title: A new photoreactive antagonist cross-links to the N-terminal domain of the gonadotropin-releasing hormone receptor.
abstract: A new photoreactive gonadotropin-releasing hormone (GnRH) antagonist [Ac-(4-azidobenzoyl)-D-Lys1, D-4-Cl-Phe2, D-Trp3, D-Arg6, D-Ala10]GnRH (PAnt-1) was synthesized and shown to bind covalently to mouse and human GnRH receptors after ultraviolet irradiation. PAnt-1 exhibited high binding affinity (Ki = 3.1 +/- 0.8 nM), and high crosslinking efficiency as shown by loss of 78% of binding sites following crosslinking at saturating concentration. Crosslinking resulted in irreversible receptor blockade as shown by inhibition of GnRH-stimulated inositol phosphate production. PAnt-1 has a photoreactive group at residue 1 of the peptide, a region believed to be critical in determining antagonist versus agonist properties of GnRH analogues. The attachment site of PAnt- to the receptor was localized between residues 11 and 19 of the extracellular N-terminal domain of the receptor by peptide mapping studies using natural sequence differences between human, mouse and sheep GnRH receptors, as well as a panel of GnRH receptor constructs with a series of engineered protease cleavage sites. A disulphide bridge between Cys14 and Cys200 was cleaved during crosslinking, suggesting that Cys14 is the crosslinked residue. These results suggest that peptide GnRH antagonists bind to the receptor with the N-terminal end of the peptide positioned in a site comprising the constrained regions of the N-terminal domain and second extracellular loop in the vicinity of the Cys14-Cys200 disulphide bridge.
authors.affiliation: Department of Chemical Pathology, University of Cape Town Medical School, South Africa.
authors.forename: D
authors.initials: D
authors.lastname: Assefa
authors.forename: A J
authors.initials: AJ
authors.lastname: Pawson
authors.forename: C A
authors.initials: CA
authors.lastname: McArdle
authors.forename: R P
authors.initials: RP
authors.lastname: Millar
authors.forename: C A
authors.initials: CA
authors.lastname: Flanagan
authors.forename: R
authors.initials: R
authors.lastname: Roeske
authors.forename: J S
authors.initials: JS
authors.lastname: Davidson
mesh_terms: D000345:Affinity Labels / Q000138:chemical synthesis / Q000493:pharmacokinetics*; D000595:Amino Acid Sequence; D000818:Animals; D001665:Binding Sites; D001667:Binding, Competitive; D019556:COS Cells; D002460:Cell Line; D003432:Cross-Linking Reagents; D007987:Gonadotropin-Releasing Hormone / Q000031:analogs & derivatives* / Q000138:chemical synthesis / Q000493:pharmacokinetics / Q000494:pharmacology; D006801:Humans; D007295:Inositol Phosphates / Q000378:metabolism; D007700:Kinetics; D051379:Mice; D008958:Models, Molecular; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D017433:Protein Structure, Secondary; D011869:Radioligand Assay; D011966:Receptors, LHRH / Q000737:chemistry / Q000378:metabolism*; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D012756:Sheep; D014162:Transfection
chemical_list: D000345:Affinity Labels; D003432:Cross-Linking Reagents; C402742:GnRH, acetyl(4-azidobenzoyl)-Lys(1)-4-chloro-Phe(2),Trp(3),Arg(6),Ala(10)-; D007295:Inositol Phosphates; D011966:Receptors, LHRH; D011994:Recombinant Proteins; D007987:Gonadotropin-Releasing Hormone