Phosphorylation of the receptor for PTH and PTHrP is required for internalization and regulates receptor signaling.
We have previously shown that agonist-dependent phosphorylation of the PTH/PTHrP receptor occurs on its carboxyl-terminal tail. Using site-directed mutagenesis, phosphopeptide mapping, and direct sequencing of cyanogen bromide-cleaved fragments of phosphoreceptors, we report here that PTH-dependent phosphorylation occurs on the serine residues at positions 491, 492, 493, 495, 501, and 504, and that the serine residue at position 489 is required for phosphorylation. When these seven sites were mutated to alanine residues, the mutant receptor was no longer phosphorylated after PTH stimulation. The phosphorylation-deficient receptor, stably expressed in LLCPK-1 cells, was impaired in PTH-dependent internalization and showed an increased sensitivity to PTH stimulation; the EC(50) for PTH-stimulated cAMP accumulation was decreased by 7-fold. Furthermore, PTH stimulation of the phosphorylation-deficient PTH/PTHrP receptor caused a sustained elevation in intracellular cAMP levels. These data indicate that agonist-dependent phosphorylation of the PTH/PTHrP receptor plays an important role in receptor function.
Tawfeek
Hesham A W
HA
Endocrine Unit, Massachusetts General Hospital and Harvard Medical School, Boston, Massachusetts 02114, USA.
Qian
Fang
F
Abou-Samra
Abdul B
AB
D000595:Amino Acid Sequence; D000818:Animals; D019556:COS Cells / Q000187:drug effects; D000242:Cyclic AMP / Q000378:metabolism; D008969:Molecular Sequence Data; D009154:Mutation; D010281:Parathyroid Hormone / Q000378:metabolism / Q000494:pharmacology; D044162:Parathyroid Hormone-Related Protein; D010766:Phosphorylation; D011506:Proteins / Q000378:metabolism*; D018016:Receptors, Parathyroid Hormone / Q000235:genetics / Q000378:metabolism*; D015398:Signal Transduction
D010281:Parathyroid Hormone; D044162:Parathyroid Hormone-Related Protein; D011506:Proteins; D018016:Receptors, Parathyroid Hormone; D000242:Cyclic AMP