Abstract
The nucleotide receptor P2Y1 regulates a variety of physiological processes and is involved in platelet aggregation. Using human P2Y1-receptors C-terminally fused with a fluorescent protein, we studied the role of potential receptor phosphorylation sites in receptor internalization and β-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 μM 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. P2Y1-receptor stimulated β-arrestin-2-yellow fluorescent protein (YFP) translocation followed the same pattern as receptor internalization. Hence, no β-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 β-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. P2Y1-receptor internalization in HEK-293 cells was not blocked by inhibitors of PKC and calmodulin-dependent protein kinase. Thus, we conclude that P2Y1-receptor desensitization and internalization are mediated by different phosphorylation sites and kinases.
Footnotes
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This work was supported by the Deutsche Forschungsgemeinschaft-Sonderforschungsbereich “Regulatory Membrane Proteins” [Grant SFB-487 TP-A1].
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Article, publication date, and citation information can be found at http://molpharm.aspetjournals.org.
doi:10.1124/mol.109.060467
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ABBREVIATIONS:
- GPCR
- G-protein-coupled receptor
- 2-MeSADP
- 2-methylthio-adenosine 5′-diphosphate
- CaM
- calmodulin-dependent
- DMEM
- Dulbecco′s modified Eagle′s medium
- FRET
- fluorescence resonance energy transfer
- GFP
- green fluorescent protein
- CFP
- cyan fluorescent protein
- YFP
- yellow fluorescent protein
- eCFP
- enhanced cyan fluorescent protein
- Gö-6850
- {2-[1-(3-dimethylaminopropyl)-1H-indol-3-yl]-3-(1H-indol-3-yl)-maleimide}
- Gö-6976
- [12-(2-cyanoethyl)-6,7,12,13-tetrahydro-13-methyl-5-oxo-5H-indolo(2,3-a), (3,4-c)-carbazole]
- Gö-6983
- {2-[1-(3-dimethylaminopropyl)-5-methoxyindol-3-yl]-3-(1H-indol-3-yl) maleimide}
- GRK
- G protein-coupled receptor kinase
- KN-62
- {1-[N,O-bis-(5-isoquinolinesulfonyl)-N-methyl-l-tyrosyl]-4-phenylpiperazine}
- KN-93
- {2-[N-(2-hydroxyethyl)]-N-(4-methoxybenzenesulfonyl) amino-N-(4-cholocinnamyl)-N-methylbenzylamine)}
- PKC
- protein kinase C
- PMA
- phorbol 12-myristate 13-acetate
- ROI
- region of interest
- PBS
- phosphate-buffered saline
- ANOVA
- analysis of variance
- HEK
- human embryonic kidney
- HA
- hemagglutinin
- ELISA
- enzyme-linked immunosorbent assay.
- Received August 20, 2009.
- Accepted September 9, 2009.
- © 2009 The American Society for Pharmacology and Experimental Therapeutics
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