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

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.