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 February 2, 2015; First 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 1

Processing, complexing, and localization of PC1 and PC2.

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Processing, complexing, and localization of PC1 and PC2. (A) Immunoblot ...
(A) Immunoblot (IB) of endogenous human PC1 and PC2 derived from membrane fractions of a renal cortical tubule epithelial (RCTE) cell line. Samples were untreated (Un) or treated with EndoH (+E) or PNGase F (+P) and detected with an antibody against N-terminal PC1 (7e12; PC1 NT) or PC2 (YCE2). A nonspecific protein (Supplemental Figure 1, A and B) is indicated (n.s.). N-terminal glycoproducts, EndoH resistant (NTR) and EndoH sensitive (NTS), were resolved and were both reduced to the size of the aa backbone with PNGase F treatment (~330 kDa). All of PC2 was sensitive to EndoH. Representative blots are shown from 3 independent experiments. (B) IPs with a PC1 CT (BD3) or PC2 (YCE2) antibody from RCTE cells followed by deglycosylation detected with PC1 NT or YCE2 (PC2). PKD1–/– epithelial cells (9-12 cells; PKD1–/–) and IP with irrelevant antibody (IgG) were used as negative controls. The PC1 and PC2 complex was formed in the ER (EndoH sensitive), since PC2 coimmunoprecipitated all PC1 glycoforms, including PC1-FL, even in high-salt (500 mM NaCl) conditions. Representative blots are shown from 3 independent experiments. (C) Maturation of PC1-NTR was affected by 2 μg/ml swainsonine (+Sw) treatment. A 72-hour swainsonine treatment reduced the PC1-NTR molecular weight but did not affect PC1-NTS, PC1-FL, or PC2, indicating that only PC1, but not PC2, traffics through the Golgi apparatus. Representative blots are shown from 3 independent experiments. (D) Schematic of PC1 cleavage and glycosylation showing the size of the FL and the 2 GPS/GAIN N-terminal cleavage products, NTS and NTR.