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Molecular Mechanism of Beta-Arrestin-dependent ERK activation Downstream of Protease-activated Receptor-2


β-arrestins, originally discovered in the context of G protein-coupled receptor (GPCR) desensitization and internalization, also function in signaling of these receptors independently of G protein coupling. These novel functions involve the roles for β-arrestins as scaffolds. It has been reported that β-arrestins interact with a number of binding partners including trafficking proteins, cytosolic kinases, cytoskeletal proteins, and non-receptor tyrosine kinase. Downstream of protease-activated receptor-2 (PAR-2), β-arrestin scaffolds the components of the ERK cascade, Raf-1, MEK1, and ERK1/2 with the receptor at the plasma membrane, leading to activation of cytoplasmic/membrane ERK1/2 signaling independent of G-protein coupling. Furthermore, we previously demonstrated that stimulation of PAR-2 resulted in prolonged activation of ERK1/2 in pseudopodia in a β-arrestin-dependent manner and β-arrestins were required in PAR-2 mediated ERK1/2 activation at the membrane and cell migration in metastatic tumor cell lines, suggesting β-arrestin-dependent ERK1/2 activation might play a role in cell motility. Although a number of recent studies reported that β-arrestins are required for ERK1/2 activation independently of G protein coupling, molecular mechanism of β-arrestin- mediated ERK1/2 activation via c-Raf has remained unclear. We hypothesized that the ability of β-arrestins to scaffold and prolong MAPK signaling at the membrane, is dependent upon precise molecular interactions that are facilitated by interaction of β-arrestins with PAR-2. To investigate the hypothesis, we determined the sites/domains in β-arrestin-1 that interact with components of the ERK module (c-Raf, MEK1, and ERK1/2) both in vitro and in cells using GST pull down assay, sandwich immunoassay, and co-immunoprecipitation and investigated the role of these identified regions of β-arrestin for interaction and activation of ERK1/2 using truncated mutants of β-arrestin or β-arrestin mutants lacking domains. In addition, a mysterious link in functional mechanism of β-arrestin-mediated ERK1/2 activation is how c-Raf relieves autoinhibition engendering a conformation change from a closed, inactive state to an open, active state, without small GTPase Ras, which is critical for c-Raf activation in the classical G protein dependent ERK activation. We hypothesized that β-arrestins behave similar to Ras for β-arrestin-dependent ERK activation to relieve autoinhibition of c-Raf. We have proved that β-arrestin-1 binds to the regulatory domain of c-Raf where Ras was indentified to bind. Therefore, binding of β-arrestin-1 to c-Raf might ensure formation of scaffolding complex containing β-arrestin-1 and the ERK cascade and play a critical role to activate c-Raf in β-arrestin-dependent ERK1/2 activation.

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