Agonist Induced-Phosphorylation of G 11 Protein Reduces Coupling to 5-HT2A Receptors

We previously demonstrated that 24-h treatment with ( )-1(2,5-dimethoxy-4-iodophenyl)-2-aminopropane HCl (DOI) causes phosphorylation of G 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 [ I]( )-DOI-labeled highaffinity binding sites (46%) than the reduction of [H]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 [I]( )-DOI binding in DOI-treated cells. These results suggest that DOI treatment causes downregulation of 5-HT2A receptors and reductions in G proteincoupled 5-HT2A receptors. In contrast, in cells transfected with the phosphorylation state mimic G 11S154D, GTP S-sensitive [I]( )-DOI binding was decreased by 48%; however, there was no significant difference in the KD and Bmax values of [I]( )-DOI-labeled receptors. The receptor binding experiments suggest that phosphorylation of G 11 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 G 11 to 5-HT2A receptors. To determine whether DOI increases phosphorylation of G q/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 G q/11 proteins by 51% in the frontal cortex. These data suggest that DOI causes phosphorylation of G q/11 in vivo and could thereby contribute to the desensitization of 5-HT2A receptors. Serotonin (5-HT) 2A receptors play important roles in the central nervous system (Roth et al., 1986; 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 downregulation 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 This work was supported by United States Public Health Service Grants MH068612, DA013669, and NS034153. Article, publication date, and citation information can be found at http://jpet.aspetjournals.org. doi:10.1124/jpet.107.122317. 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; GTP S, guanosine 5 -3-O-(thio)triphosphate; MDL 100,907, 4-piperidinemethanol, 1-[2-[4-fluorophenyl]ethyl]-(2,3-dimethoxyphenyl)-, ( R). 0022-3565/07/3231-248–256$20.00 THE JOURNAL OF PHARMACOLOGY AND EXPERIMENTAL THERAPEUTICS Vol. 323, No. 1 Copyright © 2007 by The American Society for Pharmacology and Experimental Therapeutics 122317/3258862 JPET 323:248–256, 2007 Printed in U.S.A. 248 at A PE T Jornals on A uust 7, 2017 jpet.asjournals.org D ow nladed from 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 G 11 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 G q/11 protein. Using site-directed mutagenesis, we found that mutation of serine 154 to alanine on G 11 protein reduced the desensitization of 5-HT2A receptor signaling and prevented the increase in phosphorylation of G q/11 protein caused by DOI. Mutation of G 11 protein serine 154 to aspartic acid, a phosphorylation mimic, directly reduced 5-HT2A receptor signaling. In contrast, mutation of G q 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 G q/11 protein in rat brain. Now we report the outcome of a time-course study to determine the impact of DOI on phosphorylation of G q/11 protein and desensitization of 5-HT2A receptor signaling in rat frontal cortex. In rat frontal cortex, G 11 protein mRNA is more abundant than G q protein mRNA (Tanaka et al., 2000), suggesting that increased phosphorylation of G 11 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 G q/11 protein, a serotonin reuptake inhibitor, fluoxetine, which increases 5-HT2A receptor signaling. We further hypothesize that phosphorylation of G 11 protein reduces 5-HT2A receptor signaling by altering the interactions of G 11 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 GTP S [guanosine 5 -3-O-(thio)triphosphate], and suggested that an alteration at the receptor or an alteration in coupling of 5-HT2A receptors to G q/11 protein occurs with agonist treatment (Damjanoska et al., 2004). To determine the impact of phosphorylation of G 11 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 [I]( )-DOI, which labels high-affinity state of the 5-HT2A receptor, and [ H]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 GTP S (tetrasodium salt solution) on [I]( )-DOI binding. Materials and Methods