Irreversible Activation of the Gonadotropin-Releasing Hormone Receptor by Photoaffinity Cross-Linking:  Localization of Attachment Site to Cys Residue in N-Terminal Segment

Abstract

Photoaffinity cross-linking with [azidobenzoyl-d-Lys6]GnRH leads to irreversible activation of the gonadotropin-releasing hormone (GnRH) receptor. In order to localize the cross-linking site, the disulfide bridge structure was initially probed by mutagenesis. A consistent pattern of changes in the ability of GnRH to stimulate signal transduction after Ser substitutions of extracellularly located Cys residues indicated that Cys14 in the N-terminal domain is connected to Cys200 in the second extracellular loop, while Cys196 in this loop is connected to the highly conserved Cys114 at the extracellular end of transmembrane helix 3. Protein chemical analysis of radioactive fragments of cross-linked GnRH receptor following deglycosylation and enzymatic digest with endoproteinase Glu-C and trypsin before and after introduction or elimination of potential protease cleavage sites indicated that 125I[azidobenzoyl-d-Lys6]GnRH cross-links to a segment comprising residues 12-18 of the N-terminal domain. The existence of the Cys114-Cys196 bridge was directly confirmed as a labeled fragment, including that Cys114 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 reaction disconnected the disulfide bridge between Cys14 and Cys200 and indicates 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 peptide leads to continued activation of the receptor presumably through covalent binding to Cys14 in the N-terminal domain of the receptor.

The Functional Microdomain in Transmembrane Helices 2 and 7 Regulates Expression, Activation, and Coupling Pathways of the Gonadotropin-releasing Hormone Receptor

“…1). This pattern resembles that reported for the photoaffinity-labeled GnRH receptor (28). To increase sensitivity of detection and facilitate comparison of band intensity, all receptors were deglycosylated prior to immunoblot analysis.…”

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

“…This was followed by minor manual adjustments ensuring that the most highly conserved residues, motifs, and the disulfide bridge Cys were located at the same place as in the rhodopsin structure. The experimentally identified disulfide bond between Cys 14 , in the N-terminal domain and Cys 200 in the second extracellular loop (ECL 2) [27] was also incorporated into the model as an additional constraint of the receptor structure. The MODELLER-generated models with the highest values of the 3D-profile score were selected for refinement according to the experimentally identified receptor inter-and intra-molecular interactions.…”

Activation of G-protein-coupled receptors: a common molecular mechanism

“…In the metabotrophic glutamate receptors, the frizzled family and the secretin receptor family, either deletion or mutation of the large, N-terminal, ligand-binding domain results in constitutive activity. This indicates that the activity of TM domain is constrained by the EC domain in the native receptors in absence of the ligand [25][26][27][28][29][30][31][32][33][34][35][36][37][38][39][40][41][42][43].…”