OFD1 and Flotillins Are Integral Components of a Ciliary Signaling Protein Complex Organized by Polycystins in Renal Epithelia and Odontoblasts

Mutation of the X-linked oral-facial-digital syndrome type 1 (OFD1) gene is embryonic lethal in males and results in craniofacial malformations and adult onset polycystic kidney disease in females. While the OFD1 protein localizes to centriolar satellites, centrosomes and basal bodies, its cellular function and how it relates to cystic kidney disease is largely unknown. Here, we demonstrate that OFD1 is assembled into a protein complex that is localized to the primary cilium and contains the epidermal growth factor receptor (EGFR) and domain organizing flotillin proteins. This protein complex, which has similarity to a basolateral adhesion domain formed during cell polarization, also contains the polycystin proteins that when mutant cause autosomal dominant polycystic kidney disease (ADPKD). Importantly, in human ADPKD cells where mutant polycystin-1 fails to localize to cilia, there is a concomitant loss of localization of polycystin-2, OFD1, EGFR and flotillin-1 to cilia. Together, these data suggest that polycystins are necessary for assembly of a novel flotillin-containing ciliary signaling complex and provide a molecular rationale for the common renal pathologies caused by OFD1 and PKD mutations.     

Identification and characterization of polycystin-2, the PKD2 gene product.

PKD2, the second gene for the autosomal dominant polycystic kidney disease (ADPKD), encodes a protein, polycystin-2, with predicted structural similarity to cation channel subunits. However, the function of polycystin-2 remains unknown. We used polyclonal antisera specific for the intracellular NH(2) and COOH termini to identify polycystin-2 as an approximately 110-kDa integral membrane glycoprotein. Polycystin-2 from both native tissues and cells in culture is sensitive to Endo H suggesting the continued presence of high-mannose oligosaccharides typical of pre-middle Golgi proteins. Immunofluorescent cell staining of polycystin-2 shows a pattern consistent with localization in the endoplasmic reticulum. This finding is confirmed by co-localization with protein-disulfide isomerase as determined by double indirect immunofluorescence and co-distribution with calnexin in subcellular fractionation studies. Polycystin-2 translation products truncated at or after Gly(821) retain their exclusive endoplasmic reticulum localization while products truncated at or before Glu(787) additionally traffic to the plasma membrane. Truncation mutants that traffic to the plasma membrane acquire Endo H resistance and can be biotinylated on the cell surface in intact cells. The 34-amino acid region Glu(787)-Ser(820), containing two putative phosphorylation sites, is responsible for the exclusive endoplasmic reticulum localization of polycystin-2 and is the site of specific interaction with an as yet unidentified protein binding partner for polycystin-2. The localization of full-length polycystin-2 to intracellular membranes raises the possibility that the PKD2 gene product is a subunit of intracellular channel complexes.     

Molecular pathogenesis of autosomal dominant polycystic kidney disease

Autosomal dominant polycystic kidney disease (ADPKD) is one of the commonest inherited human disorders yet remains relatively unknown to the wider medical, scientific and public audience. ADPKD is characterised by the development of bilateral enlarged kidneys containing multiple fluid-filled cysts and is a leading cause of end-stage renal failure (ESRF). ADPKD is caused by mutations in two genes: PKD1 and PKD2. The protein products of the PKD genes, polycystin-1 and polycystin-2, form a calcium-regulated, calcium-permeable ion channel. The polycystin complex is implicated in regulation of the cell cycle via multiple signal transduction pathways as well as the mechanosensory function of the renal primary cilium, an enigmatic cellular organelle whose role in normal physiology is still poorly understood. Defects in cilial function are now documented in several other human diseases including autosomal recessive polycystic kidney disease, nephronophthisis, Bardet-Biedl syndrome and many animal models of polycystic kidney disease. Therapeutic trials in these animal models of polycystic kidney disease have identified several promising drugs that ameliorate disease severity. However, elucidation of the function of the polycystins and the primary cilium will have a major impact on our understanding of renal cystic diseases and will create exciting new opportunities for the design of disease-specific therapies.     

The gating of polycystin signaling complex

Mutations in either polycystin-2 (PC2) or polycystin-1 (PC1) proteins cause severe, potentially lethal, kidney disorders (autosomal dominant polycystic kidney disease, ADPKD) and multiple extrarenal disease phenotypes. PC2, a member of the transient receptor potential channel superfamily and PC1, an orphan membrane receptor of largely unknown function, are thought to be part of a common signalling pathway. Here, I show that co-assembly of full-length PC with PC2 forms an ion channel signalling complex in which PCI regulates PC2 channel gating through a structural rearrangement of the polycystin complex (Delmas et al., 2004a). These polycystin complexes function either as a receptor-cation channel or as a G-protein-coupled receptor. Thus, PC acts as a prototypical membrane receptor that regulates G-proteins and plasmalemmal PC2, a bimodal mechanism that may account for the multifunctional roles of polycystin proteins in various cell types. Genetic alteration of polycystin proteins such as those occurring in kidney diseases may impede polycystin signalling, thereby providing a likely mechanistic explanation to the pathogenesis of ADPKD. Our proposed mechanism may also be paradigmatic for the function of polycystin orthologues and other polycystin-related proteins in a variety of nonrenal cell types, including sperm, muscle cells and sensory neurons.     

Polycystin-1 and polycystin-2 are both required to amplify inositol-trisphosphate-induced Ca2+ release

Autosomal dominant polycystic kidney disease is caused by loss-of-function mutations in the PKD1 or PKD2 genes encoding respectively polycystin-1 and polycystin-2. Polycystin-2 stimulates the inositol trisphosphate (IP(3)) receptor (IP(3)R), a Ca(2+)-release channel in the endoplasmic reticulum (ER). The effect of ER-located polycystin-1 is less clear. Polycystin-1 has been reported both to stimulate and to inhibit the IP(3)R. We now studied the effect of polycystin-1 and of polycystin-2 on the IP(3)R activity under conditions where the cytosolic Ca(2+) concentration was kept constant and the reuptake of released Ca(2+) was prevented. We also studied the interdependence of the interaction of polycystin-1 and polycystin-2 with the IP(3)R. The experiments were done in conditionally immortalized human proximal-tubule epithelial cells in which one or both polycystins were knocked down using lentiviral vectors containing miRNA-based short hairpins. The Ca(2+) release was induced in plasma membrane-permeabilized cells by various IP(3) concentrations at a fixed Ca(2+) concentration under unidirectional (45)Ca(2+)-efflux conditions. We now report that knock down of polycystin-1 or of polycystin-2 inhibited the IP(3)-induced Ca(2+) release. The simultaneous presence of the two polycystins was required to fully amplify the IP(3)-induced Ca(2+) release, since the presence of polycystin-1 alone or of polycystin-2 alone did not result in an increased Ca(2+) release. These novel findings indicate that ER-located polycystin-1 and polycystin-2 operate as a functional complex. They are compatible with the view that loss-of-function mutations in PKD1 and in PKD2 both cause autosomal dominant polycystic kidney disease.     

PKD1 induces p21(waf1) and regulation of the cell cycle via direct activation of the JAK-STAT signaling pathway in a process requiring PKD2

Autosomal dominant polycystic kidney disease is characterized by cyst formation in the kidney and other organs and results from mutations of PKD1 or PKD2. Previous studies suggest that their gene products have an important role in growth regulation. We now show that expression of polycystin-1 activates the JAK-STAT pathway, thereby upregulating p21(waf1) and inducing cell cycle arrest in G0/G1. This process requires polycystin-2, a channel protein, as an essential cofactor. Mutations that disrupt polycystin-1/2 binding prevent activation of the pathway. Mouse embryos lacking Pkd1 have defective STAT1 phosphorylation and p21(waf1) induction. These results suggest that one function of the polycystin-1/2 complex is to regulate the JAK/STAT pathway and explain how mutations of either gene can result in dysregulated growth.     

Tuberin-dependent membrane localization of polycystin-1: a functional link between polycystic kidney disease and the TSC2 tumor suppressor gene

The PKD1 gene accounts for 85% of autosomal dominant polycystic kidney disease (ADPKD), the most common human genetic disorder. Rats with a germline inactivation of one allele of the Tsc2 tumor suppressor gene developed early onset severe bilateral polycystic kidney disease, with similarities to the human contiguous gene syndrome caused by germline codeletion of PKD1 and TSC2 genes. Polycystic rat renal cells retained two normal Pkd1 alleles but were null for Tsc2 and exhibited loss of lateral membrane-localized polycystin-1. In tuberin-deficient cells, intracellular trafficking of polycystin-1 was disrupted, resulting in sequestration of polycystin-1 within the Golgi and reexpression of Tsc2 restored correct polycystin-1 membrane localization. These data identify tuberin as a determinant of polycystin-1 functional localization and, potentially, ADPKD severity.     

Submembraneous microtubule cytoskeleton: interaction of TRPP2 with the cell cytoskeleton

TRPP2, also called polycystin-2, the gene product of PKD2, is a membrane protein defective in 10-15% of cases of autosomal dominant polycystic kidney disease. Mutations in PKD2 are also associated with extrarenal disorders, such as hepatic cystogenesis and cardiovascular abnormalities. TRPP2 is a Ca-permeable nonselective cation channel present in the endoplasmic reticulum and plasma membrane, as well as in cilia of renal epithelial and embryonic nodal cells, in which it likely forms part of a flow sensor. Recent studies have identified a number of TRPP2-interacting proteins, of which many are cytoskeletal components. Work from our and other laboratories indicates that cytoskeletal partner proteins seem to play important, albeit highly complex, roles in the regulation of TRPP2 expression, localization and channel function. This minireview covers current knowledge about cytoskeletal interactions with TRPP2, and suggests that mutations in proteins of the TRPP2-cytoskeleton complex may be implicated in the pathogenesis of autosomal dominant polycystic kidney disease.     

Polycystin-1L2 is a novel G-protein-binding protein

Mutations in genes encoding polycystin-1 (PC1) and polycystin-2 cause autosomal dominant polycystic kidney disease. The polycystin protein family is composed of Ca2+-permeable pore-forming subunits and receptor-like integral membrane proteins. Here we describe a novel member of the polycystin-1-like subfamily, polycystin-1L2 (PC1L2), encoded by PKD1L2, which has various alternative splicing forms with two translation initiation sites. PC1L2 short form starts in exon 12 of the long form. The longest open reading frame of PKD1L2 short form, determined from human testis cDNA, encodes a 1775-amino-acid protein and 32 exons, whereas the long form is predicted to encode a 2460-residue protein. Both forms have a small receptor for egg jelly domain, a G-protein-coupled receptor proteolytic site, an LH2/PLAT, and 11 putative transmembrane domains, as well as a number of rhodopsin-like G-protein-coupled receptor signatures. RT-PCR analysis shows that the short form, but not the long form, of human PKD1L2 is expressed in the developing and adult heart and kidney. Furthermore, by GST pull-down assay we observed that PC1L2 and polycystin-1L1 are able to bind to specific G-protein subunits. We also show that PC1 C-terminal cytosolic domain binds to Galpha12, Galphas, and Galphai1, while it weakly interacts with Galphai2. Our results indicate that both PC1-like molecules may act as G-protein-coupled receptors.     

Polycystin-2 regulates proliferation and branching morphogenesis in kidney epithelial cells

Autosomal dominant polycystic kidney disease (ADPKD) is characterized by the formation of multiple fluid-filled cysts that expand over time and destroy the renal architecture. Loss or mutation of polycystin-1 or polycystin-2, the respective proteins encoded by the ADPKD genes PKD1 and PKD2, is associated with most cases of ADPKD. Thus, the polycystin proteins likely play a role in cell proliferation and morphogenesis. Recent studies indicate that polycystin-1 is involved in these processes, but little is known about the role played by polycystin-2. To address this question, we created a number of related cell lines variable in their expression of polycystin-2. We show that the basal and epidermal growth factor-stimulated rate of cell proliferation is higher in cells that do not express polycystin-2 versus those that do, indicating that polycystin-2 acts as a negative regulator of cell growth. In addition, cells not expressing polycystin-2 exhibit significantly more branching morphogenesis and multicellular tubule formation under basal and hepatocyte growth factor-stimulated conditions than their polycystin-2-expressing counterparts, suggesting that polycystin-2 may also play an important role in the regulation of tubulogenesis. Cells expressing a channel mutant of polycystin-2 proliferated faster than those expressing the wild-type protein, but exhibited blunted tubule formation. Thus, the channel activity of polycystin-2 may be an important component of its regulatory machinery. Finally, we show that polycystin-2 regulation of cell proliferation appears to be dependent on its ability to prevent phosphorylated extracellular-related kinase from entering the nucleus. Our results indicate that polycystin-2 is necessary for the proper growth and differentiation of kidney epithelial cells and suggest a possible mechanism for the cyst formation seen in ADPKD2.     

Post-translational modifications of the polycystin proteins

Autosomal dominant polycystic kidney disease (ADPKD) is the most common inherited cause of kidney failure and affects up to 12 million people worldwide. Germline mutations in two genes, PKD1 or PKD2, account for almost all patients with ADPKD. The ADPKD proteins, polycystin-1 (PC1) and polycystin-2 (PC2), are regulated by post-translational modifications (PTM), with phosphorylation, glycosylation and proteolytic cleavage being the best described changes. A few PTMs have been shown to regulate polycystin trafficking, signalling, localisation or stability and thus their physiological function. A key challenge for the future will be to elucidate the functional significance of all the individual PTMs reported to date. Finally, it is possible that site-specific mutations that disrupt PTM could contribute to cystogenesis although in the majority of cases, confirmatory evidence is awaited.     

A modified mammalian tandem affinity purification procedure to prepare functional polycystin-2 channel

The tandem affinity purification (TAP) procedure was initially developed as a tool for rapid purification of native protein complexes expressed at their natural levels in yeast cells. This purification procedure was also applied to study interactions between soluble proteins in mammalian cells. In order to apply this procedure to mammalian membrane proteins, we created a modified TAP tag expression vector and fused with the PKD2 gene, encoding a membrane cation channel protein, polycystin-2, mutated in 15% of autosomal dominant polycystic kidney disease. We generated epithelial Madin-Darby canine kidney cell line stably expressing TAP-tagged polycystin-2, improved the subsequent steps for membrane protein release and stability, and succeeded in purifying this protein. Using patch clamp electrophysiology, we detected specific polycystin-2 channel activities when the purified protein was reconstituted into a lipid bilayer system. Thus, this modified TAP procedure provides a powerful alternative to functionally characterize membrane proteins, such as ion channels, transporters and receptors, using cell-free system derived from mammalian cells.     

A tale of two tails: ciliary mechanotransduction in ADPKD

Autosomal dominant polycystic kidney disease (ADPKD) is a common lethal genetic disorder, characterized by the progressive development of fluid-filled cysts in the kidney, pancreas and liver, and anomalies of the cardiovascular system. Mutations in PKD1 and PKD2, which encode the transmembrane proteins polycystin-1 (PC1) and polycystin-2 (PC2) respectively, account for almost all cases of ADPKD. However, the mechanisms by which abnormalities in PKD1 and PKD2 lead to aberrant kidney development remain unknown. Recent progress in the understanding of ADPKD has focused on primary cilia, which act as sensory transducers in renal epithelial cells. New evidence shows that a mechanosensitive signal, cilia bending, activates the PC1-PC2 channel complex. When working properly, this functional complex elicits a transient Ca(2+) influx, which is coupled to the release of Ca(2+) from intracellular stores.     

Transport function of the naturally occurring pathogenic polycystin-2 mutant, R742X

Most patients with autosomal dominant polycystic kidney disease (ADPKD) harbor mutations truncating polycystin-1 (PC1) or polycystin-2 (PC2), products of the PKD1 and PKD2 genes, respectively. A third member of the polycystin family, polycystin-L (PCL), was recently shown to function as a Ca(2+)-modulated nonselective cation channel. More recently, PC2 was also shown to be a nonselective cation channel with comparable properties to PCL, though the membrane targeting of PC2 likely varies with cell types. Here we show that PC2 expressed heterologously in Xenopus oocytes is targeted to intracellular compartments. By contrast, a truncated form of mouse PC2 corresponding to a naturally occurring human mutation R742X is targeted predominantly to the plasma membrane where it mediates K(+), Na(+), and Ca(2+) currents. Unlike PCL, the truncated form does not display Ca(2+)-activated transport activities, possibly due to loss of an EF-hand at the C-terminus. We propose that PC2 forms ion channels utilizing structural components which are preserved in the R742X form of the protein. Implications for epithelial cell signaling are discussed.     

Annexin A5 interacts with polycystin-1 and interferes with the polycystin-1 stimulated recruitment of E-cadherin into adherens junctions

Polycystin-1 is the gene product of PKD1, the first gene identified to be causative for the condition of autosomal dominant polycystic kidney disease (ADPKD). Mutations in PKD1 are responsible for the majority of ADPKD cases worldwide. Polycystin-1 is a protein of the transient receptor potential channels superfamily, with 11 transmembrane spans and an extracellular N-terminal region of approximately 3109 amino acid residues, harboring multiple putative ligand binding domains. We demonstrate here that annexin A5 (ANXA5), a Ca(2+) and phospholipid binding protein, interacts with the N-terminal leucine-rich repeats of polycystin-1, in vitro and in a cell culture model. This interaction is direct and specific and involves a conserved sequence of the ANXA5 N-terminal domain. Using Madin-Darby canine kidney cells expressing polycystin-1 in an inducible manner we also show that polycystin-1 colocalizes with E-cadherin at cell-cell contacts and accelerates the recruitment of intracellular E-cadherin to reforming junctions. This polycystin-1 stimulated recruitment is significantly delayed by extracellular annexin A5.     

Identification of a new target molecule for a cascade therapy of polycystic kidney

Autosomal dominant polycystic kidney disease is a systemic disorder that primary affects the kidney which is characterized by the formation of fluid-filled cysts in both kidneys that leads to progressive renal failure. Mutated genes, polycystin-1 and polycystin-2, are identified, and evidence has emerged that polycystins are ion channels or regulators of ion channels. In spite of extensive characterization of polycystins, how polycystin channel signaling may be involved in cyst formation in ADPKD is still unclear. We found a mutant mouse which exhibits polycystic kidney and bone deformity in the course of making a transgenic mouse carrying the Drosophila sex-lethal gene. We identified a mutated gene Makorin1 by positional cloning. Makorin1 carries a typical RING-finger motif, suggesting that Makorin1 belongs to ubiquitinase E3 family. Makorin1 would open a new avenue to understand pathogenesis of polycystic kidney, and become a new therapeutic target of polycystic kidney.     

Polycystin-1 distribution is modulated by polycystin-2 expression in mammalian cells

Mutations in PKD1 and PKD2, the genes that encode polycystin-1 and polycystin-2 respectively, account for almost all cases of autosomal dominant polycystic kidney disease. Although the polycystins are believed to interact in vivo, the two proteins often display dissimilar patterns and gradients of expression during development. In an effort to understand this apparent discrepancy, we investigated how changes in polycystin-2 expression can affect the subcellular localization of polycystin-1. We show that, when polycystin-1 is expressed alone in a PKD2 null cell line, it localizes to the cell surface, as well as to the endoplasmic reticulum. When co-expressed with polycystin-2, however, polycystin-1 is not seen at the cell surface and co-localizes completely with polycystin-2 in the endoplasmic reticulum. The localization of a polycystin-1 fusion protein was similarly affected by changes in its level of expression relative to that of polycystin-2. This phenomenon was observed in populations as well as in individual COS-7 cells. Our data suggest that the localization of polycystin-1 can be regulated via the relative expression level of polycystin-2 in mammalian cells. This mechanism may help to explain the divergent patterns and levels of expression observed for the polycystins, and may provide clues as to how the function of these two proteins are regulated during development.     

PKD1 inhibits cancer cells migration and invasion via Wnt signaling pathway in vitro

The approximately 14 kb mRNA of the polycystic kidney disease gene PKD1 encodes a large ( approximately 460 kDa) protein, termed polycystin-1 (PC-1), that is responsible for autosomal dominant polycystic kidney disease (ADPKD). The unique organization of its multiple adhesive domains (16 Ig-like domains/PKD domains) suggests that it may play an important role in cell-cell/cell-matrix interactions. Here we demonstrated that PKD1 promoted cell-cell and cell-matrix interactions in cancer cells, indicating that PC-1 is involved in the cell adhesion process. Furthermore in this study, we showed that PKD1 inhibited cancer cells migration and invasion. And we also showed that PC-1 regulated these processes in a process that may be at least partially through the Wnt pathway. Collectively, our data suggest that PKD1 may act as a novel member of the tumor suppressor family of genes.