Structure-function studies of visual arrestin

Vanderbilt University, Nashville TN

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Abstract

DESCRIPTION (provided by applicant): The decrease of cell responsiveness to a persistent stimulus, usually termed desensitization, is a widespread biological phenomenon. Visual amplification cascade (and 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 tight binding of arrestin to light-activated phosphorylated rhodopsin (P-Rh*). The crucial role of arrestin binding in signal shut-off is well established. However, many aspects of the molecular mechanisms that govern arrestin-receptor interaction in different types of photoreceptor cells remain to be elucidated. The objectives of this proposal are to elucidate the molecular mechanism responsible for preferential binding of rod arrestin to P-Rh* and the structural basis of arrestin transition into its active high-affinity rhodopsin-binding state. The role of arrestin dimerization in its expression and function in photoreceptors will also be studied in vitro and in vivo with the use of mutants with an enhanced and reduced propensity for self-association. Rods and cones express different arrestin proteins that quench signaling by rhodopsin and cone visual pigments (iodopsins), respectively. The molecular mechanism of cone arrestin activation will be compared to that of a better studied rod arrestin. The elements of rod and cone arrestins responsible for their preference for rhodopsin and iodopsins, respectively, will be identified, and their role in the transition of both arrestin proteins into a high-affinity receptor-binding state will be elucidated. Already constructed and new constitutively active" arrestin mutants that bind with high affinity to both P-Rh* and Rh* will be used to study the kinetics of signal shut-off and recovery in rods. Several congenital vision disorders are associated with excessive rhodopsin signaling in rods. Constitutively active arrestin mutants with an enhanced ability to shut-off this signaling appear to be logical tools for gene therapy of these disorders. The therapeutic potential of these mutants will be tested in models of these disorders, in particular in mice expressing rhodopsin that lacks rhodopsin kinase phosphorylation sites and in rhodopsin kinase knock-out mice, to find out whether the compensatory change in arrestin can normalize their response kinetics and prevent light-dependent retinal degeneration.