Development of a photoreactive parathyroid hormone antagonist to probe antagonist-receptor bimolecular interaction

Parathyroid hormone (PTH) and PTH-related protein (PTHrP) exert their calci otropic activities by binding to a specific seven transmembrane-helix-conta ining G protein-coupled receptor mainly located in bone and kidney cells. I n order tb map in detail the nature of hormone-receptor interaction, we are employing 'photoaffinity scanning' of the bimolecular interface. To this e nd, we have developed photoreactive benzophenone (BP)-containing PTH analog s which can be specifically and efficiently cross-linked to the human (h) P TH/PTHrP receptor. In this report, we describe the photocross-linking of a BP-containing PTH antagonist, [Nle(8,18),D-2-Nal(12),Lys(13)(epsilon-Bp),2- Nal(23),Tyr(34)]bPTH(7-34)NH2 (ANT) to the recombinant hPTH/PTHrP receptor stably expressed in human embryonic kidney cells (HEK-293, clone C-21). Thi s photoreactive antagonist has high affinity for the hPTH/PTHrP receptor an d inhibits agonist-induced cyclase activity and intracellular calcium relea se. The photo-induced cross-linking of the radioiodinated antagonist (I-125 -ANT) to the recombinant hPTH/PTHrP receptor followed by SDS-PAGE analysis reveals a single radiolabeled band of approximate to 85kDa, similar to that observed after cross-linking of a radioiodinated BP-containing agonist. Th e formation of this covalent I-125-ANT - hPTH/PTHrP receptor conjugate is c ompeted dose-dependently by a variety of unlabelled PTH- and PTHrP-derived agonists and antagonists. This is the first report of a specific and effici ent photocross-linking of a radioiodinated PTH antagonist to the hPTH/PTHrP receptor. Therefore, it provides the opportunity to study directly the nat ure of the bimolecular interaction of PTH antagonist with the hPTH/PTHrP re ceptor.