Agonist-dependent phosphorylation of the parathyroid hormone/parathyroid hormone-related peptide receptor.
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.
Qian
F
F
Endocrine Unit, Massachusetts General Hospital and Harvard Medical School, Boston, Massachusetts 02114, USA.
Leung
A
A
Abou-Samra
A
A
D000595:Amino Acid Sequence; D000818:Animals; D019556:COS Cells; D003488:Cyanogen Bromide / Q000494:pharmacology; D003593:Cytoplasm / Q000378:metabolism; D015536:Down-Regulation; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D010281:Parathyroid Hormone / Q000378:metabolism*; D010449:Peptide Mapping; D010766:Phosphorylation; D051381:Rats; D044168:Receptor, Parathyroid Hormone, Type 1; D018016:Receptors, Parathyroid Hormone / Q000235:genetics / Q000378:metabolism*; D011994:Recombinant Proteins / Q000378:metabolism
D010281:Parathyroid Hormone; D044168:Receptor, Parathyroid Hormone, Type 1; D018016:Receptors, Parathyroid Hormone; D011994:Recombinant Proteins; D003488:Cyanogen Bromide