A highly conserved aspartic acid (Asp-155) anchors the terminal amine moiety of tryptamines and is involved in membrane targeting of the 5-HT(2A) serotonin receptor but does not participate in activation via a "salt-bridge disruption" mechanism.
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-HT(2A) 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-HT(2A) agonists. Interestingly, the D155E mutation interfered with the membrane targeting of the 5-HT(2A) 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-HT(2A) 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.
Kristiansen
K
K
Department of Biochemistry, Case Western Reserve University Medical School, Cleveland, OH 44106-4935, USA.
Kroeze
W K
WK
Willins
D L
DL
Gelber
E I
EI
Savage
J E
JE
Glennon
R A
RA
Roth
B L
BL
D000818:Animals; D001692:Biological Transport; D019556:COS Cells; D002462:Cell Membrane / Q000378:metabolism; D006868:Hydrolysis; D008958:Models, Molecular; D016297:Mutagenesis, Site-Directed; D010716:Phosphatidylinositols / Q000378:metabolism; D044402:Receptor, Serotonin, 5-HT2A; D011985:Receptors, Serotonin / Q000737:chemistry* / Q000378:metabolism; D013134:Spiperone / Q000378:metabolism; D013329:Structure-Activity Relationship; D014363:Tryptamines / Q000737:chemistry*
D010716:Phosphatidylinositols; D044402:Receptor, Serotonin, 5-HT2A; D011985:Receptors, Serotonin; D014363:Tryptamines; D013134:Spiperone