Evidence for a model of agonist-induced activation of 5-hydroxytryptamine 2A serotonin receptors that involves the disruption of a strong ionic interaction between helices 3 and 6.
5-Hydroxytryptamine 2A (5-HT2A) receptors are essential for the actions of serotonin (5-hydroxytryptamine (5-HT)) on physiological processes as diverse as vascular smooth muscle contraction, platelet aggregation, perception, and emotion. In this study, we investigated the molecular mechanism(s) by which 5-HT activates 5-HT2A receptors using a combination of approaches including site-directed mutagenesis, molecular modeling, and pharmacological analysis using the sensitive, cell-based functional assay R-SAT. Alanine-scanning mutagenesis of residues close to the intracellular end of H6 of the 5-HT2A receptor implicated glutamate Glu-318(6.30) in receptor activation, as also predicted by a newly constructed molecular model of the 5-HT2A receptor, which was based on the x-ray structure of bovine rhodopsin. Close examination of the molecular model suggested that Glu-318(6.30) could form a strong ionic interaction with Arg-173(3.50) of the highly conserved "(D/E)RY motif" located at the interface between the third transmembrane segment and the second intracellular loop (i2). A direct prediction of this hypothesis, that disrupting this ionic interaction by an E318(6.30)R mutation would lead to a highly constitutively active receptor with enhanced affinity for agonist, was confirmed using R-SAT. Taken together, these results predict that the disruption of a strong ionic interaction between transmembrane helices 3 and 6 of 5-HT2A receptors is essential for agonist-induced receptor activation and, as recently predicted by ourselves (B. L. Roth and D. A. Shapiro (2001) Expert Opin. Ther. Targets 5, 685-695) and others, that this may represent a general mechanism of activation for many, but not all, G-protein-coupled receptors.
Shapiro
David A
DA
Department of Biochemistry, Case Western Reserve University Medical School, Cleveland, Ohio 44106-4935, USA.
Kristiansen
Kurt
K
Weiner
David M
DM
Kroeze
Wesley K
WK
Roth
Bryan L
BL
D016475:3T3 Cells; D000818:Animals; D006860:Hydrogen Bonding; D051379:Mice; D008958:Models, Molecular; D016296:Mutagenesis; D011485:Protein Binding; D044402:Receptor, Serotonin, 5-HT2A; D011985:Receptors, Serotonin / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D017366:Serotonin Receptor Agonists / Q000737:chemistry / Q000494:pharmacology*
D044402:Receptor, Serotonin, 5-HT2A; D011985:Receptors, Serotonin; D017366:Serotonin Receptor Agonists