IRREVERSIBLE ACTIVATION OF THE GONADOTROPIN-RELEASING-HORMONE RECEPTOR BY PHOTOAFFINITY CROSS-LINKING - LOCALIZATION OF ATTACHMENT SITE TO CYS RESIDUE IN N-TERMINAL SEGMENT

Photoaffinity cross-linking with [azidobenzoyl-D-Lys(6)]GnRH leads to irreversible activation of the gonadotropin-releasing hormone (GnRH) r eceptor. In order to localize the cross-linking site, the disulfide br idge structure was initially probed by mutagenesis. A consistent patte rn of changes in the ability of GnRH to stimulate signal transduction after Ser substitutions of extracellularly located Cys residues indica ted that Cys14 in the N-terminal domain is connected to Cys200 in the second extracellular loop, while Cys196 in this loop is connected to t he highly conserved Cys114 at the extracellular end of transmembrane h elix 3. Protein chemical analysis of radioactive fragments of cross-li nked GnRH receptor following deglycosylation and enzymatic digest with endoproteinase Glu-C and trypsin before and after introduction or eli mination of potential protease cleavage sites indicated that I-125[azi dobenzoyl-D-Lys(6)] GnRH cross-links to a segment comprising residues 12-18 of the N-terminal domain. The existence of the Cys114-Cys196 bri dge was directly confirmed as a labeled fragment, including that Cys11 4 was resolvable only under reducing conditions. The observation that the cross-linked N-terminal enzymatic fragments had identical apparent size under non-reducing conditions shows that,the cross-linking react ion disconnected the disulfide bridge between Cys14 and Cys200 and ind icates that Cys14 is probably the residue involved in cross-linking of the ligand. It is concluded that covalent tethering of GnRH through a photoreactive side chain located at position 6 in the middle of the p eptide leads to continued activation of the receptor presumably throug h covalent binding to Cys14 in the N-terminal domain of the receptor.