Agonist induced-phosphorylation of Gα11 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(alpha 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 B-max of [I-125](+/-)-DOI-labeled high-affinity binding sites (46%) than the reduction of [H-3] ketanserin binding sites (25%). Although the K D values are not altered, there is a smaller amount of GTP gamma S [ guanosine 5 '-3-O-(thio)triphosphate]- sensitive [I-125](+/-)-DOI binding in DOI-treated cells. These results suggest that DOI treatment causes down-regulation 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(alpha 11) S154D, GTP gamma S-sensitive [I-125](+/-)-DOI binding was decreased by 48%; however, there was no significant difference in the K D and B-max values of [I-125](+/-)-DOI-labeled receptors. The receptor binding experiments suggest that phosphorylation of G(alpha 11) on serine 154 reduces coupling of 5-HT2A receptors, whereas DOI causes down-regulation of 5-HT2A receptors in addition to the phosphorylationinduced uncoupling of G(alpha 11) to 5-HT2A receptors. To determine whether DOI increases phosphorylation of G(alpha 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 E-max concentration of 5-HT by 40% and increased phosphorylation of G(alpha q/11) proteins by 51% in the frontal cortex. These data suggest that DOI causes phosphorylation of G(alpha q/11) in vivo and could thereby contribute to the desensitization of 5-HT2A receptors.