DIRECT MAPPING OF AN AGONIST-BINDING DOMAIN WITHIN THE PARATHYROID-HORMONE PARATHYROID HORMONE-RELATED PROTEIN-RECEPTOR BY PHOTOAFFINITY CROSS-LINKING

Parathyroid hormone (PTH) and PTH-related protein (PTHrP) are calciotr opic hormones interacting with a shared seven-transmembrane domain G p rotein-coupled receptor, which is located predominantly in bone and ki dney. To map the interface of the bimolecular interaction between horm one and receptor, we designed and radioiodinated a bioactive, photorea ctive PTH agonist, e(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 conta ins a photoreactive benzophenone moiety attached to the side chain of Lys(13). All other lysyl residues are substituted by argynyls. The ana log photocrosslinks specifically to the recombinant hPTH/PTHrP recepto r stably transfected into human embryonic kidney cells (HEK-293/C-21 c ells, approximate to 400,000 receptors per cell), generating a diffuse approximate to 87-kDa band on SDS/PAGE autoradiography. To identify t he ''contact domain'' within the hPTH/PTHrP receptor involved in bindi ng of I-125-all-R-K13, the radiolabeled band containing the ligand-rec eptor 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 brom ide; Route B, cyanogen bromide followed by endo-N-glycosydase F. The i dentified 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, locate d at the C-terminal region of the N-terminal extracellular domain.