Polycystin-1, the PKD1 gene product, is in a complex containing E-cadherin and the catenins

Abstract

Autosomal dominant polycystic kidney disease (ADPKD) is a common human genetic disease characterized by cyst formation in kidney tubules and other ductular epithelia. Cells lining the cysts have abnormalities in cell proliferation and cell polarity. The majority of ADPKD cases are caused by mutations in the PKD1 gene, which codes for polycystin-1, a large integral membrane protein of unknown function that is expressed on the plasma membrane of renal tubular epithelial cells in fetal kidneys. Because signaling from cell-cell and cell-matrix adhesion complexes regulates cell proliferation and polarity, we speculated that polycystin-1 might interact with these complexes. We show here that polycystin-1 colocalized with the cell adhesion molecules E-cadherin and alpha-, beta-, and gamma-catenin. Polycystin-1 coprecipitated with these proteins and comigrated with them on sucrose density gradients, but it did not colocalize, coprecipitate, or comigrate with focal adhesion kinase, a component of the focal adhesion. We conclude that polycystin-1 is in a complex containing E-cadherin and alpha-, beta-, and gamma-catenin. These observations raise the question of whether the defects in cell proliferation and cell polarity observed in ADPKD are mediated by E-cadherin or the catenins.

Figure 1

Confocal images of sections of human fetal kidney subjected to double-stain indirect immunofluorescence with antibodies to polycystin-1 (red) and to junctional proteins (green). Each horizontal series shows, from left to right, the green channel (junctional protein), the red channel (polycystin-1), and the total fluorescence (dual-scanned image). Colocalization is seen in the right-hand panels as yellow staining. (a-c) Double staining for polycystin-1 and E-cadherin, where (a) is E-cadherin, (b) is polycystin-1, and (c) is the dual-scanned image. (d-f) Double staining for α-catenin, where (d) is α-catenin, (e) is polycystin-1, and (f) is the dual-scanned image. (g-i) Double staining for β-catenin, where (g) is β-catenin, (h) is polycystin-1, and (i) is the dual-scanned image. (j-l) Double staining for γ-catenin, where (j) is γ-catenin, (k) is polycystin-1, and (l) is the dual-scanned image. In all these images, polycystin-1 and E-cadherin or the catenins are colocalized (yellow) at the apical and lateral aspects of the epithelial cells. There are areas where distinct green or red staining can be seen, showing that not all of the polycystin-1 colocalizes with the junctional proteins. These images were acquired with the ×100 lens. (m-o) Confocal images of antibodies to polycystin-1 (red) and β-catenin (green) incubated in the presence of the peptide against which the polycystin-1 antibody was raised. The peptide blocked polycystin-1 staining (n) but did not affect β-catenin staining (m, o). Some precipitated anti-rabbit antibody is seen as red dots (n). These images were acquired with the ×40 lens.

Figure 2

Expression of polycystin-1 in HPAC cells. (a) RT-PCR with PKD1-specific primers yielded the predicted 564-bp product (PKD1, – RNase). RT-PCR with human β-actin primers from 2 exons yielded the mRNA-specific product of 661 bp (actin, – RNase). The sizes of the markers on the left are 1,000, 700, 500, and 300 bp. Treatment of the RNA template with RNase A before RT-PCR eliminated both the actin and the PKD1 products (+ RNase). (b) Polycystin-1 was precipitated from [³⁵S]methionine/cysteine–labeled HPAC cells as described, displayed on a 4–10% linear gradient gel, and viewed with a PhosphorImager analysis system (Molecular Dynamics). IM + peptide: precipitation with immune serum in the presence of the immunizing peptide. IM: precipitation with immune serum. PI: precipitation with preimmune serum. Note that the polycystin polypeptide of approximately 500 kDa is precipitated only by immune serum and only in the absence of peptide.

Figure 3

Coimmunoprecipitation of polycystin-1 with junctional complex proteins. (a) Immunoprecipitation of HPAC cell lysates with affinity-purified antibody to polycystin-1. Each panel is an immunoblot probed with antibody to the protein named at the top of the panel. In each panel, the left lane is an aliquot of the cell lysate (lysate). The middle and right lanes were immunoprecipitated with affinity-purified antibody against polycystin-1 in the absence (–) or presence (+) of the immunizing peptide. Each set of samples was probed with antibody to E-cadherin, α-catenin, β-catenin, γ-catenin, β₁-integrin, or FAK. Note that E-cadherin and the catenins coprecipitated with polycystin-1 only in the absence of competing peptide. The doublet of 120 kDa and 140 kDa in the lysate probed with antibody to β₁-integrin represents reduced (140 kDa) and unreduced (120 kDa) β₁-integrin. Results are representative of at least 5 independent experiments. (b) Double immunoprecipitation of metabolically labeled HPAC lysates with antibodies to E-cadherin or β-catenin (first immunoprecipitation) followed by affinity-purified antibody to polycystin-1 (second immunoprecipitation). The left pair of lanes shows the first immunoprecipitation with monoclonal antibody to β-catenin (β-cat) or E-cadherin (E-cad). Note the high-molecular-weight polypeptide that is precipitated as part of the complex. The center pair of lanes shows the reimmunoprecipitation of the complex with affinity-purified antibody to polycystin-1. The high-molecular-weight polypeptide seen in the left panels was reprecipitated by polycystin antibody and is therefore polycystin-1. Note that some of the E-cadherin/catenin complex remains associated with polycystin-1 (arrows). The right pair of lanes shows control experiments performed on aliquots of the same HPAC lysate. The lane marked “PKD1” was precipitated with affinity-purified antibody to polycystin-1. The lane marked “IgG” was precipitated with the same concentration of nonimmune rabbit anti-mouse IgG. This figure is a single gel. The results are representative of 3 independent experiments.

Figure 4

Sucrose density gradient centrifugation of HPAC cell lysates. Top: Distribution of polycystin-1, β-catenin, and actin within the gradient. Polycystin-1 was detected by immunoprecipitation and PhosphorImager analysis (Molecular Dynamics). Immunoblotting was used to detect β-catenin and actin. Results are representative of 3 independent experiments. Bottom: Distribution of β-catenin, FAK, and complexes of β-catenin and polycystin-1 within gradient. The distributions of β-catenin and FAK were determined by immunoblotting. The distributions of complexes containing polycystin-1 and either β-catenin or FAK were determined by immunoprecipitating each fraction of the gradient with affinity-purified antibodies to polycystin-1 and probing immunoblots of the precipitates with antibodies to β-catenin (IP: polycystin-1, blot: β-catenin) or FAK (IP: polycystin-1, blot: FAK). No FAK was coprecipitated with polycystin-1. Results are representative of 3 independent experiments.

Figure 5

Confocal images of sections of human fetal kidney stained with antibody to polycystin-1 (red) and either β₁-integrin (a) or FAK (green) (b). Only the dual-scanned images are shown. The areas where polycystin-1 colocalized with β₁-integrin (a) are yellow. Polycystin-1 was expressed only in the epithelial structures of the ureteric bud and the S-shaped body (arrowheads), whereas β₁-integrin was ubiquitously expressed in both the epithelia (yellow staining due to colocalization with polycystin-1) and the surrounding mesenchyme (green staining, because polycystin-1 is not expressed in the mesenchyme). The absence of yellow staining in (b) shows that polycystin-1 did not colocalize with FAK.