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/

Low resolution mutational studies have indicated that the amino-termin al extracellular domain of the rat parathyroid hormone (PTH)/PTH-relat ed protein (PTHrP) receptor (rP1R) interacts with the carboxyl-termina l portion of PTH-(1-34) or PTHrP-(1-36), To further define ligand-rece ptor 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, radioiodin ated [Bpa23]PTHrP covalently and specifically bound to the rP1R, CNBr cleavage of the broad approximate to 80-kDa complex yielded a radiolab eled approximate to 9-kDa nonglycosylated protein band that could pote ntially 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 CN Br digestion, a broad protein band of approximate to 46 kDa, which was reduced to a sharp band of approximate to 20 kDa upon deglycosylation , CNBr digestion of complexes formed with two additional rP1R double m utants (rP1R-M63I/L40M and rP1R-M63I/L41M) yielded non-glycosylated pr otein bands that were approximate to 6 kDa in size, indicating that [B pa23]PTHrP cross-links to amino acids 23-40 of the rP1R, This segment overlaps a receptor region previously identified by deletion mapping t o be important for ligand binding. Alanine scanning of this region rev ealed two residues, Thr(33) and Gln(37), as being functionally involve d in ligand binding. Thus, the convergence of photoaffinity cross-link ing and mutational data demonstrates that the extreme amino-terminus o f the rP1R participates in ligand binding.