Molecular mechanisms of arrestin function

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DESCRIPTION (Verbatim from the Applicant's Abstract): The decrease of cell responsiveness to a persistent stimulus, usually termed desensitization, is a widespread biological phenomenon. Signaling by a wide variety of G protein-coupled receptors (GPCRs) is attenuated by a two-step mechanism: receptor phosphorylation by a specific kinase, followed by tight binding of an arrestin protein to activated phosphorylated receptor. Arrestin binding terminates the signaling via G protein, tags receptor for internalization, and in some cases initiates an additional signaling cascade via Src kinase. Internalized receptor is either recycled back to the plasma membrane (resensitization) or transported to lysosomes and degraded (down-regulation). It is well established that arrestins play a key role in desensitization and trafficking of various GPCRs. However, the molecular mechanisms that dictate arrestins' remarkable selectivity toward activated phosphorylated receptors, determine receptor specificity of different arrestin proteins, and regulate arrestins' interaction with a variety of other partners in the cell remain to be elucidated. The objectives of this proposal are to identify the elements of b-arrestin and arrestin3 involved in their interaction with receptors and determine which of these elements dictate arrestins' receptor specificity. We propose to elucidate the mechanism of arrestin transition from its basal inactive state into high-affinity receptor binding state. A combination of mutagenesis, site-directed spin labeling, and X-ray crystallography will be used for this purpose. Various arrestin mutants will be tested in vitro, in cell culture, and in Xenopus oocytes. Arrestin mutants with special functional characteristics will be constructed, such as "constitutively active" arrestins that bind to phosphorylated and unphosphorylated receptors, and arrestins with enhanced specificity for certain receptors. These mutants will be used to study the mechanisms of receptor trafficking in cells. Excessive signaling by certain GPCRs causes a variety of disorders, including several forms of cancer. Arrestin mutants with enhanced specificity for these receptors and enhanced capability to attenuate such faulty signaling promise to become useful tools for gene therapy of these disorders.