Phosphorylation of G_α11Protein Contributes to Agonist-Induced Desensitization of 5-HT_2AReceptor Signaling

Abstract

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 (Ϫ)-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 32 P 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 G␣ 11 significantly reduced DOI-stimulated phosphorylation of G␣ 11 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 G␣ 11 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 G␣ 11 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.

Sustained treatment with a 5-HT2A receptor agonist causes functional desensitization and reductions in agonist-labeled 5-HT2A receptors despite increases in receptor protein levels in rats

“…Posttranslational modifications to 5-HT 2A receptors (Gray, et al, 2003), G αq/11 proteins (Shi, et al, 2007b;Shi, et al, 2007a) or both could alter the interaction of 5-HT 2A receptors with G αq/11 proteins and thereby alter agonist binding. Our previous studies in frontal cortex of rats treated with DOI as well as in cells in culture, suggest that sustained treatment with DOI increases the phosphorylation of G α11 protein and thereby reduces coupling to 5-HT 2A receptors (Shi et al, 2007a;Shi et al, 2007b). Similar mechanisms may be at work in the PVN, but unfortunately due to the small size of the nucleus, we are unable to analyze phosphorylation of G α11 protein using our current techniques.…”

Phosphorylation of GαsInfluences Its Association with the μ-Opioid Receptor and Is Modulated by Long-Term Morphine Exposure

“…Phosphorylation of G␣ i suppresses the hormonal inhibition of adenylyl cyclase (AC) in human platelet membranes (Katada et al, 1985) and ␦-opioid receptor mediated inhibition of AC activity in NG108-15 cells (Strassheim and Malbon, 1994). Recently, G␣ 11 protein phosphorylation has been demonstrated to contribute to diminishing 5-HT2A receptor signaling (Shi et al, 2007). In addition, tyrosine phosphorylation of purified recombinant G␣ s by immunecomplexed pp60c-src enhances rates of ␤-adrenergic receptor-mediated binding of guanosine 5Ј-O-(2-[ 35 S]thio)triphosphate (GTP␥S) as well as receptor-stimulated steady-state rate of GTP hydrolysis by G s (Hausdorff et al, 1992).…”

Chronic olanzapine activates the Stat3 signal transduction pathway and alters expression of components of the 5-HT2A receptor signaling system in rat frontal cortex

“…An increase in G αq/11 proteins would be expected when an increase rather than a decrease in 5-HT 2A receptor signaling is detected and therefore the increase in membrane-associated G αq/11 proteins could not contribute to the desensitization response caused by chronic olanzapine. Other studies in our laboratory suggest that phosphorylation of G α11 protein contributes to desensitization of 5-HT 2A receptor signaling induced by DOI treatment (Shi, et al, 2007) and could mitigate the effects of increased levels of G α proteins. We are currently determining whether phosphorylation of G α11 protein occurs with chronic olanzapine treatment as well.…”

“…In this scenario, total 5-HT 2A receptor protein levels could increase despite a decrease in agonist binding if the post-translational modification directly reduced the number of receptors that can bind to the agonist or indirectly altered agonist binding by interfering with the ability of the receptor to bind to Gα proteins, and thus maintaining the receptors in a low affinity state. Phosphorylation of 5-HT 2A receptors has been described in cells in culture (Gray, et al, 2003) and we have demonstrated phosphorylation of G α11 protein that causes desensitization of 5-HT 2A receptor signaling (Shi, et al, 2007). Considering that the 5-HT 2A receptor antibody epitope is based on the primary amino acid sequence, it is feasible to obtain differences in levels of 5-HT 2A receptors detected by immunoblot analysis versus changes detected by radioligand binding studies.…”