Open AccessDirect mapping of an agonist-binding domain within the parathyroid hormone/parathyroid hormone-related protein receptor by photoaffinity crosslinking
Author(s) -
Alice T. Zhou,
Roberto Bessalle,
Alessandro Bisello,
Chizu Nakamoto,
Michael Rosenblatt,
Larry J. Suva,
Michael Chorev
Publication year - 1997
Publication title -
proceedings of the national academy of sciences of the united states of america
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 5.011
H-Index - 771
eISSN - 1091-6490
pISSN - 0027-8424
DOI - 10.1073/pnas.94.8.3644
Subject(s) - chemistry , parathyroid hormone , photoaffinity labeling , receptor , parathyroid hormone receptor , cyanogen bromide , agonist , parathyroid hormone related protein , biochemistry , stereochemistry , hormone receptor , peptide sequence , biology , calcium , organic chemistry , cancer , breast cancer , gene , genetics
Parathyroid hormone (PTH) and PTH-related protein (PTHrP) are calciotropic hormones interacting with a shared seven-transmembrane domain G protein-coupled receptor, which is located predominantly in bone and kidney. To map the interface of the bimolecular interaction between hormone and receptor, we designed and radioiodinated a bioactive, photoreactive PTH agonist,125 I-[Nle8,18 ,Lys13 (ɛ-p -(3-I-Bz)Bz),l -2-Nal23 ,Arg26,27 ,Tyr34 ]bPTH-(1–34)NH2 (125 I-all-R-K13). This ligand contains a photoreactive benzophenone moiety attached to the side chain of Lys13 . All other lysyl residues are substituted by argynyls. The analog photocrosslinks specifically to the recombinant hPTH/PTHrP receptor stably transfected into human embryonic kidney cells (HEK-293/C-21 cells, ≈400,000 receptors per cell), generating a diffuse ≈87-kDa band on SDS/PAGE autoradiography. To identify the “contact domain” within the hPTH/PTHrP receptor involved in binding of125 I-all-R-K13, the radiolabeled band containing the ligand–receptor conjugate was subjected to chemical and enzymatic cleavage. Two independent pathways of sequential digestion were used: Route A, lysyl endopeptidase C, then endo-N-glycosidase F, followed by cyanogen bromide; Route B, cyanogen bromide followed by endo-N -glycosydase F. The identified domain is in contact with position 13 in125 I-all-R-K13 and corresponds to amino acids 173–189 of the hPTH/PTHrP receptor, located at the C-terminal region of the N-terminal extracellular domain.