Polycystin-1 maturation requires polycystin-2 in a dose-dependent manner
Vladimir G. Gainullin, … , Cynthia J. Hommerding, Peter C. Harris
Vladimir G. Gainullin, … , Cynthia J. Hommerding, Peter C. Harris
Published January 9, 2015
Citation Information: J Clin Invest. 2015;125(2):607-620. https://doi.org/10.1172/JCI76972.
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Research Article

Polycystin-1 maturation requires polycystin-2 in a dose-dependent manner

Abstract

Autosomal dominant polycystic kidney disease (ADPKD) is a common inherited nephropathy responsible for 4%–10% of end-stage renal disease cases. Mutations in the genes encoding polycystin-1 (PC1, PKD1) or polycystin-2 (PC2, PKD2) cause ADPKD, and PKD1 mutations are associated with more severe renal disease. PC1 has been shown to form a complex with PC2, and the severity of PKD1-mediated disease is associated with the level of the mature PC1 glycoform. Here, we demonstrated that PC1 and PC2 first interact in the ER before PC1 cleavage at the GPS/GAIN site and determined that PC2 acts as an essential chaperone for PC1 maturation and surface localization. The chaperone function of PC2 was dependent on the presence of the distal coiled-coil domain and was disrupted by pathogenic missense mutations. In Pkd2–/– mice, complete loss of PC2 prevented PC1 maturation. In Pkd2 heterozygotes, the 50% PC2 reduction resulted in a nonequimolar reduction (20%–25%) of the mature PC1 glycoform. Interbreeding between various Pkd1 and Pkd2 models revealed that animals with reduced levels of functional PC1 and PC2 in the kidney exhibited severe, rapidly progressive disease, illustrating the importance of complexing of these proteins for function. Our results indicate that PC2 regulates PC1 maturation; therefore, mature PC1 levels are a determinant of disease severity in PKD2 as well as PKD1.

Authors

Vladimir G. Gainullin, Katharina Hopp, Christopher J. Ward, Cynthia J. Hommerding, Peter C. Harris

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Figure 8

Deletion of CC2 and missense mutations in PC2 influence PC1 maturation.

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Deletion of CC2 and missense mutations in PC2 influence PC1 maturation. ...
(A) Diagram of WT and mutant human PC2 used in the cotransfection experiments (B and C) showing the positions of domains, deletions, and aa substitutions. (B) mCherry-PC1 cotransfected with WT GFP-PC2 or PC2 lacking the C-terminal cytoplasmic tail (GFP-PC2-L703X) into RCTE cells. Detection of PC1 NT (mCherry) revealed EndoH-resistant PC1-NTR (arrow) only in WT-PC2, but not in PC2-L703X, cotransfected cells. Detection of PC2 (GFP) shows that a portion of PC2-L703X was EndoH resistant (R; arrow). Representative blots are shown from 3 independent experiments. (C) Cotransfection of various GFP-PC2 mutants and mCherry-PC1 into RCTE cells showing the effect of PC2 mutations on the PC1 glycosylation pattern and PC2 products. Deletion of the EF hand and coiled coil 1 (delEF+CC1) did not disrupt PC1 maturation, but deletion of coiled coil 2 (delCC2) greatly reduced the level of the PC1-NTR product. Pathogenic missense substitutions in PC2, especially p.R322Q and p.W414G, also had a negative effect on PC1 maturation. Representative blots are shown from 3 independent experiments. (D) Quantification of the ratio of PC1-NTR to NTS glycoforms obtained from the cotransfection experiments with WT and mutant PC2 GFP constructs (C and Supplemental Figure 5A). Deletion mutants removing CC2 but not EF+CC1 largely disrupted PC1 maturation, while missense mutations also significantly disrupted maturation. n = 3 for all except R322W (n = 2) and D511V (n = 4). Quartile box plots represent the median, quartiles, and minimum/maximum range, with the mean of each group in parentheses. P values are shown as compared with GFP-PC2-WT (control) with the Student’s t test; ****P < 0.0001.