Importance of the amino terminus in secretin family G protein-coupled receptors - Intrinsic photoaffinity labeling establishes initial docking constraints for the calcitonin receptor

被引:43
|
作者
Dong, MQ
Pinon, DI
Cox, RF
Miller, LJ
机构
[1] Mayo Clin Scottsdale, Dept Mol Pharmacol & Expt Therapeut, Scottsdale, AZ 85259 USA
[2] GlaxoSmithKline, Res Triangle Pk, NC 27709 USA
关键词
D O I
10.1074/jbc.M305719200
中图分类号
Q5 [生物化学]; Q7 [分子生物学];
学科分类号
071010 ; 081704 ;
摘要
The calcitonin receptor is a member of the class B family of G protein-coupled receptors, closely related to secretin and parathyroid hormone receptors. Although mechanisms of ligand binding have been directly explored for those receptors, current knowledge of the molecular basis of calcitonin binding to its receptor is based only on receptor mutagenesis. In this work we have utilized the more direct approach of photoaffinity labeling to explore spatial approximations between distinct residues within calcitonin and its receptor. For this we have developed two human calcitonin analogues incorporating a photolabile p-benzoyl-L-phenylalanine residue in the mid-region and carboxyl-terminal half of the peptide in positions 16 and 26, respectively. Both probes specifically bound to the human calcitonin receptor with high affinity and were potent stimulants of cAMP accumulation in calcitonin receptor-bearing human embryonic kidney 293 cells. They covalently labeled the calcitonin receptor in a saturable and specific manner. Further purification, deglycosylation, specific chemical and enzymatic cleavage, and sequencing of labeled wild type and mutant calcitonin receptors identified the sites of labeling for the position 16 and 26 probes as receptor residues Phe(137) and Thr(30), respectively. Both were within the extracellular amino terminus of the calcitonin receptor, with the former adjacent to the first transmembrane segment and the latter within the distal amino-terminal tail of the receptor. These data are consistent with affinity labeling of other members of the class B G protein-coupled receptors using analogous probes and may suggest a common ligand binding mechanism for this family.
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收藏
页码:1167 / 1175
页数:9
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