A new photoreactive antagonist cross-links to the N-terminal domain of the gonadotropin-releasing hormone receptor

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.