Molecular mechanisms of P2Y1 and P2Y12 receptor trafficking

Margaret Rose Cunningham, Shaista Nisar, Robert Pope, Suart Mundell. University of Bristol, School of Physiology and Pharmacology, BS8 1TD, Bristol, United Kingdom.

P2Y1 and P2Y12 purinergic G-protein coupled receptors (GPCRs) play a critical role in ADP-mediated platelet function. In order to maintain platelet responsiveness to ADP, P2Y receptors are rapidly internalised and recycled following agonist exposure (Mundell et al., 2008). An essential part of this recycling process involves tightly co-ordinated events; the molecular mechanism of which remains to be studied in detail. Various motifs within the intracellular domains of GPCRs have been shown to facilitate in the regulation of receptor trafficking. Interestingly, both P2Y1 and P2Y12 receptors express a type 1 PDZ ligand at their C-terminal extremity. Such internal PDZ ligands have been identified in other GPCRs as critical determinants in receptor signalling and recycling. Here we investigated PDZ-dependent regulation of P2Y receptor trafficking.

The ability of key platelet PDZ proteins, NHERF1, NHERF2 and NHERF3 to bind to the PDZ ligand within P2Y1 and P2Y12 was determined. GST pulldown was carried out using GST fusion proteins of the P2Y receptor C-terminal tail with the PDZ ligand either intact or removed. Protein interaction was confirmed using co-immunoprecipitation in human 1321N1 astrocytoma cells expressing HA-tagged P2Y1 and P2Y12 receptors. Changes in cell surface expression were determined using ELISA.

Exciting data from our laboratory has identified that P2Y1 and P2Y12 both interact with the Na+/H+ exchange regulatory factor (NHERF) isoforms, NHERF1 and NHERF2 but not NHERF3 (Nisar et al., 2010, submitted). NHERF1 was shown to interact with P2Y12 under basal conditions with interaction enhanced following agonist exposure with ADP (10µM, 5 mins). Experiments using NHERF1 siRNA identified that the presence of NHERF1 was essential for agonist-induced P2Y12 receptor internalization (Percentage of total cell surface receptor following 30 mins ADP (10µM) = 88.95 ± 1.98, NHERF1 siRNA = 103.32 ± 3.85). Removal of the PDZ ligand resulted in a loss of NHERF1 binding and a loss of basal interaction with P2Y12 but did not affect P2Y12 receptor internalization (Percentage loss of receptor following 30 mins ADP (10µM) for WT = 17.76 ± 5.6, ΔPDZ = 22.29 ± 0.94). Despite removal of the PDZ ligand, NHERF1 was still able to interact with P2Y12 following agonist exposure. Interaction between NHERF1 and arrestin 2 was observed in our studies. Experiments using arrestin 2 siRNA identified that arrestin 2 was required for agonist-dependent interaction between P2Y12 and NHERF1. This work identifies for the first time an arrestin 2-NHERF1 complex which may be responsible for agonist dependent, PDZ-independent P2Y12 internalization.

Work is currently under way to identify the structural determinants that underlie interaction between NHERF1, β-arrestin and P2Y12. Furthermore the potential for further PDZ proteins to interact with P2Y1 and P2Y12 are being explored to identify associated roles in the trafficking of these purinergic GPCRs.

Mundell et al. 2008 J Thromb Haemost.

Nisar et al., 2010 submitted to JCB