Targeting By mLin-7 Binding Partners In Renal Epithelia

描述(改编自申请者摘要)：细胞靶向 表面蛋白到上皮细胞顶端或基底侧的膜 对正常的肾脏功能至关重要。最近的研究表明，一个重要的 蛋白质-蛋白质相互作用结构域在这种极化靶向中的作用 蛋白质。这些工作主要集中在PSD-95IDlg/ZO-1(PDZ)的作用上。 结构域蛋白。在大脑中，PDZ结构域蛋白结合到羧基末端 以及受体，并将这些蛋白质聚集在突触中。PDZ 结构域蛋白可能在细胞靶向方面扮演类似的角色。 上皮细胞中的表面蛋白。在秀丽隐杆线虫中，有三个基因lin-2， LIN-7和LIN-10对于蠕虫的基侧靶向很重要 表皮生长因子受体。我们的团队已经确定了这些PDZ结构域的哺乳动物同源物 蛋白质，在基底外侧检测到稳定的Mlin-2和Mlin-7复合体 肾上皮细胞表面。哺乳动物LIN-7的PDZ结构域可与 甜菜碱γ-氨基丁酸转运蛋白-I的羧基末端 (BGT-L)，并可能在BGT-1保留在 肾上皮细胞的基底外侧表面。然而，确切的机制是 Mlin-7的目标是基地侧面，目前还不清楚。最近的研究表明 与Mlin-2结合可能在这一过程中发挥重要作用，但 与Mlin-7氨基末端结合的其他蛋白质也可能是 很重要。在本提案中，我们将重点介绍其中两个新确定的 Mlin-7结合蛋白，我们称之为PALS 1和PALS 2。就像Mlin-2这些 蛋白质几乎完全由蛋白质-蛋白质相互作用结构域组成 包括PDZ、Src同源物3和具有可变结构的鸟苷酸激酶结构域 氨基末端区域。我们还确定了一个名为L27的新域名 在这些蛋白质中重复的结构域，并介导与 Mlin-7。我们已经演示了Pals 1和Pals2的不同目标 肾上皮细胞，并已开始表征它们与 截然不同的结合伙伴。我们假设这些不同的结合伙伴 控制PALS蛋白的差异靶向并影响其功能 靶向蛋白，如BGT-L。为了检验这些假设，我们将检验 不同蛋白质相互作用结构域在细胞表面的作用 靶向这些蛋白并确定与帕尔西相关的新蛋白 和Pals2。我们还将研究Mlin-7及其结合的重要性 关于BGT-1运输机的目标和活动的合作伙伴。这部作品 将为Mlin-7系统在肾脏中的功能提供新的见解 并增加我们对潜在的基本机制的理解 极化细胞中的蛋白质靶向。

DESCRIPTION (Adapted from the Applicant's Abstract): The targeting of cell surface proteins to apical or basolateral membranes of epithelial cells is crucial for proper renal function. Recent studies have suggested an important role for protein-protein interaction domains in this polarized targeting of proteins. Much of this work has focused on the role of PSD-95IDlg/ZO-1 (PDZ) domain proteins. In the brain, PDZ domain proteins bind to the carboxy-terminus of channels as well as receptors and cluster these proteins in synapses. PDZ domain proteins are likely to play a similar role in the targeting of cell surface proteins in epithelia. In Caenorhabditis elegans, three genes lin-2, lin-7 and lin-10, are important for the basolateral targeting of the worm EGF-Receptor. Our group has identified mammalian homologues of these PDZ domain proteins and detected a stable complex of mLin-2 and mLin-7 at the basolateral surface of renal epithelia. The PDZ domain of mammalian Lin-7 can bind to the carboxy-terminal tail of the betaine gamma amino butyric acid transporter-i (BGT-l) and may have an important role in the retention of BGT-1 at the basolateral surface of renal epithelia. However the exact mechanism that targets mLin-7 to the basolateral surface is unclear. Recent studies indicate that binding to mLin-2 may have an important role in this process but additional proteins that bind to the amino-terminus of mLin-7 may also be important. In this proposal we will focus on two of these newly identified mLin-7 binding proteins that we call Pals 1 and Pals2. Like mLin-2 these proteins are composed almost exclusively of protein-protein interaction domains including PDZ, Src Homology 3 and Guanylate Kinase domains with variable amino-terminal regions. We have also identified a new domain called the L27 domain that is repeated in these proteins and mediates the interaction with mLin-7. We have demonstrated differential targeting of Pals 1 and Pals2 in kidney epithelial cells and have begun to characterize their association with distinct binding partners. We hypothesize that these distinct binding partners control the differential targeting of the Pals proteins and affect the function of targeted proteins such as BGT-l. To test these hypotheses we will examine the role of the different protein-protein interaction domains on cell surface targeting of these proteins and identify new proteins that associate with Palsi and Pals2. We will also examine the importance of mLin-7 and its binding partners on the targeting and activity of the BGT- 1 transporter. This work will allow new insights into the function of the mLin-7 system in renal epithelia and increase our understanding of the basic mechanisms underlying protein targeting in polarized cells.