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Molecular Pharmacology

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Research ArticleArticle

Agonist-Induced Internalization of the P2Y2 Receptor

Susan M. Sromek and T. Kendall Harden
Molecular Pharmacology September 1998, 54 (3) 485-494; DOI: https://doi.org/10.1124/mol.54.3.485
Susan M. Sromek
Department of Pharmacology and Curriculum in Neurobiology, University of North Carolina School of Medicine, Chapel Hill, North Carolina 27599
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T. Kendall Harden
Department of Pharmacology and Curriculum in Neurobiology, University of North Carolina School of Medicine, Chapel Hill, North Carolina 27599
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Abstract

The Gq/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.

Footnotes

    • Received December 19, 1997.
    • Accepted May 13, 1998.
  • Send reprint requests to: T. Kendall Harden, Ph.D., CB# 7365, Department of Pharmacology, School of Medicine, University of North Carolina, Chapel Hill, NC 27599-7365. E-mail: tkh{at}med.unc.edu

  • This research was supported by United States Public Health Service Grants HL34322 and GM38213.

  • The American Society for Pharmacology and Experimental Therapeutics
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Molecular Pharmacology: 54 (3)
Molecular Pharmacology
Vol. 54, Issue 3
1 Sep 1998
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Research ArticleArticle

Agonist-Induced Internalization of the P2Y2 Receptor

Susan M. Sromek and T. Kendall Harden
Molecular Pharmacology September 1, 1998, 54 (3) 485-494; DOI: https://doi.org/10.1124/mol.54.3.485

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Research ArticleArticle

Agonist-Induced Internalization of the P2Y2 Receptor

Susan M. Sromek and T. Kendall Harden
Molecular Pharmacology September 1, 1998, 54 (3) 485-494; DOI: https://doi.org/10.1124/mol.54.3.485
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