Article

Polycystin-2 Expression Is Regulated by a PC2-binding Domain in the Intracellular Portion of Fibrocystin

Departmentof Medicine, Vanderbilt University, Nashville, Tennessee 37232, USA.
Journal of Biological Chemistry (Impact Factor: 4.57). 11/2008; 283(46):31559-31566. DOI: 10.1074/jbc.M805452200

ABSTRACT

Autosomal dominant (ADPKD) and autosomal recessive (ARPKD) polycystic kidney disease are caused by mutations in Pkd1/Pkd2 and Pkhd1, which encode polycystins (PCs) and fibrocystin/polyductin (FPC). Our recent study reported that a deficiency in FPC increases
the severity of cystic disease in Pkd2 mutants and down-regulates PC2 in vivo, but the precise molecular mechanism of these effects is unknown (Kim, I., Fu, Y., Hui, K., Moeckel, G., Mai, W., Li, C.,
Liang, D., Zhao, P., Ma, J., Chen, X.-Z., George, A. L., Jr., Coffey, R. J., Feng, Z. P., and Wu, G. (2008) J. Am. Soc. Nephrol. 19, 455–468). In this study, through the use of deletion and mutagenesis strategies, we identified a PC2-binding domain in
the intracellular C terminus of FPC and an FPC-binding domain in the intracellular N terminus of PC2. These binding domains
provide a molecular basis for the physical interaction between PC2 and FPC. In addition, we also found that physical interaction
between the binding domains of PC2 and FPC is able to prevent down-regulation of PC2 induced by loss of FPC. In vivo, we generated a mouse model of ADPKD with hypomorphic Pkd2 alleles (Pkd2nf3/nf3) and show that PC2 down-regulation is accompanied by a phenotype similar to that of Pkhd1–/– mice. These findings demonstrate a common mechanism underlying cystogenesis in ADPKD and ARPKD and provide insight into the
molecular relationship between PC2 and FPC.

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    • "Its longest reading frame (ORF) comprises 66 exons, and encodes a large (4074 amino acids) single-pass transmembrane protein fibrocystin/polyductin. Although the exact function of fibrocystin in the formation of the ARPKD phenotype remains unclear, localization of fibrocystin in primary cilia and its interaction with cation channel polycystin-2 suggests its participation in signaling pathways, disruption of which can cause defects in morphogenesis and maintenance of tubular architecture in the organs8910. The severity of the ARPKD phenotype and its formation in the perinatal period result in a strong demand for reliable prenatal diagnosis. "
    [Show abstract] [Hide abstract] ABSTRACT: Background Autosomal recessive polycystic kidney disease (ARPKD) is an early-onset form of polycystic kidney disease that often leads to devastating outcomes for patients. ARPKD is caused by mutations in the PKHD1 gene, an extensive gene that encodes for the ciliary protein fibrocystin/polyductin. Next-generation sequencing is presently the best option for molecular diagnosis of ARPKD. Our aim was to set up the first study of ARPKD patients from the Czech Republic, to determine the composition of their mutations and genotype-phenotype correlations, along with establishment of next-generation sequencing of the PKHD1 gene that could be used for the diagnosis of ARPKD patients. Methods Mutational analysis of the PKHD1 gene was performed in 24 families using the amplicon-based next-generation sequencing (NGS) technique. In patients without 2 causal mutations identified by NGS, subsequent MLPA analysis of the PKHD1 gene was carried out. Results Two underlying mutations were detected in 54 % of families (n = 13), one mutation in 13 % of families (n = 3), and in 33 % of families (n = 8) no mutation could be detected. Overall, seventeen different mutations (5 novel) were detected, including deletion of one exon. The detection rate in our study reached 60 % in the entire cohort of patients; but 90 % in the group of patients who fulfilled all clinical criteria of ARPKD, and 42 % in the group of patients with unknown kidney pathology. The most frequent mutation was T36M, accounting for nearly 21 % of all identified mutations. Conclusions Next-generation sequencing of the PKHD1 gene is a very useful method of molecular diagnosis in patients with a full clinical picture of ARPKD, and it has a high detection rate. Furthermore, its relatively low costs and rapidity allow the molecular genetic analysis of patients without the full clinical criteria of ARPKD, who might also have mutations in the PKHD1 gene.
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    • "Western blot analyses were performed using protocols similar to those described previously [18,22]. Briefly, proteins from cultured cells or tissues were extracted in lysis buffer (0.5% NP-40, 5% Sodium deoxycholate, 50 mM NaCl, 10 mM Tris-HCl (pH 7.5), 1% BSA), homogenized and centrifuged. "
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    • "On the other hand, mice carrying one Pkd1 as well as one Pkd2 mutant allele are viable and show accelerated renal cystic disease compared to the single gene phenotypes [29]. Also hypomorphic mouse models for polycystic kidney disease that express Pkd1 or Pkd2 at a reduced level are viable, although they develop severe PKD [30] [31] [32]. Extra-renal manifestations such as cysts in liver and pancreas [21–23,28,33–35], and aortic dissecting aneurysms have been reported as well [30] [36]. "
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