Charged extracellular residues, conserved throughout a G-protein-coupled receptor family, are required for ligand binding, receptor activation, and cell-surface expression

Stuart R Hawtin, John Simms, Matthew Conner, Zoe Lawson, Rosemary A Parslow, Julie Trim, Andrew Sheppard, Mark Wheatley

Research output: Contribution to journalArticle

40 Citations (Scopus)

Abstract

For G-protein-coupled receptors (GPCRs) in general, the roles of extracellular residues are not well defined compared with residues in transmembrane helices (TMs). Nevertheless, extracellular residues are important for various functions in both peptide-GPCRs and amine-GPCRs. In this study, the V(1a) vasopressin receptor was used to systematically investigate the role of extracellular charged residues that are highly conserved throughout a subfamily of peptide-GPCRs, using a combination of mutagenesis and molecular modeling. Of the 13 conserved charged residues identified in the extracellular loops (ECLs), Arg(116) (ECL1), Arg(125) (top of TMIII), and Asp(204) (ECL2) are important for agonist binding and/or receptor activation. Molecular modeling revealed that Arg(125) (and Lys(125)) stabilizes TMIII by interacting with lipid head groups. Charge reversal (Asp(125)) caused re-ordering of the lipids, altered helical packing, and increased solvent penetration of the TM bundle. Interestingly, a negative charge is excluded at this locus in peptide-GPCRs, whereas a positive charge is excluded in amine-GPCRs. This contrasting conserved charge may reflect differences in GPCR binding modes between peptides and amines, with amines needing to access a binding site crevice within the receptor TM bundle, whereas the binding site of peptide-GPCRs includes more extracellular domains. A conserved negative charge at residue 204 (ECL2), juxtaposed to the highly conserved disulfide bond, was essential for agonist binding and signaling. Asp(204) (and Glu(204)) establishes TMIII contacts required for maintaining the beta-hairpin fold of ECL2, which if broken (Ala(204) or Arg(204)) resulted in ECL2 unfolding and receptor dysfunction. This study provides mechanistic insight into the roles of conserved extracellular residues.

Original languageEnglish
Pages (from-to)38478-38488
Number of pages11
JournalJournal of Biological Chemistry
Volume281
Issue number50
DOIs
Publication statusPublished - 15 Dec 2006

Fingerprint

Cell Surface Receptors
G-Protein-Coupled Receptors
Chemical activation
Ligands
Amines
Viperidae
Peptides
Molecular modeling
Binding Sites
Lipids
Vasopressin Receptors
Mutagenesis
Disulfides

Keywords

  • Amino Acid Sequence
  • Base Sequence
  • Cell Membrane
  • DNA Primers
  • Enzyme-Linked Immunosorbent Assay
  • Ligands
  • Models, Molecular
  • Molecular Sequence Data
  • Protein Binding
  • Radioligand Assay
  • Receptors, G-Protein-Coupled
  • Sequence Homology, Amino Acid
  • Journal Article
  • Research Support, Non-U.S. Gov't

Cite this

Charged extracellular residues, conserved throughout a G-protein-coupled receptor family, are required for ligand binding, receptor activation, and cell-surface expression. / Hawtin, Stuart R; Simms, John; Conner, Matthew; Lawson, Zoe; Parslow, Rosemary A; Trim, Julie; Sheppard, Andrew; Wheatley, Mark.

In: Journal of Biological Chemistry, Vol. 281, No. 50, 15.12.2006, p. 38478-38488.

Research output: Contribution to journalArticle

Hawtin, Stuart R ; Simms, John ; Conner, Matthew ; Lawson, Zoe ; Parslow, Rosemary A ; Trim, Julie ; Sheppard, Andrew ; Wheatley, Mark. / Charged extracellular residues, conserved throughout a G-protein-coupled receptor family, are required for ligand binding, receptor activation, and cell-surface expression. In: Journal of Biological Chemistry. 2006 ; Vol. 281, No. 50. pp. 38478-38488.
@article{bd41a6566fe147109a4cf483a6b0861b,
title = "Charged extracellular residues, conserved throughout a G-protein-coupled receptor family, are required for ligand binding, receptor activation, and cell-surface expression",
abstract = "For G-protein-coupled receptors (GPCRs) in general, the roles of extracellular residues are not well defined compared with residues in transmembrane helices (TMs). Nevertheless, extracellular residues are important for various functions in both peptide-GPCRs and amine-GPCRs. In this study, the V(1a) vasopressin receptor was used to systematically investigate the role of extracellular charged residues that are highly conserved throughout a subfamily of peptide-GPCRs, using a combination of mutagenesis and molecular modeling. Of the 13 conserved charged residues identified in the extracellular loops (ECLs), Arg(116) (ECL1), Arg(125) (top of TMIII), and Asp(204) (ECL2) are important for agonist binding and/or receptor activation. Molecular modeling revealed that Arg(125) (and Lys(125)) stabilizes TMIII by interacting with lipid head groups. Charge reversal (Asp(125)) caused re-ordering of the lipids, altered helical packing, and increased solvent penetration of the TM bundle. Interestingly, a negative charge is excluded at this locus in peptide-GPCRs, whereas a positive charge is excluded in amine-GPCRs. This contrasting conserved charge may reflect differences in GPCR binding modes between peptides and amines, with amines needing to access a binding site crevice within the receptor TM bundle, whereas the binding site of peptide-GPCRs includes more extracellular domains. A conserved negative charge at residue 204 (ECL2), juxtaposed to the highly conserved disulfide bond, was essential for agonist binding and signaling. Asp(204) (and Glu(204)) establishes TMIII contacts required for maintaining the beta-hairpin fold of ECL2, which if broken (Ala(204) or Arg(204)) resulted in ECL2 unfolding and receptor dysfunction. This study provides mechanistic insight into the roles of conserved extracellular residues.",
keywords = "Amino Acid Sequence, Base Sequence, Cell Membrane, DNA Primers, Enzyme-Linked Immunosorbent Assay, Ligands, Models, Molecular, Molecular Sequence Data, Protein Binding, Radioligand Assay, Receptors, G-Protein-Coupled, Sequence Homology, Amino Acid, Journal Article, Research Support, Non-U.S. Gov't",
author = "Hawtin, {Stuart R} and John Simms and Matthew Conner and Zoe Lawson and Parslow, {Rosemary A} and Julie Trim and Andrew Sheppard and Mark Wheatley",
year = "2006",
month = "12",
day = "15",
doi = "10.1074/jbc.M607639200",
language = "English",
volume = "281",
pages = "38478--38488",
journal = "Journal of Biological Chemistry",
issn = "0021-9258",
publisher = "American Society for Biochemistry and Molecular Biology",
number = "50",

}

TY - JOUR

T1 - Charged extracellular residues, conserved throughout a G-protein-coupled receptor family, are required for ligand binding, receptor activation, and cell-surface expression

AU - Hawtin, Stuart R

AU - Simms, John

AU - Conner, Matthew

AU - Lawson, Zoe

AU - Parslow, Rosemary A

AU - Trim, Julie

AU - Sheppard, Andrew

AU - Wheatley, Mark

PY - 2006/12/15

Y1 - 2006/12/15

N2 - For G-protein-coupled receptors (GPCRs) in general, the roles of extracellular residues are not well defined compared with residues in transmembrane helices (TMs). Nevertheless, extracellular residues are important for various functions in both peptide-GPCRs and amine-GPCRs. In this study, the V(1a) vasopressin receptor was used to systematically investigate the role of extracellular charged residues that are highly conserved throughout a subfamily of peptide-GPCRs, using a combination of mutagenesis and molecular modeling. Of the 13 conserved charged residues identified in the extracellular loops (ECLs), Arg(116) (ECL1), Arg(125) (top of TMIII), and Asp(204) (ECL2) are important for agonist binding and/or receptor activation. Molecular modeling revealed that Arg(125) (and Lys(125)) stabilizes TMIII by interacting with lipid head groups. Charge reversal (Asp(125)) caused re-ordering of the lipids, altered helical packing, and increased solvent penetration of the TM bundle. Interestingly, a negative charge is excluded at this locus in peptide-GPCRs, whereas a positive charge is excluded in amine-GPCRs. This contrasting conserved charge may reflect differences in GPCR binding modes between peptides and amines, with amines needing to access a binding site crevice within the receptor TM bundle, whereas the binding site of peptide-GPCRs includes more extracellular domains. A conserved negative charge at residue 204 (ECL2), juxtaposed to the highly conserved disulfide bond, was essential for agonist binding and signaling. Asp(204) (and Glu(204)) establishes TMIII contacts required for maintaining the beta-hairpin fold of ECL2, which if broken (Ala(204) or Arg(204)) resulted in ECL2 unfolding and receptor dysfunction. This study provides mechanistic insight into the roles of conserved extracellular residues.

AB - For G-protein-coupled receptors (GPCRs) in general, the roles of extracellular residues are not well defined compared with residues in transmembrane helices (TMs). Nevertheless, extracellular residues are important for various functions in both peptide-GPCRs and amine-GPCRs. In this study, the V(1a) vasopressin receptor was used to systematically investigate the role of extracellular charged residues that are highly conserved throughout a subfamily of peptide-GPCRs, using a combination of mutagenesis and molecular modeling. Of the 13 conserved charged residues identified in the extracellular loops (ECLs), Arg(116) (ECL1), Arg(125) (top of TMIII), and Asp(204) (ECL2) are important for agonist binding and/or receptor activation. Molecular modeling revealed that Arg(125) (and Lys(125)) stabilizes TMIII by interacting with lipid head groups. Charge reversal (Asp(125)) caused re-ordering of the lipids, altered helical packing, and increased solvent penetration of the TM bundle. Interestingly, a negative charge is excluded at this locus in peptide-GPCRs, whereas a positive charge is excluded in amine-GPCRs. This contrasting conserved charge may reflect differences in GPCR binding modes between peptides and amines, with amines needing to access a binding site crevice within the receptor TM bundle, whereas the binding site of peptide-GPCRs includes more extracellular domains. A conserved negative charge at residue 204 (ECL2), juxtaposed to the highly conserved disulfide bond, was essential for agonist binding and signaling. Asp(204) (and Glu(204)) establishes TMIII contacts required for maintaining the beta-hairpin fold of ECL2, which if broken (Ala(204) or Arg(204)) resulted in ECL2 unfolding and receptor dysfunction. This study provides mechanistic insight into the roles of conserved extracellular residues.

KW - Amino Acid Sequence

KW - Base Sequence

KW - Cell Membrane

KW - DNA Primers

KW - Enzyme-Linked Immunosorbent Assay

KW - Ligands

KW - Models, Molecular

KW - Molecular Sequence Data

KW - Protein Binding

KW - Radioligand Assay

KW - Receptors, G-Protein-Coupled

KW - Sequence Homology, Amino Acid

KW - Journal Article

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

U2 - 10.1074/jbc.M607639200

DO - 10.1074/jbc.M607639200

M3 - Article

VL - 281

SP - 38478

EP - 38488

JO - Journal of Biological Chemistry

JF - Journal of Biological Chemistry

SN - 0021-9258

IS - 50

ER -