Journal
AMERICAN JOURNAL OF PHYSIOLOGY-CELL PHYSIOLOGY
Volume 288, Issue 6, Pages C1342-C1356Publisher
AMER PHYSIOLOGICAL SOC
DOI: 10.1152/ajpcell.00315.2004
Keywords
purinergic receptor; recycling; dynamin; clathrin; cervix; epithelium
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Funding
- NHLBI NIH HHS [HL-41618, P01 HL041618] Funding Source: Medline
- NIA NIH HHS [R01 AG015955-09, R01 AG015955, AG-15955] Funding Source: Medline
- NICHD NIH HHS [HD-29924] Funding Source: Medline
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The objective of this study was to understand the mechanisms involved in P2X(7) receptor activation. Treatments with ATP or with the P2X(7) 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 P2X(7) receptor than in human embryonic kidney ( HEK)-293 cells expressing exogenous P2X(7) receptor (HEK-293- hP2X(7)-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- hP2X(7)-R cells ATP also increased binding of the 85-kDa form to G protein-coupled receptor kinase (GRK)-3, beta-arrestin-2, and dynamin, and it stimulated beta-arrestin-2 redistribution into submembranous regions of the cell. These results suggest a novel mechanism for P2X(7) receptor action, whereby activation involves a GRK-3-, beta-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.
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