Identification of ERK2‐arrestin‐3 interaction sites

Arrestins play a major role in receptor desensitization and internalization by binding active phosphorylated G protein‐coupled receptors (GPCR) and blocking G protein activation. Recent studies show that arrestins also function as scaffolds in mediating the activation of several mitogen‐activated protein kinases (MAPKs) through assembly of a multiprotein complex, and in vitro biochemical data show that arrestin‐2 and arrestin‐3 promote ERK2 activation. However, the mechanism of arrestin‐dependent recruitment of MAPKs into a signaling complex is still largely unknown. The objective of this work has been to characterize the ERK2 binding sites on arrestin‐3 using the site‐directed spin labeling (SDSL) electron paramagnetic resonance (EPR) spectroscopy technique. By attaching a spin label at different sites on arrestin‐3, we were able to monitor the local mobility changes upon ERK2 binding. We also conducted EPR on each arrestin‐3 mutant in the presence of both activated, phosphorylated rhodopsin (model GPCR), and ERK2, based on evidence that receptor‐arrestin complexes can promote ERK2 binding to arrestin‐3. Collectively, our data identify several sites on arrestin‐3 that are involved in ERK2 binding, providing the first mapping of the ERK2 footprint on arrestin‐3. These data are the first demonstration of a direct ERK2‐arrestin interaction within a complex. Supported by NIH GM081756.