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.
Defects in development lead to congenital anomalies of the kidney and urinary tract (CAKUT). At present, little is understood about the cause of these malformations. We are studying the role of a protein called uroplakin 3a, which when mutated results in CAKUT. Using zebrafish larvae, which have a simple urinary tract, and cultured mammalian kidney cells we are studying how uroplakin 3a contributes to urinary tract development by ensuring the proper differentiation of urinary tract-associated epithelial cells.
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