Role of Uroplakins in Urinary Tract Development and CAKUT

描述(由申请人提供)：先天性肾脏和尿路异常(CAKUT)是每500名活产中就有一名发生的发育障碍，但这些畸形的细胞和分子基础尚未揭示。UPK3a是CAKUT的靶基因，编码I型跨膜蛋白UPK3a(UPK3a)。UPK3a胞浆区的Pro残基取代Leu(P273L)导致肾发育不全和其他尿路缺陷。然而，UPK3a在尿路发育中的作用(S)以及该蛋白突变导致CAKUT的原因尚不清楚。以斑马鱼幼体为研究对象，我们发现由于PAR极性复合体蛋白(Par3、Par6、aPKC？)表达的改变，UPK3a基因的表达缺失导致了尿路(即前肾)功能障碍。Ezrin激活和微绒毛形成缺陷。此外，我们最近的遗传和生化研究表明，UPK3a/Upk3l可能通过与aPKC？结合而发挥作用。然后通过促进PKC之间的互动？Ezrin位于原肾小管上皮细胞的顶端。基于这些观察，我们认为尿路发育依赖于UPK3a促进UEEC分化的能力，而UPK3a(例如P273L)的突变扰乱了这一功能，导致CAKUT。在我们的第一个目标中，我们将使用生物化学来进一步定义UPK3a和Upk3l中与PAR复合蛋白结合的细胞质基序，反之亦然。我们还将确定PAR复合体在原肾小管细胞顶膜上的募集是否依赖于与UPK3a/Upk3l的相互作用。此外，我们将在通常不表达UPK3a/Upk3l的MDCK细胞中异位表达UPK3a/Upk3l，并评估这对PAR复合体招募的影响。在第二个目标中，我们将定义Ezrin如何与UPK3a/Upk3l相互作用，以及这种相互作用是否对微绒毛的形成至关重要。我们将进一步 定义是否存在PKC？负责磷酸化Ezrin-T567，这是Ezrin激活的关键步骤。在第三个目的中，我们将使用MDCK细胞作为模型系统来探索P273L突变是否改变了UPK3a的内质网退出和顶面递送。我们还将使用生化和核磁共振光谱来确定UPK3a的C-末端是否包含结构，该结构是否被P273L突变干扰，以及该突变是否影响与其结合伙伴的相互作用。这项拟议的工作非常重要，因为它将为下尿路发育和上皮分化的基本机制、UPK3a在这些过程中的作用以及CAKUT的分子和细胞基础提供新的基本见解。

DESCRIPTION (provided by applicant): Congenital anomalies of the kidney and urinary tract (CAKUT) are developmental disorders that occur in 1 out of every 500 live births, yet the cellular and molecular basis of these malformations has not been revealed. UPK3a, which encodes the type I transmembrane protein uroplakin 3a (UPK3a), is one gene targeted in CAKUT. Substitution of a Pro residue for a Leu (P273L) in the cytoplasmic domain of UPK3a leads to renal adysplasia and other urinary tract defects. However, the function(s) of UPK3a during urinary tract development, and the reason why mutations in this protein lead to CAKUT are not known. Using zebrafish larvae, we find that loss of expression of the UPK3a ortholog Upk3l leads to urinary tract (i.e., pronephros) dysfunction as a result of altered expression of Par polarity complex proteins (Par3, Par6, aPKC?) and defects in ezrin activation and microvilli formation. Moreover, our recent genetic and biochemical studies demonstrate that UPK3a/Upk3l may exert its influence by binding to aPKC? and then by fostering interactions between aPKC? and ezrin at the apical pole of pronephric tubule epithelial cells. Based on these observations, we propose that urinary tract development is dependent on the ability of UPK3a to promote UEEC differentiation, and mutations in UPK3a (e.g., P273L) perturb this function, leading to CAKUT. In our first aim we will use biochemistry to further define the cytoplasmic motifs in UPK3a and Upk3l that bind to the Par complex proteins and vice-versa. We will also define whether recruitment of the Par complex to the apical membrane of pronephric tubule cells depends on interactions with UPK3a/Upk3l. Furthermore, we will ectopically express UPK3a/Upk3l in MDCK cells, which do not normally express these proteins, and assess what impact this has on Par complex recruitment. In the second aim we will define how ezrin interacts with UPK3a/Upk3l and whether this interaction is critical for microvilli formation. We will further define whether aPKC? is responsible for phosphorylating ezrin-T567, a critical step in ezrin activation. In the third aim we will use MDCK cells as a model system to explore whether the P273L mutation alters ER exit and apical surface delivery of UPK3a. We will also use biochemistry and NMR spectroscopy to define whether the C-terminal tail of UPK3a contains structure, whether this structure is perturbed by the P273L mutation, and whether this mutation affects interactions with its binding partners. The proposed work is important because it will provide fundamental new insights into the basic mechanisms of lower urinary tract development and epithelial differentiation, into the role of UPK3a in these processes, and into the molecular and cellular basis of CAKUT.