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，在通过基因靶向消除Dishevelled-1基因而产生的小鼠品系中描述了感觉运动门控和社会互动的缺陷。 Dishevelled-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。 这一证据和其他证据表明，Dishevelled，以及可能更普遍的Wnt通路，可能通过影响成年中枢神经系统内细胞的发育或活性来影响复杂的行为。 为了理解这是如何发生的，有必要鉴定哺乳动物脑细胞中与Dishevelled-1相互作用的蛋白质。 目前，与Dishevelled相互作用的蛋白质仍然完全未知。 小鼠虽然非常易于遗传、神经解剖和行为研究，但不太适合直接进行生化实验。 本提案的具体目的是确定蛋白质的相互作用与Dishevelled利用酵母双杂交系统和生化温顺的青蛙胚胎。来自非洲爪蟾（非洲爪蛙）的Dishevelled蛋白将被分成与Dishevelled-1和其它Dishevelled同源物高度保守的亚结构域。 与这些亚结构域相互作用的蛋白质将从非洲爪蟾胚胎文库中分离出来，并在非洲爪蟾胚胎中研究它们与Dishevelled和Wnt途径的相互作用。 通过计算机检索遗传数据库，从其他研究者处或通过PCR从遗传文库中获得确认的Dishevelled相互作用物的哺乳动物同源物。将检测这些哺乳动物同系物与酵母和非洲爪蟾系统中相应哺乳动物Dishevelled蛋白的相互作用。 将产生Dishevelled相互作用物的多克隆抗体，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。 使用从非洲爪蟾获得的数据作为指导，在正常和Dishevelled-1基因敲除小鼠的大脑中研究Dishevelled-interactors的活动，加工和定位的变化，特别关注大脑区域，如丘脑核，杏仁核和海马，在其他研究中显示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Dishevelled-interactors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.