Structure-function studies of visual arrestin

Vanderbilt University, Nashville TN

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Abstract

The decrease of cell responsiveness to a persistent stimulus, usually termed desensitization, is a widespread biological phenomenon. Visual amplification cascade (and the signaling by other G protein-coupled receptors) is attenuated by a two-step mechanism: phosphorylation of light-activated rhodopsin (Rh*) by rhodopsin kinase followed by arrestin binding to light-activated phosphorylated rhodopsin (P-Rh*). Arrestin binding terminates transducin-mediated signaling, playing an important role in the recovery of photoreceptor cells. The main objective of this proposal is to elucidate how the fine molecular mechanisms of visual arrestin function translate into its timely binding to rhodopsin, subsequent dissociation from phosphoopsin, its translocation into rod outer segment in the light and its movement to the inner segment in the dark. Using site-directed mutagenesis, direct binding assay, spin labeling of arrestin and rhodopsin followed by EPR spectroscopy, and X-ray crystallography we will identify arrestin and rhodopsin residues participating in their interaction. We will elucidate the number of rhodopsin-attached phosphates necessary for tight arrestin binding and the role of arrestin and rhodopsin dimerization in their interaction. We will use custom-designed arrestin mutants expressed in transgenic mice to study the kinetics of signal shut-off and recovery in rods, the physiological role of arrestin self-association and function of its light-dependent translocation. Several congenital disorders are associated with excessive rhodopsin signaling. We have created phosphorylation-independent "super-arrestins" binding with high affinity to P-Rh* and Rh*, that appear to be logical tools for gene therapy of these disorders. The feasibility of using "super-arrestins" as tools for correcting response kinetics and preventing retinal degeneration in mouse models with the loss of rhodopsin phosphorylation sites or rhodopsin kinase will be also tested.