Agonist induced-phosphorylation of Galpha11 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 Galpha11 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 [125I](+/-)-DOI-labeled high-affinity binding sites (46%) than the reduction of [3H]ketanserin binding sites (25%). Although the KD values are not altered, there is a smaller amount of GTPgammaS [guanosine 5'-3-O-(thio)triphosphate]-sensitive [125I](+/-)-DOI binding in DOI-treated cells. These results suggest that DOI treatment causes down-regulation of 5-HT2A receptors and reductions in G protein-coupled 5-HT2A receptors. In contrast, in cells transfected with the phosphorylation state mimic G(alpha11)S154D, GTPgammaS-sensitive [125I](+/-)-DOI binding was decreased by 48%; however, there was no significant difference in the KD and Bmax values of [125I](+/-)-DOI-labeled receptors. The receptor binding experiments suggest that phosphorylation of Galpha11 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 Galpha11 to 5-HT2A receptors. To determine whether DOI increases phosphorylation of Galphaq/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 Galphaq/11 proteins by 51% in the frontal cortex. These data suggest that DOI causes phosphorylation of Galphaq/11 in vivo and could thereby contribute to the desensitization of 5-HT2A receptors.
Shi
Ju
J
Department of Pharmacology and Experimental Therapeutics, Loyola University Chicago, Stritch School of Medicine, Maywood, Illinois, USA.
Damjanoska
Katerina J
KJ
Singh
Rakesh K
RK
Carrasco
Gonzalo A
GA
Garcia
Francisca
F
Grippo
Angela J
AJ
Landry
Michelle
M
Sullivan
Nicole R
NR
Battaglia
George
G
Muma
Nancy A
NA
D000662:Amphetamines / Q000494:pharmacology*; D000818:Animals; D018929:Cell Culture Techniques; D002460:Cell Line; D005473:Fluoxetine / Q000494:pharmacology; D005625:Frontal Lobe / Q000187:drug effects / Q000378:metabolism; D043802:GTP-Binding Protein alpha Subunits, Gq-G11 / Q000096:biosynthesis / Q000378:metabolism*; D007650:Ketanserin / Q000494:pharmacology; D008297:Male; D010766:Phosphorylation; D011485:Protein Binding; D051381:Rats; D017207:Rats, Sprague-Dawley; D044402:Receptor, Serotonin, 5-HT2A / Q000235:genetics; D058826:Serotonin 5-HT2 Receptor Agonists; D017366:Serotonin Receptor Agonists / Q000494:pharmacology*; D017367:Selective Serotonin Reuptake Inhibitors / Q000494:pharmacology; D015398:Signal Transduction; D014162:Transfection; D010738:Type C Phospholipases / Q000378:metabolism
D000662:Amphetamines; D005473:Fluoxetine; D043802:GTP-Binding Protein alpha Subunits, Gq-G11; D007650:Ketanserin; D044402:Receptor, Serotonin, 5-HT2A; D058826:Serotonin 5-HT2 Receptor Agonists; D017366:Serotonin Receptor Agonists; D017367:Selective Serotonin Reuptake Inhibitors; D010738:Type C Phospholipases; C015952:4-iodo-2,5-dimethoxyphenylisopropylamine