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Spatial proximity between a photolabile residue in position 19 of salmon calcitonin and the amino terminus of the human calcitonin receptor.
- Source :
-
The Journal of biological chemistry [J Biol Chem] 2004 Feb 20; Vol. 279 (8), pp. 6720-9. Date of Electronic Publication: 2003 Nov 17. - Publication Year :
- 2004
-
Abstract
- Calcitonins are 32-amino acid peptide hormones with both peripheral and central actions mediated via specific cell surface receptors, which belong to the class II subfamily of G protein-coupled receptors. Understanding receptor function, particularly in terms of ligand recognition by calcitonin receptors, may aid in the rational design of calcitonin analogs with increased potency and improved selectivity. To directly identify sites of proximity between calcitonin and its receptor, we carried out photoaffinity labeling studies followed by protein digestion and mapping of the radiolabeled photoconjugated receptor. A fully active salmon calcitonin analog [Arg(11,18),Bpa19]sCT, incorporating a photolabile p-benzoyl-L-phenylalanine into position 19 of the ligand, has been used to demonstrate spatial proximity between residue 19 of the peptide and the amino-terminal extracellular domain of the receptor. Cyanogen bromide cleavage together with endoproteinase Asp-N digestion indicated that binding was predominantly to the region delimited by receptor residues Cys134 and Met187. Binding to this fragment was supported further by cyanogen bromide-digestion of receptors that were mutated to remove the predicted cleavage site at Met133 (M133A, M133L). Binding within the 54-amino acid fragment was refined further by digestion with endoproteinase Lys-C to the 8-amino acid region corresponding to Cys134-Lys141. These results provide the first direct demonstration of a contact domain between salmon calcitonin and its receptor and will contribute toward modeling of the calcitonin-receptor interface.
- Subjects :
- Amino Acid Sequence
Animals
COS Cells
Calcitonin chemistry
Cell Membrane metabolism
Cross-Linking Reagents pharmacology
Cyanogen Bromide pharmacology
Cyclic AMP chemistry
Cysteine chemistry
DNA, Complementary metabolism
Dose-Response Relationship, Drug
Endopeptidases pharmacology
Ligands
Lysine chemistry
Metalloendopeptidases pharmacology
Methionine chemistry
Molecular Sequence Data
Mutagenesis
Mutation
Oxygen metabolism
Peptides chemistry
Phenylalanine chemistry
Photoaffinity Labels pharmacology
Point Mutation
Precipitin Tests
Protein Binding
Protein Structure, Tertiary
Salmon metabolism
Transfection
Phenylalanine analogs & derivatives
Receptors, Calcitonin chemistry
Subjects
Details
- Language :
- English
- ISSN :
- 0021-9258
- Volume :
- 279
- Issue :
- 8
- Database :
- MEDLINE
- Journal :
- The Journal of biological chemistry
- Publication Type :
- Academic Journal
- Accession number :
- 14623894
- Full Text :
- https://doi.org/10.1074/jbc.M307214200