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.

Figure 8. Loss of PC1-CTF expression in the absence of PC2.

(A) Conditionally immortalized renal epithelial cell lines from Pkd2^fl/fl Pkd1^F/H-BAC (Tg248) mice before and after Pkd2 inactivation in vitro by transient expression of Cre recombinase. Immunostaining showing loss of PC2 (green) after Cre expression with preserved tight junction formation (ZO-1) in both cell lines. Scale bar: 5 μm. (B) Immunoblots using anti-PC2 (YCC2) showing PC2 expression in Pkd2^fl/fl Pkd1^F/H-BAC cells and an absence of PC2 in 2 Pkd2^–/– Pkd1^F/H-BAC cell lines. PC2-HA, cell lysate overexpressing PC2. Cell line names: SF4, Pkd2^fl/fl Pkd1^F/H-BAC; SF3a, SF3b, 2 independent Pkd2^–/– Pkd1^F/H-BAC cell lines. NS, nonspecific band used as a loading control. (C) Markedly reduced level in PC1-CTF in Pkd2^–/–-null cells compared with that in Pkd2^fl/fl cells that still express PC2. PC1-CTF and PC1-FL detected by IP followed by IB with anti-HA. (D, left panel) No change in PC1-NTF levels was detected by anti-FLAG IP and IB in the presence (Pkd2^fl/fl) or absence (Pkd2^–/–) of PC2 expression. Right panel: Same gel reprobed with anti-HA to show the relative migration of PC1-NTF. *, nonspecific band. (E) Immunoblots of control Pkd2^fl/fl Pkd1^F/H-BAC cells (left lane) and Pkd2^–/– Pkd1^F/H-BAC cells reoverexpressing WT PC2-Myc or COOH-terminal truncated PC2^L703-Myc, showing that only full-length PC2 reconstituted PC1-CTF expression levels. Expression of PC2 had no effect on PC1-FL expression. (F) Reoverexpression of the channel-dead pathogenic PC2^D511V missense mutant also reconstituted the expression levels of PC1-CTF in Pkd2^–/– Pkd1^F/H-BAC cells.

Figure 7. Altered trafficking of PC1 in vivo.

(A) Presence of endo H–resistant PC1-CTF in tissues (arrow). PC1 from brain tissue lysates of Pkd1^F/H-BAC (Tg8) mice was subjected to IP using anti-HA and treated by endo H, PNGase F, or incubated with reaction buffer alone (C), followed by IB with anti-HA. (B and C) Distribution of PC1 (B) and PC1^L3040H (C) in density fractions from tissue lysates. Lung tissue lysates from Pkd1^F/H-BAC (Tg8) (B) and Pkd1^L3040H-BAC (Tg7^L3040H) (C) mice were fractionated by density-gradient centrifugation on 5% to 25% linear iodixanol density gradients. Equal volumes of 16 fractions were loaded in each lane: fraction 1 is the top of the gradient; fraction 16 is the bottom. Samples were analyzed by IB with antibodies against FLAG, HA, calnexin, NaK-ATPase, and PC2. Left and right panels represent separate gels. FLAG, HA, calnexin, and NaK-ATPase were detected on the same gels; PC2 was detected on a separate gel, in which fractions 5 and 6 were combined into a single lane. (D) Fractions 1 and 12 from B were run alongside fraction 11 from C and subjected to IB with anti-FLAG to confirm the differential migration of the PC1-NTF bands detected in B and PC1-FL detected in C.

Figure 6. The PC1^L3040H mutation results in loss of function in vivo.

(A) Immunoblots showing expression of PC1^L3040H as uncleaved full-length (FL) protein in lung and kidney from Pkd1^L3040H-BAC transgenic founders Tg7^L3040H and Tg46^L3040H. Pkd1^F/H-BAC (Tg248) shows normal cleavage at the GPS, and the PC1-CTF appears as 2 bands due to alternative splicing. Total tissue lysates were subjected to IP and IB with anti-HA. Panels represent noncontiguous lanes from a single gel. (B) Tissue expression of PC1^L3040H from 2-week-old Pkd1^L3040H-BAC (Tg46^L3040H) transgenic mice. PC1^L3040H was subjected to IP and IB with anti-HA. The 2 left panels represent noncontiguous lanes from a single gel; right panel represents a separate gel. (C) Pkd1^–/– embryos at E16.5 had indistinguishable lethal phenotypes including edema (arrows) with or without the Pkd1^L3040H-BAC transgene, indicating that PC1^L3040H did not reconstitute PC1 function. (D and E) Representative postnatal cystic kidneys showing failure of PC1^L3040H to complement loss of PC1 function. (D) Pkd1^fl/fl Ksp-Cre mice at P15 and (E) Pkd1^fl/fl Pkhd1-Cre mice at P14 without (left) or with (right) Pkd1^L3040H-BAC transgenes Tg46^L3040H and Tg7^L3040H, respectively. Scale bars: 1 mm. (F and G) Expression of PC1^L3040H protein in kidney tissues from (F) Pkd1^fl/fl Ksp-Cre Pkd1^L3040H–BAC (Tg46^L3040H) cystic (Cy) and Pkd1^fl/fl Pkd1^L3040H–BAC noncystic (WT) mice at P5 and from (G) a pair of littermate Pkd1^fl/– Pkhd1-Cre Pkd1^L3040H–BAC (Tg7^L3040H) polycystic mice at P15 showing that expression of the uncleaved protein was present in the cystic kidney tissues despite a failure to rescue the PKD. PC1^L3040H was detected by IP and IB with anti-HA. Panels represent noncontiguous lanes from a single gel.

Figure 5. PC1 expression in Pkd1^F/H-BAC transgenic mouse lines.

(A and B) Membrane-enriched protein fractions prepared from kidneys of 2-week-old mice descended from 4 independent transgenic founders (Tg248, Tg276, Tg8, and Tg14; transgene copy number is in parentheses) analyzed by IB with anti-HA (A) and anti-FLAG (B). WT, nontransgenic littermate; PC1, cell lysate overexpressing PC1. Tubulin served as a loading control. Panels represent noncontiguous lanes on a single gel. (C) Evidence of alternative splicing of PC1 expressed from the mouse Pkd1^F/H-BAC transgene. Kidney tissue lysates from Tg248 underwent IP with anti-HA and were treated with PNGase F or buffer without enzyme (C) prior to IB with anti-HA. Arrows indicate CTF forms with differing migration after PNGase F. (D) Membrane-enriched protein fractions from the indicated organs of a 2-week-old Tg248 mouse underwent IB using anti-HA to show the relative tissue expression of transgenically expressed PC1 (left panel). IP followed by IB of PC1 using anti-HA (right panel) shows detectable levels of PC1 in organs in which IB alone was not sufficiently sensitive. Panels represent noncontiguous lanes on a single gel. (E) Pkd1^F/H-BAC kidney tissue (Pkd1^F/H kidney), MEFs (Pkd1^F/H MEFs), and nontransgenic (WT MEFs) cell lysates subjected to IB with anti-HA show expression and GPS cleavage of PC1 in MEFs. (F) PC1 from the Pkd1^F/H-BAC was expressed in cilia of MEFs from Tg248 mice. Green, anti-HA; red, anti–acetylated α-tubulin. The rightmost panels in F represent a merge of the images at left. Scale bars: 5 μm.

Figure 4. PC2 cilia–trafficking mutations.

(A and B) PC2^W414G retained channel activity comparable to that of WT PC2. (A) Electrophysiological experiments were performed under asymmetrical conditions (500 mM CsCl on the cis side, 250 mM CsCl on the trans side; 7–10 μM Ca²⁺; n = 10). Downward deflections represent channel openings. Dashed line to the right of each current trace represents the open state; solid line represents the closed state. Current traces were obtained at a holding potential of –20 mV, filtered at 400 Hz. (B) Current voltage curves showing current amplitudes for WT PC2 and PC2^W414G obtained at the indicated voltages. Error bars represent the mean ± SEM. (C) Ciliated cells stably overexpressing Myc epitope–tagged PC2^W414G (green); cilia marked by anti–acetylated α-tubulin (red). There was no PC2^W414G expression in cilia (arrows). (D) The channel-dead missense variant PC2^D511V with a Myc epitope tag was expressed in cilia (arrows). (E) Truncated Myc epitope–tagged PC2^L703X showed strong cilia expression, whereas (F) the chimeric mutant PC2^W414G;L703X, introducing the PC2^W414G mutation on the PC2^L703X backbone, completely abrogated cilia location despite stable expression of the protein product. The rightmost panel for each series of images in C–F represents a merge of the images at left. Scale bars: 5 μm.

Figure 3. Functional consequences of human pathogenic variants of PC1.

(A) Missense variants outside of the predicted GAIN/REJ domain (PC1C^210G, PC1^V685D, PC1^G1160S) did not affect GPS cleavage of PC1. The GAIN/REJ mutant PC1^L2811P abrogated GPS cleavage, whereas the benign polymorphism PC1^C2085R 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.

Figure 2. Expression of PC1 in cilia.

(A) PC1 expression detected by the COOH-terminal HA epitope and NH₂-terminal FLAG epitope in cilia (arrows) of LLC-PK₁ cells. Bottom row of panels show triple labeling of a single cell using rabbit anti-FLAG and preconjugated mouse anti–β-tubulin and anti-HA antibodies. Merged images are shown at right. (B) PC1 did not colocalize with basal bodies, marked by γ-tubulin (arrows). (C) PC1^L3040H expressed in LLC-PK₁ cells did not traffic to cilia. Inserts are ×2 magnified reconstructions from image stacks used to verify that cell bodies belonging to the respective cilia and basal bodies were expressing PC1^L3040H. (D) GPS cleavage was normal in the truncation mutants of PC1, as shown by IB with anti-HA (left panel). TPC1^Y4100X-CTF showed no endo-H–resistant species (right panel). The 3 panels were run on separate gels. (E–L) Cilia trafficking of truncated forms of PC1. (E–H) The PC1^R4218X variant orthologous to the PC1^R4228X human mutation and (I and J) the PC1^R4204X truncation placed before the coiled-coil domain were both expressed in cilia. (K and L) PC1^R4100X, truncating the entire COOH terminus, was not expressed in cilia. The COOH terminus of PC1 was detected with anti-HA (E, G, I, K, and L), and the NH₂ terminus was detected with anti-FLAG (F, H, and J). Cilia axonemes were marked by anti–acetylated tubulin (E, F, I, J, and K), and basal bodies were marked by anti–γ-tubulin (G, H, and L). The rightmost panel for each series of images in A–C and E–L represents a merge of the images at left. Scale bars: 5 μm.

Figure 1. N-glycosylation and surface expression of PC1.

(A) LLC-PK₁ cell lysates expressing PC1 with NH₂-terminal triple-FLAG and COOH-terminal triple-HA epitope tags underwent IP with either nonimmune IgG (IgG) or anti-HA (HA) and IB with anti-HA (left) and anti-FLAG (right). The 2 panels are from a single gel and show PC1-NTF (NTF), PC1-CTF (CTF), and uncleaved full-length PC1 (FL). (B) PC1 IP followed by digestion with endo H, PNGase F (PNG F), or reaction buffer (Control). Panels at right are magnifications of the left panel (original magnification, ×2). PC1-CTF shows endo H–resistant (+) and –sensitive (*) products. PC1-FL shows complete endo H sensitivity. (C) IP of PC1 by anti-FLAG coimmunoprecipitated the endo H–sensitive fraction of PC1-CTF, indicative of the intracellular PC1-NTF/PC1-CTF pool. (D) Shedding of PC1-NTF into the culture medium may explain the absence of a detectable cell-surface PC1-NTF/PC1-CTF complex. Medium was either run directly (No IP) or subjected to IP prior to IB. Lysate, total cell lysate. (E) LLC-PK₁ cells grown on semipermeable supports selectively labeled with biotin on the apical or basal surface. Total lysates (left panel) and streptavidin-precipitated biotinylated proteins (right panel) were run on a single gel and analyzed by IB with anti-HA (upper 2 panels) and anti–Na-K-ATPase (lower panel). PC1-CTF was biotinylated on the apical surface. Na-K-ATPase served as a positive control for the basal surface. (F) GPS cleavage–deficient PC1^L3040H was entirely endo H sensitive, suggesting that it did not traffic past the middle Golgi apparatus in cells; the 2 panels show noncontiguous lanes on a single gel.