Format

Send to

Choose Destination
See comment in PubMed Commons below
Mol Pharmacol. 2009 Dec;76(6):1162-71. doi: 10.1124/mol.109.060467. Epub 2009 Sep 9.

beta-Arrestin-2 interaction and internalization of the human P2Y1 receptor are dependent on C-terminal phosphorylation sites.

Author information

  • 1Department of Pharmacology and Toxicology, University of Wuerzburg, Versbacher Str. 9, 97078 Wuerzburg, Germany.

Abstract

The nucleotide receptor P2Y(1) regulates a variety of physiological processes and is involved in platelet aggregation. Using human P2Y(1)-receptors C-terminally fused with a fluorescent protein, we studied the role of potential receptor phosphorylation sites in receptor internalization and beta-arrestin-2 translocation by means of confocal microscopy. Three receptor constructs were generated that lacked potential phosphorylation sites in the third intracellular loop, the proximal C terminus, or the distal C terminus. The corresponding receptor constructs were expressed in human embryonic kidney (HEK)-293 cells and stimulated with 100 muM ADP. Rapid receptor internalization was observed for the wild-type receptor and from those constructs mutated in the third intracellular loop and the proximal C terminus. However, the construct lacking phosphorylation sites at the distal C terminus did not show receptor internalization upon stimulation. The microscopic data were validated by HA-tagged receptor constructs using a cell surface enzyme-linked immunosorbent assay. P2Y(1)-receptor stimulated beta-arrestin-2-yellow fluorescent protein (YFP) translocation followed the same pattern as receptor internalization. Hence, no beta-arrestin-2-YFP translocation was observed when the distal C-terminal phosphorylation sites were mutated. Individual mutations indicate that residues Ser352 and Thr358 are essential for receptor internalization and beta-arrestin-2-YFP translocation. In contrast, protein kinase C (PKC)-mediated receptor desensitization was not affected by mutation of potential phosphorylation sites in the distal C terminus but was prevented by mutation of potential phosphorylation sites in the proximal C terminus. P2Y(1)-receptor internalization in HEK-293 cells was not blocked by inhibitors of PKC and calmodulin-dependent protein kinase. Thus, we conclude that P2Y(1)-receptor desensitization and internalization are mediated by different phosphorylation sites and kinases.

[PubMed - indexed for MEDLINE]
Free full text
PubMed Commons home

PubMed Commons

0 comments
How to join PubMed Commons

    Supplemental Content

    Full text links

    Icon for HighWire
    Loading ...
    Write to the Help Desk