Differential regulation of the uridine nucleotide-activated P2Y4 and P2Y6 receptors. SER-333 and SER-334 in the carboxyl terminus are involved in agonist-dependent phosphorylation desensitization and internalization of the P2Y4 receptor

J Biol Chem. 2001 Apr 13;276(15):11939-48. doi: 10.1074/jbc.M009909200. Epub 2000 Dec 12.

Abstract

Agonist-promoted regulation of the uridine nucleotide-activated human P2Y4 receptor (P2Y4-R) and P2Y6 receptor (P2Y6-R) was studied. Incubation of P2Y4-R-expressing 1321N1 human astrocytoma cells with the cognate agonist UTP resulted in rapid desensitization of the inositol phosphate response and a 50% loss of cell surface receptors. In contrast, incubation of P2Y6-R-expressing cells with the cognate agonist UDP caused neither rapid desensitization nor rapid loss of cell surface receptors. Removal of UTP from the medium of UTP-pretreated cells resulted in rapid and complete recovery of surface P2Y4-R even after 12 h of agonist treatment. Although extended incubation with UDP also caused a loss of surface P2Y6-R, rapid recovery of surface P2Y6-R did not occur following removal of agonist. Pharmacological studies indicated that neither protein kinase C nor other Ca(2+)-activated kinases were involved in agonist-promoted desensitization or loss of surface P2Y4-R or P2Y6-R. Mutational analyses were carried out to identify domains involved in agonist-dependent regulation of P2Y4-R. Sequential truncation of the carboxyl-terminal domain revealed that sequence between amino acids 332 and 343 was necessary for UTP-promoted desensitization and internalization. Further mutational analyses of the three serines in this domain confirmed that Ser-333 and Ser-334 play a major role in these agonist-promoted changes in P2Y4-R. Experiments were carried out with [(32)P]P(i)-labeled cells to ascertain the role of phosphorylation in regulation of P2Y4-R. Incubation with UTP for 2 min caused a marked increase in phosphorylation of both the wild-type P2Y4-R and the P2Y4-343 truncation mutant. In contrast, no UTP-promoted phosphorylation of the P2Y4-332 truncation mutant was observed. Taken together, these results demonstrate differential regulation of uridine nucleotide-activated P2Y4-R and P2Y6-R and indicate that Ser-333 and Ser-334 in the carboxyl terminus of P2Y4-R are important for UTP-dependent phosphorylation, desensitization, and loss of surface receptors.

Publication types

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

MeSH terms

  • Amino Acid Sequence
  • Base Sequence
  • Cell Membrane / metabolism
  • DNA Primers
  • Endocytosis*
  • Humans
  • Molecular Sequence Data
  • Phosphorylation
  • Purinergic P2 Receptor Agonists
  • Receptors, Purinergic P2 / chemistry
  • Receptors, Purinergic P2 / metabolism*
  • Sequence Homology, Amino Acid
  • Serine / metabolism*
  • Tumor Cells, Cultured
  • Uridine Triphosphate / pharmacology*

Substances

  • DNA Primers
  • Purinergic P2 Receptor Agonists
  • Receptors, Purinergic P2
  • purinoceptor P2Y4
  • purinoceptor P2Y6
  • Serine
  • Uridine Triphosphate