We are interested in the structural basis of the function of arrestin proteins. G protein-coupled receptors (GPCRs) are the largest known family of signaling molecules, with ~800-3,200 subtypes in mammalian species. Arrestins bind activated phosphorylated GPCRs, shutting down their signaling via G proteins (desensitization), targeting receptors for internalization, and redirecting the signaling to mitogen-activated protein (MAP) kinases, Src family kinases, ubiquitin ligases, and other G protein-independent pathways.
In collaboration with various crystallographers we solved crystal structures of all four subtypes of vertebrate arrestin proteins in the basal conformation, as well as the structure of the arrestin-rhodopsin complex. We showed that arrestins act as molecular coincidence detectors: their preference for active phosphorylated GPCRs is ensured by the presence of two sensors, the phosphate sensor and the active receptor sensor, both of which must be triggered for arrestin to spring into action and bind GPCR with high affinity. We elucidated the mechanism of action of the arrestin phosphate sensor. This allowed us to construct arrestin mutants that bind the active form of their cognate GPCRs regardless of receptor phosphorylation. These “super-arrestins” may prove useful for gene therapy of disorders associated with excessive signaling by various GPCRs that range from night blindness and retinal degeneration to several forms of cancer. We found that transgenic expression of phosphorylation-independent mutants of visual arrestin prevents retinal degeneration in mouse models with deficits in rhodopsin phosphorylation. We also found that phosphorylation-independent non-visual arrestins dramatically change receptor trafficking in cells and the ultimate fate of the internalized receptor.
Mammals have only four arrestins, two of which are expressed in photoreceptor cells in the retina. Thus the remaining two non-visual arrestins interact with hundreds of different GPCRs. We have identified the residues responsible for the preferential binding of arrestins to particular receptors and we are constructing mutants specific for individual subtypes or small groups of receptors, focusing on disease-causing GPCR mutants. These receptor-specific non-visual arrestins are necessary for therapeutic use of enhanced non-visual arrestins and for rewiring of cell signaling by connecting particular receptors to the signaling pathways we want.
The arrestin-receptor complex often serves as a nucleus of the “signalosome” where receptor-bound arrestin recruits and activates a variety of signaling proteins, including the components of the trafficking machinery, small G proteins and their regulators, ubiquitin ligases, c-Src and related kinases, etc. The complex also facilitates the signaling in MAP kinase cascades acting as a scaffold by directly recruiting MAPKKK and MAPK. We are identifying the interaction sites for the non-receptor binding partners of arrestin proteins with the ultimate goal of constructing arrestins that can selectively couple specific receptors with the signaling pathways of our choosing. These custom-designed arrestins will be useful tools for experimental manipulation of cell signaling and have potential therapeutic value in a variety of disorders associated with faulty signaling due to genetic or acquired defects. In the process we constructed peptide mini-scaffold that facilitates JNK3 activation in the cell without fulfilling any other arrestin functions, as well as dominant-negative arrestin-3 mutant that suppresses JNK signaling in cells. We are testing the ability of JNK-activating arrestin-3-derived peptides to serve as anti-cancer agents, currently using neuroblastoma model.
We are identifying novel arrestin binding partners and elucidating biological role of arrestins in various cellular processes.
We believe that the combination of different approaches ranging from hard-core biochemistry and X-ray crystallography to cell culture and transgenic animals is necessary to answer biologically relevant questions concerning various facets of arrestin function. Structure-function information also paves the way to devising novel therapeutic approaches.