β-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 ␤-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 M 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 HAtagged receptor constructs using a cell surface enzyme-linked immunosorbent assay. P2Y 1 -receptor stimulated ␤-arrestin-2-yellow fluorescent protein (YFP) translocation followed the same pattern as receptor internalization. Hence, no ␤-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 ␤-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.The P2Y-receptor family comprises eight different subtypes (P2Y 1 , P2Y 2 , P2Y 4 , P2Y 6 , P2Y 11 , P2Y 12 , P2Y 13 , and P2Y 14 ), which can further be subdivided into two subgroups based on their G-protein-coupling specificity (von Kü gelgen, 2006;Abbracchio et al., 2006). One group consist of the P2Y 1 , P2Y 2 , P2Y 4 , P2Y 6 , and P2Y 11 receptor and couples mainly to G q -proteins, whereas the second group consisting of the P2Y 12 , P2Y 13 , and P2Y 14 receptor mainly couples to G i -proteins. These receptors mediate the action of extracellular nucleotides on cellular signaling. The P2Y 1 receptor is activated by ADP and has been shown to play a major role in the initiation of platelet activation and aggregation (Abbracchio et al., 2006;Gachet, 2006). Besides expression in platelets, the P2Y 1 receptor is expressed in epithelial and endothelial cells and in immune cells and osteoclasts; thus, the receptor offers a diverse therapeutic potential (Jacobson et al., 2002;Abbracchio et al., 2006;Burnstock, 2006). Significant mutagenesis efforts have led to a ...

Desensitization and Internalization of Endothelin Receptor A

“…It was claimed for a long time that receptor phosphorylation is essential for arrestin binding (40). In this context, a mutant of the human P2Y1 receptor lacking Ser-352 and Thr-358 in the distal portion of the C-terminal tail failed to induce the translocation of arrestin and consequently receptor internalization in HEK-293 cells (41). This contrasts with our data suggesting a phosphorylation-independent internalization mechanism of ET A .…”

Differential Signaling of the Endogenous Agonists at the β2-Adrenergic Receptor

“…Various parameters were investigated to assess the interaction of ␤ 2 AR with ␤-arrestin2 (35,47,48): ␤-arrestin2-YFP translocation to the cell surface, FRET between ␤ 2 AR-YFP and ␤-arrestin2-CFP, and ␤ 2 AR internalization. Although generally in agreement with the extent of ␤ 2 AR conformational change, these results were not identical.…”

P2 receptors and platelet function

“…On the one hand, it is thought that the phenomenon of platelet refractoriness to ADP is due to selective desensitization and internalization of the P2Y 1 receptor, while the P2Y 12 receptor remains functional with the ability of ADP to induce amplification of the platelet aggregation induced by other agonists [89][90][91]. Desensitization of the P2Y 1 receptor has been shown to be dependent on receptor C-terminal phosphorylation sites, β-arrestin-2 interaction and protein kinase C (PKC) activity [92,93]. The in vivo consequence is that under conditions of platelets refractory to stimulation by ADP, the P2Y 12 receptor remains functional and able to promote their reactivity at sites of injury, thus preventing loss of haemostatic function.…”