A novel mutation in the DRY motif of the P2Y12 receptor results in chronic bleeding in a patient

Stuart Mundell1, Kathryn Garner1, Shaista Nisar1, Steve Watson2, Yatin Patel3, Steve Austin4. 1School of Physiology and PharmacologyUniversity of Bristol, University Walk, Bristol, BS8 1TD, UK, 2School of Clinical and Experimental Medicine Institute of Biomedical Research, University of Birmingham, Birmingham B15 2TT, UK, 3Kings College London, Rayne Institute, St. Thomas’ Hospital, London, UK, 4Centre for Haemostasis and Thrombosis, St. Thomas’ Hospital, London, UK

Introduction: Platelets play a central role in the development of arterial thrombosis in heart disease. ADP is regarded as a central mediator of haemostasis and thrombosis mediating its actions through two G protein-coupled receptors (GPCRs), the P2Y1 and P2Y12 receptors (P2Y12R). As part of the Genotyping and Phenotyping of Platelets (GAPP) consortium we have identified a number of mutations in receptor genes that could contribute to bleeding tendency in patients, including mutations in the P2Y12R (Daly et al., 2009 and Nisar et al., 2011). Recently we have identified a patient, with a chronic bleeding disorder expressing a novel missense mutation (R122C) in their P2Y12R. Importantly this mutation is found within the DRY motif of this receptor which in other GPCRs plays a critical role in regulating conformational states. We therefore examined the consequences of this mutation upon P2Y12R function in both the patient’s platelets and cell lines.

Methods: Platelet function was assessed by measuring platelet aggregation responses in platelet rich plasma (PRP) whilst functional responses to the P2Y12R were assessed by measuring VASP phosphorylation. In cell line studies HA-tagged wild type or R122C-P2Y12R were expressed in HEK293, human 1321N1 astrocytoma or CHO cells. Receptor trafficking was studied using ELISA and confocal microscopy, to quantify and visualise receptor trafficking, respectively. Protein interactions were investigated by co-immunoprecipitation and immunoblotting. P2Y12R activity was assessed as previously described (Daly et al., 2009).

Results: Aggregation responses to all doses of ADP (1-20 uM) were abnormal in the R122C patient, with secondary aggregation being consistently absent, whilst ADP-dependent VASP phosphorylation was reduced from 83% in a control sample to 10% in the patient following ADP stimulation demonstrating P2Y12 dysfunction in the patient’s platelets. Initial cell line studies indicated that versus wild type receptor the R122C variant expressed poorly at the cell surface and had significantly compromised ADP responses. Further studies revealed that although the R122C variant could express at the cell surface this mutant displayed a high degree of agonist-independent constitutive internalization versus the wild type. Further study revealed that following its surface expression and internalization the R122C variant unlike the wild type P2Y12R trafficked to lysosomes.

Conclusions: We have identified a novel P2Y12R defect associated with patient bleeding. Further our studies demonstrate that as with other GPCRs the DRY motif of the P2Y12R is critical in maintaining the receptor in a basal non-activated state.

References

Daly ME, Dawood BB, Lester WA, Peake IR, Rodeghiero F, Goodeve AC, Makris M, Wilde JT, Mumford AD, Watson SP and Mundell SJ (2009) Identification and characterisation of a novel P2Y12 variant in a patient diagnosed with type 1 von Willebrand disease in the European MCMDM-1VWD study. Blood 113, 4110-3.

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.