The functions of the two proteins defective in autosomal dominant polycystic kidney disease, polycystin-1 and polycystin-2, have not been fully clarified, but it has been hypothesized that they may heterodimerize to form a "polycystin complex" involved in cell adhesion. In this paper, we demonstrate for the first time the existence of a native polycystin complex in mouse kidney tubular cells transgenic for PKD1, non-transgenic kidney cells, and normal adult human kidney. Polycystin-1 is heavily N-glycosylated, and several glycosylated forms of polycystin-1 differing in their sensitivity to endoglycosidase H (Endo H) were found; in contrast, native polycystin-2 was fully Endo H-sensitive. Using highly specific antibodies to both proteins, we show that polycystin-2 associates selectively with two species of full-length polycystin-1, one Endo H-sensitive and the other Endo H-resistant; importantly, the latter could be further enriched in plasma membrane fractions and coimmunoprecipitated with polycystin-2. Finally, a subpopulation of this complex co-localized to the lateral cell borders of PKD1 transgenic kidney cells. These results demonstrate that polycystin-1 and polycystin-2 interact in vivo to form a stable heterodimeric complex and suggest that disruption of this complex is likely to be of primary relevance to the pathogenesis of cyst formation in autosomal dominant polycystic kidney disease.
Autosomal dominant polycystic kidney disease (ADPKD) largely results from mutations in the PKD1 gene leading to hyperproliferation of renal tubular epithelial cells and consequent cyst formation. Rodent models of PKD suggest that the multifunctional hormone insulin-like growth factor-1 (IGF-1) could play a pathogenic role in renal cyst formation. In order to test this possibility, conditionally immortalized renal epithelial cells were prepared from normal individuals and from ADPKD patients with known germline mutations in PKD1. All patient cell lines had a decreased or absence of polycystin-1 but not polycystin-2. These cells had an increased sensitivity to IGF-1 and to cyclic AMP, which required phosphatidylinositol-3 (PI3)-kinase and the mitogen-activated protein kinase, extracellular signal-regulated protein kinase (ERK) for enhanced growth. Inhibition of Ras or Raf abolished the stimulated cell proliferation. Our results suggest that haploinsufficiency of polycystin-1 lowers the activation threshold of the Ras/Raf signalling system leading to growth factor-induced hyperproliferation. Inhibition of Ras or Raf activity may be a therapeutic option for decreasing tubular cell proliferation in ADPKD.
PKD2 is mutated in 15% of patients with autosomal dominant polycystic kidney disease. Polycystin-2 (PC2), the PKD2 protein, is a non-selective Ca(2+)-permeable cation channel which may function at the cell surface and ER. Nevertheless, the factors that regulate the dynamic translocation of PC2 between the ER and other compartments are not well understood. Constitutive phosphorylation of PC2 at a single C-terminal site (Ser(812)) has been previously reported. As we were unable to abolish phospholabelling of PC2 in HEK293 cells by site-directed mutagenesis of Ser(812) or all five predicted phosphorylation sites in the C-terminus, we hypothesized that PC2 could also be phosphorylated at the N-terminus. In this paper, we report the identification of a new phosphorylation site for PC2 within its N-terminal domain (Ser(76)) and demonstrate that this residue is phosphorylated by glycogen synthase kinase 3 (GSK3). The consensus recognition sequence for GSK3 (Ser(76)/Ser(80)) is evolutionarily conserved down to lower vertebrates. In the presence of specific GSK3 inhibitors, the lateral plasma membrane pool of endogenous PC2 redistributes into an intracellular compartment in MDCK cells without any change in primary cilia localization. Finally, co-injection of wild-type but not a S76A/S80A mutant PKD2 capped mRNA could rescue the cystic phenotype induced by an antisense morpholino oligonucleotide to pkd2 in zebrafish pronephric kidney. We conclude that surface localization of PC2 is regulated by phosphorylation at a unique GSK3 site in its N-terminal domain in vivo and in vitro. This site is functionally significant for the maintenance of normal glomerular and tubular morphology.
Abstract. The PKD1 protein, polycystin-1, is a large transmembrane protein of uncertain function and topology. To study the putative functions of polycystin-1, conditionally immortalized kidney cells transgenic for PKD1 were generated and an interaction between transgenic polycystin-1 and endogenous polycystin-2 has been recently demonstrated in these cells. This study provides the first functional evidence that transgenic polycystin-1 directly mediates cell-cell adhesion. In non-permeabilized cells, polycystin-1 localized to the lateral cell borders with N-terminal antibodies but not with a C-terminal antibody; there was a clear difference in surface intensity between transgenic and non-transgenic cells. Compared with non-transgenic cells, transgenic cells showed a dramatic increase in resistance to the disruptive effect of a polycystin-1 antibody raised to the PKD domains of polycystin-1 (IgPKD) in both cell adhesion and cell aggregation assays. The differential effect on cell adhesion between transgenic and nontransgenic cells could be reproduced using recombinant fusion proteins encoding non-overlapping regions of the IgPKD domains. In contrast, antibodies raised to other extracellular domains of polycystin-1 had no effect on cell adhesion. Finally, the specificity of this finding was confirmed by the lack of effect of IgPKD antibody on cell adhesion in a PKD1 cystic cell line deficient in polycystin-1. These results demonstrate that one of the primary functions of polycystin-1 is to mediate cell-cell adhesion in renal epithelial cells, probably via homophilic or heterophilic interactions of the PKD domains. Disruption of cell-cell adhesion during tubular morphogenesis may be an early initiating event for cyst formation in ADPKD.Autosomal dominant polycystic kidney disease (ADPKD), the most common inherited human renal disease (incidence, 1 in 1000 live births) is caused by mutations in two genes, PKD1 (85%) and PKD2 (15%). It is a systemic disorder characterized by the formation of fluid-filled cysts mainly in the kidney but also commonly in the liver and pancreas. ADPKD is also associated with an increased incidence of non-cystic manifestations, including hypertension, cardiac valve abnormalities, diverticular disease, and intracranial aneurysms.Since the identification of PKD1 and PKD2, investigation into the putative functions of the two ADPKD proteins, polycystin-1 and polycystin-2, has been intense, but a consensus on their likely physiologic functions has not been reached. Several possible mutational mechanisms underlying cyst formation including haploinsufficiency and a two-hit model have been proposed, but it is not clear how mutations in either gene lead to cyst formation (1). Potentially more than one mechanism could be operative.Polycystin-1 is a large (Ͼ460 kD) heavily glycosylated integral membrane protein (2). It is predicted to have a large N-terminal extracellular domain (approximately 2500 aa), 11 transmembrane domains, and a short C-terminal cytoplasmic tail (3). The extracellular region a...
Autosomal dominant polycystic kidney disease (ADPKD), the most common inherited cause of kidney failure, is caused by mutations in either PKD1 (85%) or PKD2 (15%). The PKD2 protein, polycystin-2 (PC2 or TRPP2), is a member of the transient receptor potential (TRP) superfamily and functions as a non-selective calcium channel. PC2 has been found to form oligomers in native tissues suggesting that it may form functional homo-or heterotetramers with other subunits, similar to other TRP channels. Our experiments unexpectedly revealed that PC2 mutant proteins lacking the known C-terminal dimerization domain were still able to form oligomers and co-immunoprecipitate full-length PC2, implying the possible existence of a proximal dimerization domain. Using yeast two-hybrid and biochemical assays, we have mapped an alternative dimerization domain to the N terminus of PC2 (NT2-1-223, L224X). Functional characterization of this domaindemonstrated that it was sufficient to induce cyst formation in zebrafish embryos and inhibit PC2 surface currents in mIMCD3 cells probably by a dominant-negative mechanism. In summary, we propose a model for PC2 assembly as a functional tetramer which depends on both C-and N-terminal dimerization domains. These results have significant implications for our understanding of PC2 function and disease pathogenesis in ADPKD and provide a new strategy for studying PC2 function.
Mutations in PKD1 (85%) or PKD2 (15%) account for almost all cases of autosomal dominant polycystic kidney disease (ADPKD). The ADPKD proteins, termed as polycystin-1 (PC1) and polycystin-2 (PC2), interact via their C-termini to form a receptor-ion channel complex whose function and regulation are not fully understood. Here, we report the first phosphorylated residue (Ser(829)) in PC2, whose dephosphorylation is mediated by PC1 binding through the recruitment of protein phosphatase-1 alpha (PP1α). Using a new phosphospecific antibody (pPC2) to this site, we demonstrate that Ser(829) is phosphorylated by Protein kinase A (PKA) but remains constitutively phosphorylated in cells and tissues lacking PC1. cAMP increased pSer(829) basolateral localization in MDCK cells in a time dependent manner and was essential for pronephric development in Xenopus embryos. When constitutively expressed, a complex phenotype associated with enhanced ATP-dependent ER Ca(2+) release and loss of growth suppression was observed in cycling cells. These results reveal a reciprocal functional link between PC1 and PC2 which is critically dependent on their interaction. Unopposed cAMP stimulated hyperphosphorylation of PC2 in the absence of functional PC1 could contribute to cyst initiation in PKD1 patients and represents a new molecular paradigm in understanding ADPKD pathogenesis.
Autosomal dominant polycystic kidney disease (ADPKD) is the most common inherited human renal disease and is caused by mutations in two genes, PKD1 (85%) and PKD2 (15%). Cyst epithelial cells are characterised by a complex cellular phenotype including changes in proliferation, apoptosis, basement membrane composition and apicobasal polarity. Since polycystin 1 (PC1), the PKD1 protein, has been located in the basolateral membrane of kidney epithelial cells, we hypothesised that it might have a key role in mediating or stabilising cell-cell interactions. In non-ciliated L929 cells, stable or transient surface expression of the PC1 extracellular domain was sufficient to confer an adhesive phenotype and stimulate junction formation. In MDCK cells, we found that PC1 was recruited to the lateral membranes coincident with E-cadherin within 30 minutes after a `calcium switch'. Recruitment of both proteins was significantly delayed when cells were treated with a PC1 blocking antibody raised to the PKD domains. Finally, PC1 and E-cadherin could be coimmunoprecipitated together from MDCK cells. We conclude that PC1 has a key role in initiating junction formation via initial homophilic interactions and facilitates junction assembly and the establishment of apicobasal polarity by E-cadherin recruitment.
Predicting long-term outcome after breast-cancer diagnosis remains problematic, particularly for patients with clinically small, axillary lymph node- negative tumours. Evidence suggests that the lectin Helix pomatia agglutinin (HPA) identifies oligosaccharides associated with poor-prognosis cancer. Our aim was to identify oligosaccharides that bind HPA in aggressive breast cancers. Breast-cancer cell lines (MCF-7, BT-549 and BT-20) and a cell line from human milk (HBL-100), which showed a range of HPA-binding intensities, were used to extract HPA-binding glycoproteins. Oligosaccharides were released using anhydrous hydrazine and separated on a range of HPLC matrices. We investigated whether HPA-binding oligosaccharides from cell lines were present in human breast-cancer tissues, using 69 breast-cancer specimens from patients with between 5 and 10 years' follow-up. A monosialylated oligosaccharide was over-expressed in the cell line that bound HPA strongly. Further analysis by normal-phase HPLC showed that the 2-aminobenzamide-conjugated oligosaccharide had a hydrodynamic volume of 4.58 glucose units (HPAgly1). Increased expression of HPAgly1 was associated with HPA staining of breast-cancer specimens (Student's t-test p = 0.025). Analysis of oligosaccharide levels and disease-free survival after treatment for breast cancer indicated a shorter disease-free interval for patients with elevated levels of HPAgly1. This is the first time that histochemical lectin staining has been correlated with biochemical mapping of oligosaccharides. Using this approach, we have identified a monosialylated HPA lectin-binding oligosaccharide present in breast-cancer cells grown in vitro which is elevated in breast-cancer specimens that bind the lectin.
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