmodulin dependent kinase (CaMK) consensus site in G₁₁ significantly reduced DOI-stimulated phosphorylation of G₁₁ and DOI-induced desensitization of 5-HT_2A receptor signaling. Inhibition of PKC and CaMK attenuated phosphorylation of G_q/11 proteins and DOI-induced desensitization of 5-HT_2A receptors. Expression of G₁₁ S154D, a phosphorylation mimic, reduced DOI-stimulated inositol phosphate accumulation. DOI treatment for 24 h also produced heterologous desensitization, as indicated by decreased bradykinin-stimulated inositol phosphate accumulation. These data suggest that phosphorylation of G₁₁ protein by PKC and CaMK contributes to agonistinduced homologous desensitization of 5-HT_2A receptor signaling as well as heterologous desensitization. The phosphorylation of G protein represents a novel mechanism involved in regulation of receptor signaling and agonist-induced desensitization of G protein-coupled receptors.

Desensitization can occur as a result of receptor uncoupling from G proteins, internalization (sequestration of the receptor away from the cell surface), or down-regulation (reduced ligand-bound receptor); each has been reported for 5-HT_2A receptors. Internalization of 5-HT_2A receptors was induced by agonist stimulation in vivo and in cell culture models, although the mechanisms involved in internalization of 5-HT_2A receptors are cell type-specific (Grotewiel and Sanders-Bush, 1994; Gray et al., 2001; Hanley and Hensler, 2002). Blockade of receptor internalization prevented agonist-induced desensitization of endogenous 5-HT_2A receptors in C6 glioma cells but not in human embryonic kidney 293 cells transfected with 5-HT_2A receptors. Receptor binding and autoradiographic studies have shown a 40% average decrease in agonist and antagonist binding in several brain regions, including cortical areas after sustained agonist treatment (Buckholtz et al., 1988; McKenna et al., 1989; Smith et al., 1999; Anji et al., 2000; Valdez et al., 2002). McKenna et al. (1989) found a greater reduction in the B_max of the high affinity (±)-1-(2,5-dimethoxy-4-iodophenyl)-2-aminopropane HCl-labeled receptors than the reduction in B_max of ketanserin-labeled 5-HT_2A receptors after long-term agonist treatment. The greater decrease in the DOI-labeled receptors would reflect a reduction in G proteins coupling to 5-HT_2A receptors.

Our recent in vivo data are consistent with a reduction in G protein coupling. We previously found a decrease in serotonin (5-HT)-stimulated phospholipase C (PLC) activity in the frontal cortex of rats after 4 to 7 days of DOI treatment (Damjanoska et al., 2004). However, sustained DOI treatment had no effect on guanosine 5'-O-(3-thio)triphosphatestimulated PLC activity, suggesting that an alteration at the receptor or an alteration in receptor-G protein interaction mediates the desensitization of 5-HT_2A receptors. In addition, this desensitization cannot fully be explained by an alteration in the levels of G_q and G₁₁ proteins (Damjanoska et al., 2004).

Previous studies suggested that second messenger-dependent kinases, such as protein kinase C (PKC) and Ca²⁺-calmodulin dependent kinase (CaMK) are important in desensitization of 5-HT_2A receptors in cell culture models. Short-term studies demonstrated that PKC is important in 5-HT_2A receptor desensitization in Chinese hamster ovary cell lines that stably express human 5-HT_2A receptors (Berg et al., 2001) and HEK293 cells (Bhattacharyya et al., 2002). PKC inhibitors and CaMK inhibitors prevent agonist-induced 5-HT_2A receptor system desensitization in some (Berg et al., 2001) but not all cultured cells (Hanley and Hensler, 2002). Although kinases can mediate desensitization of 5-HT_2A receptor signaling, the target for phosphorylation is not known. Among the many potential targets, phosphorylation of 5-HT_2A receptors and G_q/11 proteins could lead to desensitization. Mutation of two serine residues to alanine residues on 5-HT_2A receptors attenuated agonist-induced desensitization of 5-HT_2A receptors; however, mutation of serine and threonine residues in PKC consensus sites had no effect on agonist-induced desensitization (Gray et al., 2003). Furthermore, in a variant of Chinese hamster lung fibroblasts, 5-HT_2A receptors are not phosphorylated after PKC-mediated 5-HT_2A receptor desensitization (Vouret-Craviari et al., 1995), suggesting that PKC-catalyzed phosphorylation of another protein such as G_q or G₁₁ proteins could be involved. In addition, CaMK has been shown to be involved in agonist-induced desensitization of 5-HT_2A receptor signaling (Gray et al., 2001), although it is also not clear which protein or proteins this kinase phosphorylates to mediate the desensitization of 5-HT_2A receptor signaling. To summarize these previous studies, PKC and CaMK are necessary for desensitization of 5-HT_2A receptor signaling, but it is not clear which proteins are phosphorylated by these kinases to mediate the desensitization response. G_q/11 proteins could be the necessary substrate for desensitization, because each contains several consensus sites for phosphorylation by either CaMK or PKC subtypes (, , , µ, ).

Few studies have investigated alterations in the signaling pathway downstream of 5-HT_2A receptors that may contribute to receptor desensitization. In the present study, we found that sustained treatment with DOI caused an increase in phosphorylation of G_q/11 proteins and desensitization of 5-HT_2A receptor signaling in A1A1v cells. Mutation of serine 154 to alanine in the G₁₁ subunit, a PKC and CaMK site, significantly attenuated DOI-induced desensitization of 5-HT_2A receptor signaling in A1A1v cells. These data suggest that phosphorylation of G₁₁ proteins contributes to sustained agonist-induced desensitization of 5-HT_2A receptor signaling.

	Materials and Methods

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Materials. Dulbecco's modified Eagle's medium (DMEM), Lipofectamine Plus, and [³²P]phosphate were supplied by Invitrogen (Carlsbad, CA). DOI and phorbol 12-myristate 13-acetate (PMA) were purchased from Sigma (St. Louis, MO). KN-93 was supplied by Tocris Cookson Inc. (Ellisville, MO). [myo-³H]Inositol was supplied by PerkinElmer Life and Analytical Sciences (Boston, MA). QuikChange site-directed mutagenesis kit was purchased from Stratagene (La Jolla, CA). All other reagents were of the highest grade available.

Site-Directed Mutagenesis and Plasmid Construction. The mammalian expression vector pcDNA 3.1(+) containing a cytomegalovirus promoter was purchased from Invitrogen. The pcDNA3.1(+) clones for human wild-type G₁₁, and G_q were obtained from the UMR cDNA Resource Center (http://www.cdna.org). The sequence similarity between rat G₁₁ and human G₁₁ is 96.1%. The sequence similarity between rat G_q and human G_q is 99.4%. G₁₁ contains five consensus PKC and/or CaMK phosphorylation sites (Ser154, Ser156, Ser268, Thr54, and Thr76). G_q contains four consensus PKC and/or CaMK phosphorylation sites (Ser154, Ser268, Thr54, and Thr76). These consensus sites are identical in rat and human proteins. The codons in G_q and G₁₁ genes that encode these serine or threonine residues were mutated to codons that encode alanine residues using the QuikChange site-directed mutagenesis kit from Stratagene (La Jolla, CA). Mutation of a serine or threonine to alanine is an approach commonly used to examine the role of phosphorylated residues. Codon 154 in G₁₁ was mutated to aspartic acid to mimic serine phosphorylation. DNA constructs used for transfection were purified from TOP10 Escherichia coli (Invitrogen) using Bio-Rad Quantum Prep Plasmid Miniprep kit (Bio-Rad Laboratories, Hercules, CA) according to the manufacturer's protocol. All DNA constructs were verified by DNA sequencing. Sequencing is performed on an ABI Prism 3100 4-capillary automated genetic analyzer (Agencourt Bioscience Corporation, Beverly, MA) by Loyola University Medical Center Core Facility.

Cell Culture and Transfections. A1A1v neuronal cells endogenously express the 5-HT_2A 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 DMEM supplemented with 10% fetal bovine serum in a humidified atmosphere containing 5% CO₂. Serum was heat-inactivated and charcoaltreated to remove monoamines (Berg et al., 1994; Scalzitti et al., 1998). Cells were plated onto 24-well plates at a density of 2 x 10⁴ cells/well. Cells were transiently transfected with one of the following cDNAs: either pcDNA3.1(+) empty vector, wild-type G_q, G₁₁, mutant G_q, or mutant G₁₁ using Lipofectamine Plus (Invitrogen) according to the manufacturer's recommendations. Total DNA of 4 µg/dish or 0.15 µg/well was used in each transfection. Overexpression of proteins was verified 48 h after transfection by Western blots. Samples containing 10 µg of protein were separated by SDS-polyacrylamide gel electrophoresis, and immunodetection was performed with either anti-G₁₁ (1:500; Santa Cruz Biotechnology, Santa Cruz, CA) or anti-G_q (1:500; Santa Cruz Biotechnology).

To determine the percentage of cells transiently transfected, cells were transiently transfected with either G₁₁-EE (Glu-Glu)-tagged (UMR, Rolla, MO) or pcDNA 3.1-EGFP (gift from Dr. Rory A. Fisher, University of Iowa, Iowa City, IA). Cells were transfected with G₁₁-EE tagged and 48 h later used for immunocytochemistry to determine transfection efficiency. Expression of G₁₁-EE was detected by fluorescein isothiocyanate-labeled Glu-Glu monoclonal antibody (1:500; Covance Research Products, Princeton, NJ). Vectashield mounting medium with 4,6-diamidino-2-phenylindole (Vector Laboratories, Inc., Burlingame, CA) was used to label cell nuclei, and the transfection ratio was determined by comparing fluorescein isothiocyanate- or green fluorescent protein-labeled cells with the total number of 4,6-diamidino-2-phenylindole-labeled cells.

Immunoprecipitation of G_q/11 Proteins. After treatment, cells grown in 100-mm dishes were washed twice in cold Tris-buffered saline, pH 7.4, and lysed in Tris assay buffer (50 mM Tris-HCl, 10 mM EGTA, 100 mM NaCl, 0.5% Triton-X100, 1 mM dithiothreitol, 50 mM NaF, 2 mM activated sodium orthovanadate, and protease inhibitor cocktail (Sigma) 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. After sonication and 30-min rotation at 4°C, the homogenate was centrifuged for 15 min at 20,000g. 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, Rockford, IL).

For immunoprecipitation of G_q/11 proteins, 1000 to 1500 µg of protein from each sample was brought up to a total 800-µl volume with a Tris assay buffer. Within an assay, the same amount of protein was used for all samples. The samples were then precleared using 25 µl of recombinant protein G (rProtein G) agarose (Invitrogen, Carlsbad, CA) for 1 h. The samples were centrifuged for 10 min at 10,600g, and the supernatant was incubated overnight at 4°C with either 2 µg of G_q/11 antibody (Santa Cruz Biotechnology) or 2 µg of normal rabbit IgG (Santa Cruz Biotechnology) as a control for nonspecific binding. The immuno-complexes were precipitated using 30 µl of the rProtein G agarose at 4°C for 1 h. The agarose-immuno complexes were washed three times using the Tris assay buffer and centrifuged after each wash at 1100g for 3 min. After the last wash, the agarose-immuno complex was resuspended in 2x electrophoresis sample buffer containing bromphenol blue and heated for 10 min at 100°C. The samples were centrifuged at 15,300g for 5 min, and the supernatant containing the G_q/11 proteins was removed.

The immunoprecipitated G_q/11 proteins were resolved by loading 15 µl of supernatant from each sample onto an SDS-polyacrylamide gel containing 0.1% SDS, 10% acrylamide/bisacrylamide (30:0.2), 4.6 M urea, and 375 mM Tris, pH 8.7. The proteins were electrophoretically transferred from the gels onto nitrocellulose membranes. The membranes were incubated at room temperature in blocking buffer (TBS solution containing 5% bovine serum albumin and 0.1% Tween 20) for 1 h and then incubated overnight at 4°C with phospho-Ser/Thr/Tyr antibody (1:200; Spring Bioscience, Fremont, CA) diluted in a TBS solution containing 5% bovine serum albumin and 0.1% Tween 20. The membranes were washed after the overnight incubation, followed by 1-h incubation at room temperature with a horseradish peroxidase-labeled anti-mouse antibody (1:50,000; Jackson ImmunoResearch Laboratories, Inc., West Grove, PA) diluted in the same TBS/bovine serum albumin solution as the primary antibody. Levels of G_q/11 proteins were examined to verify equal loading of protein in each lane [using G_q/11 antibody (1:500) and a horseradish peroxidase-labeled anti-rabbit antibody (1:100,000); Santa Cruz Biotechnology].

Films were analyzed densitometrically using the Scion Image program (Scion Corp., Frederick, MD). For normalization, the integrated optical density (IOD) of phosphorylated G_q/11 protein bands on each film was divided by the mean IOD of saline-treated animals and by the IOD of the respective G_q/11 protein bands (independent of their phosphorylation state). The G_q/11 protein usually resolved as a doublet (i.e., two bands very close together); however, on some films, a single larger band was evident. Both bands or the single larger band was included in the measurement of the G_q/11 protein bands.

In Vivo Labeling with [³²P]phosphate. For in vivo phosphorylation experiments, A1A1v cells were cultured and transfected as described above. Twenty-four hours after transfection, cells were labeled with [³²P]phosphate (1 mCi/100-mm dish) for 24 h, treated with the indicated amount of DOI during [³²P]phosphate labeling, and subjected to immunoprecipitation, gel electrophoresis, and analyses with autoradiography. Cells from two 100-mm dishes were combined for each sample. Levels of G_q/11 proteins were examined to verify the loading of protein in each lane. For normalization, the IOD of phosphorylated G_q/11 protein bands was divided by the IOD of the respective G_q/11 protein bands.

Phosphoinositol Hydrolysis. Cells were plated into 24-well plates with DMEM and 10% dialyzed fetal bovine serum. Approximately 18 to 24 h before the assay, cells were labeled with 1 µCi/ml [myo-³H]inositol in serum-free and inositol-free medium. PI hydrolysis assays were performed as described by Berg et al. (1994). In brief, cells were washed with Hanks' balanced salt solution containing 20 mM LiCl₂ and 20 mM HEPES, pH 7.4. After a 15-min preincubation in Hanks' balanced salt solution, the stimulation of PI hydrolysis was initiated by addition of DOI or bradykinin at 37°C. Reaction was stopped after 30 min by the addition of ice-cold 10 mM formic acid. The accumulation of total ³H-labeled inositol phosphates (IP) (inositol monophosphate, inositol bisphosphate, and inositol triphosphate) was determined by ion exchange chromatography.

Statistical Analyses. All data are presented as group mean ± S.E.M. The data were analyzed using a one- or two-way ANOVA followed by a Newman-Keul's post hoc analysis. GB-STAT software (Dynamic Microsystems, Inc., Silver Spring, MD) was used for all statistical analyses. The data for the [³²P]phosphate labeling resulted in unequal variances between groups, so the Wilcoxon MannWhitney U test was used as a nonparametric test in place of a Student's t test. Inhibition data were analyzed using Prism software (GraphPad Software, Inc. San Diego, CA). A probability level of p < 0.05 was considered to be statistically significant for all statistical tests.

	Results

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Identification of Possible Phosphorylation Sites in G_q and G₁₁ Proteins. Possible phosphorylation sites on G_q and G₁₁ proteins were searched for based on consensus sequences for various protein kinases and the amino acid sequences of G_q and G₁₁ proteins in the Swiss-Pro database using programs available on http://scansite.mit.edu. We found that G_q and G₁₁ proteins contain several consensus sites for phosphorylation by CaMK and for a number of PKC subtypes (, , , µ, ) (Fig. 1).

View larger version (37K): [in this window] [in a new window]   Fig. 1. Possible phosphorylation sites on Gq and G11 proteins. Consensus sites for possible phosphorylation of Gq and G11proteins were identified using programs available on http://scansite.mit.edu. Possible Ser/Thr phosphorylation sites are underlined, *, possible PKC (, , µ, ) site; , possible CaMK phosphorylation site.

View larger version (33K): [in this window] [in a new window]   Fig. 2. Effect of sustained DOI treatment (100 nM, 24 h) on IP accumulation and levels of phosphorylated Gq/11 proteins in A1A1v cells. A, treatment of A1A1v cells with 100 nM DOI for 24 h resulted in a significant attenuation in IP accumulation stimulated by 100 µM DOI compared with vehicle group. Data shown are the mean of triplicate determinations of a single representative experiment. Basal IP accumulation was 37.7 ± 2.1 and 39.5 ± 3.6 dpm for vehicle- and DOI-treated A1A1v cells, respectively. B, 5-HT2A receptor antagonist MDL 100,907 caused a dose-dependent (0.5-100 nM) inhibition of DOI-stimulated IP accumulation in A1A1v cells. The concentration required for 50% inhibition is 5.35 ± 0.86 nM. C, treatment with 100 nM DOI for 24 h significantly increased phosphorylation of Gq/11 protein compared with vehicle treatment. A representative blot showing increased phosphorylation of Gq/11 proteins after DOI treatment. Gq/11 proteins were immunoprecipitated and then separated on an SDS-PAGE gel. The blot was probed with a phosphorylation-dependent antibody and then with an antibody for Gq/11 proteins. D, increased phosphorylation of Gq/11 proteins after DOI treatment was probed by [32P]phosphate labeling. Phosphorylation data represent the mean ± S.E.M. of five experiments. E, treatment of cells with 100 nM DOI for 15 or 30 min resulted in a significant attenuation in IP accumulation stimulated by 100 µM DOI compared with the vehicle-treated group. Data shown are the mean ± S.E.M. of three experiments. F, levels of phosphorylation of Gq/11 proteins after DOI treatment for 30 min were probed by [32P] phosphate labeling. Phosphorylation data represent the mean ± S.E.M. of three experiments. **, p < 0.01 using Student's t test; +, p < 0.01 using Wilcoxon Mann-Whitney U test (used because of unequal variance).

We also checked the effect of short exposures of DOI on 5-HT_2A receptor-mediated IP accumulation and levels of phosphorylation of G_q/11 proteins. As shown in Fig. 2E, treatment of A1A1v cells with 100 nM DOI for 15 or 30 min resulted in a significant attenuation in the E_max values of IP accumulation stimulated by agonist (100 µM DOI) by 39.98 and 36.06% compared with the vehicle-only group (p < 0.01), respectively. In vivo labeling of cells with [³²P]phosphate showed that there is no change of the levels of phosphorylation of G_q/11 proteins after 30-min DOI treatment compared with vehicle-treated cells (p > 0.05) (Fig. 2F).

Role of Serine and Threonine Residues of G₁₁ on DOI-Induced 5-HT_2A Receptor Desensitization. G₁₁ contains five consensus PKC and/or CaMK phosphorylation sites: Ser154, Ser156, Ser268, Thr54, and Thr76 (Fig. 1). To explore the functional consequence of phosphorylation at these residues on DOI-induced desensitization of 5-HT_2A receptors in A1A1v cells, we individually mutated each of these serine and threonine residues in the G₁₁ subunit to alanine. Expression vectors encoding full-length wild-type or mutant G₁₁ were constructed. The overexpression of wild-type or mutated G₁₁ was verified in cell homogenates 48 h after transfection by Western blot analysis (Fig. 3A). Western blot analysis revealed an increase in the level of G₁₁ subunit in wild-type or mutant G₁₁ of approximately 10- to 20-fold compared with control vector group.

View larger version (24K): [in this window] [in a new window]   Fig. 3. Effect of PKC and CaMK consensus site mutations in G11 on DOI-induced desensitization of 5-HT2A receptor signaling in A1A1v cells. A1A1v cells were transiently transfected with vector, wild-type G11, or one of five G11 mutants. A, a representative blot demonstrates overexpression of wild-type G11 and five G11 mutants 48 h after transfection. The blot was probed with G11 antibody. B, treatment with 100 nM for 24 h significantly decreased IP accumulation in cells overexpressing of wild-type and mutant G11. **, p < 0.01; *, p < 0.05 compared with cells transfected with the same construct but treated with vehicle. C, overexpression of G11 S154A attenuated desensitization induced by DOI treatment. #, p < 0.05 compared with cells transfected with wild-type G11. The data are presented as a percentage of the cells transfected with wild-type G11 and treated with vehicle and are the mean ± S.E.M. of three experiments assayed in triplicate.

We also examined the effect of these consensus site mutants on the DOI-induced desensitization of 5-HT_2A receptor signaling. As shown in Fig. 3B, 24 h of treatment with 100 nM DOI resulted in a significant decrease in the maximum IP accumulation in cells overexpressing wild-type and mutant G₁₁ proteins compared with the respective vehicletreated control group. There was no significant interaction between DOI treatment and G₁₁ protein mutation (F_6,30 = 2.16, p = 0.08). Furthermore, as shown in Fig. 3C, there was a main effect of G₁₁ protein mutation for the DOI-induced decrease in IP accumulation (F_6,15 = 4.85, p < 0.01). Overexpression of G₁₁ S154A attenuated 5-HT_2A receptor desensitization induced by 24-h pretreatment with DOI. The reduction in DOI-mediated IP accumulation in cells overexpressing G₁₁ S154A was significantly attenuated by 40.9% (p < 0.05) compared with wild-type G₁₁ (Fig. 3C).

Role of Serine and Threonine Residues of G_q on DOI-induced 5-HT_2A Receptor Desensitization. Four consensus PKC and/or CaMK phosphorylation sites were found in G_q subunit: Ser154, Ser268, Thr54, and Thr76 (Fig. 1). Each of these residues was mutated, and an expression vector was constructed. Forty-eight hours after transfection, the overexpression of wild-type or mutant G_q protein subunit was verified by Western blot (Fig. 4A). Increases in G_q subunit levels of approximately 10-fold were found in cells transfected with wild-type and mutant G_q compared with vector control.

View larger version (20K): [in this window] [in a new window]   Fig. 4. Effect of PKC and CaMK consensus site mutation in Gq on DOI-induced desensitization of 5-HT2A receptor signaling in A1A1v cells. A, a representative blot demonstrating overexpression of wild-type Gq and four Gq mutants 48 h after transfection. The blot was probed with Gq antibody. B, treatment with 100 nM for 24 h resulted in a significant decrease in IP accumulation in cells overexpressing wild-type and Gq mutant cells. **, p < 0.01; *, p < 0.05 compared with cells transfected with the same construct but treated with vehicle. Overexpression of Gq T54A, T76A, and S268A did not cause a difference in IP accumulation after agonist stimulation compared with wild-type Gq. Overexpression of Gq S154A caused an increase in IP accumulation. , p < 0.05 compared with wild-type Gq. The data are presented as a percentage of cells transfected with wild-type Gq and treated with vehicle and are the mean ± S.E.M. of three experiments assayed in triplicate. C, none of the mutant Gq proteins caused difference in the extent of DOI-induced desensitization measured using IP accumulation.

To investigate the effect of PKC and CaMK consensus site mutations in G_q on DOI-mediated desensitization of 5-HT_2A receptor signaling, A1A1v cells were first transfected with either vector, wild-type G_q, or mutated G_q. After 24 h, transfected cells were treated with either vehicle or 100 nM DOI for another 24 h. Finally, the DOI-stimulated IP accumulation was used to estimate the effect of the mutation and DOI treatment. The two-way ANOVA for mutation and DOI treatment indicated significant main effects for G_q protein mutation (F_5,24 = 4.41, p < 0.01) and DOI treatment (F_1,24 = 144.63, p < 0.001). The interaction effect between mutation and DOI treatment was not statistically significant. There was no difference in the agonist-stimulated IP accumulation between wild-type G_q and vector-transfected cells. Overexpression of G_q T54A, T76A, and S268A did not cause a difference in IP accumulation after agonist stimulation compared with wild-type G_q (Fig. 4B). It is noteworthy that overexpression of G_q S154A caused an increase in IP accumulation compared with wild-type G_q protein overexpression (p < 0.01).

We also examined the effect of these consensus site mutants on DOI-induced 5-HT_2A receptor desensitization. As shown in Fig. 4B, 24 h of 100 nM DOI treatment resulted in a significant decrease in the IP accumulation in cells overexpressing wild-type proteins and each of the mutant G_q proteins compared with respective vehicle-treated control group (F_1,24 = 144.63, p < 0.001). There was no significant interaction between DOI treatment and mutation (F_5,24 = 1.22, p = 0.33). In the one-way ANOVA, there was no main effect of G_q protein mutation on the 24 h DOI-induced decrease in IP accumulation (F_5,12 = 0.22, p = 0.95). As seen in Fig. 4C, none of the mutant G_q proteins caused change in the extent of IP accumulation produced by 24 h DOI treatment.

Expression of Phosphorylation State Mimic G₁₁S154D in A1A1v Cells Decreased the 5-HT_2A Receptor Signaling. To confirm the key role played by G₁₁ Ser154 in mediating the effect of DOI-induced 5-HT_2A receptor desensitization, we mutated G₁₁ Ser154 to aspartic acid (G₁₁S154D), which is a commonly used approach for approximating a phosphorylated residue. A1A1v cells were transfected with G₁₁S154D and 24 h later were treated with vehicle or DOI 100 nM for 24 h. We also overexpressed G₁₁S154A as a control. Expression of G₁₁S154D was confirmed by Western blotting 48 h after transfection (Fig. 5A). Overexpression of G₁₁S154D resulted in decreased DOI-induced PI accumulation compared with wild-type G₁₁ (p < 0.05) and G₁₁S154A (p < 0.05) (Fig. 5B). We also examined the effect of this mutant on DOI-induced 5-HT_2A receptor desensitization. As shown in Fig. 5B, 24 h of 100 nM DOI treatment resulted in a significant decrease in the IP accumulation in cells overexpressing wild-type G₁₁ proteins and each of the mutant G₁₁ proteins compared with respective vehicle-treated control group (F_1,31 = 116.24, p < 0.001). There was no significant interaction between DOI treatment and mutation (F_3,24 = 2.9, p = 0.051). Furthermore, after 24-h pretreatment with DOI, the DOI-induced IP accumulation in cells overexpressing G₁₁ S154D was not significantly different from wild-type G₁₁ (Fig. 5B).

View larger version (22K): [in this window] [in a new window]   Fig. 5. Expression of G11S154D in A1A1v cells decreased 5-HT2A receptor signaling. A, a representative blot demonstrates overexpression of wild-type G11, G11S154A, or G11S154D mutant 48 h after transfection. B, overexpression of G11S154D resulted in decreased DOI-induced IP accumulation compared with cells transfected with wild-type G11 and G11S154A and treated with vehicle. #, p < 0.05; **, p < 0.01 compared with cells transfected with the same construct but treated with vehicle. The data are presented as a percentage of the cells transfected with wild-type G11 and treated with vehicle and are the mean ± S.E.M. of three experiments assayed in triplicate.

Effect of Expression of G₁₁S154A on DOI-Induced Phosphorylation of G_q/11 Proteins in A1A1v Cells. To determine whether 24-h DOI treatment altered phosphorylation of G_q/11 at sites other than G₁₁ Ser154, we compared phosphorylation in cells transfected with wild-type G₁₁ or G₁₁S154A and treated with DOI or vehicle for 24 h. To normalize phosphorylation levels to the levels of G_q/11 (i.e., to normalize for equal loading of protein), we also examined the G_q/11 protein levels on Western blots. As shown in Fig. 6A, 24 h of treatment with 100 nM DOI significantly increased phosphorylated G_q/11 proteins levels to 190% of vehicle treated cells transfected with wild-type G₁₁ proteins as measured by [³²P]phosphate incorporation. Levels of phosphorylation of G_q/11 proteins were not increased in the cells transfected with G₁₁S154A after DOI treatment (Fig. 6A).

View larger version (27K): [in this window] [in a new window]   Fig. 6. Effect of expression of G11S154A on DOI-induced phosphorylation of Gq/11 proteins. A, A1A1v cells were transfected with wild-type G11 or G11S154A and treated with DOI or vehicle for 24 h. The level of phosphorylated Gq/11 protein was measured by [32P] phosphate incorporation and normalized to the levels of Gq/11 (to verify the equal loading of protein). *, p < 0.05 compared with cells transfected with the same construct but treated with vehicle. B, a representative blot demonstrating the change of the phosphorylated Gq/11 protein probed with [32P] phosphate.

Effect of Kinase Inhibitor on DOI Induced Desensitization. To determine whether second messenger-dependent kinases, such as PKC or CaMK, play a role in DOI-induced desensitization of 5-HT_2A receptors, cells were pretreated with the selective CaMK inhibitor KN-93 or PKC activator PMA. Overnight treatment with PMA causes the down-regulation of most PKC isoforms (Dempsey et al., 2000). Therefore, in our experiment, we used it as a PKC inhibitor. Different concentrations of inhibitors were tested to determine the concentration that produced inhibition of phosphorylation without causing cell death (data not shown). As shown in Fig. 7, pretreatment of cells with KN-93 (Fig. 7A) or PMA (Fig. 7B) for 24 h attenuated DOI-induced attenuation of DOI-stimulated IP accumulation by 36.9% (p < 0.01) and 23.52% (p < 0.01), respectively. Next, we measured the effects of KN-93 and PMA on the phosphorylation of G_q/11 using [³²P] phosphate incorporation. Pretreatment of cells with KN-93 or PMA for 24 h attenuated the DOI-induced increase in phosphorylation of G_q/11 proteins by 41.0% (p < 0.05) and 28.3% (p < 0.05), respectively (Fig. 7C).

View larger version (33K): [in this window] [in a new window]   Fig. 7. Inhibitors of second messenger-dependent kinases attenuate DOI-induced desensitization of 5-HT2A receptor functions and phosphorylation of Gq/11 protein. Cells were pretreated with 10 µMPMA for 24 h or 10 nM KN-93 for 30 min before the addition of 100 nM DOI for 24 h first, then incubated with kinase inhibitors during the DOI treatment. A and B, accumulation of [3H]IP was measured in triplicate after 30-min incubation with DOI. Treatment of cells with PMA alone did not alter the Emax (vehicle, 253.5 ± 54.3% above basal; PMA-treated, 216.6 ± 35.5% above basal; n = 4). Treatment of cells with KN-93 alone did not alter the Emax (vehicle, 176.8 ± 17.5% above basal; KN-93-treated, 134.1 ± 12.3% above basal; n = 5). Data plotted are expressed as Emax, percentage of respective control. Each value represents the mean ± S.E.M. of four experiments. Data were analyzed by Student's t test; **, p < 0.01. C, the levels of phosphorylated Gq/11 protein were measured by [32P] phosphate incorporation and normalized to the levels of Gq/11 (for the equal loading of Gq/11 protein). Data were analyzed by one-way ANOVA and Newman-Keuls post hoc [F3,11 = 4.5, p = 0.039]; *, p < 0.05 compared with cells treated with DOI. D, a representative blot of the phosphorylated Gq/11 protein probed with [32P]phosphate.

11

View larger version (26K): [in this window] [in a new window]   Fig. 8. Effect of sustained DOI treatment (100 nM, 24 h) on bradykinin receptor-mediated signaling. A, the concentration-response curves represent IP accumulation after 30-min incubations with various concentrations of bradykinin (10-1010-5 M) induced. We found concentration-dependent increases of IP accumulation with an EC50 at 10-8 M and an Emax at 10-5 M. Data shown are the mean of triplicate determinations in a single representative experiment. B, treatment of A1A1v cells with 100 nM DOI for 24 h resulted in a significant attenuation in IP accumulation stimulated by 10-8 M bradykinin compared with the vehicle-treated group. Data represent the mean ± S.E.M. of three experiments. Basal IP accumulations were 45.6 ± 5.5 and 48.5 ± 2.5 dpm for vehicle- and DOI-treated A1A1v cells, respectively. *, significantly different at p < 0.05 using Student's t test. C and D, cells transfected with G11S154D (4 µg/plate) for 48 h had a lower IP accumulation after stimulation with 10-8 M (C) and 10-5 M (D) bradykinin compared with cells transfected with wild-type G11. Data represent the mean ± S.E.M. of 3 experiments. *, significantly different at p < 0.05 using Student's t test; **, significantly different at p < 0.01 using Student's t test.

	Discussion

Top Abstract Materials and Methods Results Discussion References

11

Desensitization of the 5-HT_2A receptor was induced by both short-term (30-min) and prolonged (24-h) DOI exposure in our study. However, the increased phosphorylation of G_q/11 proteins occurred only with prolonged DOI exposure, not with short-term, 30-min DOI exposure. Consistent with previous reports, our results suggest that different mechanisms are involved in the production of desensitization of 5-HT_2A receptors after short-term and prolonged exposure to agonist (Roth et al., 1995; Hanley and Hensler, 2002).

To determine whether the phosphorylation of G₁₁ and G_q is necessary for agonist-induced desensitization, we overexpressed the wild-type and mutant G₁₁ and/or G_q in A1A1v cells. A recent study reported a large increase in maximal agonist-induced IP production after the overexpression of G_q protein using an inducible system that produced a 38-fold increase compared with noninduced cells (Scragg et al., 2005). However, we found no difference in DOI-stimulated IP accumulation among cells transfected with vector, overexpression of wild-type G₁₁ or G_q, and mutant G₁₁ or G_q except for the G_q S154A mutant. The difference between the results of that report and the results reported herein may be due to the lower levels of G₁₁ or G_q expression in our studies (10-20-fold). It is noteworthy that we found that DOI-induced desensitization was not completely suppressed in cells transiently transfected with G₁₁S154A. One simple explanation for this phenomenon is transfection efficiency, because only 50% of cells were transfected. Another possible explanation is that other mechanisms also contribute to the desensitization response produced by 24-h exposure to DOI. We were surprised to find that the increase in phosphorylation of G_q/11 protein was completely suppressed in cells transfected with G₁₁S154A and treated with DOI for 24 h. We would expect to see the same reduction in phosphorylation that occurred for the IP accumulation. We suspect that the sensitivity of the IP accumulation assay is greater than the sensitivity in the assay measuring phosphorylation; this difference could account for our inability to detect phosphorylation differences in cells transfected with G₁₁S154A compared with wild-type G₁₁.

Previous studies have shown that PKC and CaMK are necessary for short-term desensitization of 5-HT_2A receptor signaling, but 5-HT_2A receptors do not appear to be the necessary substrate of these kinases (Vouret-Craviari et al., 1995; Anji et al., 2001; Berg et al., 2001; Gray et al., 2003). Because Ser154 in G₁₁ is a PKC and CaMK consensus site, these previous studies are consistent with our data, suggesting that phosphorylation of G₁₁ protein is necessary for complete agonist-induced desensitization of 5-HT_2A receptor signaling. The increase in phosphorylation of G_q/11 proteins that we observed could be due to a number of mechanisms. Agonist treatment could increase activity of kinases or decrease activity of phosphatases. Both PKC and CaMK could be activated by sustained 5-HT_2A receptor agonist treatment. As shown in this report and many others, 5-HT_2A receptor stimulation leads to an increase in PLC activity (Berg et al., 2001). Thereafter, PLC-catalyzed production of diacylglycerol stimulates PKC activity. In addition, PLC-catalyzed production of inositol triphosphate leads to increases in intracellular calcium, leading to an increase in CaMK activity. Our results suggest that both PKC and CaMK play a role in increased phosphorylation of G_q/11 proteins because treatment of cells with a CaMK inhibitor (KN-93) or PKC inhibitor (overnight treatment of PMA) attenuated DOI-induced attenuation of DOI-stimulated IP accumulation and phosphorylation of G_q/11 proteins.

The data from our lab have shown that 4 and 7 days of DOI treatment are sufficient to induce a desensitization of PLC activity stimulated 5-HT in rat frontal cortex (Damjanoska et al., 2004). This treatment does not, however, induce a desensitization of guanosine 5'-O-(3-thio)triphosphate-mediated PLC activity. These differential effects suggest that desensitization of 5-HT_2A receptor signaling is due to a disruption between the receptor and the G protein and not between the G protein and the effector. Phosphorylation of G_q/11 proteins may hinder receptor and G protein interaction. Indeed, phosphorylation of tyrosine residues in G_q/11 proteins diminished the interaction of G_q/11 proteins with M1 muscarinic receptors in mouse embryo fibroblast cells (Umemori et al., 1997). Other in vitro studies suggest that some but not all G subunits are substrates for phosphorylation by protein kinase C (Fields and Casey, 1995; Kozasa and Gilman, 1996; Glick et al., 1998). Phosphorylation of the G₁₂ and G_z protein subunits inhibit the interaction of these subunits with the G heterodimer (Fields and Casey, 1995; Kozasa and Gilman, 1996); phosphorylation of G_i2 inhibits interaction with the effector enzyme (Strassheim and Malbon, 1994), and phosphorylation of G_z inhibits interaction with RGS proteins (Glick et al., 1998). Phosphorylation of the G₁₁ protein could prevent the formation of the G protein trimer or prevent the G protein trimer from associating with the receptor, or both. Caveolin-1 has also been shown to be important in the coupling of 5-HT_2A receptors to G proteins, indicating that phosphorylation of G₁₁ could potentially interfere with the interaction of G₁₁ protein with caveolin-1 (Bhatnagar et al., 2001).

In our current study, we found that 24-h DOI treatment significantly decreased bradykinin-stimulated IP accumulation at the ED₅₀ concentration compared with vehicle-treated cells (p < 0.05), but not at the E_max concentration. This DOI-mediated heterologous desensitization of bradykinin stimulated IP accumulation is pharmacologically characterized as a right-shift in the dose-response curve with no change in the maximal response (E_max). A right-shift in the dose-response curve suggests a reduction in coupling of G protein to bradykinin receptors. Consistent with phosphorylation of G₁₁ causing the heterologous desensitization of bradykinin receptor system in A1A1v cells, transfection of G₁₁S154D, the phosphorylation state mimic also produced heterologous desensitization of bradykinin receptor signaling.

It is noteworthy that overexpression of mutant G_qS154A increased the DOI-stimulated IP accumulation compared with wild-type G_q. These results are consistent with the hypothesis that the phosphorylation of G_q Ser154 may occur constitutively in A1A1v cells and decrease 5-HT_2A receptor mediated signaling. Further studies are needed to determine whether this site is constitutively phosphorylated, as the data with the G_qS154A mutant suggest. Mutation of the same site in G₁₁ resulted in diminished agonist-mediated desensitization of 5-HT_2A receptor signaling. Furthermore, expression of the phosphorylation state mimic G₁₁S154D decreased the 5-HT_2A receptor signaling. All these data support the hypothesis that the G_q/11 Ser154 site is important in the function of G_q/11 proteins and that phosphorylation of this site plays a regulatory role in signaling.

In conclusion, these studies are the first to show that agonist-induced desensitization of 5-HT_2A receptor signaling correlates with increased phosphorylation of G_q/11 proteins and that phosphorylation of G₁₁ Ser154 is necessary for the full desensitization response of the 5-HT_2A receptor system in a cell culture model. Although our assay to detect phosphorylation of G_q/11 was not able to differentiate phosphorylation of G_q and G₁₁, our results with mutant G_q/11 proteins suggest that phosphorylation of only G₁₁ was involved in the desensitization response induced by DOI. The contribution of phosphorylation of G₁₁ in the desensitization of 5-HT_2A receptor signaling underscores the importance of studying the involvement of various postsynaptic proteins in receptor signaling pathways to fully delineate the mechanisms mediating desensitization phenomena. In this study, we examined only single mutations in G_q/11 proteins. It would be interesting to determine the effects of mutants with multiple substitutions for serine and threonine residues compared with single mutations in G_q/11 proteins, because previous studies have demonstrated functional cooperativity in phosphorylation. Further studies are also needed to determine whether phosphorylation of G₁₁ is involved in antagonist-induced desensitization of 5-HT_2A receptors or in agonist-induced desensitization of 5-HT_2A receptors in animal models.

	Footnotes

 
This work was supported by United States Public Health Service grant MH068612.

ABBREVIATIONS: 5-HT, serotonin (5-hydroxytryptamine); DOI, (-)-1-(2,5-dimethoxy-4-iodophenyl)-2-aminopropane HCl; PLC, phospholipase C; PKC, protein kinase C; CaMK, Ca²⁺-calmodulin dependent kinase; DMEM, Dulbecco's modified Eagle's medium; PMA, phorbol 12-myristate 13-acetate; KN-93, 2-(N-(2-hydroxyethyl)-N-(4-methoxybenzenesulfonyl))amino-N-(4-chlorocinnamyl)-N-methylbenzylamine; TBS, Tris-buffered saline; IOD, integrated optical density; PI, phosphoinositol; IP, inositol phosphate; ANOVA, analysis of variance; MDL 100,907, (±)- -(2,3-dimethoxyphenyl)-1-[2-(4-fluorophenylethyl)]-4-piperidinemethanol; ANOVA, analysis of variance.

Address correspondence to: Dr. Nancy A. Muma, Department of Pharmacology Loyola University Chicago, Stritch School of Medicine, 2160 South First Avenue, Maywood, IL 60153. E-mail: nmuma{at}lumc.edu

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