Abstract
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 α1b-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-HT2Aserotonin 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.
Footnotes
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Send reprint requests to: Bryan L. Roth, M.D., Ph.D., Department of Biochemistry; Room W438, Case Western Reserve University Medical School, 10900 Euclid Ave., Cleveland, OH 44106-4935. E-mail:roth{at}biocserver.cwru.edu
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↵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.
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↵2 Current address: Institute of Molecular Pharmacology, Molecular Modeling Group, Alfred Kowalke Str. 4, D-10315 Berlin, Germany.
- Abbreviations:
- GPCR
- G-protein-coupled receptor
- TM
- transmembrane domain
- GnRH
- gonadotropin-releasing hormone receptor
- 5-HT
- 5-hydroxytryptamine
- IP
- inositol monophosphate
- PI
- phosphoinositide
- GFP
- green fluorescent protein
- DOM
- 4-methyl-2,5-dimethyoxyphenylisopropylamine
- DMT
- dimethyltryptamine
- Received December 3, 1999.
- Accepted March 2, 2000.
- The American Society for Pharmacology and Experimental Therapeutics
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