PROTEINS INTERACTING WITH DISHEVELLED IN VERTEBRATES

社会互动和感觉运动门控在精神分裂症和其他精神疾病中被破坏。最近，通过基因靶向消除disheveled -1基因，在小鼠品系中描述了感觉运动门控和社会互动的缺陷。disheveled -1是一种高度保守的细胞质蛋白的哺乳动物同源物，是Wnt信号通路的一部分，参与几乎所有真核生物的细胞命运决定。这和其他证据表明，disheveled，可能更广泛地说，Wnt通路可能通过影响成人中枢神经系统内细胞的发育或活动来影响复杂的行为。为了理解这是如何发生的，有必要确定哺乳动物脑细胞中与disheveled -1相互作用的蛋白质。目前，与disheveled相互作用的蛋白质仍然完全未知。老鼠虽然在遗传学、神经解剖学和行为学研究中非常容易控制，但不太适合直接进行生化实验。本提案的具体目的是利用酵母双杂交系统和生物化学可处理的青蛙胚胎来鉴定与disheveled相互作用的蛋白质。非洲爪蟾（Xenopus laevis）的disheveled蛋白将被划分为与disheveled -1和其他disheveled同源物高度保守的亚结构域。我们将从爪蟾胚胎文库中分离出与这些亚结构域相互作用的蛋白，并研究它们在爪蟾胚胎中与dishevelded和Wnt通路的相互作用。经确认的disheveld -相互作用物的哺乳动物同源物将通过计算机搜索遗传数据库进行鉴定，并从其他研究人员或通过PCR从遗传文库中获得。这些哺乳动物同源物将被测试与酵母和爪蟾系统中相应的哺乳动物散乱蛋白的相互作用。disheveled -相互作用物的多克隆抗体将被生成并用于表征其在哺乳动物中枢神经系统中的表达和发育分布。利用从非洲爪蟾获得的数据作为指导，将在正常和disheveled -1敲除小鼠的大脑中研究disheveled -1相互作用者的活动、处理和定位的变化，并特别关注大脑区域，如伏隔核、杏仁核和海马体，这些区域在其他研究中显示与感觉运动门控和社会联系有关。通过与华盛顿大学精神病学部门的同事合作，disheveled -interactors的发育表达将在人脑组织中进行表征。

Social interaction and sensorimotor gating are disrupted in Schizophrenia and other psychiatric disorders. Recently, deficits in sensorimotor gating and social interaction were described in a mouse strain created by elimination of the Dishevelled-1 gene through gene targeting. Dishevelled-1 is a mammalian homolog of a highly-conserved cytoplasmic protein that is part of the Wnt signaling pathway, involved in cell-fate determination in virtually all eukaryotes. This and other evidence suggest that Dishevelled, and possibly the Wnt pathway more generally, may affect complex behavior by influencing either the development or the activity of cells within the adult central nervous system. To understand how this happens, it is necessary to identify proteins that interact with Dishevelled-1 in mammalian brain cells. At present, proteins that interact with Dishevelled remain entirely unknown. The mouse, while extremely tractable for genetic, neuroanatomic, and behavioral studies, is less well-suited to direct biochemical experimentation. The specific aims of this proposal are to identify proteins that interact with Dishevelled by taking advantage of the yeast two-hybrid system and the biochemically-tractable frog embryo. The Dishevelled protein from Xenopus laevis (African clawed frog) will be divided into subdomains that are highly conserved with Dishevelled-1 and with other Dishevelled homologs. Proteins that interact with these subdomains will be isolated from a Xenopus embryonic library, and their interactions with Dishevelled and the Wnt pathway studied in the Xenopus embryo. Mammalian homologs of confirmed Dishevelled-interactors will be identified by computerized search of the genetic databases, and obtained either from other investigators or by PCR from genetic libraries. These mammalian homologs will be tested for interaction with the corresponding mammalian Dishevelled proteins in the yeast and Xenopus systems. Polyclonal antibodies to Dishevelled- interactors will be generated and used to characterize their expression and developmental distribution in the mammalian central nervous system. Using the data obtained from Xenopus as a guide, alterations in the activity, processing, and localization of Dishevelled-interactors will be studied in the brains of both normal and Dishevelled-1 knock-out mice, with specific focus on brain regions, such as the nucleus accumbens, amygdala, and hippocampus, shown in other studies to be involved in sensorimotor gating and social affiliation. The developmental expression of Dishevelled-interactors will be characterized in human brain tissue, through collaboration with colleagues within the Department of Psychiatry at the University of Washington.