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.

Photoaffinity scanning in the mapping of the peptide receptor interface of class II G protein—coupled receptors

“…In addition to the photoactive agonist, [Bpa 13 ] PTH(1-34), Chorev and coworkers also developed a photoactive antagonist, PTH , substituted at position 13 to directly study the nature of the bimolecular interface interaction of PTH antagonist with its receptor [86]. In this case, equivalent sites of cross-linking occurred for both peptides (R 186 in the ECD), indicating that orientation of this segment of the receptor does not alter dramatically during agonist activation of the receptor.…”

Design, synthesis, and conformational study of biologically active photolabeled analogues of the main immunogenic region of the acetylcholine receptor

“…Introduction of the amino acid p ‐benzoyl‐ L ‐phenylalanine (Bpa)11 into peptides by solid phase synthesis has allowed direct incorporation of benzophenone photoprobe at a defined position, thus bringing a significant advantage in the application of photoaffinity labeling and photoinsertion to the study of peptide–protein interactions. Benzophenone photoprobe has been successfully employed in studies of cross‐linking to several receptors,12–21 enzymes,22–26 and various proteins 11, 27, 28…”

Insights into Interactions between the α-Helical Region of the Salmon Calcitonin Antagonists and the Human Calcitonin Receptor using Photoaffinity Labeling

“…workers developed a photoactive antagonist, PTH (7-34), substituted at position 13 to directly study the nature of the bimolecular interface interaction of PTH antagonist with its receptor (65). In this case, equivalent sites of cross-linking occurred for both peptides (Arg 186 in the extracellular domain).…”