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-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.
Davidson
J S
JS
M.R.C. Regulatory Peptides Research Unit, Department of Chemical Pathology, University of Cape Town Medical School, Observatory 7925, South Africa. JAMES@CHEMPATH.UCT.AC.ZA
Assefa
D
D
Pawson
A
A
Davies
P
P
Hapgood
J
J
Becker
I
I
Flanagan
C
C
Roeske
R
R
Millar
R
R
D000595:Amino Acid Sequence; D019943:Amino Acid Substitution; D000818:Animals; D001386:Azides / Q000378:metabolism* / Q000494:pharmacology; D001665:Binding Sites; D019556:COS Cells; D003001:Cloning, Molecular; D003432:Cross-Linking Reagents; D003545:Cysteine; D007987:Gonadotropin-Releasing Hormone / Q000031:analogs & derivatives* / Q000378:metabolism* / Q000494:pharmacology; D006801:Humans; D007295:Inositol Phosphates / Q000378:metabolism; D007700:Kinetics; D051379:Mice; D008961:Models, Structural; D008969:Molecular Sequence Data; D010446:Peptide Fragments / Q000737:chemistry / Q000378:metabolism; D011487:Protein Conformation; D017433:Protein Structure, Secondary; D011966:Receptors, LHRH / Q000737:chemistry* / Q000378:metabolism*; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D012756:Sheep; D014162:Transfection
D001386:Azides; D003432:Cross-Linking Reagents; D007295:Inositol Phosphates; D010446:Peptide Fragments; D011966:Receptors, LHRH; D011994:Recombinant Proteins; D007987:Gonadotropin-Releasing Hormone; C031737:LHRH, N-epsilon-azidobenzoyl-Lys(6)-; D003545:Cysteine