Proteinase-Activated Receptor-2 and -4 Heterodimerisation

Margaret Rose Cunningham1, John Pediani2, Graeme Milligan2, Robin Plevin1
1Division of Physiology and Pharmacology, University of Strathclyde, Glasgow, G42 0NR, United Kingdom, 2Division of Biochemistry and Molecular Biology, Institute of Biomedical and Life Sciences, University of Glasgow, Glasgow, G12 8QQ, United Kingdom

Proteinase-activated receptors (PARs) are a family of G-protein-coupled receptors (GPCRs), distinguished through their mechanism of activation through irreversible proteolytic cleavage (Macfarlane et al., 2001). So far four family members have been identified, PAR-1 through to PAR-4, with work primarily elucidating downstream signalling events. However, recent evidence has emerged to suggest that members of this family can in fact form homo- and heterodimers (Leger et al., 2006 and McLaughlin et al., 2007). Previous work in our laboratory has observed dual upregulation of PAR-2 and PAR-4 in HUVECs following inflammatory challenge (Ritchie et al., 2007). Therefore, this study sought to identify if PAR-2 and PAR-4 could heterodimerise.

In this study PAR-2 and PAR-4 was C-terminal tagged with EYFP and ECFP respectively and transiently expressed in HEK293 cells. To ensure that receptor function and expression was intact, MAP kinase activation was assessed by Western blotting using phospho-specific antibodies. Receptor interaction was assessed following co-expression of PAR-2 YFP and PAR-4 CFP in HEK293 cells using single cell imaging fluorescence resonance energy transfer (FRET), using a modified ratio method for FRET quantification (RFRET).

When transiently expressed in HEK293 cells, PAR-2 YFP was expressed at the cell surface with punctate intracellular stores. Following stimulation with trypsin (50nM) and SLIGKV-OH (100μM), receptor internalisation and a transient increase (10.3±0.9 fold, n=3) in ERK activity was observed. Transient expression of PAR-4 CFP found that PAR-4 was largely retained inside the cell, with weak expression observed on the plasma membrane. Following stimulation with thrombin (3U/ml) and AYPGKF-NH2 (100μM), ERK1/2 was strongly activated (19.8±0.2 fold, n=3), however no concomitant internalisation of membrane PAR-4 was observed. FRET analysis identified that significant energy transfer was evident when PAR-2 YFP and PAR-4 CFP were co-expressed in HEK293 cells (RFRET value 1.88 ± 0.035, p<0.01, n=24). An RFRET value of 1 was indicative of the absence of energy transfer (i.e. ECFP and EYFP RFRET value 1.1 ± 0.055, p<0.01, n=24). Furthermore PAR-2/4 FRET was more pronounced inside the cell, presumably the ER/Golgi apparatus, with only a weak FRET signal on the membrane.

These initial findings suggest that the fluorescent constructs used were functional and demonstrated for the first time that PAR-2 and PAR-4 may form heterodimers when co-expressed in HEK293 cells. However, further work is required in order to further validate the potential physical interaction and elucidate the pharmacology and functional significance.

Leger A et al., (2006) Circulation 113: 1244-54
Macfarlane SR et al.,(2001) Pharmacol.Rev 53: 245-82
McLaughlin J et al., (2007) PNAS 104: 5662-67
Richie E et al., (2005) Br J Pharmacol 150: 1044-54