%0 Journal Article %1 Reiner2009 %A Reiner, S. %A Ziegler, N. %A Leon, C. %A Lorenz, K. %A von Hayn, K. %A Gachet, C. %A Lohse, M. J. %A Hoffmann, C. %D 2009 %J Mol Pharmacol %K Arrestins/metabolism/*physiology Assay Binding/drug C/physiology Calcium-Calmodulin-Dependent Calcium/analysis Catalytic Cell Confocal Domain/drug Energy Enzyme-Linked Fluorescence Humans Immunosorbent Kidney/chemistry/enzymology/metabolism Kinase Kinases/physiology Line Microscopy, P2/metabolism/*physiology Phosphorylation Protein Purinergic Resonance Transfer effects Receptor %N 6 %P 1162-71 %T beta-Arrestin-2 interaction and internalization of the human P2Y1 receptor are dependent on C-terminal phosphorylation sites %U http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=PubMed&dopt=Citation&list_uids=19741005 %V 76 %X 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.

@article{Reiner2009, 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.}, added-at = {2010-12-14T18:12:02.000+0100}, author = {Reiner, S. and Ziegler, N. and Leon, C. and Lorenz, K. and von Hayn, K. and Gachet, C. and Lohse, M. J. and Hoffmann, C.}, biburl = {https://www.bibsonomy.org/bibtex/2f274a7a2f33e15842b37113920e28e5c/pharmawuerz}, endnotereftype = {Journal Article}, interhash = {df3078a655997cdf0c0c113b2c6ab665}, intrahash = {f274a7a2f33e15842b37113920e28e5c}, issn = {1521-0111 (Electronic) 1521-0111 (Linking)}, journal = {Mol Pharmacol}, keywords = {Arrestins/metabolism/*physiology Assay Binding/drug C/physiology Calcium-Calmodulin-Dependent Calcium/analysis Catalytic Cell Confocal Domain/drug Energy Enzyme-Linked Fluorescence Humans Immunosorbent Kidney/chemistry/enzymology/metabolism Kinase Kinases/physiology Line Microscopy, P2/metabolism/*physiology Phosphorylation Protein Purinergic Resonance Transfer effects Receptor}, month = Dec, note = {Reiner, Susanne Ziegler, Nicole Leon, Catherine Lorenz, Kristina von Hayn, Kathrin Gachet, Christian Lohse, Martin J Hoffmann, Carsten Research Support, Non-U.S. Gov't United States Molecular pharmacology Mol Pharmacol. 2009 Dec;76(6):1162-71. Epub 2009 Sep 9.}, number = 6, pages = {1162-71}, shorttitle = {beta-Arrestin-2 interaction and internalization of the human P2Y1 receptor are dependent on C-terminal phosphorylation sites}, timestamp = {2010-12-14T18:20:08.000+0100}, title = {beta-Arrestin-2 interaction and internalization of the human P2Y1 receptor are dependent on C-terminal phosphorylation sites}, url = {http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=PubMed&dopt=Citation&list_uids=19741005}, volume = 76, year = 2009 }