FUNCTIONAL-PROPERTIES OF A SYNTHETIC CHICKEN PARATHYROID HORMONE-RELATED PROTEIN-1-36 FRAGMENT

The biologic activities of human parathyroid hormone-related protein [ hPTHrP(1-34] and bovine PTH [bPTH(1-34)] are remarkably similar despit e marked sequence divergence in their primary binding domain, residues 25-34. Chicken PTHrP (cPTHrP) is identical to hPTHrP through residue 21. However, in the 25-34 region, cPTHrP displays three fewer basic re sidues than hPTHrP and contains five residues not present in any other member of the PTH/PTHrP family. To assess the biologic consequences o f these structural differences, we compared the activities of syntheti c [(36)Tyr]cPTHrP(1-36)NH2 and hPTHrP(1-34)NH2 with those of bPTH(1-34 ) in avian systems (chicken renal plasma membranes and 19 day chick em bryonic bone cells) and mammalian systems [canine renal plasma membran es and rat osteosarcoma cells (UMR-106-H5)]. In both avian and mammali an systems the binding affinity of [(36)Tyr]cPTHrP(1-36)NH2 (0.8-3.4 n M) was approximately one-half that of hPTHrP(1-34)NH2 (0.4-1.1 nM). Th e potencies of [(36)Tyr]cPTHrP(1-36)NH2 and hPTHrP(1-34)NH2 for activa tion of adenylate cyclase were similar in canine renal membranes (5.2 and 6.7 nM) and chick bone cells (1.0 nM). In UMR-106 cells and chicke n renal membranes the potency of [(36)Tyr[cPTHrP(1-36)NH2 for activati on of adenylate cyclase was about one-half that of [(36)Tyr]hPTHrP(1-3 6)NH2. Binding of I-125-[(36)Tyr]cPTHrP(1-36)NH2 to chick bone cells a nd chicken renal membranes was completely displaced by bPTH(1-34) and hPTHrP(1-34)NH2: thus there was no evidence for a distinct chicken PTH rP receptor. In general, [(36)Tyr]cPTHrP(1-36)NH2 and hPTHrP(1-34)NH2 activated adenylate cyclase similarly despite their sequence differenc es in the 25-32 region. This suggests that basic residues in the 25-32 region ace not required for the peptide to assume a biologically acti ve conformation at the receptor. In cross-linking studies, both I-125- hPTHrP(1-34)NH2 and I-125-[(36)Tyr]cPTHrP(1-36)NH2 labeled a major 85 kD PTH/PTHrP receptor component in canine renal plasma membranes. I-12 5-hPTHrP(1-34)NH2 also labeled a less than or equal to 14 k) receptor fragment, whereas I-125-[(36)Tyr]cPTHrP(1-36)NH2 did not. The present results suggest that retained sequence features in the 25-32 region ma y be critical determinants of receptor binding and that sequence diffe rences in this region alter the sites of interaction of PTHrP peptides with the PTH/PTHrP receptor.