Spatial proximity between a photolabile residue in position 19 of salmon calcitonin and the amino terminus of the human calcitonin receptor.
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.
Pham
Vi
V
Howard Florey Institute of Experimental Physiology and Medicine, the University of Melbourne, Victoria 3010, Australia.
Wade
John D
JD
Purdue
Brooke W
BW
Sexton
Patrick M
PM
D000595:Amino Acid Sequence; D000818:Animals; D019556:COS Cells; D002116:Calcitonin / Q000737:chemistry; D002462:Cell Membrane / Q000378:metabolism; D003432:Cross-Linking Reagents / Q000494:pharmacology; D003488:Cyanogen Bromide / Q000494:pharmacology; D000242:Cyclic AMP / Q000737:chemistry; D003545:Cysteine / Q000737:chemistry; D018076:DNA, Complementary / Q000378:metabolism; D004305:Dose-Response Relationship, Drug; D010450:Endopeptidases / Q000494:pharmacology; D008024:Ligands; D008239:Lysine / Q000737:chemistry; D008666:Metalloendopeptidases / Q000494:pharmacology; D008715:Methionine / Q000737:chemistry; D008969:Molecular Sequence Data; D016296:Mutagenesis; D009154:Mutation; D010100:Oxygen / Q000378:metabolism; D010455:Peptides / Q000737:chemistry; D010649:Phenylalanine / Q000031:analogs & derivatives* / Q000737:chemistry; D019920:Photoaffinity Labels / Q000494:pharmacology; D017354:Point Mutation; D011233:Precipitin Tests; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D018003:Receptors, Calcitonin / Q000737:chemistry*; D012474:Salmon / Q000378:metabolism; D014162:Transfection
D003432:Cross-Linking Reagents; D018076:DNA, Complementary; D008024:Ligands; D010455:Peptides; D019920:Photoaffinity Labels; D018003:Receptors, Calcitonin; D010649:Phenylalanine; D002116:Calcitonin; C053764:benzoylphenylalanine; D008715:Methionine; D000242:Cyclic AMP; D010450:Endopeptidases; D008666:Metalloendopeptidases; C039830:peptidyl-Lys metalloendopeptidase; C058921:endoproteinase Asp-N; D008239:Lysine; D003545:Cysteine; D003488:Cyanogen Bromide; D010100:Oxygen