Effects of mutations at conserved TM II residues on ligand binding and activation of mouse 5-HT6 receptor.
An aspartate residue (Asp-72) in the transmembrane helix II of mouse 5-hydroxytryptamine-6 receptor (5-HT6) is conserved among most G protein-coupled receptors. We have examined the functional significance of this residue by site-directed mutagenesis. A single Asp --> Ala (D72A) mutation resulted in an 8-fold decrease in apparent affinity for 5-HT, and a 60-fold reduction in EC50 value of agonist-induced stimulation of adenylyl cyclase. A F69L/T70I/D72A triple mutant showed a 2-fold reduction in apparent affinity for 5-HT but complete loss of adenylyl cyclase stimulation. Binding of SB-258585 (4-iodo-N-[4-methoxy-3-(4-methylpiperazin-1-yl)phenyl]benzene-sulfonamide), a selective 5-HT6 antagonist, was mildly affected (2- to 4-fold decrease in affinity) in the two mutants. Our data suggest that Asp-72 and additional residues toward the intracellular side of TM II have a limited role in ligand binding but are critical for functional activation of the 5-HT6 receptor.
Zhang
Jiaping
J
Department of Metabolic Disorders, Merck Research Laboratories, Rahway, NJ 07065, USA.
Shen
Chun-Pyn
CP
Xiao
Jing C
JC
Lanza
Thomas J
TJ
Lin
Linus S
LS
Francis
Barbara E
BE
Fong
Tung M
TM
Chen
Richard Z
RZ
D000595:Amino Acid Sequence; D019943:Amino Acid Substitution; D000818:Animals; D002460:Cell Line; D000242:Cyclic AMP / Q000378:metabolism; D006801:Humans; D008024:Ligands; D051379:Mice; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D010879:Piperazines / Q000378:metabolism; D017434:Protein Structure, Tertiary; D011985:Receptors, Serotonin / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D012701:Serotonin / Q000378:metabolism; D012702:Serotonin Antagonists / Q000378:metabolism; D017366:Serotonin Receptor Agonists / Q000378:metabolism; D013449:Sulfonamides / Q000378:metabolism; D014162:Transfection
D008024:Ligands; D010879:Piperazines; D011985:Receptors, Serotonin; C418570:SB 258585; D012702:Serotonin Antagonists; D017366:Serotonin Receptor Agonists; D013449:Sulfonamides; C097216:serotonin 6 receptor; D012701:Serotonin; D000242:Cyclic AMP