A novel role for NHERF proteins in P2Y1 and P2Y12 receptor internalization

Shaista Nisar, Margaret Cunningham, Robert Pope, Eamonn Kelly, Stuart Mundell. University of Bristol, Physiology and Pharmacology, Bristol BS8 1TD, United Kingdom.

Background: ADP plays a key role in regulating platelet function by activation of P2Y1 and P2Y12 GPCRs. We have recently shown that P2Y receptors rapidly internalize and resensitize in human platelets (Mundell et al.,2008) however the mechanisms underlying these processes are not fully elucidated. Both the P2Y1 and P2Y12 receptors possess a type 1 PDZ ligand in their C terminal tail. For other GPCRs this motif is required for efficient receptor signalling and stabilization via interaction with Na+/H+ Exchanger Regulatory Factors (NHERFs). Here we investigated the role of NHERF1 and 2 in P2Y receptor regulation.

Methods: Human platelets and HA-tagged P2Y1 and P2Y12 receptors expressed in human 1321N1 astrocytoma cells were used in these studies. Protein interactions were investigated by co-immunoprecipitation. ELISA and confocal microscopy were used to quantify and visualise receptor trafficking, respectively.

Results: Endogenous P2Y12 receptor co-localised with NHERF1 in human platelets upon ADP stimulation. In addition the C-tail of the P2Y1 and P2Y12 receptor bound NHERF1 found in platelet lysates. In 1321N1 cells P2Y1 and P2Y12 receptors interacted with NHERF1 in an agonist-dependent manner. siRNA-inhibition of NHERF1 blocked P2Y1 and P2Y12 internalization to ADP (P2Y1: control 25.1±5.2% vs NHERF1 siRNA 7.9±5.4%; P2Y12 control 15.6±2.2% vs NHERF1 siRNA 5.9±5.3%, n = 3) but did not affect acute receptor signalling or desensitization. Surprisingly, deletion of the P2Y12 receptor PDZ ligand did not affect internalization (WT: 17.8±5.6%, PDZΔ 22.3±0.9%, n = 3), which was blocked by NHERF1 siRNA. Furthermore, mutant receptors lacking the PDZ motif retained the ability to interact with NHERF1 in an agonist-dependent manner. β-arrestins are also required for P2Y12 internalization (Mundell et al., 2006). Arrestin-2 was able to interact with NHERF1, suggesting arrestin may recruit NHERF1 to the receptor.

Conclusions: We report an interaction between NHERF1 and P2Y12 and between NHERF1 and β-arrestin. This study is the first demonstration that NHERF proteins are required for GPCR internalization. These data suggest that formation of a super-complex consisting of NHERF1, β-arrestin and P2Y12 is required for agonist-induced internalization. These findings may have important implications for alternative methods of manipulating P2Y12 receptor activity and are now under further investigation in NHERF1 knock-out mice.

Mundell, S. J., J. Luo, et al. (2006). “Distinct clathrin-coated pits sort different G protein-coupled receptor cargo.” Traffic 7(10): 1420-1431.

Mundell, S. J., J. F. Barton, et al. (2008). “Rapid resensitization of purinergic receptor function in human platelets.” J Thromb Haemost 6(8): 1393-1404.