Molecular and Cellular Endocrinology 333 (2011) 20–27 Contents lists available at ScienceDirect Molecular and Cellular Endocrinology journal homepage: www.elsevier.com/locate/mce Agonist-specific requirement for a glutamate in transmembrane helix 1 of the oxytocin receptor Denise L. Wootten a,1 , John Simms a,1 , Amelia J. Massoura a , Julie E. Trim b,2 , Mark Wheatley a,∗ a School of Biosciences, University of Birmingham, Edgbaston, Birmingham B15 2TT, UK b Ferring Research Ltd., Southampton Science Park, 1 Venture Road, Southampton, UK article info Article history: Received 6 July 2010 Received in revised form 18 November 2010 Accepted 25 November 2010 Keywords: GPCR Oxytocin receptor Oxytocin Peptide hormone abstract Defining key differences between agonist and antagonist binding to hormone receptors is important and will aid rational drug design. Glu 1.35 in transmembrane helix 1 (TM1) of the human oxytocin receptor (OTR) is absolutely conserved in all OTRs cloned to date. We establish that Glu 1.35 is critical for high affinity binding of agonists (full and partial) but is not required for antagonist binding (peptide or non-peptide). Consequently, the mutant receptor [E1.35A]OTR exhibited markedly decreased OT affinity (>1200-fold) and disrupted second messenger generation. Substitutions of Glu 1.35 by Asp, Gln or Arg were incapable of supporting wild-type OTR agonist binding or signaling. Molecular modeling revealed that Glu 1.35 projects into the receptor’s central binding crevice and provides agonist-specific contacts not utilized by antag- onists. This study explains why Glu is absolutely conserved at residue-1.35 in all receptors binding OT and related peptides, and provides molecular insight into key differences between agonist–receptor and antagonist–receptor binding modes. © 2010 Elsevier Ireland Ltd. All rights reserved. 1. Introduction The neurohypophysial peptide hormone oxytocin (OT) and oxytocin-like peptides, such as mesotocin and isotocin, facili- tate reproduction in all vertebrates (Acher et al.,1995; Parry and Bathgate, 2000; Gimpl and Fahrenholz, 2001). Even in the rela- tively simple earthworm Eisenia foetida, the OT-related peptide annetocin induces egg-laying behaviour (Oumi et al., 1996). In humans, OT mediates a wide range of central and peripheral effects (Gimpl and Fahrenholz, 2001; Opar, 2008), including increasing the frequency and intensity of uterine contraction at parturition and contraction of the mammary gland myoepithelium during lacta- tion (Gimpl and Fahrenholz, 2001; Soloff et al., 1979). The potent uterotonic role played by OT in birth has resulted in extensive use of this peptide clinically to induce and augment labor (Owen and Hauth, 1992). The physiological effects of OT are mediated Abbreviations: AVP, [arginine 8 ]vasopressin; ELISA, enzyme-linked immunosor- bent assay; GPCR, G-protein-coupled receptor; InsP, inositol phosphate; InsP3, inositol trisphosphate; OT, oxytocin; OTA, d(CH2)5Tyr(Me) 2 Thr 4 Orn 8 Tyr(NH2) 9 vasotocin; OTR, oxytocin receptor; TM, transmembrane helix. ∗ Corresponding author. Tel.: +44 121 414 3981; fax: +44 121 414 5925. E-mail address: m.wheatley@bham.ac.uk (M. Wheatley). 1 Present address: Monash Institute of Pharmaceutical Sciences, 381 Royal Parade, Parkville, Melbourne, Victoria 3052, Australia. 2 Present address: Shire Pharmaceutical Development, Hampshire International Business Park, Chineham, Basingstoke RG24 8EP, UK. by a specific oxytocin receptor (OTR) expressed by target tissues. As pregnancy approaches term there is an increase in the abun- dance of OTRs expressed by the myometrium which results in a specifically timed increased responsiveness of the uterus to OT (Fuchs et al., 1995; Kimura and Saji, 1995; Parry and Bathgate, 2000). Binding antagonists to the OTRs can effectively blockade the receptors, thereby reducing receptor availability to OT resulting in increased uterine quiescence. Both peptide antagonists such as Atosiban (d[DTyr(Et) 2 ,Thr 4 ,Orn 8 ]OT) (Valenzuela et al., 2000) and non-peptide antagonists (Pettibone and Freidinger, 1997; Hawtin et al., 2005a) have been developed for this tocolytic purpose. The OTR is a Family A (rhodopsin-like) G-protein-coupled recep- tor (GPCR) and exhibits structural features typical of this family, including seven transmembrane (TM) helices (Kimura et al., 1992). Only one OTR subtype has been cloned from humans, implying that the wide range of physiological effects of OT is mediated by a sin- gle receptor which signals primarily by coupling to phospholipase C to generate inositol trisphosphate (InsP 3 ) as second messenger (Gimpl and Fahrenholz, 2001). As agonists induce OTR signaling and antagonists do not, defin- ing differences between the agonist–OTR interaction and the antagonist–OTR interaction at the molecular level, will provide insight into OT action and may aid rational drug design. In this study we establish that a Glu in TM1 of the human OTR is critical for agonist binding and signaling but is not required for antagonist binding (peptide antagonist or non-peptide antagonist). Further- more, we demonstrate that there is a specific requirement for Glu 0303-7207/$ – see front matter © 2010 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.mce.2010.11.029