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Functional characterization of β‐arrestin 1/ERK scaffolding complex downstream of PAR2
Author(s) -
Min Jungah,
DeFea Kathryn
Publication year - 2010
Publication title -
the faseb journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.709
H-Index - 277
eISSN - 1530-6860
pISSN - 0892-6638
DOI - 10.1096/fasebj.24.1_supplement.586.2
Subject(s) - arrestin , mapk/erk pathway , microbiology and biotechnology , scaffold protein , chemistry , g protein coupled receptor , receptor , protease , hek 293 cells , biology , signal transduction , biochemistry , enzyme
β‐arrestin serves as a multifunctional scaffold protein that brings together MAPK cascade modules and spatially restricts them within the cell. Downstream of protease‐activated‐receptor‐2 (PAR‐2), ERK activity is prolonged by β‐arrestins at the plasma membrane and we hypothesize that this is dependent upon precise molecular interactions that are facilitated by interaction of the ERK module with β‐arrestins. We have shown that Raf binds directly to β‐arrestin 1 in vitro , but MEK1 and ERK2 bind indirectly to via Raf. Using bioluminescence resonance energy transfer (BRET), we have shown direct interaction of Raf with β‐arrestin‐1 in cells upon PAR‐2 activation. Furthermore, using recombinant proteins, we have determined that the C‐terminus of β‐arrestin‐1 directly binds Raf, while the N‐terminus of β‐arrestin binds Raf upon PAR‐2 activation, suggesting that the composition of the β‐arrestin/ERK scaffold is dependent upon the activating receptor. Expression of either N or C‐terminal β‐arrestin truncations partially rescued PAR‐2 stimulated ERK1/2 activation in β‐arrestin knockout cells; expression of both N and C‐terminal truncations together fully rescues PAR‐2 stimulated ERK1/2 activation. Taken together, β‐arrestin 1 recruits the ERK cascade modules via direct binding to Raf and subsequent recruitment of MEK1/2 and ERK1/2 through interactions with sites on both its N and C‐terminus.