The principal receptor-binding domain (Ser17–Val31) of parathyroid hormone (PTH) is predicted to form an amphiphilic α-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 Arg20 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 Trp23, Leu24, and Leu28, which together form the hydrophobic face of the predicted amphiphilic α-helix. Glu substitutions at Arg25, Lys26, and Lys27 (which forms the hydrophilic face of the helix) caused 4–10-fold reductions in affinity for both receptors. Thus, the side chains of Arg20, together with those composing the hydrophobic face of the ligand's putative amphiphilic α-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.
Role of Amino Acid Side Chains in Region 17–31 of Parathyroid Hormone (PTH) in Binding to the PTH Receptor