Agonist-induced internalization of the P2Y2 receptor.
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.
Sromek
S M
SM
Department of Pharmacology and Curriculum in Neurobiology, University of North Carolina School of Medicine, Chapel Hill, North Carolina 27599, USA.
Harden
T K
TK
D001254:Astrocytoma / Q000378:metabolism / Q000648:ultrastructure; D004797:Enzyme-Linked Immunosorbent Assay; D000939:Epitopes / Q000378:metabolism; D019084:Fluorescent Antibody Technique, Indirect; D006388:Hemagglutinins / Q000378:metabolism; D006801:Humans; D007700:Kinetics; D018613:Microscopy, Confocal; D009711:Nucleotides / Q000494:pharmacology; D058910:Purinergic P2 Receptor Agonists; D018048:Receptors, Purinergic P2 / Q000378:metabolism*; D058473:Receptors, Purinergic P2Y2; D014407:Tumor Cells, Cultured
D000939:Epitopes; D006388:Hemagglutinins; D009711:Nucleotides; C546385:P2RY2 protein, human; D058910:Purinergic P2 Receptor Agonists; D018048:Receptors, Purinergic P2; D058473:Receptors, Purinergic P2Y2