Phosphorylation of the cytoplasmic tail of the PTH/PTHrP receptor.
Activation of the G protein-coupled receptor for parathyroid hormone (PTH)/PTH-related protein (PTHrP) produces homologous desensitization of receptor signaling. We have shown recently that the opossum PTH/PTHrP receptor stably expressed in human embryonic kidney (HEK) 293 cells is phosphorylated upon agonist binding and upon activation of serine/threonine protein kinases (PKA and PKC), an event which for some G protein-coupled receptors has been linked to desensitization. To locate the sites of phosphorylation, mutated forms of the opossum PTH/PTHrP receptor were stably expressed in HEK 293 cells, and ligand-stimulated receptor phosphorylation was evaluated. The five serine and threonine residues of the third cytoplasmic loop of the receptor were not required for receptor phosphorylation. Basal and ligand-induced phosphorylation were, however, completely abolished upon deletion of all but the 16 juxtamembrane residues of the cytoplasmic C-terminal tail of the receptor, even though this truncated receptor resembled the wild-type receptor in its level of expression based on Western blotting and radioligand binding. To identify further the phosphorylation sites, the 129 amino acid C-terminal tail of the rat PTH/PTHrP receptor was expressed in E. coli as a recombinant glutathione S-transferase fusion protein. Elimination of a single PKA consensus site in the tail (serine 491) resulted in > or = 90% loss of PKA-mediated phosphorylation, identifying this as the preferential site for PKA, with two other sites (serine 473 and/or 475) being minor sites. Phosphorylation by PKC occurred largely in the proximal portion of the tail, whereas beta-adrenergic receptor kinase 1 (beta ARK1) phosphorylated more distally in the tail. The ability of these kinases to phosphorylate the PTH/PTHrP receptor at distinct sites on the cytoplasmic tail may allow differential regulation of receptor signaling and trafficking.
Blind
E
E
Endocrine Unit, Veterans Affairs Medical Center, University of California, San Francisco, USA.
Bambino
T
T
Huang
Z
Z
Bliziotes
M
M
Nissenson
R A
RA
D000595:Amino Acid Sequence; D000818:Animals; D002460:Cell Line; D006801:Humans; D008969:Molecular Sequence Data; D044162:Parathyroid Hormone-Related Protein; D010766:Phosphorylation; D011506:Proteins / Q000378:metabolism*; D051381:Rats; D044168:Receptor, Parathyroid Hormone, Type 1; D018016:Receptors, Parathyroid Hormone / Q000235:genetics / Q000378:metabolism*; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D015398:Signal Transduction
C470121:PTHLH protein, human; D044162:Parathyroid Hormone-Related Protein; D011506:Proteins; D044168:Receptor, Parathyroid Hormone, Type 1; D018016:Receptors, Parathyroid Hormone; D011993:Recombinant Fusion Proteins