Agonist-induced internalization of the P2Y2 receptor

The G(q)/phospholipase C-linked human P2Y(2) receptor was tagged at its amino terminus with the hemagglutinin A (HA) epitope sequence (P2Y(2)-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 P2Y(2)-HA receptors using an anti-HA antibody. Incubation of cells with P2Y(2) 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 P2Y(2) receptors was demonstrated directly by prelabeling PXY2-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 similar to 1 hr. Incubation of P2Y(2)-HA receptor-expressing cells with P2Y(2) receptor agonists also resulted in receptor-specific desensitization of nucleotide-promoted inositol phosphate accumulation. This loss of responsiveness occurred move 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 P2Y(2) receptor-specific desensitization. These results illustrate that the P2Y(2) receptor undergoes agonist-promoted movement to an intracellular compartment. This receptor internalization is not required for agonist-induced desensitization.