Direct binding of β‐arrestin‐1 with c‐Raf regulates spatial activity of ERK1/2

β‐arrestins serve as scaffold proteins for activation of MAPK cascades by bringing them within microdomain of the cell and restricting their activities. Our lab showed that downstream of protease activated‐receptor‐2 (PAR‐2), β‐arrestins lead to prolonged activation of ERK1/2 at the membrane. 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 initial recruitment of β‐arrestins with PAR‐2. We have shown that cRaf and ERK2 bind directly to β‐arrestin 1 in vitro , but it appeared that each of MAPK component has different affinities for interaction. Using bioluminescence resonance energy transfer (BRET), we have shown direct interaction of cRaf with β‐arrestin‐1 in cells upon PAR‐2 activation. Using recombinant proteins, we have determined that the N‐ and C‐termini of β‐arrestin‐1 are involved in direct binding to cRaf and ERK2 with different apparent affinities. Furthermore, we have shown that cRaf and ERK1/2 interact with β‐arrestin‐1 within its 100–163 amino acids and 1–99 amino acids respectively and these interactions are enhanced by PAR‐2 activation. In addition to these domains, other domains seemed to be constitutively involved in the interactions, suggesting multiple interacting sites exist within β‐arrestin‐1 and the composition of the β‐arrestin/ERK scaffold is dependent upon the activating receptor. Taken together, β‐arrestin 1 recruits the ERK cascade modules via direct binding to them and probably direct binding of β‐arrestin 1 to cRaf is critical for function of β‐arrestin 1/ERK scaffold.