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

Cunningham, M. R., McIntosh, K. A., Pediani, J. D. , Robben, J., Cooke, A. E., Nilsson, M., Gould, G. W. , Mundell, S., Milligan, G. and Plevin, R. (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. (doi:10.1074/jbc.M111.315911)

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Publisher's URL: http://dx.doi.org/10.1074/jbc.M111.315911

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 beta-COP1. Analysis of the PAR(4) protein sequence identified an arginine-based (RXR) ER retention sequence located within intracellular loop-2 (R(183)AR -> 3 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 beta-COP1 binding and facilitation of interaction with the chaperone protein 14-3-3 zeta. 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.

Item Type:Articles
Status:Published
Refereed:Yes
Glasgow Author(s) Enlighten ID:Gould, Professor Gwyn and Milligan, Professor Graeme and Robben, Dr Joris and Pediani, Dr John and Plevin, Prof Robin
Authors: Cunningham, M. R., McIntosh, K. A., Pediani, J. D., Robben, J., Cooke, A. E., Nilsson, M., Gould, G. W., Mundell, S., Milligan, G., and Plevin, R.
College/School:College of Medical Veterinary and Life Sciences > Institute of Molecular Cell and Systems Biology
Journal Name:Journal of Biological Chemistry
Journal Abbr.:J Biol Chem.
Publisher:American Society for Biochemistry and Molecular Biology, Inc.
ISSN:0021-9258
ISSN (Online):1083-351X
Published Online:12 March 2012
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Project CodeAward NoProject NamePrincipal InvestigatorFunder's NameFunder RefLead Dept
510631The organisational structure of class A GPCRs: implications for function and drug designGraeme MilliganMedical Research Council (MRC)G0900050RI NEUROSCIENCE & PSYCHOLOGY