ATP stimulates GRK-3 phosphorylation and beta-arrestin-2-dependent internalization of P2X7 receptor.
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.
Feng
Ying-Hong
YH
Department of Pharmacology, Uniformed Services University of the Health Sciences, Bethesda, Maryland, USA.
Wang
Liqin
L
Wang
Qifang
Q
Li
Xin
X
Zeng
Robin
R
Gorodeski
George I
GI
D000255:Adenosine Triphosphate / Q000502:physiology*; D019390:Arrestins / Q000502:physiology*; D002460:Cell Line; D002462:Cell Membrane / Q000378:metabolism; D002966:Clathrin / Q000502:physiology; D003600:Cytosol / Q000502:physiology; D034281:Dynamins / Q000502:physiology; D054770:G-Protein-Coupled Receptor Kinase 3; D015870:Gene Expression; D006801:Humans; D010766:Phosphorylation; D017346:Protein Serine-Threonine Kinases / Q000378:metabolism*; D018048:Receptors, Purinergic P2 / Q000378:metabolism*; D058486:Receptors, Purinergic P2X7; D000071560:beta-Arrestin 2; D000071557:beta-Arrestins
C000605254:ARRB2 protein, human; D019390:Arrestins; D002966:Clathrin; C546468:P2RX7 protein, human; D018048:Receptors, Purinergic P2; D058486:Receptors, Purinergic P2X7; D000071560:beta-Arrestin 2; D000071557:beta-Arrestins; D000255:Adenosine Triphosphate; D017346:Protein Serine-Threonine Kinases; D054770:G-Protein-Coupled Receptor Kinase 3; C517848:GRK3 protein, human; D034281:Dynamins
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