Agonist-dependent phosphorylation of the parathyroid hormone/parathyroid hormone-related peptide receptor

Biochemistry. 1998 May 5;37(18):6240-6. doi: 10.1021/bi9726281.


Native PTH/PTHrP receptors in ROS 17/2.8 cells are downregulated after PTH treatment. Since downregulation may involve receptor phosphorylation, we examined PTH/PTHrP receptor phosphorylation in ROS 17/2.8 cells and we mapped the agonist-induced phosphorylation sites using recombinant PTH/PTHrP receptors expressed in LLCPK-1 and COS-7 cells. The data show that the PTH/PTHrP receptor is rapidly phosphorylated in ROS 17/2.8 cells with a maximum occurring at 20 min. The phosphorylation was dose-dependent; it occurred with PTH concentrations that are known to downregulate the PTH/PTHrP receptor in ROS 17/2.8 cells. The time course and the dose requirement for phosphorylation were similar in ROS 17/2.8 cells, which express native PTH/PTHrP receptors, and in LLCPK-1 cells stably transfected with the PTH/PTHrP receptor cDNA. PTH/PTHrP receptor phosphorylation in ROS 17/2.8, COS-7, and LLCPK-1 cells was also stimulated with forskolin and phorbol myristate acetate (PMA). Additionally, in LLCPK-1 cells, which express native clacitonin receptors, PTH/PTHrP receptor phosphorylation was stimulated by calcitonin. These data suggest involvement of second messenger-stimulated kinases in PTH/PTHrP receptor phosphorylation. However, staurosporine, which fully blocked the effects of PMA, forskolin, and clacitonin, partially decreased the effects of PTH on PTH/PTHrP receptor phosphorylation. These data indicate involvement of other kinase(s) in PTH-induced PTH/PTHrP receptor phosphorylation. CNBr cleavage of recombinant receptors expressed in COS-7 cells combined with site-directed mutagenesis revealed that the phosphorylated residues of the PTH/PTHrP receptor map to two regions of the carboxyl-terminal tail located between residues A480 and M499 and residues M499 and M553. These data indicate that the PTH/PTHrP receptor is phosphorylated after PTH stimulation on two regions of the carboxyl-terminal tail and that agonist-dependent phosphorylation involves both staurosporine-sensitive and -insensitive kinases.

Publication types

  • Research Support, U.S. Gov't, P.H.S.

MeSH terms

  • Amino Acid Sequence
  • Animals
  • COS Cells
  • Cyanogen Bromide / pharmacology
  • Cytoplasm / metabolism
  • Down-Regulation
  • Molecular Sequence Data
  • Mutagenesis, Site-Directed
  • Parathyroid Hormone / metabolism*
  • Peptide Mapping
  • Phosphorylation
  • Rats
  • Receptor, Parathyroid Hormone, Type 1
  • Receptors, Parathyroid Hormone / genetics
  • Receptors, Parathyroid Hormone / metabolism*
  • Recombinant Proteins / metabolism


  • Parathyroid Hormone
  • Receptor, Parathyroid Hormone, Type 1
  • Receptors, Parathyroid Hormone
  • Recombinant Proteins
  • Cyanogen Bromide