Polycystin-1 Expression Research Articles

Polycystin-1 Negatively Regulates Polycystin-2 Expression via the Aggresome/Autophagosome Pathway

Mutations of the PKD1 and PKD2 genes, encoding polycystin-1 (PC1) and polycystin-2 (PC2), respectively, lead to autosomal dominant polycystic kidney disease. Interestingly, up-regulation or down-regulation of PKD1 or PKD2 leads to polycystic kidney disease in animal models, but their interrelations are not completely understood. We show here that full-length PC1 that interacts with PC2 via a C-terminal coiled-coil domain regulates PC2 expression in vivo and in vitro by down-regulating PC2 expression in a dose-dependent manner. Expression of the pathogenic mutant R4227X, which lacks the C-terminal coiled-coil domain, failed to down-regulate PC2 expression, suggesting that PC1-PC2 interaction is necessary for PC2 regulation. The proteasome and autophagy are two pathways that control protein degradation. Proteins that are not degraded by proteasomes precipitate in the cytoplasm and are transported via histone deacetylase 6 (HDAC6) toward the aggresomes. We found that HDAC6 binds to PC2 and that expression of full-length PC1 accelerates the transport of the HDAC6-PC2 complex toward aggresomes, whereas expression of the R4227X mutant fails to do so. Aggresomes are engulfed by autophagosomes, which then fuse with the lysosome for degradation; this process is also known as autophagy. We have now shown that PC1 overexpression leads to increased degradation of PC2 via autophagy. Interestingly, PC1 does not activate autophagy generally. Thus, we have now uncovered a new pathway suggesting that when PC1 is expressed, PC2 that is not bound to PC1 is directed to aggresomes and subsequently degraded via autophagy, a control mechanism that may play a role in autosomal dominant polycystic kidney disease pathogenesis.

Immunohistochemical study of polycystin-1 in dentigerous cysts.

The alterations involved in step-wise transformation of a dental follicle to dentigerous cyst (DC) is not clearly known. Primary cilium and its protein have been hypothesized to be associated with DC. Mutation of a ciliary protein, polycystin-1 (PC1) is associated with autosomal dominant polycystic kidney disease. This study was performed to assess the immunohistochemical expression of PC1 between DC and postfunctional follicular tissue (PFFT). Thirty-one consecutive PFFT and 15 DC formed the study group. The PFFT and DC tissues were stained with antibody against PC1. Statistical Package for Social Service was used to analyze data. Descriptive statistics and Student's Chi-square test were appropriately used. P≤0.05 was taken as significant. Fifteen DC (100%) and 7 (22.58%) PFFT were positive for PC1. The difference was statistically significant (P=0.000). PC1 expression was observed in the cytoplasm with varying intensity. All PC1 positive epithelial cells' cytoplasm stained diffusely. Abnormal cytoplasmic expression of PC1 in all positive epithelial lining indicates that the PC1 probably is associated with cystic transformation.

Pathological changes in primary cilia: A novel mechanism of graft cholangiopathy caused by prolonged cold preservation in a rat model of orthotopic liver transplantation.

To study the impairment of cholangiocyte primary cilia caused by prolonged cold preservation and its correlation with graft cholangiopathy after orthotopic liver transplantation (OLT). Subjects were 60 male Wistar rats that were divided into 2 groups: a control group (n = 30) receiving a donor liver preserved for 1 h and a study group (n = 30) receiving a donor liver preserved for 12 h. A two-cuff method was used to establish the OLT model, and the hepatic artery and bile ducts were reconstructed using stents. Samples were collected 2, 8, and 16 weeks after surgery, and 5 samples were collected from each group at each time point. Serum biochemical indicators were measured, morphological changes in intrahepatic bile ducts and cholangiocyte primary cilia were observed using an optical microscope and scanning electronic microscope, respectively, and the ciliary marker (α-tubulin) and membrane proteins (PC-1, TRPV4, and P2Y12) were detected using immunofluorescence analysis and Western blotting. In the study group, phlogocytes infiltrated around bile ducts and bile ducts proliferated markedly at 8 weeks. At 16 weeks, the biliary structures were indistinct and some bile ducts disappeared, a large amount of collagen was deposited, numerous phlogocytes infiltrated around ducts, some biliary epithelial cells (BECs) were deformed or dead, and primary cilia disappeared. In the control group, the intrahepatic bile ducts and BECs were nearly intact and the primary cilia were present. In the study group, the expression of α-tubulin, polycystin-1 (PC-1), TRPV4, and P2Y12 in bile ducts disappeared completely after 8 weeks. In the control group, expression of the marker and proteins decreased at 2 weeks and increased slightly after 8 weeks. These results suggest that the study group had dysfunctional primary cilia at the start of OLT and that this dysfunction was irreversible. In the control group, the primary cilia defects and subsequent biliary injury were temporary. Thus, prolonged cold preservation of a donor liver may cause graft cholangiopathy by altering the integrity and functions of cholangiocyte primary cilia.

Polycystin 2 is involved in the nitric oxide production in responding to oscillating fluid shear in MLO-Y4 cells

As a mechano-calcium channel, polycystin2 (PC2) play an important role in the response of renal epithelial cells to fluid flow shear stress. In bone tissue, osteocytes are well known as the main mechanosensory cells, and sensitive to fluid flow stimulus in vitro. In the study, we investigated the effects of oscillating fluid flow (OFF, 2h, 1Hz, 1.0Pa) on the release of Nitric Oxide (NO) and ProstaglandinE2 (PGE2), and the role of PC2 on the release. Our findings demonstrate that PC2 expression increases after 2h of OFF, and silencing PC2 by RNAi inhibits downstream NO production and iNOS expression, but does not affect the response of PGE2 to OFF.

Reduced ciliary polycystin-2 in induced pluripotent stem cells from polycystic kidney disease patients with PKD1 mutations.

Heterozygous mutations in PKD1 or PKD2, which encode polycystin-1 (PC1) and polycystin-2 (PC2), respectively, cause autosomal dominant PKD (ADPKD), whereas mutations in PKHD1, which encodes fibrocystin/polyductin (FPC), cause autosomal recessive PKD (ARPKD). However, the relationship between these proteins and the pathogenesis of PKD remains unclear. To model PKD in human cells, we established induced pluripotent stem (iPS) cell lines from fibroblasts of three ADPKD and two ARPKD patients. Genetic sequencing revealed unique heterozygous mutations in PKD1 of the parental ADPKD fibroblasts but no pathogenic mutations in PKD2. Undifferentiated PKD iPS cells, control iPS cells, and embryonic stem cells elaborated primary cilia and expressed PC1, PC2, and FPC at similar levels, and PKD and control iPS cells exhibited comparable rates of proliferation, apoptosis, and ciliogenesis. However, ADPKD iPS cells as well as somatic epithelial cells and hepatoblasts/biliary precursors differentiated from these cells expressed lower levels of PC2 at the cilium. Additional sequencing confirmed the retention of PKD1 heterozygous mutations in iPS cell lines from two patients but identified possible loss of heterozygosity in iPS cell lines from one patient. Furthermore, ectopic expression of wild-type PC1 in ADPKD iPS-derived hepatoblasts rescued ciliary PC2 protein expression levels, and overexpression of PC1 but not a carboxy-terminal truncation mutant increased ciliary PC2 expression levels in mouse kidney cells. Taken together, these results suggest that PC1 regulates ciliary PC2 protein expression levels and support the use of PKD iPS cells for investigating disease pathophysiology.

Abstract 19326: Microrna Regulating Protein Bicc1 Causes Dilated Cardiomyopathy Through Modulation of Polycystin-2

An ENU-induced mutation in the RNA binding protein Bicc1 was recovered in a mutant named Destro. Destro exhibits complex congenital cardiovascular defects (CHD) associated with heterotaxy, with some mutants also exhibiting kidney, biliary ductal and pancreatic cysts. The mutation was identified by whole genome sequencing as Bicc1c.606+2T>C, which caused an 18 amino acid in-frame deletion in the K homology domain required for RNA recognition. Previous studies showed Bicc1 mutations can cause polycystic kidney disease (PKD) via its role in regulating microRNA-17 (miR-17) activity, and the altered expression of polycystin-2 (Pkd2), a known miR-17 target. Destro mutants without CHD or laterality defects are postnatal viable, but die prematurely at 4-5 months of age with severe runting, even in the absence of PKD. Transthoracic echocardiography showed cardiac function in such animals declined markedly with age. This was associated with increase in LV chamber size/body weight ratio and the elevated expression of heart failure markers such as Nppa, Nppb, and osteopontin. As Pkd2 was recently shown to interact with ryanodine receptor, RyR2 to modulate Ca(2+) signaling in cardiomyocytes, we propose Bicc1 mutation can cause cardiomyopathy independent of defects associated with the kidney. Consistent with this hypothesis, we noted all homozygous Bicc1 mutants surviving to adulthood succumb to heart failure associated with dilated cardiomyopathy, but yet show no evidence of PKD. Overall, these findings indicate a novel role for the RNA binding protein Bicc1 in cardiac pathology involving dilated cardiomyopathy. Experiments are underway to examine the role of microRNA regulation of Pkd2 on cardiac pathology in the Bicc1 mutants. Based on these observations, we suggest patients with PKD may have additional risk for cardiomyopathy of an independent etiology from any underlying kidney disease.

The primary cilium conducts chondrocyte mechanotransduction

In several cell types fluid-flow deflection of primary cilia initiates a mechanotransduction pathway via polycystin 1 and 2 (PC1/2). In articular cartilage the chondrocyte primary cilium extends into cartilage matrix. Mechanical signals including compression and fluid flow trigger mechanosensitive regulation of matrix synthesis underpinning tissue homeostasis. Here we tested the hypothesis that the cilium plays a key role in chondrocyte mechanotransduction that includes ATP release, ATP-induced calcium transients and the subsequent regulation of matrix synthesis. These studies used murine chondrocytes with a hypomorphic mutation of Tg737, (encoding IFT88) which abolishes cilia growth. 3D agarose culture allowed compressive loading of WT and Tg737 chondrocytes followed by quantification of ATP release with a luciferase assay, calcium transients by Fluo-4 imaging, and matrix synthesis by qPCR and biochemical assay. Additionally, expression of purinergic receptors (P2R) and polycystins was assessed by western blot and immunocytochemistry. Compression of WT chondrocytes increased calcium transients and matrix production. By contrast this mechanosensitive behaviour was completely abolished in Tg737 cells. However mechanosensitive ATP release was present in both WT and Tg737 cells suggesting that IFT88 and the cilium are required for purinergic reception. Indeed exogenous ATP elicited calcium transients in WT but not in Tg737 cells. P2R expression profiles showed no global differences but polycystin-1 expression was altered in ORPK. We conclude that IFT88 plays a critical role in ATP-induced calcium signalling and is therefore essential to chondrocyte mechanotransduction. Furthermore, this suggests that IFT88 and the cilium may be fundamentally important for purinergic-calcium signalling pathways.

Autosomal dominant polycystic kidney disease is associated with central and nephrogenic defects in osmoregulation

Autosomal dominant polycystic kidney disease (ADPKD) is associated with a urine-concentrating defect attributed to renal cystic changes. As PKD genes are expressed in the brain, altered central release of arginine vasopressin could also play a role. In order to help determine this we measured central and nephrogenic components of osmoregulation in 10 adults and 10 children with ADPKD, all with normal renal function, and compared them to 20 age- and gender-matched controls. Overnight water deprivation caused a lower rise in urine osmolality in the patients with ADPKD than controls, reflecting an impaired release of vasopressin and a peripheral defect in the patients. The reactivity of plasma vasopressin to water deprivation, as found in controls, was blunted in the patients with the latter showing lower urine osmolality for the same range of plasma vasopressin. The maximal urine osmolality correlated negatively with total kidney volume. Defective osmoregulation was confirmed in the children with ADPKD but was unrelated to number of renal cysts or kidney size. Thus, patients with ADPKD have an early defect in osmoregulation, with a blunted release of arginine vasopressin. This reflects expression of polycystins in hypothalamic nuclei that synthesize vasopressin, and this should be considered when evaluating treatments targeting the vasopressin pathway in ADPKD.

A large coronary aneurysm and its probable precursor lesions in a patient with autosomal dominant polycystic kidney disease: An implication for the process of aneurysmogenesis

Coronary artery aneurysms are rare complications of autosomal dominant polycystic kidney disease (ADPKD), and their pathogenesis remains poorly understood. We report an autopsy case of a 64-year-old ADPKD patient with an asymptomatic, large (4 cm in diameter) saccular aneurysm arising from the left circumflex (LCX) branch of the coronary artery with only mild atherosclerotic changes. Autopsy also revealed small, focal defects of media with or without microaneurysm formation in the LCX, mesenteric and renal arteries, and a fibromuscular dysplasia-like lesion with microaneurysm in the common iliac artery. Since polycystin-1 and -2 are expressed in arterial smooth-muscle cells, these findings imply that abnormal polycystin expression in ADPKD initially causes the focal medial defects, some of which might later progress to microaneurysms and then overt aneurysms. To the best of our knowledge, this is the first description of the pathologic findings of an ADPKD-associated coronary aneurysm and its probable precursor lesions in arteries.

Mechanical strain stimulates hedgehog signalling in adult articular chondrocytes and reduces primary cilia length

Mechanical strain stimulates hedgehog signalling in adult articular chondrocytes and reduces primary cilia length

Distribution and function of polycystin-2 in mouse retinal ganglion cells

The polycystin family of transient receptor potential (TRP) channels form Ca2+ regulated cation channels with distinct subcellullar localizations and functions. As part of heteromultimeric channels and multi-protein complexes, polycystins control intracellular Ca2+ signals and more generally the translation of extracellular signals and stimuli to intracellular responses. Polycystin-2 channels have been cloned from retina, but their distribution and function in retinal ganglion cells (RGCs) have not yet been established. In the present study, we determined cellular and subcellular localization as well as functional properties of polycystin-2 channels in RGCs. Polycystin-2 expression and distribution in RGCs was assessed by immunohistochemistry on vertical cryostat section of mouse retina as well as primary cultured mouse RGCs, using fluorescence microscopy. Biophysical and pharmacological properties of polycystin-2 channels isolated from primary cultured RGCs were determined using planar lipid bilayer electrophysiology. We detected polycystin-2 immunoreactivity both in the ganglion cell layer as well as in primary cultured RGCs. Subcellular analysis revealed strong cytosolic localization pattern of polycystin-2. Polycystin-2 channel current was Ca2+ activated, had a maximum slope conductance of 114 pS, and could be blocked in a dose-dependent manner by increasing concentrations of Mg2+. The cytosolic localization of polycystin-2 in RGCs is in accordance with its function as intracellular Ca2+ release channel. We conclude that polycystin-2 forms functional channels in RGCs, of which biophysical and pharmacological properties are similar to polycystin-2 channels reported for other tissues and organisms. Our data suggest a potential role for polycystin-2 in RGC Ca2+ signaling.

It’s not all in the cilium, but on the road to it: Genetic interaction network in polycystic kidney and liver diseases and how trafficking and quality control matter

Autosomal dominant polycystic liver disease results from mutations in PRKCSH or SEC63. The respective gene products, glucosidase IIb and SEC63p, function in protein translocation and quality control pathways in the endoplasmic reticulum. Here we show that glucosidase IIb and Sec63p are required in mice for adequate expression of a functional complex of the polycystic kidney disease gene products, polycystin-1 and polycystin-2. We find that polycystin-1 is the rate-limiting component of this complex and that there is a dose–response relationship between cystic dilation and levels of functional polycystin-1 following mutation of Prkcsh or Sec63.Reduced expression of polycystin-1 also serves to sensitize the kidney to cyst formation resulting from mutations in Pkhd1, the recessive polycystic kidney disease gene. Finally, we show that proteasome inhibition increases steady-state levels of polycystin-1 in cells lacking glucosidase IIb and that treatment with a proteasome inhibit or reduces cystic disease in orthologous gene models of human autosomal dominant polycystic liver.

Polycystin-2 Expression and Function in Adult Mouse Lacrimal Acinar Cells

Lacrimal glands regulate the production and secretion of tear fluid. Dysfunction of lacrimal gland acinar cells can ultimately result in ocular surface disorders, such as dry eye disease. Ca(2+) homeostasis is tightly regulated in the cellular environment, and secretion from the acinar cells of the lacrimal gland is regulated by both cholinergic and adrenergic stimuli, which both result in changes in the cytosolic Ca(2+) concentration. We have previously described the detailed intracellular distribution of inositol-1,4,5-trisphosphate receptors (IP(3)Rs), and ryanodine receptors (RyRs) in lacrimal acinar cells, however, little is known regarding the expression and distribution of the third major class of intracellular Ca(2+) release channels, transient receptor potential polycystin family (TRPP) channels. Studies were performed in adult lacrimal gland tissue of Swiss-Webster mice. Expression, localization, and intracellular distribution of TRPP Ca(2+) channels were investigated using immunocytochemistry, immunohistochemistry, and electron microscopy. The biophysical properties of single polycystin-2 channels were investigated using a planar lipid bilayer electrophysiology system. All channel-forming isoforms of TRPP channels (polycystin-2, polycystin-L, and polycystin-2L2) were expressed in adult mouse lacrimal gland. Subcellular analysis of immunogold labeling revealed strongest polycystin-2 expression on the membranes of the endoplasmic reticulum, Golgi, and nucleus. Biophysical properties of lacrimal gland polycystin-2 channels were similar to those described for other tissues. The expression of TRPP channels in lacrimal acinar cells suggests a functional role of the proteins in the regulation of lacrimal fluid secretion under physiological and disease conditions, and provides the basis for future studies focusing on physiology and pharmacology.

A genetic interaction network of five genes for human polycystic kidney and liver diseases defines polycystin-1 as the central determinant of cyst formation

Autosomal dominant polycystic liver disease results from mutations in PRKCSH or SEC63. The respective gene products, glucosidase IIβ and SEC63p, function in protein translocation and quality control pathways in the endoplasmic reticulum. Here we show that glucosidase IIβ and Sec63p are required in mice for adequate expression of a functional complex of the polycystic kidney disease gene products, polycystin-1 and polycystin-2. We find that polycystin-1 is the rate-limiting component of this complex and that there is a dose-response relationship between cystic dilation and levels of functional polycystin-1 following mutation of Prkcsh or Sec63. Reduced expression of polycystin-1 also serves to sensitize the kidney to cyst formation resulting from mutations in Pkhd1, the recessive polycystic kidney disease gene. Finally, we show that proteasome inhibition increases steady-state levels of polycystin-1 in cells lacking glucosidase IIβ and that treatment with a proteasome inhibitor reduces cystic disease in orthologous gene models of human autosomal dominant polycystic liver disease.

Calreticulin mediated control of polycystin-2 expression

Polycystin-2 or transient receptor potential polycystic 2 (TRPP2) is a membrane glycoprotein that is encoded for by the gene pkd-2, which accounts for ˜15% of autosomal dominant polycystic kidney disease. TRPP2 is an independent non selective cation channel localized to either the plasma membrane or the endoplasmic reticulum (ER) that is involved in diverse cellular functions including control of cell cycle, cell wall synthesis, left-right symmetry, cardiac & renal development and mating behavior. In addition, it interacts with polycystin-1 that regulates different cell signaling pathways including JAK/STAT. As a trans-membrane protein, polycystin-2 is expressed, folded and matured in the endoplasmic reticulum. To date no data is available about the nature of endoplasmic reticulum which is responsible for the proper folding of polycystin-2. This led us to the hypothesis that calreticulin, an endoplasmic reticular calcium binding chaperone protein, is involved in stabilizing and transporting polycystin-2 to the plasma membrane. To test this hypothesis we examined changes in polycystin expression and localization in wild type and calreticulin deficient cells using western blot analysis, and immunocytochemistry. Furthermore, western blot and immunohistochemical analyses were used to examine changes in polycystin expression and localization upon calreticulin over expression in vascular smooth muscle and endothelial cells of transgenic mice. Our data showed that over expression of calreticulin in either vascular smooth muscle cells or endothelial cells of transgenic mice results in the development of multiple clear cysts in the kidney cortex of these mice. Histopathological analysis of these kidneys resembles those of human polycystic kidney disease. Furthermore, loss of calreticulin function resulted in altered polycystin-2 expression in the mouse embryonic fibroblast cell lines. However, there were no significant changes in the localization of polycystin-2 protein in calreticulin deficient cells when compared to the wild type cells. Our data supports a possible role for calreticulin in the expression of polycystin-2. Further research is warranted to elucidate the role of calreticulin as a chaperone or regulator of calcium homeostasis in the expression of polycystin-2.

Fluid Shear Stress Induces Renal Epithelial Gene Expression through Polycystin-2-Dependent Trafficking of Extracellular Regulated Kinase

Background: The cilium and cilial proteins have emerged as principal mechanosensors of renal epithelial cells responsible for translating mechanical forces into intracellular signals. Polycystin-2 (PC-2), a cilial protein, regulates flow/shear-induced changes in intracellular Ca²⁺ ([Ca²⁺]_i) and recently has been implicated in the regulation of mitogen-activated protein (MAP) kinases. We hypothesize that fluid shear stress (FSS) activates PC-2 which regulates MAP kinase and, in turn, induces MAP kinase-dependent gene expression, specifically, monocyte chemoattractant protein-1 (MCP-1). Methods: To test this, PC-2 expression was constitutively reduced in a murine inner medullary collecting duct (IMCD3) cell line, and the expression of FSS-induced MCP-1 expression and MAP kinase signaling compared between the parental (PC-2-expressing) and PC-2-deficient IMCD3 cells. Results: FSS induces MAP kinase signaling and downstream MCP-1 mRNA expression in wild-type IMCD3 cells, while inhibitors of MAP kinase prevented the FSS-induced MCP-1 mRNA response. In contradistinction, FSS did not induce MCP-1 mRNA expression in PC-2-deficient cells, but did increase activation of the upstream MAP kinases. Wild-type cells exposed to FSS augmented the nuclear abundance of activated MAP kinase while PC-2-deficient cells did not. Conclusions: PC-2 regulates FSS-induced MAP kinase trafficking into the nucleus of CD cells.

Expression and distribution of transient receptor potential (TRP) channels in bladder epithelium

The urothelium is proposed to be a sensory tissue that responds to mechanical stress by undergoing dynamic membrane trafficking and neurotransmitter release; however, the molecular basis of this function is poorly understood. Transient receptor potential (TRP) channels are ideal candidates to fulfill such a role as they can sense changes in temperature, osmolarity, and mechanical stimuli, and several are reported to be expressed in the bladder epithelium. However, their complete expression profile is unknown and their cellular localization is largely undefined. We analyzed expression of all 33 TRP family members in mouse bladder and urothelium by RT-PCR and found 22 specifically expressed in the urothelium. Of the latter, 10 were chosen for closer investigation based on their known mechanosensory or membrane trafficking functions in other cell types. Western blots confirmed urothelial expression of TRPC1, TRPC4, TRPV1, TRPV2, TRPV4, TRPM4, TRPM7, TRPML1, and polycystins 1 and 2 (PKD1 and PKD2) proteins. We further defined the cellular and subcellular localization of all 10 TRP channels. TRPV2 and TRPM4 were prominently localized to the umbrella cell apical membrane, while TRPC4 and TRPV4 were identified on their abluminal surfaces. TRPC1, TRPM7, and TRPML1 were localized to the cytoplasm, while PKD1 and PKD2 were expressed on the apical and basolateral membranes of umbrella cells as well as in the cytoplasm. The cellular location of TRPV1 in the bladder has been debated, but colocalization with neuronal marker calcitonin gene-related peptide indicated clearly that it is present on afferent neurons that extend into the urothelium, but may not be expressed by the urothelium itself. These findings are consistent with the hypothesis that the urothelium acts as a sentinel and by expressing multiple TRP channels it is likely it can detect and presumably respond to a diversity of external stimuli and suggest that it plays an important role in urothelial signal transduction.

Protein kinase D-mediated phosphorylation of polycystin-2 (TRPP2) is essential for its effects on cell growth and calcium channel activity.

PKD2 is mutated in 15% of patients with autosomal dominant polycystic kidney disease. The PKD2 protein, polycystin-2 or TRPP2, is a nonselective Ca2+-permeable cation channel that has been shown to function at several locations, including primary cilia, basolateral membrane, and at the endoplasmic reticulum (ER). Nevertheless, the factors that regulate the channel activity of polycystin-2 are not well understood. Polycystin-2 has been shown previously to be regulated by phosphorylation at two serine residues (Ser812 and Ser76) with distinct functional consequences. Here, we report the identification of a previously unrecognized phosphorylation site within the polycystin-2 C terminus (Ser801), and we demonstrate that it is phosphorylated by protein kinase D. Phosphorylation at this site was significantly increased in response to serum and epidermal growth factor stimulation. In nonciliated Madin-Darby canine kidney I cells, inducible expression of polycystin-2 inhibited cell proliferation compared with wild-type cells. Mutagenesis at Ser801 abolished these effects and reduced ATP-stimulated Ca2+ release from ER stores. Finally, we show that a pathogenic mutation (S804N) within the consensus kinase recognition sequence abolished Ser801 phosphorylation. These results suggest that growth factor-stimulated, protein kinase D-mediated phosphorylation of polycystin-2 is essential for its ER channel function and links extracellular stimuli to its effects on cell growth and intracellular calcium regulation.

Identification of polycystin-1 and Gα12 binding regions necessary for regulation of apoptosis

Most patients with autosomal dominant polycystic kidney disease (ADPKD) harbor mutations in PKD1, the gene for polycystin-1 (PC1), a transmembrane protein with a cytoplasmic C-terminus that interacts with numerous signaling molecules, including Gα12. The functions of PC1 and the mechanisms of cyst development leading to renal failure are complex. Recently, we reported that PC1 expression levels modulate activity of Gα12-stimulated apoptosis (Yu et al., J. Biol. Chem . 2010 285(14):10243-51) . Herein, a mutational analysis of Gα12 and PC1 was undertaken to identify regions required for their interaction and ability to modulate apoptosis. A set of Gα12 mutations with systematic replacement of six amino acids with NAAIRS was tested for binding to the PC1 C-terminus in GST pulldowns. Additionally, a series of deletions within the PC1 C-terminus was examined for binding to Gα12. We identified 3 NAAIRS substitutions in Gα12 that completely abrogated binding, and identified a previously described 74 amino acid Gαi/o binding domain in the PC1 C-terminus as necessary for Gα12 interaction. The functional consequences of uncoupling PC1/Gα12 binding were studied in apoptosis assays utilizing HEK293 cells with inducible PC1 overexpression. Gα12 mutants deficient in PC1 binding were refractory to PC1 inhibition of Gα12-stimulated apoptosis. Likewise, deletion of the Gα12-interacting sequence from the PC1 cytoplasmic domain abrogated its inhibition of Gα12-stimulated apoptosis. Based on the crystal structure of Gα12, the PC1 interaction sites are likely to reside on exposed regions within the G protein helical domain. These structural details should facilitate the design of reagents to uncouple PC1/Gα12 signaling in ADPKD.

Identification of a Polycystin-1 Cleavage Product, P100, That Regulates Store Operated Ca2+ Entry through Interactions with STIM1

Autosomal Dominant Polycystic Kidney Disease (ADPKD) is a genetic disorder resulting in large kidney cysts and eventual kidney failure. Mutations in either the PKD1 or PKD2/TRPP2 genes and their respective protein products, polycystin-1 (PC1) and polycystin-2 (PC2) result in ADPKD. PC2 is known to function as a non-selective cation channel, but PC1's function and the function of PC1 cleavage products are not well understood. Here we identify an endogenous PC1 cleavage product, P100, a 100 kDa fragment found in both wild type and epitope tagged PKD1 knock-in mice. Expression of full length human PC1 (FL PC1) and the resulting P100 and C-Terminal Fragment (CTF) cleavage products in both MDCK and CHO cells significantly reduces the store operated Ca2+ entry (SOCE) resulting from thapsigargin induced store depletion. Exploration into the roles of P100 and CTF in SOCE inhibition reveal that P100, when expressed in Xenopus laevis oocytes, directly inhibits the SOCE currents but CTF does not, nor does P100 when containing the disease causing R4227X mutation. Interestingly, we also found that in PC1 expressing MDCK cells, translocation of the ER Ca2+ sensor protein STIM1 to the cell periphery was significantly altered. In addition, P100 Co-immunoprecipitates with STIM1 but CTF does not. The expression of P100 in CHO cells recapitulates the STIM1 translocation inhibition seen with FL PC1. These data describe a novel polycystin-1 cleavage product, P100, which functions to reduce SOCE via direct inhibition of STIM1 translocation; a function with consequences for ADPKD.