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Ralph Witzgall - One of the best experts on this subject based on the ideXlab platform.
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Phosphorylation of C-terminal Polycystin-2 influences the interaction with PIGEA14: a QCM study based on solid supported membranes.
Biochemical and biophysical research communications, 2013Co-Authors: Daniela Morick, Ralph Witzgall, Helen Hoffmeister, Michaela Schatz, Raphael Hubrich, Anya Krefft, Claudia SteinemAbstract:Abstract Polycystin-2 (PC2) trafficking has been proposed to be a result of the interaction of PIGEA14 with PC2 as a function of the phosphorylation state of PC2. Here, we investigated the interaction of PIGEA14 with the C-terminal part of Polycystin-2 wild type (cPC2wt) and the pseudophosphorylated mutant (cPC2S812D) to first, quantify the binding affinity between cPC2 and PIGEA14 and second, to elucidate the influence of PC2 phosphorylation on PIGEA14 binding. Solid supported membranes composed of octanethiol/1,2-dioleoyl- sn -glycero-3-phosphocholine doped with the receptor lipid DOGS–NTA–Ni were used to attach PIGEA14 to the membrane via its hexahistidine tag. By means of the quartz crystal microbalance technique, binding affinities as well as kinetic constants of the interaction were extracted in a label-free manner by applying the scaled particle theory. The results show that the dissociation constant of cPC2 to PIGEA14 is in the 10 nM regime providing strong evidence of a very specific interaction of cPC2 with PIGEA14. The interaction of cPC2wt is twofold larger than that of cPC2S812D. The moderate higher binding affinity of cPC2wt to PIGEA14 is discussed in light of PC2 trafficking to the plasma membrane.
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Polycystin-2 takes different routes to the somatic and ciliary plasma membrane
The Journal of cell biology, 2011Co-Authors: Helen Hoffmeister, Anna Cedzich, Karin Babinger, Sonja Gürster, Christine Meese, Karin Schadendorf, Larissa Osten, Uwe De Vries, Anne Rascle, Ralph WitzgallAbstract:Polycystin-2 (also called TRPP2), an integral membrane protein mutated in patients with cystic kidney disease, is located in the primary cilium where it is thought to transmit mechanical stimuli into the cell interior. After studying a series of Polycystin-2 deletion mutants we identified two amino acids in loop 4 that were essential for the trafficking of Polycystin-2 to the somatic (nonciliary) plasma membrane. However, Polycystin-2 mutant proteins in which these two residues were replaced by alanine were still sorted into the cilium, thus indicating that the trafficking routes to the somatic and ciliary plasma membrane compartments are distinct. We also observed that the introduction of dominant-negative Sar1 mutant proteins and treatment of cells with brefeldin A prevented the transport into the ciliary plasma membrane compartment, whereas metabolic labeling experiments, light microscopical imaging, and high-resolution electron microscopy revealed that full-length Polycystin-2 did not traverse the Golgi apparatus on its way to the cilium. These data argue that the transport of Polycystin-2 to the ciliary and to the somatic plasma membrane compartments originates in a COPII-dependent fashion at the endoplasmic reticulum, that Polycystin-2 reaches the cis side of the Golgi apparatus in either case, but that the trafficking to the somatic plasma membrane goes through the Golgi apparatus whereas transport vesicles to the cilium leave the Golgi apparatus at the cis compartment. Such an interpretation is supported by the finding that mycophenolic acid treatment resulted in the colocalization of Polycystin-2 with GM130, a marker of the cis-Golgi apparatus. Remarkably, we also observed that wild-type Smoothened, an integral membrane protein involved in hedgehog signaling that under resting conditions resides in the somatic plasma membrane, passed through the Golgi apparatus, but the M2 mutant of Smoothened, which is constitutively located in the ciliary but not in the somatic plasma membrane, does not. Finally, a dominant-negative form of Rab8a, a BBSome-associated monomeric GTPase, prevented the delivery of Polycystin-2 to the primary cilium whereas a dominant-negative form of Rab23 showed no inhibitory effect, which is consistent with the view that the ciliary trafficking of Polycystin-2 is regulated by the BBSome.
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The human Polycystin-2 protein represents an integral membrane protein with 6 membrane-spanning domains and intracellular NH2- and COOH-termini
Biochemical Journal, 2010Co-Authors: Helen Hoffmeister, Anne Rascle, Anna Rachel Gallagher, Ralph WitzgallAbstract:PKD2 is one of the two genes mutated in autosomal-dominant polycystic kidney disease (ADPKD). The protein product of PKD2, Polycystin-2, functions as a non-selective cation channel in the endoplasmic reticulum and possibly at the plasma membrane. Hydrophobicity plots and its assignment to the TRP family of cation channels suggest that Polycystin-2 contains 6 transmembrane domains and that both the NH2- and COOH-termini extend into the cytoplasm. However, no experimental evidence for this model has been provided so far. To determine the orientation of the different loops of Polycystin-2, we truncated Polycystin-2 within the predicted loops 1 to 5 and tagged the constructs at the COOH-terminus with a HA-epitope. After transient expression and selective membrane permeabilization, immunofluorescence staining for the HA epitope revealed that loops 1, 3 and 5 extend into the lumen of the endoplasmic reticulum or the extracellular space, while loops 2 and 4 extend into the cytoplasm. This approach also confirmed the cytoplasmic orientation of the NH2- and COOH-termini of Polycystin-2. In accordance with the immunofluorescence data, protease protection assays from microsomal preparations yielded protected fragments when Polycystin-2 was truncated in loops 1, 3 and 5 while no protected fragments could be detected when Polycystin-2 was truncated in loops 2 and 4. Our data therefore provide the first experimental evidence for the topological orientation of Polycystin-2.
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The human Polycystin-2 protein represents an integral membrane protein with six membrane-spanning domains and intracellular N- and C-termini.
The Biochemical journal, 2010Co-Authors: Helen Hoffmeister, Anne Rascle, Anna Rachel Gallagher, Ralph WitzgallAbstract:PKD2 is one of the two genes mutated in ADPKD (autosomaldominant polycystic kidney disease). The protein product of PKD2, Polycystin-2, functions as a non-selective cation channel in the endoplasmic reticulum and possibly at the plasma membrane. Hydrophobicity plots and its assignment to the TRP (transient receptor potential) family of cation channels suggest that Polycystin-2 contains six transmembrane domains and that both the N- and C-termini extend into the cytoplasm. However, no experimental evidence for this model has so far been provided. To determine the orientation of the different loops of Polycystin2, we truncated Polycystin-2 within the predicted loops 1–5 and tagged the constructs at the C-terminus with an HA (haemagglutinin) epitope. After transient expression and selective membrane permeabilization, immunofluorescence staining for the HA epitope revealed that loops 1, 3 and 5 extend into the lumen of the endoplasmic reticulum or the extracellular space, whereas loops 2 and 4 extend into the cytoplasm. This approach also confirmed the cytoplasmic orientation of the N- and C-termini of Polycystin-2. In accordance with the immunofluorescence data, protease protection assays from microsomal preparations yielded protected fragments when Polycystin-2 was truncated in loops 1, 3 and 5, whereas no protected fragments could be detected when Polycystin-2 was truncated in loops 2 and 4. The results of the present study therefore provide the first experimental evidence for the topological orientation of Polycystin-2.
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Ca2+-dependent Conformational Changes in a C-terminal Cytosolic Domain of Polycystin-2
The Journal of biological chemistry, 2009Co-Authors: Frank Schumann, Ralph Witzgall, Helen Hoffmeister, Reto Bader, M Schmidt, Hans Robert KalbitzerAbstract:The PKD1 and PKD2 genes are the genes that are mutated in patients suffering from autosomal dominant polycystic kidney disease. The human PKD2 gene codes for a 968-amino acid long membrane protein called Polycystin-2 that represents a cation channel whose activity can be regulated by Ca2+ ions. By CD, fluorescence, and NMR spectroscopy, we have studied a 117-amino acid-long fragment of the cytoplasmic domain of Polycystin-2, Polycystin-2-(680–796) that was proposed to contain a Ca2+-binding site. NMR structure determination reveals the existence of two Ca2+-binding sites in Polycystin-2-(680–796) arranged in a typical and an atypical EF-hand motif. In the absence of Ca2+ the protein forms a dimer that is dissociated by Ca2+ binding. This dissociation may be related to the Ca2+ inactivation observed earlier. The calcium affinity of the protein was determined by fluorescence and NMR spectroscopy. At 293 K, the KD values for the high and low affinity sites are 55 μm and 179 μm, respectively.
Horacio F Cantiello - One of the best experts on this subject based on the ideXlab platform.
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Structural interaction and functional regulation of Polycystin-2 by filamin.
PloS one, 2012Co-Authors: Qian Wang, Horacio F Cantiello, Xiao-qing Dai, Zuocheng Wang, Maria Del Rocio Cantero, Ji Shen, Xingzhen ChenAbstract:Filamins are important actin cross-linking proteins implicated in scaffolding, membrane stabilization and signal transduction, through interaction with ion channels, receptors and signaling proteins. Here we report the physical and functional interaction between filamins and Polycystin-2, a TRP-type cation channel mutated in 10–15% patients with autosomal dominant polycystic kidney disease. Yeast two-hybrid and GST pull-down experiments demonstrated that the C-termini of filamin isoforms A, B and C directly bind to both the intracellular N- and C-termini of Polycystin-2. Reciprocal co-immunoprecipitation experiments showed that endogenous Polycystin-2 and filamins are in the same complexes in renal epithelial cells and human melanoma A7 cells. We then examined the effect of filamin on Polycystin-2 channel function by electrophysiology studies with a lipid bilayer reconstitution system and found that filamin-A substantially inhibits Polycystin-2 channel activity. Our study indicates that filamins are important regulators of Polycystin-2 channel function, and further links actin cytoskeletal dynamics to the regulation of this channel protein.
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regulation by calcium of the trp channel Polycystin 2 trpp2
Biophysical Journal, 2009Co-Authors: Maria Del Rocio Cantero, Horacio F CantielloAbstract:• Polycystin-2 (PC2, TRPP2) is a member of the TRP (transient receptor potential) superfamily of cation channels. Like other members of this superfamily, PC2 permeates Ca2+, which is involved in both signal transduction, and Ca2+ entry. Previously, we showed that PC2 is normally active at intracellularly high Ca2+ concentrations (10-15 μM). Little is known, however, about the role intracellular Ca2+ plays in PC2 channel function. Here, we explored the role of physiological concentrations of intracellular Ca2+ in PC2-mediated channel function in reconstituted apical membranes from term human syncytiotrophoblast (hST). Addition of either EGTA (1 mM) or BAPTA (2 mM) to reach low intracellular Ca2+ (<5 nM) at the cytoplasmic side, elicited a complete PC2 channel inhibition. A dose response elicited by addition of increasing cytoplasmic Ca2+ showed that Ca2+ activated PC2 with an apparent half activating concentration of 4.78 nM and a Hill coefficient of ∼5. Conversely, extracellular Ca2+ concentrations, between 0.5 mM and 5 mM, had a stimulatory effect on PC2 channel activity while higher external concentrations (10-90 mM) were inhibitory. Further, this activating mechanism was not intrinsic to the PC2 channel but instead seems to be mediated by PC2-associated proteins. Channel function of the in vitro translated PC2 protein with Ca2+ concentrations of 10-15 mM was non-responsive to lowering cytoplasmic Ca2+ with either EGTA or BAPTA. Our data are consistent with a regulatory role of both cytoplasmic and external Ca2+ in PC2 channel function, which does not involve putative Ca2+-binding sites on the channel protein, but instead external sites to the channel protein.
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Regulation by Calcium of the TRP Channel Polycystin-2 (TRPP2)
Biophysical Journal, 2009Co-Authors: Maria Del Rocio Cantero, Horacio F CantielloAbstract:• Polycystin-2 (PC2, TRPP2) is a member of the TRP (transient receptor potential) superfamily of cation channels. Like other members of this superfamily, PC2 permeates Ca2+, which is involved in both signal transduction, and Ca2+ entry. Previously, we showed that PC2 is normally active at intracellularly high Ca2+ concentrations (10-15 μM). Little is known, however, about the role intracellular Ca2+ plays in PC2 channel function. Here, we explored the role of physiological concentrations of intracellular Ca2+ in PC2-mediated channel function in reconstituted apical membranes from term human syncytiotrophoblast (hST). Addition of either EGTA (1 mM) or BAPTA (2 mM) to reach low intracellular Ca2+ (
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A modified mammalian tandem affinity purification procedure to prepare functional Polycystin-2 channel.
FEBS letters, 2004Co-Authors: Xiao-qing Dai, Horacio F Cantiello, Patrick Y Shen, Edward Karpinski, Xingzhen ChenAbstract: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.
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Polycystin-2 as a signal transducer.
Advances in experimental medicine and biology, 2004Co-Authors: Horacio F Cantiello, Gustavo A. Timpanaro, Nicolás Montalbetti, Silvia González-perrettAbstract:The human syncytiotrophoblast (hST) is a differentiated syncytial epithelium that covers the villous tree of the maternal-facing surface of the human placenta. The hST is covered by apical microvilli which are bathed by the maternal blood. This brush border epithelial membrane displays a number of transport properties including the ability to selectively transfer ions. Ion channels in hST allow the permeation of cations such as K and Ca, and anions such as Cl. 9 We recently identified the Ca-permeable, non-selective cation channel of hST as being a functional Polycystin-2, the gene product of one of the ADPKD-causing genes, PKD2. Little is known, however, about the mechanisms that control and regulate ion channel activity, in particular Polycystin-2, in this syncytial epithelium. The chorionic villous tree presents an intricate structure which is continuously growing by branching during gestation. This process requires a dynamic cytoskeleton. The hST apical membrane is supported by an intricate network of cytoskeletal structures. The apical cytoskeleton in hST encompasses a supramolecular structure known as the "syncytioskeletal layer" of a potentially supporting nature. Major cytoskeletal components including microtubules, 13 intermediate filaments, and actin-based networks 18 have been identified which may have distinct and interactive roles in the developing placenta. Apical hST microvilli have highly organized actin filaments, and apical hST membrane vesicles retain prominent microfilamental structures associated with the presence of structured actin. Thus, the microvillous actin cytoskele-
Stefan Somlo - One of the best experts on this subject based on the ideXlab platform.
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disrupting Polycystin 2 ef hand ca2 affinity does not alter channel function or contribute to polycystic kidney disease
Journal of Cell Science, 2020Co-Authors: Thuy N. Vien, Stefan Somlo, Peter C Harris, Jessica M Smith, Ke Dong, Matteus Krappitz, Vladimir G Gainullin, Sorin V Fedeles, Paul G. DecaenAbstract:Approximately 15% of autosomal dominant polycystic kidney disease (ADPKD) is caused by variants in PKD2. PKD2 encodes Polycystin-2, which forms an ion channel in primary cilia and endoplasmic reticulum (ER) membranes of renal collecting duct cells. Elevated internal Ca2+ modulates Polycystin-2 voltage dependent gating and subsequent desensitization— two biophysical regulatory mechanisms that control its function at physiological membrane potentials. Here, we refute the hypothesis that Ca2+ occupancy of the polycytsin-2 intracellular EF hand is responsible for these forms of channel regulation, and if disrupted, results in ADPKD. We identify and introduce mutations that attenuate Ca2+-EF hand affinity but find channel function is unaltered in the primary cilia and ER membranes. We generated two new mouse strains that harbor distinct mutations that abolish Ca2+-EF hand association but do not result in a PKD phenotype. Our findings suggest additional Ca2+ binding sites within Polycystin-2 or Ca2+-dependent modifiers are responsible for regulating channel activity.
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regulation of ryanodine receptor dependent calcium signaling by Polycystin 2
Proceedings of the National Academy of Sciences of the United States of America, 2007Co-Authors: Georgia I. Anyatonwu, Stefan Somlo, Manuel Estrada, Xin Tian, Barbara E. EhrlichAbstract:Mutations in Polycystin-2 (PC2) cause autosomal dominant polycystic kidney disease. A function for PC2 in the heart has not been described. Here, we show that PC2 coimmunoprecipitates with the cardiac ryanodine receptor (RyR2) from mouse heart. Biochemical assays showed that the N terminus of PC2 binds the RyR2, whereas the C terminus only binds to RyR2 in its open state. Lipid bilayer electrophysiological experiments indicated that the C terminus of PC2 functionally inhibited RyR2 channel activity in the presence of calcium (Ca2+). Pkd2−/− cardiomyocytes had a higher frequency of spontaneous Ca2+ oscillations, reduced Ca2+ release from the sarcoplasmic reticulum stores, and reduced Ca2+ content compared with Pkd2+/+ cardiomyocytes. In the presence of caffeine, Pkd2−/− cardiomyocytes exhibited decreased peak fluorescence, a slower rate of rise, and a longer duration of Ca2+ transients compared with Pkd2+/+. These data suggest that PC2 is important for regulation of RyR2 function and that loss of this regulation of RyR2, as occurs when PC2 is mutated, results in altered Ca2+ signaling in the heart.
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Polycystin-2 Regulates Proliferation and Branching Morphogenesis in Kidney Epithelial Cells
The Journal of biological chemistry, 2005Co-Authors: David H. Grimm, Stefan Somlo, Yiqiang Cai, Anil Karihaloo, Lloyd G. Cantley, Michael J. CaplanAbstract:Abstract 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.
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Polycystin-1 distribution is modulated by Polycystin-2 expression in mammalian cells.
The Journal of biological chemistry, 2003Co-Authors: David H. Grimm, Stefan Somlo, Lin Geng, Yiqiang Cai, Veronique Chauvet, Vanathy Rajendran, Raoul Zeltner, Ellis D. Avner, William E. Sweeney, Michael J. CaplanAbstract: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.
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Altered Expression Pattern of Polycystin-2 in Acute and Chronic Renal Tubular Diseases
Journal of the American Society of Nephrology : JASN, 2002Co-Authors: Nicholas Obermüller, Stefan Somlo, Yiqiang Cai, Bettina Kränzlin, R. Brent Thomson, Norbert Gretz, Wilhelm Kriz, Ralph WitzgallAbstract:Polycystin-2 represents one of so far two proteins found to be mutated in patients with autosomal-dominant polycystic kidney disease. Evidence obtained from experiments carried out in cell lines and with native kidney tissue strongly suggests that Polycystin-2 is located in the endoplasmic reticulum. In the kidney, Polycystin-2 is highly expressed in cells of the distal and connecting tubules, where it is located in the basal compartment. It is not known whether the expression of Polycystin-2 in the kidney changes or whether it can be manipulated under certain instances. Therefore, the distribution of Polycystin-2 under conditions leading to acute and chronic renal failure was analyzed. During ischemic acute renal failure, which affects primarily the S3 segment of the proximal tubule, a pronounced upregulation of Polycystin-2 and a predominantly combined homogeneous and punctate cytoplasmic distribution in damaged cells was observed. After thallium-induced acute injury to thick ascending limb cells, Polycystin-2 staining assumed a chicken wire-like pattern in damaged cells. In the (cy/+) rat, a model for autosomal-dominant polycystic kidney disease in which cysts originate predominantly from the proximal tubule, Polycystin-2 immunoreactivity was lost in some distal tubules. In kidneys from (pcy/pcy) mice, a model for autosomal-recessive polycystic kidney disease in which cyst formation primarily affects distal tubules and collecting ducts, a minor portion of cyst-lining cells cease to express Polycystin-2, whereas in the remaining cells, Polycystin-2 is retained in their basal compartment. Data show that the expression and cellular distribution of Polycystin-2 in different kinds of renal injuries depends on the type of damage and on the nephron-specific response to the injury. After ischemia, Polycystin-2 may be upregulated by the injured cells to protect themselves. It is unlikely that Polycystin-2 plays a role in cyst formation in the (cy/+) rat and in the (pcy/pcy) mouse.
Barbara E. Ehrlich - One of the best experts on this subject based on the ideXlab platform.
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decreased Polycystin 2 levels result in non renal cardiac dysfunction with aging
PLOS ONE, 2016Co-Authors: Ivana Y Kuo, Sophie L Duong, Lily Nguyen, Barbara E. EhrlichAbstract:Mutations in the gene for Polycystin 2 (Pkd2) lead to polycystic kidney disease, however the main cause of mortality in humans is cardiac related. We previously showed that 5 month old Pkd2+/- mice have altered calcium-contractile activity in cardiomyocytes, but have preserved cardiac function. Here, we examined 1 and 9 month old Pkd2+/- mice to determine if decreased amounts of functional Polycystin 2 leads to impaired cardiac function with aging. We observed changes in calcium handling proteins in 1 month old Pkd2+/- mice, and these changes were exacerbated in 9 month old Pkd2+/- mice. Anatomically, the 9 month old Pkd2+/- mice had thinner left ventricular walls, consistent with dilated cardiomyopathy, and the left ventricular ejection fraction was decreased. Intriguingly, in response to acute isoproterenol stimulation to examine β-adrenergic responses, the 9 month old Pkd2+/- mice exhibited a stronger contractile response, which also coincided with preserved localization of the β2 adrenergic receptor. Importantly, the Pkd2+/- mice did not have any renal impairment. We conclude that the cardiac-related impact of decreased Polycystin 2 progresses over time towards cardiac dysfunction and altered adrenergic signaling. These results provide further evidence that Polycystin 2 provides a critical function in the heart, independent of renal involvement.
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structure of the ef hand domain of Polycystin 2 suggests a mechanism for ca2 dependent regulation of Polycystin 2 channel activity
Proceedings of the National Academy of Sciences of the United States of America, 2010Co-Authors: Edward T. Petri, Barbara E. Ehrlich, Titus J. Boggon, Andjelka S Celic, Scott D Kennedy, Michael E HodsdonAbstract:The C-terminal cytoplasmic tail of Polycystin-2 (PC2/TRPP2), a Ca2+-permeable channel, is frequently mutated or truncated in autosomal dominant polycystic kidney disease. We have previously shown that this tail consists of three functional regions: an EF-hand domain (PC2-EF, 720–797), a flexible linker (798–827), and an oligomeric coiled coil domain (828–895). We found that PC2-EF binds Ca2+ at a single site and undergoes Ca2+-dependent conformational changes, suggesting it is an essential element of Ca2+-sensitive regulation of PC2 activity. Here we describe the NMR structure and dynamics of Ca2+-bound PC2-EF. Human PC2-EF contains a divergent non-Ca2+-binding helix-loop-helix (HLH) motif packed against a canonical Ca2+-binding EF-hand motif. This HLH motif may have evolved from a canonical EF-hand found in invertebrate PC2 homologs. Temperature-dependent steady-state NOE experiments and NMR R1 and R2 relaxation rates correlate with increased molecular motion in the EF-hand, possibly due to exchange between apo and Ca2+-bound states, consistent with a role for PC2-EF as a Ca2+-sensitive regulator. Structure-based sequence conservation analysis reveals a conserved hydrophobic surface in the same region, which may mediate Ca2+-dependent protein interactions. We propose that Ca2+-sensing by PC2-EF is responsible for the cooperative nature of PC2 channel activation and inhibition. Based on our results, we present a mechanism of regulation of the Ca2+ dependence of PC2 channel activity by PC2-EF.
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Polycystin-2 Contains an Unpaired EF-hand Motif which May Serve as a Ca2+-Sensitive Regulator of Polycystin-2 Channel Activity
Biophysical Journal, 2010Co-Authors: Andjelka S. Ćelić, Edward T. Petri, Titus J. Boggon, Michael E Hodsdon, Barbara E. EhrlichAbstract:Autosomal dominant polycystic kidney disease (ADPKD) is the most common, monogenic cause of kidney failure in humans. Most cases of ADPKD are linked with mutations in Polycystin-1 (PC1) and Polycystin-2 (PC2). PC2 is a calcium (Ca2+) permeable channel in the TRP channel family. Deletion of the C-terminus of PC2 alters Ca2+-signaling; the most common pathogenic mutations in PC2 are premature truncations. We previously showed that this tail consists of three functional regions: an unpaired EF-hand domain (PC2-EF), an oligomeric coiled coil domain, and a linker connecting them. We hypothesize that the EF-hand serves as a Ca2+-sensor/switch, and show that PC2 undergoes Ca2+-induced conformational changes by NMR, CD, and SAXS. We have solved the NMR structure of Ca2+-bound PC2-EF and have identified residues with chemical shift changes upon Ca2+-titration. PC2-EF contains a novel unpaired EF-hand fold which may have evolved from a canonical paired EF-hand found in invertebrate PC2 homologs. Human PC2-EF contains a divergent helix-loop-helix in place of a second EF-hand. Temperature dependent steady state NOE experiments and NMR linewidth measurements indicate increased molecular motion in the EF-hand consistent with a proposed role for PC2-EF as a Ca2+-sensitive regulator. Structure-based sequence conservation analysis reveals a conserved hydrophobic pocket in this region, where PC2-EF may mediate Ca2+-dependent protein interactions. Using results of our structural studies we have examined the role of the EF-hand and coiled coil on PC2 channel function in single-channel lipid bilayers. Our results suggest that the coiled coil regulates PC2 by serving as an homo-oligomerization motif, whereas the EF-hand modulates the Ca2+-dependence of PC2 channel activity. Based on our results we propose a mechanism of regulation of the Ca2+-dependence of PC2 channel activity by PC2-EF.
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Analysis of the cytoplasmic interaction between Polycystin-1 and Polycystin-2
American journal of physiology. Renal physiology, 2009Co-Authors: Jozefina Casuscelli, Barbara E. Ehrlich, Stefan Schmidt, Brenda Degray, Edward T. Petri, Andjelka S. Ćelić, Ewa Folta-stogniew, Titus J. BoggonAbstract:Autosomal dominant polycystic kidney disease (ADPKD) arises following mutations of either Pkd1 or Pkd2. The proteins these genes encode, Polycystin-1 (PC1) and Polycystin-2 (PC2), form a signaling ...
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Analysis of the cytoplasmic interaction between Polycystin-1 and Polycystin-2
American journal of physiology. Renal physiology, 2009Co-Authors: Jozefina Casuscelli, Barbara E. Ehrlich, Stefan Schmidt, Brenda Degray, Edward T. Petri, Andjelka S. Ćelić, Ewa Folta-stogniew, Titus J. BoggonAbstract:Autosomal dominant polycystic kidney disease (ADPKD) arises following mutations of either Pkd1 or Pkd2. The proteins these genes encode, Polycystin-1 (PC1) and Polycystin-2 (PC2), form a signaling complex using direct intermolecular interactions. Two distinct domains in the C-terminal tail of PC2 have recently been identified, an EF-hand and a coiled-coil domain. Here, we show that the PC2 coiled-coil domain interacts with the C-terminal tail of PC1, but that the PC2 EF-hand domain does not. We measured the K0.5 of the interaction between the C-terminal tails of PC1 and PC2 and showed that the direct interaction of these proteins is abrogated by a PC1 point mutation that was identified in ADPKD patients. Finally, we showed that overexpression of the PC1 C-terminal tail in MDCK cells alters the Ca2+ response, but that overexpression of the PC1 C-terminal tail containing the disease mutation does not. These results allow a more detailed understanding of the mechanism of pathogenic mutations in the cytoplasmic regions of PC1 and PC2.
Peter Koulen - One of the best experts on this subject based on the ideXlab platform.
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Distribution and function of Polycystin-2 in mouse retinal ganglion cells.
Neuroscience, 2011Co-Authors: Simon Kaja, Oloruntoyin A. Mafe, Ruby A. Parikh, Prasanthi Kandula, Chanakyaram A. Reddy, Elaine V. Gregg, Hua Xin, Peter Mitchell, Michael A. Grillo, Peter KoulenAbstract:Abstract The Polycystin family of transient receptor potential (TRP) channels form Ca 2+ regulated cation channels with distinct subcellullar localizations and functions. As part of heteromultimeric channels and multi-protein complexes, Polycystins control intracellular Ca 2+ 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 Ca 2+ activated, had a maximum slope conductance of 114 pS, and could be blocked in a dose-dependent manner by increasing concentrations of Mg 2+ . The cytosolic localization of Polycystin-2 in RGCs is in accordance with its function as intracellular Ca 2+ 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 Ca 2+ signaling.
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Polycystin-2 Expression and Function in Adult Mouse Lacrimal Acinar Cells
Investigative ophthalmology & visual science, 2011Co-Authors: Simon Kaja, Elaine V. Gregg, Jill D. Hilgenberg, Volodymyr Rybalchenko, Wanda E. Medina-ortiz, Peter KoulenAbstract:Exocytosis from the lacrimal gland is tightly regulated.1,2 Stress, aging, and genetic predisposition can contribute to lacrimal gland dysfunction resulting in lack of or inadequate tear fluid secretion, as manifested for instance in dry eye disease and Sjogren's syndrome.3 As a result of an aging society, the annual societal cost associated with dry eye disease is an estimated $55.4 million.4 A better understanding of the basic principles regulating lacrimal gland function is critical to the development of novel therapies for dry eye disease. The intracellular Ca2+ concentration in a cell is tightly controlled and local and temporal increases govern a large number of cellular processes,5,6 including tear secretion.2 Ca2+ can enter the intracellular environment of the cell either from the extracellular space through voltage-, ligand-, store-, or second messenger-activated Ca2+ channels,5,6 or from intracellular stores such as the endoplasmic reticulum (ER). Release of Ca2+ from intracellular stores can be mediated by three main groups of intracellular Ca2+ channels: inositol-1,4,5-trisphosphate receptors (IP3Rs), ryanodine receptors (RyRs), and Polycystin family transient receptor potential (TRPP) channels. The group of IP3Rs consists of three subtypes; all are ligand-gated ion channels, regulated by both their endogenous ligand IP3 and Ca2+.6,7 The three known isoforms of RyRs share high sequence homology with IP3Rs and form tetrameric complexes.6 TRPPs comprise eight proteins, three of which have been shown to be ion channels: Polycystin-2, Polycystin-like (PCL), and Polycystin-2L2.8–10 In addition, mechanisms that control Ca2+ uptake into intracellular stores or extrusion of Ca2+ into the extracellular space contribute critically to Ca2+ homeostasis and the shape of Ca2+ transients.5,6 Mechanistically, lacrimal fluid secretion is controlled by differential modulation of the intracellular Ca2+ concentration through two distinct pathways involving both cholinergic and adrenergic stimuli.1,2 Experimental evidence suggests that cholinergic stimuli activate an intracellular pathway dependent on intracellular Ca2+ release from IP3Rs,11 whereas β-adrenergic stimulation results in activation of RyRs.11–13 We have previously described the expression of both IP3R and RyR isoforms in mouse lacrimal gland acinar cells14 and been able to correlate their differential distribution patterns with their proposed functions in lacrimal acinar cells.14 However, to date there is no knowledge as to whether the intracellular Ca2+ channels of the TRPP group of channels are expressed in the lacrimal gland, and/or functionally contribute to tear secretion. Given their expression patterns and role in Ca2+ signaling in other tissues, we hypothesized that TRPP Ca2+ channels are functionally expressed in the acinar cells of the mouse lacrimal gland. We here for the first time describe the expression and differential subcellular distribution of all three TRPP channels, Polycystin-2, PCL, and Polycystin-2L2, in lacrimal acinar cells. Furthermore, we isolated Polycystin-2 channels from adult lacrimal gland and identified their biophysical properties.
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Polycystin-2 accelerates Ca2+ release from intracellular stores in Caenorhabditis elegans.
Cell calcium, 2005Co-Authors: Peter Koulen, R. Scott Duncan, Jiyuan Liu, Nancy E. Cohen, Jo-ann S. Yannazzo, Nathalie Mcclung, Courtney L. Lockhart, Michael Branden, Matthew BuechnerAbstract:Abstract Polycystin-2, a member of the TRP family of calcium channels, is encoded by the human PKD2 gene. Mutations in that gene can lead to swelling of nephrons into the fluid-filled cysts of polycystic kidney disease. In addition to expression in tubular epithelial cells, human Polycystin-2 is found in muscle and neuronal cells, but its cell biological function has been unclear. A homologue in Caenorhabditis elegans is necessary for male mating behavior. We compared the behavior, calcium signaling mechanisms, and electrophysiology of wild-type and pkd-2 knockout C. elegans. In addition to characterizing PKD-2-mediated aggregation and mating behaviors, we found that Polycystin-2 is an intracellular Ca2+ release channel that is required for the normal pattern of Ca2+ responses involving IP3 and ryanodine receptor-mediated Ca2+ release from intracellular stores. Activity of Polycystin-2 creates brief cytosolic Ca2+ transients with increased amplitude and decreased duration. Polycystin-2, along with the IP3 and ryanodine receptors, acts as a major calcium-release channel in the endoplasmic reticulum in cells where rapid calcium signaling is required, and Polycystin-2 activity is essential in those excitable cells for rapid responses to stimuli.
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Polycystin 2 is an intracellular calcium release channel
Nature Cell Biology, 2002Co-Authors: Sayoko Nishimura, Ralph Witzgall, Peter Koulen, Barbara E. Ehrlich, Yoshiko Maeda, Lin Geng, Stefan SomloAbstract:Polycystin-2, the product of the gene mutated in type 2 autosomal dominant polycystic kidney disease (ADPKD), is the prototypical member of a subfamily of the transient receptor potential (TRP) channel superfamily, which is expressed abundantly in the endoplasmic reticulum (ER) membrane. Here, we show by single channel studies that Polycystin-2 behaves as a calcium-activated, high conductance ER channel that is permeable to divalent cations. Epithelial cells overexpressing Polycystin-2 show markedly augmented intracellular calcium release signals that are lost after carboxy-terminal truncation or by the introduction of a disease-causing missense mutation. These data suggest that Polycystin-2 functions as a calcium-activated intracellular calcium release channel in vivo and that polycystic kidney disease results from the loss of a regulated intracellular calcium release signalling mechanism.
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Polycystin 2 is an intracellular calcium release channel
Nature Cell Biology, 2002Co-Authors: Sayoko Nishimura, Ralph Witzgall, Peter Koulen, Barbara E. Ehrlich, Yoshiko Maeda, Lin Geng, Stefan SomloAbstract:Polycystin-2, the product of the gene mutated in type 2 autosomal dominant polycystic kidney disease (ADPKD), is the prototypical member of a subfamily of the transient receptor potential (TRP) channel superfamily, which is expressed abundantly in the endoplasmic reticulum (ER) membrane. Here, we show by single channel studies that Polycystin-2 behaves as a calcium-activated, high conductance ER channel that is permeable to divalent cations. Epithelial cells overexpressing Polycystin-2 show markedly augmented intracellular calcium release signals that are lost after carboxy-terminal truncation or by the introduction of a disease-causing missense mutation. These data suggest that Polycystin-2 functions as a calcium-activated intracellular calcium release channel in vivo and that polycystic kidney disease results from the loss of a regulated intracellular calcium release signalling mechanism.