The N-terminal juxtamembrane segment of the V1a vasopressin receptor provides two independent epitopes required for high-affinity agonist binding and signaling

Stuart R Hawtin, Victoria J Wesley, John Simms, Cymone C H Argent, Khalid Latif, Mark Wheatley

Research output: Contribution to journalArticle

19 Citations (Scopus)

Abstract

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.

Original languageEnglish
Pages (from-to)2871-2871
Number of pages11
JournalMolecular and Cellular Endocrinology
Volume19
Issue number11
DOIs
Publication statusPublished - Nov 2005

Fingerprint

Vasopressin Receptors
Epitopes
Arginine Vasopressin
G-Protein-Coupled Receptors
Posterior Pituitary Hormones
Mutation
Mutagenesis
Alanine
Pharmacology

Keywords

  • Amino Acid Motifs
  • Amino Acid Sequence
  • Amino Acid Substitution
  • Animals
  • Arginine
  • Arginine Vasopressin
  • Cells, Cultured
  • Glutamic Acid
  • Humans
  • Models, Molecular
  • Molecular Sequence Data
  • Mutagenesis
  • Protein Structure, Secondary
  • Receptors, Vasopressin
  • Signal Transduction
  • Journal Article
  • Research Support, Non-U.S. Gov't

Cite this

The N-terminal juxtamembrane segment of the V1a vasopressin receptor provides two independent epitopes required for high-affinity agonist binding and signaling. / Hawtin, Stuart R; Wesley, Victoria J; Simms, John; Argent, Cymone C H; Latif, Khalid; Wheatley, Mark.

In: Molecular and Cellular Endocrinology, Vol. 19, No. 11, 11.2005, p. 2871-2871.

Research output: Contribution to journalArticle

Hawtin, Stuart R ; Wesley, Victoria J ; Simms, John ; Argent, Cymone C H ; Latif, Khalid ; Wheatley, Mark. / The N-terminal juxtamembrane segment of the V1a vasopressin receptor provides two independent epitopes required for high-affinity agonist binding and signaling. In: Molecular and Cellular Endocrinology. 2005 ; Vol. 19, No. 11. pp. 2871-2871.
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T1 - The N-terminal juxtamembrane segment of the V1a vasopressin receptor provides two independent epitopes required for high-affinity agonist binding and signaling

AU - Hawtin, Stuart R

AU - Wesley, Victoria J

AU - Simms, John

AU - Argent, Cymone C H

AU - Latif, Khalid

AU - Wheatley, Mark

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AB - 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.

KW - Amino Acid Motifs

KW - Amino Acid Sequence

KW - Amino Acid Substitution

KW - Animals

KW - Arginine

KW - Arginine Vasopressin

KW - Cells, Cultured

KW - Glutamic Acid

KW - Humans

KW - Models, Molecular

KW - Molecular Sequence Data

KW - Mutagenesis

KW - Protein Structure, Secondary

KW - Receptors, Vasopressin

KW - Signal Transduction

KW - Journal Article

KW - Research Support, Non-U.S. Gov't

U2 - 10.1210/me.2005-0148

DO - 10.1210/me.2005-0148

M3 - Article

VL - 19

SP - 2871

EP - 2871

JO - Molecular Endocrinology

JF - Molecular Endocrinology

SN - 0303-7207

IS - 11

ER -