Altered trafficking and stability of polycystins underlie polycystic kidney disease
Yiqiang Cai, … , Barbara E. Ehrlich, Stefan Somlo
Yiqiang Cai, … , Barbara E. Ehrlich, Stefan Somlo
Published November 3, 2014
Citation Information: J Clin Invest. 2014;124(12):5129-5144. https://doi.org/10.1172/JCI67273.
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Research Article Nephrology

Altered trafficking and stability of polycystins underlie polycystic kidney disease

Abstract

The most severe form of autosomal dominant polycystic kidney disease occurs in patients with mutations in the gene (PKD1) encoding polycystin-1 (PC1). PC1 is a complex polytopic membrane protein expressed in cilia that undergoes autoproteolytic cleavage at a G protein–coupled receptor proteolytic site (GPS). A quarter of PKD1 mutations are missense variants, though it is not clear how these mutations promote disease. Here, we established a cell-based system to evaluate these mutations and determined that GPS cleavage is required for PC1 trafficking to cilia. A common feature among a subset of pathogenic missense mutations is a resulting failure of PC1 to traffic to cilia regardless of GPS cleavage. The application of our system also identified a missense mutation in the gene encoding polycystin-2 (PC2) that prevented this protein from properly trafficking to cilia. Using a Pkd1-BAC recombineering approach, we developed murine models to study the effects of these mutations and confirmed that only the cleaved form of PC1 exits the ER and can rescue the embryonically lethal Pkd1-null mutation. Additionally, steady-state expression levels of the intramembranous COOH-terminal fragment of cleaved PC1 required an intact interaction with PC2. The results of this study demonstrate that PC1 trafficking and expression require GPS cleavage and PC2 interaction, respectively, and provide a framework for functional assays to categorize the effects of missense mutations in polycystins.

Authors

Yiqiang Cai, Sorin V. Fedeles, Ke Dong, Georgia Anyatonwu, Tamehito Onoe, Michihiro Mitobe, Jian-Dong Gao, Dayne Okuhara, Xin Tian, Anna-Rachel Gallagher, Zhangui Tang, Xiaoli Xie, Maria D. Lalioti, Ann-Hwee Lee, Barbara E. Ehrlich, Stefan Somlo

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Figure 3

Functional consequences of human pathogenic variants of PC1.

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Functional consequences of human pathogenic variants of PC1. (A) Missens...
(A) Missense variants outside of the predicted GAIN/REJ domain (PC1C210G, PC1V685D, PC1G1160S) did not affect GPS cleavage of PC1. The GAIN/REJ mutant PC1L2811P abrogated GPS cleavage, whereas the benign polymorphism PC1C2085R had no effect. Cell lysates were subjected to IP and IB with anti-HA. Each panel was run on a separate gel. (B–F) Cilia trafficking of the PC1 variants in A. Pathogenic mutants in the REJ/GAIN domain (B) or NH2 terminus (D and E) of PC1 did not traffic to cilia, whereas a pathogenic mutant in the fifth PKD domain (F) trafficked to cilia similarly to WT protein. (C) The nonpathogenic variant was expressed in cilia. PC1 variants were expressed in LLC-PK1 cells and IF performed using anti–acetylated α-tubulin (red) and anti-FLAG (green) antibodies. Arrows show cilia in x-y images; inserts are 3D reconstructions from z-stacks that have been rotated to show cilia above the plane of the cell bodies and were used to verify that cell bodies belonging to the respective cilia were expressing PC1 variants (green). Original magnification (inserts), ×2. The rightmost panel for each series of images B–F represents a merge of the images at left. Scale bars: 5 μm.