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, ¹²⁵I-[Nle^8.18,Lys¹³ (ε-p-(3-I-Bz)Bz),L-2- Nal²³,Arg^26,27,Tyr³⁴]bPTH-(1-34)NH₂ (¹²⁵I-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 of ¹²⁵I-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 ¹²⁵I-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.