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Vol. 293, Issue 3, 735-746, June 2000

A Highly Conserved Aspartic Acid (Asp-155) Anchors the Terminal Amine Moiety of Tryptamines and Is Involved in Membrane Targeting of the 5-HT2A Serotonin Receptor But Does Not Participate in Activation via a "Salt-Bridge Disruption" Mechanism1

Kurt Kristiansen2 , Wesley K. Kroeze, David L. Willins, Edward I. Gelber, Jason E. Savage, Richard A. Glennon and Bryan L. Roth

Departments of Biochemistry (K.K., W.K.K., E.I.G., J.E.S., B.L.R.), Psychiatry (D.L.W., B.L.R.), and Neurosciences (B.L.R.), Case Western Reserve University Medical School, Cleveland, Ohio; and Department of Medicinal Chemistry, Medical College of Virginia, Richmond, Virginia (R.A.G.)

Discovering the molecular and atomic mechanism(s) by which G-protein-coupled receptors (GPCRs) are activated by agonists remains an elusive goal. Recently, studies examining two representative GPCRs (rhodopsin and alpha 1b-adrenergic receptors) have suggested that the disruption of a putative "salt-bridge" between highly conserved residues in transmembrane (TM) helix III, involving aspartate or glutamate, and helix VII, involving a basic residue, results in receptor activation. We have tested whether this is a general mechanism for GPCR activation by constructing a model of the 5-hydroxytryptamine (5-HT)2A receptor and characterizing several mutations at the homologous residues (Asp-155 and Asn-363) of the 5-HT2A serotonin receptor. All of the mutants (D155A, D155N, D155E, D155Q, and S363A) resulted in receptors with reduced basal activity; in no case was evidence for constitutive activity revealed. Structure-function studies with tryptamine analogs and various Asp-155 mutants demonstrated that Asp-155 interacts with the terminal, and not indole, amine moiety of 5-HT2A agonists. Interestingly, the D155E mutation interfered with the membrane targeting of the 5-HT2A receptor, and an inverse relationship was discovered when comparing receptor activation and targeting for a series of Asp-155 mutants. This represents the first known instance in which a charged residue located in a putative TM helix alters the membrane targeting of a GPCR. Thus, for 5-HT2A receptors, the TMIII aspartic acid (Asp-155) is involved in anchoring the terminal amine moiety of indole agonists and in membrane targeting and not in receptor activation by salt-bridge disruption.

1 This study was supported in part by National Institutes of Health Grants RO1MH57635, Research Scientist Development Award KO2MH01366, a gift from the Heffter Research Foundation, and a National Alliance for Research on Schizophrenia and Depression Independent Investigator Award (to B.L.R.). D.L.W. was supported in part by a National Alliance for Research on Schizophrenia and Depression Young Investigator Award.

2 Current address: Institute of Molecular Pharmacology, Molecular Modeling Group, Alfred Kowalke Str. 4, D-10315 Berlin, Germany.

0022-3565/00/2933-0735$03.00/0
THE JOURNAL OF PHARMACOLOGY AND EXPERIMENTAL THERAPEUTICS
Copyright © 2000 by The American Society for Pharmacology and Experimental Therapeutics


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