UC Riverside

Beta-arrestins are well-known mediators involved in both activation and termination of G-protein-coupled receptor (GPCR) signaling pathways. Beta-arrestins transduce signals by binding to, and either activating or inhibiting, various downstream signaling proteins, leading to a variety of cellular responses, which can occur independent of the heterotrimeric G-protein coupling. Since about 40% of currently used therapeutics target GPCRs, understanding the functions of beta-arrestins is an important biological question. We established a model that shows beta-arrestins mediate the actin cytoskeletal reorganization downstream of protease-activated receptor-2 (PAR-2) by temporally and spatially controlling cofilin activation. Cofilin severs actin filaments and is controlled by opposing actions of LIM kinase (LIMK) (which inactivates it by phosphorylation on Ser3) and cofilin-specific phosphatase, chronophin (CIN) that activates it. My study elucidated how beta-arrestin-1 can interact and regulate LIMK and cofilin in PAR-2-enhanced actin dynamics. First, beta-arrestin-1 bound and inhibited LIMK activity directly. At least two specific binding sites on beta-arrestin-1 to LIMK1 or to cofilin were identified within residues 1 to 99 and 183-418 by using multiple recombinant beta-arrestin-1 truncations and a deletion mutant (residues 146 to 182 were deleted) in sandwich immunoassays. The deletion mutant showed high binding affinity and direct inhibition of LIMK1, which suggested that if beta-arretsin-1 is induced into the appropriate conformation for exposing the amino and carboxyl binding regions, beta-arrestin-1 can directly bind and inhibit LIMK1 activity. Beta-arrestin-1 may protect cofilin from phosphorylation and inactivation, too. Results from spot peptide arrays confirmed the amino region of beta-arrestin-1 was the specific binding site for LIMK1. Co-immunoprecipitation and bioluminescence resonance energy transfer assay further proved the interaction of beta-arrestin-1 and LIMK1 in cells. The absence of beta-arrestins disrupted the cofilin activation and caused abnormal development of growth cones and mature dendritic spines in mouse hippocampus. This dissertation demonstrated that the specific regions in beta-arrestin-1 scaffold and regulate the kinase activity of LIMK1. Thus, beta-arrestin-1 contributes to the spatial regulation of cofilin activation by sequestering cofilin with CIN while inhibiting LIMK at cell protrusions.