Contact PD/PI: Shriver, Thomas J. PROJECT SUMMARY Arrestins are an important class of secondary messenger proteins involved in signal transduction at G protein- coupled receptors (GPCRs) throughout the human body. They underpin cascades associated with nociception, planning, mood regulation, anxiety, and depression – all higher order functions misregulated in individuals with addiction disorders. Despite arrestins’ involvement in many clinically important processes, our understanding of their function on a molecular basis still lags behind the “canonical” secondary messengers at GPCRs, G proteins. This is due in large part to the complicated regulation and activity of arrestins, which were initially thought to simply downregulate GPCR signaling through endocytosis. A far more sophisticated view has evolved in recent years, showing that an interplay between extracellular ligand, receptor, local membrane environment, and intracellular kinases (GRKs) all play a role in directing arrestin recruitment. Due to the multicomponent nature of these interactions and their inherently dynamic nature, structural studies revealing the detailed molecular workings of arrestin signaling regulation have been cumbersome. Recent advances in nuclear magnetic resonance (NMR) have enabled high resolution structures of some GPCR/arrestin complexes, but a significant barrier remaining is recapitulating the complex regulation of arrestin recruitment via GRKs. The spatial and temporal resolution of GRK phosphorylation allows these receptors to fine tune their selection of arrestin signaling pathway but also creates an incredibly complicated landscape for structural biologists. This difficulty is lessened if GRKs are not always required for arrestin recruitment, an idea that is finding traction within the GPCR community. Recent studies have shown that the cannabinoid 2 receptor (CB2R) can recruit arrestins in GRK knock-down cell lines and that this recruitment is sensitive to mutations in acidic glutamate and aspartate residues within the receptor C-terminus. Removal of the putative requirement of GRKs will facilitate NMR techniques uniquely capable of delineating the individual molecular components and dynamics of the interactions between GPCRs and arrestins. Such techniques will require advanced statistical treatment which is accessible to me through multiple Northwestern classes. My project seeks to show that CB2R is a suitable target for this simplification strategy and that this methodology is translational in nature, allowing similar approaches at other GPCR targets. I hypothesize that there exists a spectrum of GRK dependence for arrestin recruitment across GPCR phylogeny, enabling a reduction in system complexity for biochemical study through appropriate receptor selection. An understanding of such interactions may eventually allow the development of novel pharmaceuticals designed using dynamics-informed structural approaches.