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).
Recent mutagenesis and cross-linking studies suggest that three regions of the PTH-1 receptor play important roles in ligand interaction: (i) the extreme NH(2)-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 Phe(184), Arg(186), Leu(187), and Ile(190) as important determinants of maximum binding of (125)I-labeled bovine PTH-(1-34) and (125)I-labeled bovine PTH-(3-34) and determinants of responsiveness to the NH(2)-terminal analog, PTH-(1-14) in cAMP stimulation assays. Alanine mutations at these four sites augmented the ability of the COOH-terminal peptide [Glu(22), Trp(23)]PTHrP-(15-36) to inhibit the cAMP response induced by PTH-(1-34). At Phe(184) and Leu(187), 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 Phe(184) and Leu(187) in the PTH-1 receptor plays an important role in determining functional interaction with the 3-14 portion of PTH.
Carter
P H
PH
Endocrine Unit, Massachusetts General Hospital and Harvard Medical School Boston, Massachusetts 02114, USA.
Shimizu
M
M
Luck
M D
MD
Gardella
T J
TJ
D019943:Amino Acid Substitution; D000818:Animals; D002417:Cattle; D000242:Cyclic AMP / Q000378:metabolism; D004305:Dose-Response Relationship, Drug; D007930:Leucine / Q000378:metabolism*; D016297:Mutagenesis, Site-Directed; D010649:Phenylalanine / Q000378:metabolism*; D017433:Protein Structure, Secondary; D051381:Rats; D018016:Receptors, Parathyroid Hormone / Q000235:genetics / Q000378:metabolism*; D019379:Teriparatide / Q000378:metabolism*
D018016:Receptors, Parathyroid Hormone; D019379:Teriparatide; D010649:Phenylalanine; D000242:Cyclic AMP; D007930:Leucine