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 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 Galphaq-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, beta-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 (PAR2deltaST363/6A), which is unable to interact with 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 activates ERK1/2 by a PKC-dependent and probably a ras-independent pathway, PAR2(deltaST363/6A) appears to activate ERK1/2 by a ras-dependent pathway, resulting in increased cell proliferation. Thus, formation of a signaling complex comprising PAR2, beta-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.
DeFea
K A
KA
Department of Surgery, University of California, San Francisco, San Francisco, California 94143-0660, USA. defea@itsa.ucsf.edu
Zalevsky
J
J
Thoma
M S
MS
Déry
O
O
Mullins
R D
RD
Bunnett
N W
NW
D000818:Animals; D019390:Arrestins / Q000502:physiology*; D002118:Calcium / Q000378:metabolism; D002455:Cell Division; D002460:Cell Line; D002467:Cell Nucleus / Q000502:physiology / Q000648:ultrastructure; D003600:Cytosol / Q000502:physiology / Q000648:ultrastructure; D004705:Endocytosis; D004789:Enzyme Activation; D006801:Humans; D007700:Kinetics; D018613:Microscopy, Confocal; D019950:Mitogen-Activated Protein Kinase 1 / Q000378:metabolism*; D048052:Mitogen-Activated Protein Kinase 3; D020928:Mitogen-Activated Protein Kinases / Q000378:metabolism*; D008954:Models, Biological; D016296:Mutagenesis; D051381:Rats; D044464:Receptor, PAR-2; D018179:Receptors, Thrombin / Q000235:genetics / Q000502:physiology*; D011994:Recombinant Proteins / Q000378:metabolism; D014162:Transfection; D000071557:beta-Arrestins
D019390:Arrestins; D044464:Receptor, PAR-2; D018179:Receptors, Thrombin; D011994:Recombinant Proteins; D000071557:beta-Arrestins; D019950:Mitogen-Activated Protein Kinase 1; D048052:Mitogen-Activated Protein Kinase 3; D020928:Mitogen-Activated Protein Kinases; D002118:Calcium