Evidence for a ligand interaction site at the amino-terminus of the parathyroid hormone (PTH)/PTH-related protein receptor from cross-linking and mutational studies.

The Journal of Biological Chemistry
M MannstadtH Jüppner

Abstract

Low resolution mutational studies have indicated that the amino-terminal extracellular domain of the rat parathyroid hormone (PTH)/PTH-related protein (PTHrP) receptor (rP1R) interacts with the carboxyl-terminal portion of PTH-(1-34) or PTHrP-(1-36). To further define ligand-receptor interactions, we prepared a fully functional photoreactive analog of PTHrP, [Ile5,Bpa23,Tyr36]PTHrP-(1-36)-amide ([Bpa23]PTHrP, where Bpa is p-benzoyl-L-phenylalanine). Upon photolysis, radioiodinated [Bpa23]PTHrP covalently and specifically bound to the rP1R. CNBr cleavage of the broad approximately 80-kDa complex yielded a radiolabeled approximately 9-kDa non-glycosylated protein band that could potentially be assigned to rP1R residues 23-63, Tyr23 being the presumed amino-terminus of the receptor. This assignment was confirmed using a mutant rP1R (rP1R-M63I) that yielded, upon photoligand binding and CNBr digestion, a broad protein band of approximately 46 kDa, which was reduced to a sharp band of approximately 20 kDa upon deglycosylation. CNBr digestion of complexes formed with two additional rP1R double mutants (rP1R-M63I/L40M and rP1R-M63I/L41M) yielded non-glycosylated protein bands that were approximately 6 kDa in size, indicating that [Bpa...Continue Reading

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Citations

Dec 20, 2002·Biopolymers·Cecilia G Unson
Mar 19, 2002·Biophysical Chemistry·Luca MonticelliDale F Mierke
Jun 1, 2002·Molecular and Cellular Endocrinology·Anne E NelsonBruce G Robinson
Jun 9, 2001·Trends in Endocrinology and Metabolism : TEM·T J Gardella, H Jüppner
May 8, 2008·Biochemistry·Paul MonaghanMichael Rosenblatt
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Mar 8, 2006·Clinical Chemistry and Laboratory Medicine : CCLM·Indra Ramasamy
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Nov 13, 2004·Journal of Bone and Mineral Research : the Official Journal of the American Society for Bone and Mineral Research·Naoto ShimizuThomas J Gardella
Apr 9, 2008·Cell Biochemistry and Function·Majed S Alokail, Margaret J Peddie
Feb 8, 2005·Biochemical and Biophysical Research Communications·Robert C GensureHarald Jüppner
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Oct 27, 2006·The Biochemical Journal·Brian J HolleranRichard Leduc
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