ABSTRACT

Parathyroid hormone (PTH) and parathyroid hormone-related peptide (PTHrP) bind to a common PTH/PTHrP receptor. To explore structure-function relations in these ligands, we synthesized and functionally evaluated PTH-PTHrP hybrid peptides in which the homologous 1-14 portions were exchanged. Hybrid-2, PTH-(1-14)-PTHrP-(15-34)NH, bound to LLC-PK1 cells expressing the cloned rat PTH/PTHrP receptor with high affinity (IC 7 nM). In contrast, hybrid-1, PTHrP(1-14)-PTH-(15-34)NH, bound with much weaker affinity (IC 8,700 nM). Thus, the 1-14 region of PTHrP is incompatible with the 15-34 region of PTH. The carboxyl-terminal incompatibility site was identified as residues 19-21 (Glu-Arg-Val in PTH and Arg-Arg-Arg in PTHrP); extending the amino-terminal PTHrP sequence to residue 21 but not to 18 cured the hybrid's binding defect. The amino-terminal incompatibility site was identified as position 5 (Ile in PTH and His in PTHrP), because Ile-hybrid-1 bound with high affinity (IC 20 nM). The importance of these identified residues in the native ligands was established by evaluating the effects of substitutions at these sites in a series of PTH and PTHrP analog peptides. Overall, the results are consistent with the hypothesis that, in both PTH and PTHrP, the 1-14 and 15-34 domains interact when binding to the receptor and that residues 5, 19, and 21 contribute either directly or indirectly to this interaction.

FOOTNOTES

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The abbreviations used are: PTH, parathyroid hormone; PTHrP, parathyroid hormone-related peptide; hPTH, human PTH; hPTHrP, human PTHrP; bPTH, bovine PTH; ROS, rat osteosarcoma cells; HPLC, high pressure liquid chromatography.

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