Structural requirements for conserved arginine of parathyroid hormone

Arg-20 is one of two residues conserved in all peptides known to activate t he parathyroid hormone (PTH) receptor. Previous studies have failed to find any naturally encoded analogues of residue 20 that had any adenylyl cyclas e (AC) stimulating activity. In this work we have studied substitutions of Arg-20 with nonencoded amino acids and conformationally constrained analogu es with side chains mimicking that of Arg. No analogue had more than 20% of the AC-stimulating ability of the natural Arg-20-bearing peptide. In desce nding order of activity, the most active analogues had (S)-4-piperidyl(N-am idino)glycine (PipGly), norleucine (Nle), citrulline (Cit), or ornithine (O rm) at residue 20. Analogues with Arg-20 substituted with L-4-piperidyl-(N- amidino)alanine, Lys, Glu, Ala, Gln, (S)-2-amino-4-[(2-amino)pyrimidinyl]bu tanoic acid, or L-(4-guanidino)phenylalanine had very low or negligible act ivity. Low or negligible activities of Lys or Orn analogues suggested ionic interactions play a minor role in the Arg interaction with the receptor. T he conformational constraints imposed by the PipGly ring had a negative eff ect on its ability to substitute for Arg. The side-chain H-bonding potentia l of the Cit ureimido group was likely an important factor in its mimicry o f Arg. The increase in amphiphilicity, as demonstrated by its greater high- performance liquid chromatographic retention, and increased alpha -helix. a s shown by circular dichroic spectroscopy, likely contributed to the activi ty of the Nle-20 analogue. The data demonstrated that specific H-bonding, h ydrophobicity of the side chain, stabilization of alpha -helix, and possibl y specific cation positioning were all important in the interaction of Arg- 20 with receptor groups.