Structural Determinants for Ligand-Receptor Conformational Selection in a Peptide G Protein-coupled Receptor

Abstract

G protein coupled receptors (GPCRs) modulate the majority of physiological processes through specific intermolecular interactions with structurally diverse ligands and activation of differential intracellular signaling. A key issue yet to be resolved is how GPCRs developed selectivity and diversity of ligand binding and intracellular signaling during evolution. We have explored the structural basis of selectivity of naturally occurring gonadotropin-releasing hormones (GnRHs) from different species in the single functional human GnRH receptor. We found that the highly variable amino acids in position 8 of the naturally occurring isoforms of GnRH play a discriminating role in selecting receptor conformational states. The human GnRH receptor has a higher affinity for the cognate GnRH I but a lower affinity for GnRH II and GnRHs from other species possessing substitutions for Arg 8 . The latter were partial agonists in the human GnRH receptor. Mutation of Asn 7.45 in transmembrane domain (TM) 7 had no effect on GnRH I affinity but specifically increased affinity for other GnRHs and converted them to full agonists. Using molecular modeling and site-directed mutagenesis, we demonstrated that the highly conserved Asn 7.45 makes intramolecular interactions with a highly conserved Cys 6.47 in TM 6, suggesting that disruption of this intramolecular interaction induces a receptor conformational change which allosterically alters ligand specific binding sites and changes ligand selectivity and signaling efficacy. These results reveal GnRH ligand and receptor structural elements for conformational selection, and support co-evolution of GnRH ligand and receptor conformations.

GNRH1 mutations in patients with idiopathic hypogonadotropic hypogonadism

“…Substitutions of histidine, glutamine, leucine, serine, tyrosine, or tryptophan at this position markedly reduce the ability of GnRH to bind and activate mammalian GnRH receptors (34,35). Lu et al (35) propose that the human GnRH receptor has evolved to specifically recognize this arginine residue, and they have identified a residue in the human GnRH receptor (Asn 7.45 ) that confers this specificity.…”

Role of the Transmembrane Domain 4/Extracellular Loop 2 Junction of the Human Gonadotropin-releasing Hormone Receptor in Ligand Binding and Receptor Conformational Selection

“…Binding of GnRH I and II to the GnRH receptor may cause different intramolecular interactions to be broken, allowing the receptor to adopt varied conformations and thus enabling divergent signaling pathways. We have termed this ligand-induced selective signaling (3,8,9).…”

“…Binding of GnRH I and II to the GnRH receptor may cause different intramolecular interactions to be broken, allowing the receptor to adopt varied conformations and thus enabling divergent signaling pathways. We have termed this ligand-induced selective signaling (3,8,9).Investigations into the structure of the GnRH receptor and identification of ligand binding contacts between GnRH and its receptor will enable refinement of molecular models for structure-based drug design. Previous site-directed mutagenesis studies and modeling have identified multiple contact sites of the GnRH receptor with its ligands (2, 3, 10 -12).…”

Diversity of actions of GnRHs mediated by ligand-induced selective signaling

“…These natural GnRHs in which Arg 8 is substituted by a neutral amino acid are partial agonists in the activation of inositol phosphate generation but potent antiproliferative agents (our unpublished data). Mutation of Cys 279 (6.47) to Ala or Tyr (a mutation causing infertility in man) and Asn 315 (7.45) to Ala increases ligand binding affinity for GnRHs from other species and converts them to full agonists [76]. Recent studies have shown that partial agonists stabilize a receptor active conformation differing from that of full agonists, with different signaling capability [6,109].…”

“…GnRH ligandinduction of new receptor intra-and intermolecular interactions appears to be a fundamental component for GnRH receptor activation. The delineation of the structural elements for ligand and receptor conformational selection could have implications for the development of novel ligands that selectively activate certain signaling pathways, with improved therapeutic application [76].…”