Sorting Nexin 27 (SNX27) regulation of NHERF1 localization in P2Y12 receptor trafficking and platelet function

MR Cunningham, PJ Cullen, SJ Mundell. University of Bristol, Bristol, UK

The PDZ-motif at the extreme cytoplasmic tail of many proteins supports protein interactions essential for their traffic and function (1). In human platelets the integrity of the P2Y12R-PDZ-motif is essential for effective receptor traffic (2) through interactions with the PDZ motif-binding protein NHERF1 (3). The aim of this study was to identify other PDZ binding proteins and functionally characterize their role in relation to P2Y12R function. A GST- P2Y12R/LC-mass spectrometry approach using platelet cell lysates was carried out to identify potential P2Y12R interacting partners. Here we characterize the role of another PDZ domain protein, sorting nexin 27 (SNX27) in P2Y12R activity. Co-immunoprecipitation data revealed basal association between P2Y12R and SNX27 with a loss of interaction observed post-ADP treatment (ADP 10µM, 5 mins, 48.96 ± 13.1% of basal interaction, n=3, p0.0232). In SNX27-suppressed cells, rapid P2Y12R internalization was reduced (ADP 10µM, 5 mins, 88.9 ± 22.1% inhibition of internalization, n=5, p0.0319). This deficit coincided with endocytic retention of NHERF1 (as visualized by IF) and subsequent loss of P2Y12R/NHERF1 interaction. Whilst P2Y12R surface expression remained unchanged in SNX27 siRNA treated cells (95.84 ±8.7% of control, n=5, p0.656), Western blotting revealed elevated protein levels of oligomeric P2Y12R. This increase in P2Y12R oligomer expression in cell lines was accompanied by an increase in receptor sensitivity to ADP (EC50 40.4 ±1.21nM versus 4.43 ± 0.87nM in control versus SNX27 siRNA treated cells, n=4). P2Y12R oligomerisation is important for ADP/P2Y12-mediated signal transduction with ADP action via P2Y receptors pivotal in platelet adhesion and thrombus formation (4). Global knockout of the SNX27 gene in mice is deleterious to postnatal growth and survival (5) therefore endogenous P2Y12R protein expression was explored in SNX27 heterozygous mice (SNX27+/- mice). Immunoblotting using a P2Y12R-specific antibody identified clear dimer (~70k Da) and oligomeric P2Y12R (~250 kDa) protein bands in isolated mouse platelets. Reduced SNX27 levels in SNX27+/- mice resulted in a loss of P2Y12R dimers which coincided with elevated levels of oligomeric P2Y12R, which was consistent with cell line studies. The ability of SNX27+/- mice to form thrombi in vivo was tested using a ferric chloride-induced carotid artery injury model to measure the accumulation of thrombus at the site of injury over time (6). Despite overall end-stage thrombus size being comparable, the size of thrombi formed in SNX27 +/- mice was significantly greater at earlier time points (SNX27+/+ mice 1.57 x106 ± 5.6x105 (AU), SNX27+/- mice 3.5x106 ± 8.8x105 (AU) 5 minutes post-injury, n=10, two-way ANOVA, p<0.01). This data suggests that SNX27+/- mice were able to form a thrombus more quickly following vessel wall injury. In summary, we reveal a novel regulatory role for SNX27 in P2YR function with potential implications in the setting of thrombosis.

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