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*

*Corresponding author for this work

Research output: Contribution to journalArticle

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 V1a 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), Arg116 (ECL1), Arg125 (top of TMIII), and Asp204 (ECL2) are important for agonist binding and/or receptor activation. Molecular modeling revealed that Arg125 (and Lys 125) stabilizes TMIII by interacting with lipid head groups. Charge reversal (Asp125) 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. Asp204 (and Glu204) establishes TMIII contacts required for maintaining the α-hairpin fold of ECL2, which if broken (Ala204 or Arg204) resulted in ECL2 unfolding and receptor dysfunction. This study provides mechanistic insight into the roles of conserved extracellular residues.

Original languageEnglish
Pages (from-to)38487-38488
Number of pages2
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
Peptides
Molecular modeling
Binding Sites
Lipids
Vasopressin Receptors
Mutagenesis
Disulfides

Bibliographical note

© The American Society for Biochemistry and Molecular Biology, Inc. THE JOURNAL OF BIOLOGICAL CHEMISTRY VOL. 281, NO. 50, pp. 38478 –38488, December 15, 2006

Cite this

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. 38487-38488.
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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. 38487-38488.

Research output: Contribution to journalArticle

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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

N1 - © The American Society for Biochemistry and Molecular Biology, Inc. THE JOURNAL OF BIOLOGICAL CHEMISTRY VOL. 281, NO. 50, pp. 38478 –38488, December 15, 2006

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