Novel role for proteinase-activated receptor 2 (PAR2) in membrane trafficking of proteinase-activated receptor 4 (PAR4)

Cunningham, Margaret R. and McIntosh, Kathryn A. and Pediani, John D. and Robben, Joris and Cooke, Alexandra E. and Nilsson, Mary and Gould, Gwyn W. and Mundell, Stuart and Milligan, Graeme and Plevin, Robin (2012) Novel role for proteinase-activated receptor 2 (PAR2) in membrane trafficking of proteinase-activated receptor 4 (PAR4). Journal of Biological Chemistry, 287 (20). pp. 16656-16669. ISSN 1083-351X (https://doi.org/10.1074/jbc.M111.315911)

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Abstract

Proteinase-activated receptors 4 (PAR4) is a class A G protein-coupled receptor (GPCR) recognized through the ability of serine proteases such as thrombin and trypsin to mediate receptor activation. Due to the irreversible nature of activation, a fresh supply of receptor is required to be mobilized to the cell surface for responsiveness to agonist to be sustained. Unlike other PAR subtypes, the mechanisms regulating receptor trafficking of PAR4 remain unknown. Here, we report novel features of the intracellular trafficking of PAR4 to the plasma membrane. PAR 4 was poorly expressed at the plasma membrane and largely retained in the endoplasmic reticulum (ER) in a complex with the COPI protein subunit β-COP1. Analysis of the PAR4 protein sequence identified an arginine-based (RXR) ER retention sequence located within intracellular loop-2 (R183AR →A183AA), mutation of which allowed efficient membrane delivery of PAR4. Interestingly, co-expression with PAR2 facilitated plasma membrane delivery of PAR4, an effect produced through disruption of β-COP1 binding and facilitation of interaction with the chaperone protein 14-3-3ζ. Intermolecular FRET studies confirmed heterodimerization between PAR2 and PAR4. PAR2 also enhanced glycosylation of PAR4 and activation of PAR4 signaling. Our results identify a novel regulatory role for PAR2 in the anterograde traffic of PAR4. PAR2 was shown to both facilitate and abrogate protein interactions with PAR 4, impacting upon receptor localization and cell signal transduction. This work is likely to impact markedly upon the understanding of the receptor pharmacology of PAR4 in normal physiology and disease.

ORCID iDs

Cunningham, Margaret R. ORCID logoORCID: https://orcid.org/0000-0001-6454-8671, McIntosh, Kathryn A. ORCID logoORCID: https://orcid.org/0000-0003-0222-3585, Pediani, John D., Robben, Joris, Cooke, Alexandra E., Nilsson, Mary, Gould, Gwyn W. ORCID logoORCID: https://orcid.org/0000-0001-6571-2875, Mundell, Stuart, Milligan, Graeme and Plevin, Robin ORCID logoORCID: https://orcid.org/0000-0002-7849-1220;
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