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Development of a photoreactive parathyroid hormone antagonist to probe antagonist-receptor bimolecular interaction
- Source :
- The Journal of Peptide Research. 54:120-128
- Publication Year :
- 1999
- Publisher :
- Wiley, 1999.
-
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.
- Subjects :
- Agonist
medicine.medical_specialty
Kidney
Chemistry
medicine.drug_class
Antagonist
Parathyroid hormone
Biochemistry
Calcium in biology
law.invention
Endocrinology
medicine.anatomical_structure
law
Internal medicine
medicine
Recombinant DNA
Receptor
Cyclase activity
hormones, hormone substitutes, and hormone antagonists
Subjects
Details
- ISSN :
- 1397002X
- Volume :
- 54
- Database :
- OpenAIRE
- Journal :
- The Journal of Peptide Research
- Accession number :
- edsair.doi...........b6fbdd8c15d9377a6e5d559fd844d51a