Arrestin scaffolds NHERF1 to the P2Y12 receptor to regulate receptor internalization

Shaista Nisar, Margaret Cunningham, Robert Pope, Kunal Saxena, Eamonn Kelly, Stuart Mundell. School of Physiology and PharmacologyUniversity of Bristol, University Walk, Bristol, UK

Introduction: ADP plays a key role in regulating platelet function by activation of P2Y1 and P2Y12 G protein-coupled receptors (GPCRs). We have recently shown in a patient with mild bleeding that the PDZ-binding motif of the P2Y12 receptor (P2Y12R) is required for effective receptor traffic in human platelets (Nisar et al., 2011). For other GPCRs this motif is required for efficient receptor traffic via interaction with the PDZ-binding domain containing Na+/H+ Exchanger Regulatory Factors (NHERFs). Here we investigated the role of NHERF1 in P2Y12R regulation.

Methods: HA-tagged P2Y12R expressed in human 1321N1 astrocytoma cells and human platelets were both used in these studies. Protein interactions were investigated by co-immunoprecipitation and immunoblotting. Trafficking was studied using ELISA and confocal microscopy, to quantify and visualise receptor trafficking, respectively.

Results: Endogenous P2Y12R co-localised with NHERF1 in human platelets upon ADP stimulation. In addition the C-tail of the P2Y12R bound NHERF1 in platelet cell lysates. In 1321N1 cells P2Y12R interacted with NHERF1 in an agonist-dependent manner whilst siRNA knockdown of NHERF1 blocked P2Y12R internalisation (loss of surface receptor was 20.5±1.3% versus 5.4 ±1.9% following ADP treatment (10 µM; 30 min) in scrambled and NHERF1 siRNA knockdown cells respectively) but did not affect acute receptor signalling or desensitization. Interestingly removal of the PDZ-binding motif of the P2Y12R which reduces receptor internalization reduced agonist-independent NHERF1 / P2Y12R interaction but did not abolish agonist-dependent NHERF1 interaction. siRNA knockdown of β-arrestins which are also required for P2Y12R internalization (Mundell et al., 2006) did however reduce P2Y12R/NHERF1 interaction. Further studies demonstrated that NHERF1 and β-arrestin are able to interact in cell lines and importantly P2Y12R activation increases the level of this interaction.

Conclusions: We report for the first time a novel interaction between NHERF1 and P2Y12 and between NHERF1 and β-arrestin. This study is the first demonstration that NHERF proteins are required for agonist-dependent GPCR internalisation. Our data allow us to propose the following novel model of P2Y12R internalization. Prior to agonist stimulation there is a basal association between the P2Y12R and NHERF1 that requires the PDZ binding motif of this receptor. On receptor stimulation NHERF1 no longer directly interacts with the receptor but instead binds via β-arrestin where it plays an essential role in regulating receptor internalization. This study therefore suggests that arrestin can serve as an adaptor to promote NHERF1 interaction with a GPCR in order to facilitate effective NHERF1-dependent receptor internalization.

References

Mundell SJ, Luo J, Benovic JL, Conley PB, Poole AW (2006). Distinct clathrin-coated pits sort different G protein-coupled receptor cargo. Traffic. 7(10): 1420-1431.

Nisar S, Daly M, Federici AB, Artoni A, Mumford AD, Watson SP, Mundell SJ (2011) An intact PDZ-motif is essential for correct P2Y12 purinoceptor traffic in human platelets. Blood 118(20):5641-51.