RT Journal Article
SR Electronic
T1 Differential Activation of Gq/11 and Gi<sub>3</sub> Proteins at 5-Hydroxytryptamine<sub>2C</sub> Receptors Revealed by Antibody Capture Assays: Influence of Receptor Reserve and Relationship to Agonist-Directed Trafficking
JF Molecular Pharmacology
JO Mol Pharmacol
FD American Society for Pharmacology and Experimental Therapeutics
SP 578
OP 589
DO 10.1124/mol.62.3.578
VO 62
IS 3
A1 Didier Cussac
A1 Adrian Newman-Tancredi
A1 Delphine Duqueyroix
A1 Valérie Pasteau
A1 Mark J. Millan
YR 2002
UL http://molpharm.aspetjournals.org/content/62/3/578.abstract
AB As determined by a guanosine 5′-O-(3-[35S]thio)triphosphate ([35S]GTPγS) binding assay, which does not distinguish G protein subtypes, 5-hydroxytryptamine (5-HT) and 2(S)- 1-(6-chloro-5-fluoro-1H-indol-1-yl)-2-propanamine fumarate (Ro600175) behaved as full agonists at human 5-HT2C (h5-HT2C) receptors (VSV isoform) stably expressed in Chinese hamster ovary (CHO) cells, whereas 1–2,5-dimethoxy-4-iodophenyl-2-aminopropane (DOI),d-lysergic acid diethylamide (LSD), and lisuride exhibited partial agonist properties. After treatment with pertussis toxin to uncouple 5-HT2C receptors from Gi/Go but not Gq/11, DOI and LSD were as efficacious as 5-HT and Ro600175 in stimulating [35S]GTPγS binding, whereas lisuride still exhibited low efficacy (40%). Correspondingly, in a scintillation proximity assay employing specific antibodies against Gq/11, 5-HT, Ro600175, DOI, and LSD behaved as high-efficacy agonists, whereas lisuride showed efficacy of 36%. In contrast, when employing a specific antibody recognizing Gi3, DOI and LSD were less efficacious (80 and 30%, respectively) than 5-HT and Ro600175, and lisuride was inactive. Agonist actions were specifically mediated by h5-HT2C receptors inasmuch as the selective 5-HT2C antagonist SB242,084 blocked [35S]GTPγS binding at both Gq/11 and Gi3. Agonist potency for stimulation of Gi3 was ∼6- to 8-fold less than for Gq/11, indicating that the latter was preferentially engaged by h5-HT2C receptors. Inactivation of h5-HT2C receptors with the alkylating agentN-ethoxycarbonyl-2-ethoxy-1,2-dihydroquinoline did not modify the efficacy of 5-HT, Ro600175, and DOI at Gq/11, whereas their efficacies were substantially reduced at Gi3, indicating a greater receptor reserve for the former. Finally, the preferential activation of Gq/11 versus Gi3 by DOI, LSD, and lisuride was diminished in the presence of lower receptor number. In conclusion, h5-HT2C receptors couple to both Gq/11 and Gi3in CHO cells, and efficacy for G protein subtype activation is both ligand- and receptor reserve-dependent.