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J. Biol. Chem., Vol. 281, Issue 50, 38478-38488, December 15, 2006

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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^*

Stuart R. Hawtin¹², John Simms¹, Matthew Conner, Zoe Lawson, Rosemary A. Parslow, Julie Trim, Andrew Sheppard, and Mark Wheatley³

From the School of Biosciences, University of Birmingham, Edgbaston, Birmingham B15 2TT, United Kingdom and Ferring Research Ltd., Southampton Science Park, 1 Venture Road, Southampton SO16 7NP, United Kingdom

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), Arg¹¹⁶ (ECL1), Arg¹²⁵ (top of TMIII), and Asp²⁰⁴ (ECL2) are important for agonist binding and/or receptor activation. Molecular modeling revealed that Arg¹²⁵ (and Lys¹²⁵) 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 -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.

Received for publication, August 10, 2006 , and in revised form, September 8, 2006.

^* This work was supported by grants (to M. W.) from the Wellcome Trust, the Biotechnology and Biological Sciences Research Council, and Ferring Research. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

¹ Both authors should be considered as joint first authors.

² Present address: Novartis Pharma AG, WSJ-386.9.59, CH-4002 Basel, Switzerland.

³ To whom correspondence should be addressed: School of Biosciences, University of Birmingham, Edgbaston, Birmingham, B15 2TT, UK. Tel.: 44-121-4143981; Fax: 44-121-414-5925; E-mail: m.wheatley{at}bham.ac.uk.

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				M. Conner, S. R. Hawtin, J. Simms, D. Wootten, Z. Lawson, A. C. Conner, R. A. Parslow, and M. Wheatley Systematic Analysis of the Entire Second Extracellular Loop of the V1a Vasopressin Receptor: KEY RESIDUES, CONSERVED THROUGHOUT A G-PROTEIN-COUPLED RECEPTOR FAMILY, IDENTIFIED J. Biol. Chem., June 15, 2007; 282(24): 17405 - 17412. [Abstract] [Full Text] [PDF]