beta-Arrestin-dependent endocytosis of proteinase-activated receptor 2 is required for intracellular targeting of activated ERK1/2

Recently, a requirement for beta-arrestin-mediated endocytosis in the activ ation of extracellular signal-regulated kinases 1 and 2 (ERK1/2) by several G protein-coupled receptors (GPCRs) has been proposed. However, the import ance of this requirement for function of ERK1/2 is unknown. We report that agonists of G alpha q-coupled proteinase-activated receptor 2 (PAR2) stimul ate formation of a multiprotein signaling complex, as detected by gel filtr ation, immunoprecipitation and immunofluorescence. The complex, which conta ins internalized receptor, beta-arrestin, raf-1, and activated ERK, is requ ired for ERK1/2 activation. However, ERK1/2 activity is retained in the cyt osol and neither translocates to the nucleus nor causes proliferation. In c ontrast, a mutant PAR2 (PAR2 delta ST363/6A), which is unable to interact w ith beta-arrestin and, thus, does not desensitize or internalize, activates ERK1/2 by a distinct pathway, and fails to promote both complex formation and cytosolic retention of the activated ERK1/2. Whereas wild-type PAR2 act ivates ERK1/2 by a PKC-dependent and probably a ras-independent pathway, PA R2(delta ST363/6A) appears to activate ERK1/2 by a ras-dependent pathway, r esulting in increased cell proliferation. Thus, formation of a signaling co mplex comprising PAR2, beta-arrestin, raf-1, and activated ERK1/2 might ens ure appropriate subcellular localization of PAR2-mediated ERK activity, and thereby determine the mitogenic potential of receptor agonists.