The N-terminal juxtamembrane segment of the V1a vasopressin receptor provides two independent epitopes required for high-affinity agonist binding and signaling.
It is fundamentally important to define how agonist-receptor interaction differs from antagonist-receptor interaction. The V1a vasopressin receptor (V1aR) is a member of the neurohypophysial hormone subfamily of G protein-coupled receptors. Using alanine-scanning mutagenesis of the N-terminal juxtamembrane segment of the V1aR, we now establish that Glu54 (1.35) is critical for arginine vasopressin binding. The mutant [E54A]V1aR exhibited decreased arginine vasopressin affinity (1700-fold) and disrupted signaling, but antagonist binding was unaffected. Mutation of Glu54 had an almost identical pharmacological effect as mutation of Arg46, raising the possibility that agonist binding required a mutual interaction between Glu54 and Arg46. The role of these two charged residues was investigated by 1) substituting Glu54; 2) inserting additional Glu/Arg in transmembrane helix (TM) 1; 3) repositioning the Glu/Arg in TM1; and 4) characterizing the reciprocal mutant [R46E/E54R]V1aR. We conclude that 1) the positive/negative charges need to be precisely positioned in this N terminus/TM1 segment; and 2) Glu54 and Arg46 function independently, providing two discrete epitopes required for high-affinity agonist binding and signaling. This study explains why Glu and Arg, part of an -R(X3)L/V(X3)E(X3)L- motif, are conserved at these loci throughout this G protein-coupled receptor subfamily and provides molecular insight into key differences between agonist and antagonist binding requirements.
School of Biosciences, University of Birmingham, Edgbaston, Birmingham B15 2TT, United Kingdom.
Stuart R
SR
Hawtin
Victoria J
VJ
Wesley
John
J
Simms
Cymone C H
CC
Argent
Khalid
K
Latif
Mark
M
Wheatley
D020816:Amino Acid Motifs; D000595:Amino Acid Sequence; D019943:Amino Acid Substitution; D000818:Animals; D001120:Arginine / Q000235:genetics; D001127:Arginine Vasopressin / Q000494:pharmacology*; D002478:Cells, Cultured; D018698:Glutamic Acid / Q000235:genetics; D006801:Humans; D008958:Models, Molecular; D008969:Molecular Sequence Data; D016296:Mutagenesis; D017433:Protein Structure, Secondary; D017483:Receptors, Vasopressin / Q000819:agonists* / Q000737:chemistry* / Q000235:genetics; D015398:Signal Transduction
D017483:Receptors, Vasopressin; D001127:Arginine Vasopressin; D018698:Glutamic Acid; D001120:Arginine