ANALYSIS OF PARATHYROID HORMONES PRINCIPAL RECEPTOR-BINDING REGION BYSITE-DIRECTED MUTAGENESIS AND ANALOG DESIGN

Previous deletion studies established that the 25-34 region of PTH is important for receptor binding. We used oligonucleotide-directed mutag enesis to generate 47 different mutations in this region of human (h) PTH-(1-84) and evaluated cAMP-stimulating activity in ROS 17/2.8 cells . The hydrophobic residues Leu24 and Leu28 stood out as mutationally i ntolerant sites, while neighboring polar residues were comparatively t olerant. A series of synthetic PTH analogs was designed to test these residues further. The affinity of [Tyr34]hPTH-(1-34)NH2 for ROS 17/2.8 cells [dissociation constant (K(d)), approximately 5 nm)] was dramati cally reduced by the substitution of either Leu24 or Leu28 with Glu (K (d), approximately 20,000 and 8,000 nm, respectively). The Val31-->Glu substitution also sharply reduced affinity (K(d), approximately 200 n m). In contrast, the nearby charge-reversing change of Asp30-->Lys had no effect on binding affinity (K(d), approximately 5 nm). Similar eff ects were observed in the opossum kidney cell line. The binding of [Ty r34]hPTH-(15-34)NH2 to ROS 17/2.8 and opossum kidney cells (K(d), appr oximately 10 muM) was abolished by Glu substitutions at position 24, 2 8, or 31; the Lys30 change was without effect. These results suggest t hat the adverse effects of the Glu substitutions on receptor binding a re not due purely to the disruption of tertiary interactions with the 1-14 region. Circular dichroism spectroscopy indicated that the substi tutions do not affect local helical structure. The data suggest that L eu24, Leu28, and Val31 contribute important receptor-binding interacti ons and are consistent with the hypothesis that an amphipathic a-helix in the carboxy-terminal region of PTH-(1-34) is involved in receptor binding.