PT - JOURNAL ARTICLE AU - Kurt Kristiansen AU - Wesley K. Kroeze AU - David L. Willins AU - Edward I. Gelber AU - Jason E. Savage AU - Richard A. Glennon AU - Bryan L. Roth TI - A Highly Conserved Aspartic Acid (Asp-155) Anchors the Terminal Amine Moiety of Tryptamines and Is Involved in Membrane Targeting of the 5-HT<sub>2A</sub> Serotonin Receptor But Does Not Participate in Activation via a “Salt-Bridge Disruption” Mechanism DP - 2000 Jun 01 TA - Journal of Pharmacology and Experimental Therapeutics PG - 735--746 VI - 293 IP - 3 4099 - http://jpet.aspetjournals.org/content/293/3/735.short 4100 - http://jpet.aspetjournals.org/content/293/3/735.full SO - J Pharmacol Exp Ther2000 Jun 01; 293 AB - 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. The American Society for Pharmacology and Experimental Therapeutics