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

J Biol Chem. 2012 May 11;287(20):16656-69. doi: 10.1074/jbc.M111.315911. Epub 2012 Mar 12.

Abstract

Proteinase-activated receptors 4 (PAR(4)) 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 PAR(4) remain unknown. Here, we report novel features of the intracellular trafficking of PAR(4) 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 PAR(4) protein sequence identified an arginine-based (RXR) ER retention sequence located within intracellular loop-2 (R(183)AR → A(183)AA), mutation of which allowed efficient membrane delivery of PAR(4). Interestingly, co-expression with PAR(2) facilitated plasma membrane delivery of PAR(4), 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 PAR(2) and PAR(4). PAR(2) also enhanced glycosylation of PAR(4) and activation of PAR(4) signaling. Our results identify a novel regulatory role for PAR(2) in the anterograde traffic of PAR(4). PAR(2) 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 PAR(4) in normal physiology and disease.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • 14-3-3 Proteins / genetics
  • 14-3-3 Proteins / metabolism
  • Cell Membrane / genetics
  • Cell Membrane / metabolism*
  • Endoplasmic Reticulum / genetics
  • Endoplasmic Reticulum / metabolism
  • HEK293 Cells
  • Humans
  • Protein Binding
  • Protein Multimerization / physiology*
  • Protein Sorting Signals / physiology
  • Protein Transport / physiology
  • Receptor, PAR-2 / genetics
  • Receptor, PAR-2 / metabolism*
  • Receptors, Thrombin / genetics
  • Receptors, Thrombin / metabolism*
  • Signal Transduction / physiology*
  • Ubiquitin-Protein Ligases / genetics
  • Ubiquitin-Protein Ligases / metabolism

Substances

  • 14-3-3 Proteins
  • Protein Sorting Signals
  • Receptor, PAR-2
  • Receptors, Thrombin
  • YWHAE protein, human
  • COP1 protein, human
  • Ubiquitin-Protein Ligases
  • protease-activated receptor 4