Agonist induced-phosphorylation of Gα11 protein reduces coupling to 5-HT_2A receptors

We previously demonstrated that 24-h treatment with (-)-1-(2,5-dimethoxy-4- iodophenyl)-2-aminopropane HCl (DOI) causes phosphorylation of G _α11 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 B_max of [¹²⁵I] (±)-DOI-labeled high-affinity binding sites (46%) than the reduction of [³H]ketanserin binding sites (25%). Although the K_D values are not altered, there is a smaller amount of GTPγS [guanosine 5′-3-O-(thio)-triphosphate]-sensitive [¹²⁵I](±)-DOI binding in DOI-treated cells. These results suggest that DOI treatment causes downregulation of 5-HT_2A receptors and reductions in G protein-coupled 5-HT_2A receptors. In contrast, in cells transfected with the phosphorylation state mimic G_α11S154D, GTPγS-sensitive [¹²⁵I](±)-DOI binding was decreased by 48%; however, there was no significant difference in the K_D and B_max values of [¹²⁵I](±)-DOI-labeled receptors. The receptor binding experiments suggest that phosphorylation of G _α11 on serine 154 reduces coupling of 5-HT_2A receptors, whereas DOI causes down-regulation of 5-HT_2A receptors in addition to the phosphorylation-induced uncoupling of G_α11 to 5-HT_2A receptors. To determine whether DOI increases phosphorylation of G_αq/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 E_max concentration of 5-HT by 40% and increased phosphorylation of G _αq/11 proteins by 51% in the frontal cortex. These data suggest that DOI causes phosphorylation of G_αq/11 in vivo and could thereby contribute to the desensitization of 5-HT_2A receptors.