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© The Rockefeller University Press,
0021-9525/2000//1267 $5.00
The Journal of Cell Biology, Volume 148, Number 6,
, 2000 1267-1282
Original Article |
β-Arrestin–Dependent Endocytosis of Proteinase-Activated Receptor 2 Is Required for Intracellular Targeting of Activated Erk1/2
defea{at}itsa.ucsf.edu
Recently, a requirement for β-arrestin–mediated endocytosis in the activation of extracellular signal–regulated kinases 1 and 2 (ERK1/2) by several G protein–coupled receptors (GPCRs) has been proposed. However, the importance of this requirement for function of ERK1/2 is unknown. We report that agonists of G
q-coupled proteinase–activated receptor 2 (PAR2) stimulate formation of a multiprotein signaling complex, as detected by gel filtration, immunoprecipitation and immunofluorescence. The complex, which contains internalized receptor, β-arrestin, raf-1, and activated ERK, is required for ERK1/2 activation. However, ERK1/2 activity is retained in the cytosol and neither translocates to the nucleus nor causes proliferation. In contrast, a mutant PAR2 (PAR2
ST363/6A), which is unable to interact with β-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 activates ERK1/2 by a PKC-dependent and probably a ras-independent pathway, PAR2(ST363/6A) appears to activate ERK1/2 by a ras-dependent pathway, resulting in increased cell proliferation. Thus, formation of a signaling complex comprising PAR2, β-arrestin, raf-1, and activated ERK1/2 might ensure appropriate subcellular localization of PAR2-mediated ERK activity, and thereby determine the mitogenic potential of receptor agonists.
Key Words: subcellular localization • MAP kinase • cytosol • receptor trafficking
© 2000 The Rockefeller University Press
Abbreviations used in this paper: β2-AR, β2-adrenergic receptor;
, partition coefficient; AP, activating peptide SLIGRL/KV-NH2; EGFP, enhanced green fluorescent protein; EGFR, epidermal growth factor receptor; ERK1/2, extracellular signal–regulated kinase 1 and 2; GPCR, G protein–coupled receptor; MAPK, mitogen-activated protein kinase; MBP, myelin basic protein; PAR2, proteinase-activated receptor; pERK, phosphorylated ERK; PDB, 4-phorbol 12,13-dicanoate; PKC, protein kinase C; PTX, pertussis toxin.
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