Multiple Interactions of the Asp^2.61(98) Side Chain of the Gonadotropin-Releasing Hormone Receptor Contribute Differentially to Ligand Interaction

Abstract

Mutation of Asp(2.61(98)) at the extracellular boundary of transmembrane helix 2 of the gonadotropin-releasing hormone (GnRH) receptor decreased the affinity for GnRH. Using site-directed mutagenesis, ligand modification, and computational modeling, different side chain interactions of Asp(2.61(98)) that contribute to high-affinity binding were investigated. The conservative Asp(2. 61(98))Glu mutation markedly decreased the affinity for a series of GnRH analogues containing the native His(2) residue. This mutant showed smaller decreases in affinity for His(2)-substituted ligands. The loss of preference for His(2)-containing ligands in the mutant receptor shows that Asp(2.61(98)) determines the specificity for His(2). Analysis of the affinities of a series of position 2-substituted ligands suggests that a hydrogen bond forms between Asp(2.61(98)) and the delta NH group of His(2) and that Asp(2. 61(98)) forms a second hydrogen bond with the ligand. Substitution of Asp(2.61(98)) with an uncharged residue further decreased the affinity for all ligands and also decreased receptor expression. Computational modeling indicates an intramolecular ionic interaction of Asp(2.61(98)) with Lys(3.32(121)) in transmembrane helix 3. The uncharged, Lys(3.32(121))Gln mutation also markedly decreased agonist affinity. The modeling and the similar phenotypes of mutants with uncharged substitutions for Asp(2.61(98)) or Lys(3.32(121)) are consistent with the presence of this helix 2-helix 3 interaction. These studies support a dual role for Asp(2.61(98)): formation of an interhelical interaction with Lys(3.32(121)) that contributes to the structure of the agonist binding pocket and an interaction with His(2) of GnRH that helps stabilize agonist complexing.

A Chicken Gonadotropin-releasing Hormone Receptor That Confers Agonist Activity to Mammalian Antagonists

“…The chicken GnRH receptor that we have cloned exhibits marked pharmacological differences in its interaction with GnRH agonist and antagonist analogs and has sequence differences from the mammalian receptors that may be used to identify functional residues. 3.32(121) residues in the chicken GnRH receptor is expected, as these residues are believed to interact with the amino-(Glu(P) 1 -His 2 ) and carboxyl-terminal (Gly 10 -NH 2 ) residues of the GnRH ligands, which are conserved in the native mammalian and chicken forms of GnRH (9,11,12). As expected from biological assays, the chicken GnRH receptor does not distinguish between its cognate native ligand, [Gln 8 ]GnRH (K i 5.3 Ϯ 0.5 nM), and mammalian GnRH (K i 4.1 Ϯ 1.2 nM).…”

Salt Bridges Overlapping the Gonadotropin-Releasing Hormone Receptor Agonist Binding Site Reveal a Coincidence Detector for G Protein-Coupled Receptor Activation

“…This conformation of GnRH was manually docked in the active conformation of the receptor to comply with experimentally identified contacts between GnRH analogs and receptor residues: pGlu 1 (Sealfon et al, 1997;Flanagan et al, 2000;Hoffmann et al, 2000;Hövelmann et al, 2002;Coetsee et al, 2008;Millar et al, 2008;Stewart et al, 2008). A similar mode of GnRH docking has been proposed and experimentally validated .…”

Salt bridge integrates GPCR activation with protein trafficking

“…This observation is surprising, because many G protein coupled receptors (GPCRs) have mutants (2) or WT receptors (3) with CA. A highly conserved (4) structural feature of the GnRHR is a salt bridge between transmembrane segment (TM) 2 and TM3 (residues E 90 and K 121 in the human sequence) (5,6). Gonadotropin releasing hormone (GnRH) agonists, but not peptide antagonists, bind receptor residues D 98 and K 121 (5,6) and alter the relation between TM2 and TM3; this same relation is important for trafficking of the human GnRHR (hGnRHR) to the plasma membrane (4, 7).…”