Abstract

The G_q/phospholipase C-linked human P2Y₂receptor 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 P2Y₂receptors 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 ∼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° had no effect on the capacity of nucleotides to induce P2Y₂ receptor-specific desensitization. These results illustrate that the P2Y₂receptor undergoes agonist-promoted movement to an intracellular compartment. This receptor internalization is not required for agonist-induced desensitization.