STRUCTURE/FUNCTION STUDIES OF VISUAL ARRESTIN

描述(改编自申请人的摘要)：本报告的目的 建议阐明确保选择性的分子机制 将arrestin转化为磷酸化的光激活视紫红质 建立arrestin结合的时间和动力学之间的联系 光反应恢复的能力。利用定点突变，氨基酸 Arrestin中与视紫红质以及氨基直接相互作用的残基 参与单个分子内调控相互作用的酸， 都将被确认。在这些数据的基础上， Arrestin分子的三维结构有待定义。 特别是，arrestin的一种突变形式似乎是结构性的 现已制作完成。该突变体与Arrestin高亲和力结合 光激活视紫红质，无论其磷酸化状态如何 视紫红质。它将在转基因小鼠光感受器中表达 Arrestin空背景。对这些小鼠的生理分析将被 以确定光感受器中的限速步骤是否 闪光后的失活就是光激活的失活 视紫质或转导蛋白激活的磷酸二酯酶失活。这个 表达突变的功能后果的表征 体内的抑制素有望为未来使用这种突变体铺平道路。 用于先天性视网膜疾病的基因治疗。

DESCRIPTION (Adapted from applicant's abstract): The objectives of this proposal are to elucidate the molecular mechanism that ensures selectivity of arrestin towards phosphorylated photo-activated rhodopsin and to establish the link between the timing of arrestin binding and the kinetics of photoresponse recovery. Using site-directed mutagenesis, amino acid residues in arrestin that directly interact with rhodopsin, as well as amino acids that participate in individual intramolecular regulatory interactions, are to be identified. On the basis of these data, an approximation of the three-dimensional organization of the arrestin molecule is to be defined. In particular, a mutant form of arrestin that appears to be constitutively active has been produced. This mutant arrestin binds with high affinity to photo-activated rhodopsin regardless of the phosphorylation state of the rhodopsin. It is to be expressed in transgenic mouse photoreceptors in an arrestin null background. Physiological analyses of these mice are to be performed to determine whether the rate-limiting step in photoreceptor deactivation following a light flash is inactivation of photoactivated rhodopsin or inactivation of transducin-activated phosphodiesterase. The characterization of the functional consequences of expressing mutant arrestins in vivo is expected to pave the way for future use of such mutants for gene therapy of congenital retinal disorders.