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

• Published in: The Journal of biological chemistry 1998-07, Vol.273 (27), p.16890-16896
• Main Authors: Mannstadt, M, Luck, MD, Gardella, T J, Jueppner, H
• Format: Article
• Language: English
• Subjects: Animals; COS Cells; Cross-Linking Reagents - chemistry; Ligands; Mutagenesis, Site-Directed; Parathyroid Hormone-Related Protein; Peptide Fragments; Photoaffinity Labels; Protein Conformation; Proteins; Rats; Receptor, Parathyroid Hormone, Type 1; Receptors, Parathyroid Hormone - chemistry; Receptors, Parathyroid Hormone - genetics; Receptors, Parathyroid Hormone - metabolism
• ISSN: 0021-9258; 1083-351X
• DOI: 10.1074/jbc.273.27.16890

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, [Ile 5 ,Bpa 23 ,Tyr 36 ]PTHrP-(1–36)-amide ([Bpa 23 ]PTHrP, where Bpa is p -benzoyl- l -phenylalanine). Upon photolysis, radioiodinated [Bpa 23 ]PTHrP covalently and specifically bound to the rP1R. CNBr cleavage of the broad ≈80-kDa complex yielded a radiolabeled ≈9-kDa non-glycosylated protein band that could potentially be assigned to rP1R residues 23–63, Tyr 23 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 ≈46 kDa, which was reduced to a sharp band of ≈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 ≈6 kDa in size, indicating that [Bpa 23 ]PTHrP cross-links to amino acids 23–40 of the rP1R. This segment overlaps a receptor region previously identified by deletion mapping to be important for ligand binding. Alanine scanning of this region revealed two residues, Thr 33 and Gln 37 , as being functionally involved in ligand binding. Thus, the convergence of photoaffinity cross-linking and mutational data demonstrates that the extreme amino-terminus of the rP1R participates in ligand binding.