PAR2 regulation of PAR4 trafficking

Margaret Cunningham1,2, Kathryn McIntosh1, Graeme Milligan3, Robin Plevin1. 1Strathclyde University, 161 Cathedral Street, G4 0RE, UK, 2Bristol University, Bristol, BS8 1TD, UK, 3Glasgow University, Glasgow, G12 8QQ, UK

Proteinase activated receptor-4 (PAR4) is a class A, G-protein coupled receptor (GPCR), that is activated by means of proteolytic cleavage, by serine proteases such as thrombin and trypsin (Xu et al, 1998). Due to the irreversible nature of activation, newly synthesized pools of receptor are required to be mobilized to the cell surface in order for responsiveness to agonist to be retained (MacFarlane et al, 2001). However, unlike the other PAR family members, the mechanisms responsible for trafficking of PAR4 remain unknown. We therefore investigated the mechanisms responsible for trafficking of PAR4 to the plasma membrane.

Membrane expression of PAR4 is relatively low in most cell types and therefore proves difficult to detect. This study therefore employed techniques to transiently transfect in fluorescent tagged PAR4 constructs to study its trafficking in the keratinocyte cell line NCTC2544, Clone G cells (NCTC2544 stably expressing PAR2) or HEK293s, which have endogenous levels of PAR2. PAR4 trafficking and interaction with proteins was assessed using techniques including, Western blotting, co-immunoprecipitation, Fluorescence energy transfer (FRET), and inositol phosphate assays.

Initial experiments demonstrated that PAR4 was poorly expressed at the plasma membrane and was mainly retained in the endoplasmic reticulum. Immuno-precipitation studies revealed that in PAR2 deficient cells, PAR4 interacted with the COPI protein subunit β-COP1, which functions to retain proteins within the ER. Sequence analysis of PAR4, revealed an arginine-based (RXR) ER retention sequence located within extracellular loop 2, mutation of this sequence (R183AR – A183AA), resulted in enhanced membrane delivery of PAR4. Further experiments revealed that co-expression of PAR4 with either endogenous or transfected PAR2 enhanced plasma membrane expression, and PAR4 was demonstrated to interact with PAR2 through immuno-precipitation and FRET (PAR2/4 = 1.883 ± 0.003), however no FRET signal was detected when the fluorescent tags from the constructs were expressed alone (CFP/YFP = 1.101 ± 0.008). Interaction of PAR2 with PAR4 facilitated the disruption of the β-COP1 – PAR4 complex, allowing interaction instead with the chaperone protein 14-3-3-zeta, and the subsequent anterograde traffic of PAR4 to the plasma membrane. This resulted in enhanced PAR4 signalling, as measured through inositol phosphate accumulation.

This study identifies a novel regulatory role for PAR2 in the anterograde traffic of PAR4. PAR2 was shown to both facilitate and abrogate protein interactions of PAR4, impacting upon both receptor localization and cell signal transduction. These studies will contribute greatly to the understanding of PAR4 receptor pharmacology in both physiological and patho-physiological states.

MacFarlane, SR et al. (2001) Pharmacol Rev 53:245-82

Xu, W et al. (1998) Proc Natl Acad Sci USA, 95:6642-6