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


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.

Publication DOI:
Divisions: College of Health & Life Sciences > School of Biosciences
College of Health & Life Sciences
Additional Information: © The American Society for Biochemistry and Molecular Biology, Inc. THE JOURNAL OF BIOLOGICAL CHEMISTRY VOL. 281, NO. 50, pp. 38478 –38488, December 15, 2006
Uncontrolled Keywords: Biochemistry,Molecular Biology,Cell Biology
Publication ISSN: 1083-351X
Full Text Link:
Related URLs: http://www.scop ... tnerID=8YFLogxK (Scopus URL)
http://www.jbc. ... nt/281/50/38478 (Publisher URL)
PURE Output Type: Article
Published Date: 2006-12-15
Authors: Hawtin, Stuart R.
Simms, John (ORCID Profile 0000-0002-4675-0902)
Conner, Matthew
Lawson, Zoe
Parslow, Rosemary A.
Trim, Julie
Sheppard, Andrew
Wheatley, Mark



Version: Published Version

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