PROTEINS INTERACTING WITH DISHEVELLED IN VERTEBRATES

在精神分裂症和其他精神疾病中，社会互动和感觉运动门控被破坏。最近，通过基因打靶消除了杂乱的-1基因，创造了一种小鼠品系，描述了感觉运动门控和社会互动方面的缺陷。Dishevelled1是一种高度保守的细胞质蛋白的哺乳动物同源物，它是Wnt信号通路的一部分，参与了几乎所有真核生物的细胞命运决定。这一证据和其他证据表明，蓬头垢面，更广泛地说，Wnt途径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。为了了解这是如何发生的，有必要确定与哺乳动物脑细胞中的Disheveled-1相互作用的蛋白质。目前，与蓬头垢面相互作用的蛋白质仍然完全未知。小鼠虽然非常容易进行遗传、神经解剖学和行为研究，但不太适合进行直接的生化实验。这项提议的具体目标是通过利用酵母双杂交系统和易于生化处理的青蛙胚胎来识别与Disheveled相互作用的蛋白质。非洲爪蛙的蓬乱蛋白将被分成与Disheveled-1和其他蓬乱的同源物高度保守的亚域。与这些亚区相互作用的蛋白质将从非洲爪哇胚胎文库中分离出来，并在非洲爪哇胚胎中研究它们与Disheveled和Wnt途径的相互作用。确认的乱糟糟的相互作用子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从遗传文库通过聚合酶链式反应获得。这些哺乳动物的同源物将被测试与酵母和非洲爪哇系统中相应的哺乳动物蓬乱的蛋白质的相互作用。杂乱相互作用分子的多克隆抗体将被产生并用于表征它们在哺乳动物中枢神经系统中的表达和发育分布。以非洲爪哇获得的数据为指导，将在正常和蓬乱的-1基因敲除小鼠的大脑中研究杂乱相互作用分子的活动、处理和定位的变化，特别关注大脑区域，如伏隔核、杏仁核和海马体，在其他研究中显示这些区域参与感觉运动门控和社会联系。通过与华盛顿大学精神病学系的同事合作，将在人脑组织中表征蓬头垢面的相互作用分子的发育表达。

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.