Agonist-induced internalization of the P2Y2 receptor

Mol Pharmacol. 1998 Sep;54(3):485-94. doi: 10.1124/mol.54.3.485.

Abstract

The Gq/phospholipase C-linked human P2Y2 receptor was tagged at its amino terminus with the hemagglutinin A (HA) epitope sequence (P2Y2-HA) and stably expressed in 1321N1 human astrocytoma cells. Neither the pharmacological selectivity nor the signaling properties of the receptor were altered by the presence of the epitope. An enzyme-linked immunosorbent assay was developed to quantify cell surface levels of P2Y2-HA receptors using an anti-HA antibody. Incubation of cells with P2Y2 receptor agonists resulted in a concentration of agonist- and time-dependent decrease in cell surface immunoreactivity. Methodology for indirect immunofluorescence confocal microscopy was developed and applied to demonstrate that the agonist-promoted decreases in cell surface immunoreactivity paralleled increases in intracellular immunoreactivity. Agonist-induced internalization of P2Y2 receptors was demonstrated directly by prelabeling P2Y2-HA receptors with antibody before agonist challenge and then quantifying the movement of receptors from a cell surface to intracellular localization in the presence of agonist. Removal of agonist from the medium resulted in recovery of cell surface immunoreactivity to control levels within approximately 1 hr. Incubation of P2Y2-HA receptor-expressing cells with P2Y2 receptor agonists also resulted in receptor-specific desensitization of nucleotide-promoted inositol phosphate accumulation. This loss of responsiveness occurred more rapidly and to a greater extent than did the agonist-promoted loss of surface receptors. Inhibition of receptor internalization by reduction of temperature to 16 degrees had no effect on the capacity of nucleotides to induce P2Y2 receptor-specific desensitization. These results illustrate that the P2Y2 receptor undergoes agonist-promoted movement to an intracellular compartment. This receptor internalization is not required for agonist-induced desensitization.

Publication types

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

MeSH terms

  • Astrocytoma / metabolism
  • Astrocytoma / ultrastructure
  • Enzyme-Linked Immunosorbent Assay
  • Epitopes / metabolism
  • Fluorescent Antibody Technique, Indirect
  • Hemagglutinins / metabolism
  • Humans
  • Kinetics
  • Microscopy, Confocal
  • Nucleotides / pharmacology
  • Purinergic P2 Receptor Agonists*
  • Receptors, Purinergic P2 / metabolism*
  • Receptors, Purinergic P2Y2
  • Tumor Cells, Cultured

Substances

  • Epitopes
  • Hemagglutinins
  • Nucleotides
  • P2RY2 protein, human
  • Purinergic P2 Receptor Agonists
  • Receptors, Purinergic P2
  • Receptors, Purinergic P2Y2