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-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 [Bpa23]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, Thr33 and Gln37, 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.
Mannstadt
M
M
Endocrine Unit, Department of Medicine and Children's Service, Massachusetts General Hospital and Harvard Medical School, Boston, Massachusetts 02114, USA.
Luck
M D
MD
Gardella
T J
TJ
Jüppner
H
H
D000818:Animals; D019556:COS Cells; D003432:Cross-Linking Reagents / Q000737:chemistry; D008024:Ligands; D016297:Mutagenesis, Site-Directed; D044162:Parathyroid Hormone-Related Protein; D010446:Peptide Fragments; D019920:Photoaffinity Labels; D011487:Protein Conformation; D011506:Proteins; D051381:Rats; D044168:Receptor, Parathyroid Hormone, Type 1; D018016:Receptors, Parathyroid Hormone / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*
D003432:Cross-Linking Reagents; D008024:Ligands; D044162:Parathyroid Hormone-Related Protein; D010446:Peptide Fragments; D019920:Photoaffinity Labels; D011506:Proteins; D044168:Receptor, Parathyroid Hormone, Type 1; D018016:Receptors, Parathyroid Hormone; C054296:parathyroid hormone-related protein (1-34)