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This Article Full Text Full Text (PDF) All Versions of this Article: 288/6/C1342    most recent 00315.2004v1 Alert me when this article is cited Alert me if a correction is posted Citation Map Services Email this article to a friend Similar articles in this journal Similar articles in Web of Science Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via HighWire Citing Articles via Web of Science (21) Citing Articles via Google Scholar Google Scholar Articles by Feng, Y.-H. Articles by Gorodeski, G. I. Search for Related Content PubMed PubMed Citation Articles by Feng, Y.-H. Articles by Gorodeski, G. I.

MEMBRANE TRANSPORTERS, ION CHANNELS, AND PUMPS

ATP stimulates GRK-3 phosphorylation and -arrestin-2-dependent internalization of P2X₇ receptor

¹Department of Pharmacology, Uniformed Services University of the Health Sciences, Bethesda, Maryland; and Departments of ²Reproductive Biology, ³Physiology and Biophysics, and ⁴Oncology, Case Western Reserve University School of Medicine, Cleveland, Ohio

Submitted 6 July 2004 ; accepted in final form 26 January 2005

The objective of this study was to understand the mechanisms involved in P2X₇ receptor activation. Treatments with ATP or with the P2X₇ receptor-specific ligand 2',3'-O-(4-benzoylbenzoyl)adenosine 5'-triphosphate (BzATP) induced pore formation, but the effect was slower in CaSki cells expressing endogenous P2X₇ receptor than in human embryonic kidney (HEK)-293 cells expressing exogenous P2X₇ receptor (HEK-293-hP2X₇-R). In both types of cells Western blots revealed expression of three forms of the receptor: the functional 85-kDa form present mainly in the membrane and 65- and 18-kDa forms expressed in both the plasma membrane and the cytosol. Treatments with ATP transiently decreased the 85-kDa form and increased the 18-kDa form in the membrane, suggesting internalization, degradation, and recycling of the receptor. In CaSki cells ATP stimulated phosphorylation of the 85-kDa form on tyrosine and serine residues. Phosphorylation on threonine residues increased with added ATP, and it increased ATP requirements for phosphorylation on tyrosine and serine residues, suggesting a dominant-negative effect. In both CaSki and in HEK-293-hP2X₇-R cells ATP also increased binding of the 85-kDa form to G protein-coupled receptor kinase (GRK)-3, -arrestin-2, and dynamin, and it stimulated -arrestin-2 redistribution into submembranous regions of the cell. These results suggest a novel mechanism for P2X₇ receptor action, whereby activation involves a GRK-3-, -arrestin-2-, and dynamin-dependent internalization of the receptor into clathrin domains, followed in part by receptor degradation as well as receptor recycling into the plasma membrane.

purinergic receptor; recycling; dynamin; clathrin; cervix; epithelium

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