Arrestins were initially identified for their role in homologous desensitization and internalization of G protein–coupled receptors. Receptor-bound arrestins also initiate signaling by interacting with other signaling proteins. Arrestins scaffold MAPK signaling cascades, MAPK kinase kinase (MAP3K), MAPK kinase (MAP2K), and MAPK. In particular, arrestins facilitate ERK1/2 activation by scaffolding ERK1/2 (MAPK), MEK1 (MAP2K), and Raf (MAPK3). However, the structural mechanism underlying this scaffolding remains unknown. Here, we investigated the mechanism of arrestin-2 scaffolding of cRaf, MEK1, and ERK2 using hydrogen/deuterium exchange–mass spectrometry, tryptophan-induced bimane fluorescence quenching, and NMR. We found that basal and active arrestin-2 interacted with cRaf, while only active arrestin-2 interacted with MEK1 and ERK2. The ATP binding status of MEK1 or ERK2 affected arrestin-2 binding; ATP-bound MEK1 interacted with arrestin-2, whereas only empty ERK2 bound arrestin-2. Analysis of the binding interfaces suggested that the relative positions of cRaf, MEK1, and ERK2 on arrestin-2 likely facilitate sequential phosphorylation in the signal transduction cascade.

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