Intracellular trafficking of protease : Activated Receptor 2 (PAR2) by members of sorting nexins family

During the recent years the existing statements that G – protein coupled receptors (GPCRs) are relaying signals only from the plasma membrane have been challenged.  It has become clear that some GPCRs can also signal from intracellular compartments and the nucleus. The role and the function of these nuclear GPCRs are subject of intensive investigations.  Protease - activated receptors (PAR) are members of the GPCR family. PARs are activated by the cleavage of the N – terminus of the receptor followed by binding of the tethered ligand on to the receptor. Four PARs have been described: PAR1, PAR2, PAR3 and PAR4. PAR2 can induce mitogenic effects and participate in processes such as angiogenesis and inflammation. While many of the intracellular effects of PAR2 can be explained with its plasma membrane signalling pathway, intracrine effects of PAR2 have also been proposed. However the mechanisms by which PAR2 can induce its target gene expressions are still unknown.   The purpose of our study was to investigate whether a distinct nuclear population of PAR2 exists. We hypothesized that the roles of PAR2 at different cellular compartments are different since signalling pathways depend on subcellular context. Using confocal microscopy and Western blot techniques we were able to demonstrate the presence of a nuclear population of PAR2. Upon stimulation of the cell membrane PAR2, we observed significant translocation of the receptor from the plasma membrane to the nucleus. Using RT – PCR technique we detected diverse roles of cell surface and nuclear PAR2 on triggered gene expression.  In the current study we have attempted to reveal the mechanisms responsible for PAR2 nuclear translocation. We tested the hypothesis that members of the Sorting Nexin (SNX) family are involved in PAR2 nuclear translocation.  Sorting Nexins are a new, large group of proteins with well established cargo functions. SNX1 and SNX2 have been demonstrated to be responsible for lysosomal sorting of PAR1. SNX11 has not been studied yet, and we hypothesized that it may be another SNX involved in PAR2 signalling.       We developed stable knockdowns for SNX1, SNX2 and SNX11 in HEK293 cells.  Using immunofluorescence, Western Blot analysis and FACS assays, we determined that all three members of SNX group are interaction partners of PAR2. However only SNX11 co-localized with its partner in the nucleus and is responsible for its nuclear translocation. RT – PCR experiments on SNXs knockdowns cell lines demonstrated that PAR2 nucleus function is mostly dependent on SNX11; nevertheless SNX1 and SNX2 knockdowns can also attenuate it, suggesting that they are part of PAR2 signalling network.    In conclusion, PAR2 is being translocated from the plasma membrane to the nuclear membrane after its stimulation with SLIGKV. PAR2 nucleus translocation triggers intracellular effects different from its cell membrane signalling. SNX11 is the major factor responsible for PAR2 nuclear sorting.  Keywords: G – protein coupled receptors, Sorting Nexins, Protease - activated receptors, nuclear translocation, nuclear membrane, nuclear signalling

Keyphrases: Sorting nexins, G – Protein coupled receptors, Protease-activated receptors, Nuclear translocation, Nuclear membrane