title: beta-Arrestin-2 interaction and internalization of the human P2Y1 receptor are dependent on C-terminal phosphorylation sites.
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.
authors.affiliation: Department of Pharmacology and Toxicology, University of Wuerzburg, Versbacher Str. 9, 97078 Wuerzburg, Germany.
authors.forename: Susanne
authors.initials: S
authors.lastname: Reiner
authors.forename: Nicole
authors.initials: N
authors.lastname: Ziegler
authors.forename: Catherine
authors.initials: C
authors.lastname: Leon
authors.forename: Kristina
authors.initials: K
authors.lastname: Lorenz
authors.forename: Kathrin
authors.initials: K
authors.lastname: von Hayn
authors.forename: Christian
authors.initials: C
authors.lastname: Gachet
authors.forename: Martin J
authors.initials: MJ
authors.lastname: Lohse
authors.forename: Carsten
authors.initials: C
authors.lastname: Hoffmann
mesh_terms: D019390:Arrestins / Q000378:metabolism / Q000502:physiology*; D002118:Calcium / Q000032:analysis; D017871:Calcium-Calmodulin-Dependent Protein Kinases / Q000502:physiology; D020134:Catalytic Domain / Q000187:drug effects; D002460:Cell Line; D004797:Enzyme-Linked Immunosorbent Assay; D031541:Fluorescence Resonance Energy Transfer; D006801:Humans; D007668:Kidney / Q000737:chemistry / Q000201:enzymology / Q000378:metabolism; D018613:Microscopy, Confocal; D010766:Phosphorylation; D011485:Protein Binding / Q000187:drug effects; D011493:Protein Kinase C / Q000502:physiology; D018048:Receptors, Purinergic P2 / Q000378:metabolism / Q000502:physiology*; D058472:Receptors, Purinergic P2Y1; D000071560:beta-Arrestin 2; D000071557:beta-Arrestins
chemical_list: C000605254:ARRB2 protein, human; D019390:Arrestins; C546382:P2RY1 protein, human; D018048:Receptors, Purinergic P2; D058472:Receptors, Purinergic P2Y1; D000071560:beta-Arrestin 2; D000071557:beta-Arrestins; D011493:Protein Kinase C; D017871:Calcium-Calmodulin-Dependent Protein Kinases; D002118:Calcium