The Hydrophobic Residues Phenylalanine 184 and Leucine 187 in the Type-1 Parathyroid Hormone (PTH) Receptor Functionally Interact with the Amino-terminal Portion of PTH-(1–34)

• Published in: The Journal of biological chemistry 1999-11, Vol.274 (45), p.31955-31960
• Main Authors: Carter, Percy H., Shimizu, Masaru, Luck, Michael D., Gardella, Thomas J.
• Format: Article
• Language: English
• Subjects: Amino Acid Substitution; Animals; Cattle; Cyclic AMP - metabolism; Dose-Response Relationship, Drug; Leucine - metabolism; Mutagenesis, Site-Directed; parathyroid hormone receptors; Phenylalanine - metabolism; Protein Structure, Secondary; Rats; Receptors, Parathyroid Hormone - genetics; Receptors, Parathyroid Hormone - metabolism; Teriparatide - metabolism
• ISSN: 0021-9258; 1083-351X
• DOI: 10.1074/jbc.274.45.31955

Recent mutagenesis and cross-linking studies suggest that three regions of the PTH-1 receptor play important roles in ligand interaction: (i) the extreme NH2-terminal region, (ii) the juxtamembrane base of the amino-terminal extracellular domain, and (iii) the third extracellular loop. In this report, we analyzed the second of these segments in the rat PTH-1 receptor (residues 182–190) and its role in functional interaction with short PTH fragment analogs. Twenty-eight singly substituted PTH-1 receptors were transiently transfected into COS-7 cells and shown to be fully expressed by surface antibody binding analysis. Alanine-scanning analysis identified Phe184, Arg186, Leu187, and Ile190 as important determinants of maximum binding of 125I-labeled bovine PTH-(1–34) and125I-labeled bovine PTH-(3–34) and determinants of responsiveness to the NH2-terminal analog, PTH-(1–14) in cAMP stimulation assays. Alanine mutations at these four sites augmented the ability of the COOH-terminal peptide [Glu22,Trp23]PTHrP-(15–36) to inhibit the cAMP response induced by PTH-(1–34). At Phe184 and Leu187, hydrophobic substitutions (e.g. Ile, Met, or Leu) preserved PTH-(1–34)-mediated cAMP signaling potency, whereas hydrophilic substitutions (e.g. Asp, Glu, Lys, or Arg) weakened this response by 20-fold or more, as compared with the unsubstituted receptor's response. The results suggest that hydrophobicity at positions occupied by Phe184 and Leu187 in the PTH-1 receptor plays an important role in determining functional interaction with the 3–14 portion of PTH.