title: Development of a photoreactive parathyroid hormone antagonist to probe antagonist-receptor bimolecular interaction.
abstract: Parathyroid hormone (PTH) and PTH-related protein (PTHrP) exert their calciotropic activities by binding to a specific seven-transmembrane-helix-containing G protein-coupled receptor mainly located in bone and kidney cells. In order to map in detail the nature of hormone-receptor interaction, we are employing 'photoaffinity scanning' of the bimolecular interface. To this end, we have developed photoreactive benzophenone (BP)-containing PTH analogs which can be specifically and efficiently cross-linked to the human (h) PTH/PTHrP receptor. In this report, we describe the photocross-linking of a BP-containing PTH antagonist, [Nle8,18,D-2-Nal12,Lys13(epsilon-BP),2-Nal23,Tyr34]bPT H(7-34)NH2 (ANT) to the recombinant hPTH/PTHrP receptor stably expressed in human embryonic kidney cells (HEK-293, clone C-21). This photoreactive antagonist has high affinity for the hPTH/PTHrP receptor and inhibits agonist-induced cyclase activity and intracellular calcium release. The photo-induced cross-linking of the radioiodinated antagonist (125I-ANT) to the recombinant hPTH/PTHrP receptor followed by SDS-PAGE analysis reveals a single radiolabeled band of approximately 85kDa, similar to that observed after cross-linking of a radioiodinated BP-containing agonist. The formation of this covalent 125I-ANT - hPTH/PTHrP receptor conjugate is competed dose-dependently by a variety of unlabelled PTH- and PTHrP-derived agonists and antagonists. This is the first report of a specific and efficient photocross-linking of a radioiodinated PTH antagonist to the hPTH/PTHrP receptor. Therefore, it provides the opportunity to study directly the nature of the bimolecular interaction of PTH antagonist with the hPTH/PTHrP receptor.
authors.affiliation: Department of Medicine, Beth Israel Deaconess Medical Center and Harvard Medical School, Boston, MA 02215, USA.
authors.forename: A
authors.initials: A
authors.lastname: Bisello
authors.forename: V
authors.initials: V
authors.lastname: Behar
authors.forename: Z
authors.initials: Z
authors.lastname: Greenberg
authors.forename: L J
authors.initials: LJ
authors.lastname: Suva
authors.forename: M
authors.initials: M
authors.lastname: Rosenblatt
authors.forename: M
authors.initials: M
authors.lastname: Chorev
mesh_terms: D000595:Amino Acid Sequence; D002460:Cell Line; D006801:Humans; D007457:Iodine Radioisotopes; D015335:Molecular Probes; D008969:Molecular Sequence Data; D010281:Parathyroid Hormone / Q000037:antagonists & inhibitors* / Q000378:metabolism; D019920:Photoaffinity Labels; D011869:Radioligand Assay; D018016:Receptors, Parathyroid Hormone / Q000378:metabolism*
chemical_list: D007457:Iodine Radioisotopes; D015335:Molecular Probes; D010281:Parathyroid Hormone; D019920:Photoaffinity Labels; D018016:Receptors, Parathyroid Hormone