Mutational analysis of the receptor-activating region of human parathyroid hormone

The first 4 residues of parathyroid hormone (PTH) are highly conserved in evolution and are important for biological activity. We randomly mutated codons 1-4 of human PTH (hPTH) with degenerate oligonucleotides and, after expression in COS cells, screened the mutants for receptor binding and cAMP-stimulating activity using ROS 17/2.8 cells. This survey identified Glu⁴ and Val² as important determinants of receptor binding and activation, respectively. Positions 1 and 3 were more tolerant of substitutions indicating that these sites are less vital to hormone function. Activities of synthetic hPTH(1-34) analogs further demonstrated the importance of positions 2 and 4. The binding affinity of [Ala⁴,Tyr³⁴] hPTH(1-34)NH₂ was 100-fold reduced relative to [Tyr³⁴]hPTH(1-34)NH₂ (K_d values = 653 ± 270 and 4 ± 1 nM, respectively), and [Arg², Tyr³⁴]hPTH(1-34)NH₂ was a weak partial agonist which bound well to the ROS cell receptor (K_d = 31 ± 10 nM). The Arg² analog was nearly as potent as PTH(3-34) as an in vitro PTH antagonist in osteoblast derived cells. However, unlike PTH(3-34), [Arg²]PTH was a full agonist in opossum kidney (OK) cells. These observations suggest that the activation domains of the OK and ROS cell PTH receptors are different. Thus, amino-terminal PTH analogs may be useful as probes for distinguishing properties of PTH receptors.