Role of amino acid side chains in region 17-31 of parathyroid hormone (PTH) in binding to the PTH receptor.

The principal receptor-binding domain (Ser(17)-Val(31)) of parathyroid hormone (PTH) is predicted to form an amphiphilic alpha-helix and to interact primarily with the N-terminal extracellular domain (N domain) of the PTH receptor (PTHR). We explored these hypotheses by introducing a variety of substitutions in region 17-31 of PTH-(1-31) and assessing, via competition assays, their effects on binding to the wild-type PTHR and to PTHR-delNt, which lacks most of the N domain. Substitutions at Arg(20) reduced affinity for the intact PTHR by 200-fold or more, but altered affinity for PTHR-delNt by 4-fold or less. Similar effects were observed for Glu substitutions at Trp(23), Leu(24), and Leu(28), which together form the hydrophobic face of the predicted amphiphilic alpha-helix. Glu substitutions at Arg(25), Lys(26), and Lys(27) (which forms the hydrophilic face of the helix) caused 4-10-fold reductions in affinity for both receptors. Thus, the side chains of Arg(20), together with those composing the hydrophobic face of the ligand's putative amphiphilic alpha-helix, contribute strongly to PTHR-binding affinity by interacting specifically with the N domain of the receptor. The side chains projecting from the opposite helical face contribute weakly to binding affinity by different mechanisms, possibly involving interactions with the extracellular loop/transmembrane domain region of the receptor. The data help define the roles that side chains in the binding domain of PTH play in the PTH-PTHR interaction process and provide new clues for understanding the overall topology of the bimolecular complex.