Agonist-specific requirement for a glutamate in transmembrane helix 1 of the oxytocin receptor

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 antagonists. 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.