β-arrestins were initially identified to desensitize and internalize G-protein-coupled receptors (GPCRs). Receptor-bound β-arrestins also initiate a second wave of signaling by scaffolding mitogen-activated protein kinase (MAPK) signaling components, MAPK kinase kinase, MAPK kinase, and MAPK. In particular, β-arrestins facilitate ERK1/2 or JNK3 activation by scaffolding signal cascade components such as ERK1/2-MEK1-cRaf or JNK3-MKK4/7-ASK1. Understanding the precise molecular and structural mechanisms of β-arrestin-mediated MAPK scaffolding assembly would deepen our understanding of GPCR-mediated MAPK activation and provide clues for the selective regulation of the MAPK signaling cascade for therapeutic purposes. Over the last decade, numerous research groups have attempted to understand the molecular and structural mechanisms of β-arrestin-mediated MAPK scaffolding assembly. Although not providing the complete mechanism, these efforts suggest potential binding interfaces between β-arrestins and MAPK signaling components and the mechanism for MAPK signal amplification by β-arrestin-mediated scaffolding. This review summarizes recent developments of cellular and molecular works on the scaffolding mechanism of β-arrestin for MAPK signaling cascade.

Beta-arrestins are multifunctional proteins that are critically involved in regulating spatio-temporal aspects of GPCR signaling. The interaction of beta-arrestins with GPCRs is typically conceptualized in terms of receptor activation and phosphorylation primarily in the carboxyl-terminus. Interestingly however, there are several GPCRs that harbor majority of phosphorylation sites in their 3rd intracellular loop (ICL3) instead of carboxyl-terminus but still robustly engage beta-arrestins. Moreover, there are several 7TMRs that are now characterized as intrinsically-biased, beta-arrestin-coupled receptors (ACRs) due to lack of functional G-protein-coupling but robust beta-arrestin binding leading to functional outcomes. The molecular basis of beta-arrestin interaction and activation upon binding to these types of 7TMRs is currently elusive, and it represents a major knowledge gap in our current understanding of this signaling system. Here, we present seven cryo-EM structures of beta-arrestins in basal state, activated by the muscarinic M2 receptor (M2R) through its ICL3, and a beta-arrestin-coupled receptor known as decoy D6 receptor (D6R). These structural snapshots combined with biochemical, cellular, and biophysical experiments including HDX-MS and MD simulation provide novel insights into the ability of beta-arrestins to preferentially select specific phosphorylation patterns in the receptors, and also illuminate the structural diversity in 7TMR-beta-arrestin interaction. Surprisingly, we also observe that the carboxyl-terminus of beta-arrestin2 but not beta-arrestin1 undergoes structural transition from a beta-strand to alpha-helix upon activation by D6R, which may preclude the core-interaction with the activated receptor. Taken together, our study elucidates previously unappreciated aspects of 7TMR-beta-arrestin interaction, and provides important mechanistic clues about how the two isoforms of beta-arrestins can recognize and regulate a large repertoire of GPCRs.