PROJECT SUMMARY Arrestins were discovered as negative regulators of G protein-coupled receptor (GPCR) signaling via G proteins. New data show that the free and receptor-bound arrestins initiate signaling through MAP kinases, which regulate cell death, survival, and proliferation. For example, free and receptor-bound arrestin-3 scaffolds ASK1-MKK4/7-JNK1/2/3 cascades, promoting the activation of JNK family kinases. The two non-visual arrestins interact with ~800 different GPCRs in humans. We propose to identify arrestin elements responsible for receptor specificity, and elucidate the structural basis of the assembly of multi-protein signaling complexes (signalosomes) organized by arrestins. Our new crystal structure of arrestin-3 in the presence of an abundant cytoplasmic molecule IP6 revealed receptor-bound-like (active) conformation of arrestin-3. The ability of arrestin-3 to assume this conformation in the absence of GPCRs likely explains receptor-independent scaffolding activity of arrestin-3. We identified arrestin-3 elements critical for JNK and ERK activation, as well as conformational requirements of distinct branches of arrestin-mediated signaling. We constructed arrestin-3 mutants that bind ASK1, MKK and JNK, but do not promote JNK activation and identified short arrestin-3 peptides that enhance or suppress JNK activity in cells. We will test the potential of signaling-biased arrestins and arrestin-derived molecular tools to facilitate cell death or survival. Molecular tools that specifically increase or block pro-apoptotic signaling have therapeutic potential in disorders associated with excessive cell proliferation (e.g., cancer) or death (e.g., neurodegenerative diseases). Using arrestin-3 mutant specific for dopamine D1 receptor we showed that arrestin-mediated signaling from D1 plays a role in the behavioral sensitization to L-DOPA and development of L-DOPA-induced diskinesia in mouse model of Parkinson?s disease, but other GPCRs also contribute to these phenomena. We believe that signaling-biased arrestins and arrestinbased molecular tools with specific functional capability will help elucidate the intricacies of cellular signaling and yield novel potent therapeutic tools.