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-Lys 1, D-4-Cl-Phe 2, D-Trp 3, D-Arg 6, D-Ala 10]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 ( K i=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-1 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 Cys 14 and Cys 200 was cleaved during crosslinking, suggesting that Cys 14 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 Cys 14–Cys 200 disulphide bridge.