Role of Amino Acid Side Chains in Region 17–31 of Parathyroid Hormone (PTH) in Binding to the PTH Receptor

Abstract 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.