Direct mapping of an agonist-binding domain within the parathyroid hormone parathyroid hormone-related protein receptor by photoaffinity crosslinking

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, I-125-[Nle(8,18),Lys(13)(epsilon-p-(3-I-Bz)Bz),L-2-Nal(23), Arg(26,27),Tyr(34)]bPTH-(1-34)NH2 (I-125-all-R-K13). This ligand contains a photoreactive benzophenone moiety attached to the side chain of Lys(13). 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, approximate to 400,000 receptors per cell), generating a diffuse approximate to 87-kDa band on SDS/PAGE autoradiography. To identify the ''contact domain'' within the hPTH/PTHrP receptor involved in binding of I-125-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 in I-125-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.