RESUMO
Mutations in the genes encoding polycystin-1 (PC1) and polycystin 2 (PC2) cause autosomal dominant polycystic kidney disease. These transmembrane proteins colocalize in the primary cilia of renal epithelial cells, where they may participate in sensory processes. PC1 is also found in the apical membrane when expressed in cultured epithelial cells. PC1 undergoes autocatalytic cleavage, producing an extracellular N-terminal fragment that remains noncovalently attached to the transmembrane C-terminus. Exposing cells to alkaline solutions elutes the N-terminal fragment while the C-terminal fragment is retained in the cell membrane. Utilizing this observation, we developed a "strip-recovery" synchronization protocol to study PC1 trafficking in polarized LLC-PK1 renal epithelial cells. Following alkaline strip, a new cohort of PC1 repopulates the cilia within 30 minutes, while apical delivery of PC1 was not detectable until 3 hours. Brefeldin A (BFA) blocked apical PC1 delivery, while ciliary delivery of PC1 was BFA insensitive. Incubating cells at 20°C to block trafficking out of the trans-Golgi network also inhibits apical but not ciliary delivery. These results suggest that newly synthesized PC1 takes distinct pathways to the ciliary and apical membranes. Ciliary PC1 appears to by-pass BFA sensitive Golgi compartments, while apical delivery of PC1 traverses these compartments.
Assuntos
Membrana Celular/metabolismo , Canais de Cátion TRPP/metabolismo , Animais , Linhagem Celular , Polaridade Celular , Retículo Endoplasmático/metabolismo , Células Epiteliais/metabolismo , Rim/citologia , Sinais Direcionadores de Proteínas , Transporte Proteico , Suínos , Canais de Cátion TRPP/químicaRESUMO
AMP-activated protein kinase (AMPK) activation promotes early stages of epithelial junction assembly. AMPK activation in MDCK renal epithelial cells facilitates localization of the junction-associated proteins aPKCζ and Par3 to the plasma membrane and promotes conversion of Cdc42, a key regulator of epithelial polarization and junction assembly, to its active GTP bound state. Furthermore, Par3 is an important regulator of AMPK-mediated aPKCζ localization. Both aPKCζ and Par3 serve as intermediates in AMPK-mediated junction assembly, with inhibition of aPKCζ activity or Par3 knockdown disrupting AMPK's ability to facilitate zonula occludens (ZO-1) localization. AMPK phosphorylates the adherens junction protein afadin and regulates its interaction with the tight-junction protein zonula occludens-1. Afadin is phosphorylated at two critical sites, S228 (residing within an aPKCζ consensus site) and S1102 (residing within an AMPK consensus site), that are differentially regulated during junction assembly and that exert different effects on the process. Expression of phospho-defective mutants (S228A and S1102A) perturbed ZO-1 localization to the plasma membrane during AMPK-induced junction assembly. Expression of S228A increased the ZO-1/afadin interaction, while S1102A reduced this interaction during extracellular calcium-induced junction assembly. Inhibition of aPKCζ activity also increased the ZO-1/afadin interaction. Taken together, these data suggest that aPKCζ phosphorylation of afadin terminates the ZO-1/afadin interaction and thus permits the later stages of junction assembly.
Assuntos
Proteínas Quinases Ativadas por AMP/fisiologia , Membrana Celular/enzimologia , Junções Íntimas/enzimologia , Animais , Membrana Celular/química , Cães , Células Madin Darby de Rim Canino , Camundongos , Fosforilação/fisiologia , Proteína Quinase C/metabolismo , Junções Íntimas/química , Proteína da Zônula de Oclusão-1/metabolismoRESUMO
The Ca(2+)-sensing receptor (CaSR) belongs to the G-protein-coupled receptor superfamily and plays essential roles in divalent ion homeostasis and cell differentiation. Because extracellular Ca(2+) is essential for the development of stable epithelial tight junctions (TJs), we hypothesized that the CaSR participates in regulating TJ assembly. We first assessed the expression of the CaSR in Madin-Darby canine kidney (MDCK) cells at steady state and following manipulations that modulate TJ assembly. Next, we examined the effects of CaSR agonists and antagonists on TJ assembly. Immunofluorescence studies indicate that endogenous CaSR is located at the basolateral pole of MDCK cells. Stable transfection of human CaSR in MDCK cells further reveals that this protein co-distributes with ß-catenin on the basolateral membrane. Switching MDCK cells from low-Ca(2+) medium to medium containing a normal Ca(2+) concentration significantly increases CaSR expression at both the mRNA and protein levels. Exposure of MDCK cells maintained in low-Ca(2+) conditions to the CaSR agonists neomycin, Gd(3+) or R-568 causes the transient relocation of the tight junction components ZO-1 and occludin to sites of cell-cell contact, while inducing no significant changes in the expression of mRNAs encoding junction-associated proteins. Stimulation of CaSR also increases the interaction between ZO-1 and the F-actin-binding protein I-afadin. This effect does not involve activation of the AMP-activated protein kinase. By contrast, CaSR inhibition by NPS-2143 significantly decreases interaction of ZO-1 with I-afadin and reduces deposition of ZO-1 at the cell surface following a Ca(2+) switch from 5 µM to 200 µM [Ca(2+)]e. Pre-exposure of MDCK cells to the cell-permeant Ca(2+) chelator BAPTA-AM, similarly prevents TJ assembly caused by CaSR activation. Finally, stable transfection of MDCK cells with a cDNA encoding a human disease-associated gain-of-function mutant form of the CaSR increases the transepithelial electrical resistance of these cells in comparison to expression of the wild-type human CaSR. These observations suggest that the CaSR participates in regulating TJ assembly.
Assuntos
Sinalização do Cálcio/genética , Células Epiteliais/metabolismo , Receptores de Detecção de Cálcio/metabolismo , Junções Íntimas/metabolismo , Animais , Cálcio/metabolismo , Diferenciação Celular/genética , Membrana Celular/metabolismo , Cães , Regulação da Expressão Gênica no Desenvolvimento , Humanos , Receptores de Detecção de Cálcio/genética , Junções Íntimas/genética , TransfecçãoRESUMO
Autosomal dominant polycystic kidney disease (ADPKD) is the most prevalent potentially lethal monogenic disorder. Mutations in the PKD1 gene, which encodes polycystin-1 (PC1), account for approximately 78% of cases. PC1 is a large 462-kDa protein that undergoes cleavage in its N and C-terminal domains. C-terminal cleavage produces fragments that translocate to mitochondria. We show that transgenic expression of a protein corresponding to the final 200 amino acid (aa) residues of PC1 in two Pkd1-KO orthologous murine models of ADPKD suppresses cystic phenotype and preserves renal function. This suppression depends upon an interaction between the C-terminal tail of PC1 and the mitochondrial enzyme Nicotinamide Nucleotide Transhydrogenase (NNT). This interaction modulates tubular/cyst cell proliferation, the metabolic profile, mitochondrial function, and the redox state. Together, these results suggest that a short fragment of PC1 is sufficient to suppress cystic phenotype and open the door to the exploration of gene therapy strategies for ADPKD.
Assuntos
NADP Trans-Hidrogenase Específica para A ou B , Rim Policístico Autossômico Dominante , Canais de Cátion TRPP , Humanos , Animais , Camundongos , Modelos Animais de Doenças , Canais de Cátion TRPP/genética , Canais de Cátion TRPP/metabolismo , Rim Policístico Autossômico Dominante/genética , Rim Policístico Autossômico Dominante/patologia , Rim Policístico Autossômico Dominante/terapia , Rim/patologia , Rim/fisiologia , NADP Trans-Hidrogenase Específica para A ou B/metabolismo , Proteínas Mitocondriais/metabolismoRESUMO
Polycystin-1 (PC-1) is the product of the PKD1 gene, which is mutated in autosomal dominant polycystic kidney disease. We show that the Na,K-ATPase alpha-subunit interacts in vitro and in vivo with the final 200 amino acids of the polycystin-1 protein, which constitute its cytoplasmic C-terminal tail. Functional studies suggest that this association may play a role in the regulation of the Na,K-ATPase activity. Chinese hamster ovary cells stably expressing the entire PC-1 protein exhibit a dramatic increase in Na,K-ATPase activity, although the kinetic properties of the enzyme remain unchanged. These data indicate that polycystin-1 may contribute to the regulation of Na,K-ATPase activity in kidneys in situ, thus modulating renal tubular fluid and electrolyte transport.
Assuntos
Ouabaína/farmacologia , Proteínas , ATPase Trocadora de Sódio-Potássio/metabolismo , ATPase Trocadora de Sódio-Potássio/fisiologia , Sódio/farmacologia , Animais , Células CHO/ultraestrutura , Linhagem Celular , Cricetinae , Cricetulus , Cães , Inibidores Enzimáticos , Escherichia , Mutação , Doenças Renais Policísticas , Proteínas Recombinantes , ATPase Trocadora de Sódio-Potássio/biossíntese , ATPase Trocadora de Sódio-Potássio/efeitos dos fármacos , Canais de Cátion TRPP , TransfecçãoRESUMO
The sorting and regulation of the Na,K and H,K-ATPases requires that the pump proteins must associate, at least transiently, with kinases, phosphatases, scaffolding molecules, and components of the cellular trafficking machinery. The identities of these interacting proteins and the nature of their associations with the pump polypeptides have yet to be elucidated. We have begun a series of yeast two-hybrid screens employing structurally defined segments of pump polypeptides as baits in order to gain insight into the nature and function of these interacting proteins.
Assuntos
Proteínas de Transporte/metabolismo , Bombas de Íon/química , Bombas de Íon/metabolismo , Animais , ATPases Transportadoras de Cálcio/química , ATPases Transportadoras de Cálcio/metabolismo , Proteínas de Transporte/química , ATPase Trocadora de Hidrogênio-Potássio/química , ATPase Trocadora de Hidrogênio-Potássio/metabolismo , Proteínas de Membrana/química , Proteínas de Membrana/metabolismo , Modelos Moleculares , Fosfoproteínas Fosfatases/química , Fosfoproteínas Fosfatases/metabolismo , Fosforilação , Ligação Proteica , Conformação Proteica , Estrutura Secundária de Proteína , ATPase Trocadora de Sódio-Potássio/química , ATPase Trocadora de Sódio-Potássio/metabolismoRESUMO
Polycystin (PC)1 and PC2 are membrane proteins implicated in autosomal dominant polycystic kidney disease. A physiologically relevant cleavage at PC1's G protein-coupled receptor proteolytic site (GPS) occurs early in the secretory pathway. Our results suggest that PC2 increases both PC1 GPS cleavage and PC1's appearance at the plasma membrane. Mutations that prevent PC1's GPS cleavage prevent its plasma membrane localization. PC2 is a member of the trp family of cation channels and is an important PC1 binding partner. The effect of PC2 on PC1 localization is independent of PC2 channel activity, as tested using channel-inhibiting PC2 mutations. PC1 and PC2 can interact through their C-terminal tails, but removing the C-terminal tail of either protein has no effect on PC1 surface localization in human embryonic kidney 293 cells. Experiments in polarized LLC-PK cells show that apical and ciliary PC1 localization requires PC2 and that this delivery is sensitive to PC2 truncation. In sum, our work shows that PC2 expression is required for the movement of PC1 to the plasma and ciliary membranes. In fibroblast cells this localization effect is independent of PC2's channel activity or PC1 binding ability but involves a stimulation of PC1's GPS cleavage before the PC1 protein's surface delivery.
Assuntos
Rim/metabolismo , Rim Policístico Autossômico Dominante/genética , Processamento de Proteína Pós-Traducional , Canais de Cátion TRPP/metabolismo , Animais , Western Blotting , Membrana Celular , Cílios/metabolismo , Imunofluorescência , Células HEK293 , Humanos , Imunoprecipitação , Rim/patologia , Células LLC-PK1 , Mutação , Rim Policístico Autossômico Dominante/metabolismo , Ligação Proteica , Isoformas de Proteínas , Estrutura Terciária de Proteína , Transporte Proteico , Suínos , Canais de Cátion TRPP/química , Canais de Cátion TRPP/genéticaRESUMO
Polycystin-1 (PC1) is a large, membrane-bound protein that localizes to the cilia and is implicated in the common ciliopathy autosomal-dominant polycystic kidney disease. The physiological function of PC1 is dependent upon its subcellular localization as well as specific cleavages that release soluble fragments of its C-terminal tail. The techniques described here allow visualization and quantification of these aspects of the biology of the PC1 protein. To visualize PC1 at the plasma membrane, a live-cell surface labeling immunofluorescence protocol paired with the labeling of an internal antigen motif allows a robust detection of the surface population of this protein. This technique is modified to generate a surface enzyme-linked immunosorbent assay (ELISA), which quantitatively measures the amount of surface protein as a fraction of the total amount of the protein expressed in that cell population. These assays are powerful tools in the assessment of the small but biologically important pool of PC1 that reaches the cell surface. The C-terminal tail cleavage of PC1 constitutes an interesting modification that allows PC1 to extend its functional role into the nucleus. A reporter assay based on Gal4/VP16 luciferase can be used to quantitate the amount of PC1 C-terminal tail that reaches the nucleus. This assay can be paired with quantitative measurement of the protein expression in the cell, allowing a more complete understanding of the pattern of PC1 cleavage and the nuclear localization of the resultant.
Assuntos
Citoplasma/metabolismo , Ensaio de Imunoadsorção Enzimática , Imunofluorescência , Canais de Cátion TRPP/metabolismo , Animais , Linhagem Celular , Membrana Celular/metabolismo , Citoplasma/química , Ensaio de Imunoadsorção Enzimática/instrumentação , Ensaio de Imunoadsorção Enzimática/métodos , Imunofluorescência/instrumentação , Imunofluorescência/métodos , Genes Reporter , Humanos , Luciferases/genética , Luciferases/metabolismo , Proteínas de Membrana/química , Proteínas de Membrana/metabolismo , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Canais de Cátion TRPP/químicaRESUMO
Biodegradable polymer nanoparticles (NPs) are a promising approach for intracellular delivery of drugs, proteins, and nucleic acids, but little is known about their intracellular fate, particularly in epithelial cells, which represent a major target. Rhodamine-loaded PLGA (polylactic-co-glycolic acid) NPs were used to explore particle uptake and intracellular fate in three different epithelial cell lines modeling the respiratory airway (HBE), gut (Caco-2), and renal proximal tubule (OK). To track intracellular fate, immunofluorescence techniques and confocal microscopy were used to demonstrate colocalization of NPs with specific organelles: early endosomes, late endosomes, lysosomes, endoplasmic reticulum (ER), and Golgi apparatus. Confocal analysis demonstrated that NPs are capable of entering cells of all three types of epithelium. NPs appear to colocalize with the early endosomes at short times after exposure (approximately 2 h), but are also found in other compartments within the cytoplasm, notably Golgi and, possibly, ER, as time progressed over the period of 4-24 h. The rate and extent of uptake differed among these cell lines: at a fixed particle/cell ratio, cellular uptake was most abundant in OK cells and least abundant in Caco-2 cells. We present a model for the intracellular fate of particles that is consistent with our experimental data.
Assuntos
Células Epiteliais/metabolismo , Espaço Intracelular/metabolismo , Ácido Láctico/química , Ácido Láctico/metabolismo , Nanopartículas/química , Ácido Poliglicólico/química , Ácido Poliglicólico/metabolismo , Linhagem Celular , Humanos , Microscopia Eletrônica de Varredura , Nanopartículas/ultraestrutura , Copolímero de Ácido Poliláctico e Ácido PoliglicólicoRESUMO
We have previously reported that three residues of the fourth transmembrane segment (TM4) of the Na,K- and gastric H,K-ATPase alpha-subunits appear to play a major role in the distinct cation selectivities of these pumps [Mense, M., et al. (2000) J. Biol. Chem. 275, 1749-1756]. Substituting these three residues in the Na,K-ATPase sequence with their H,K-ATPase counterparts (L319F, N326Y, T340S) and replacing the TM3-TM4 ectodomain sequence with that of the H,K-ATPase alpha-subunit result in a pump that exhibits 50% of its maximal ATPase activity in the absence of Na(+) when the assay is performed at pH 6.0. This effect is not seen when the ectodomain alone is replaced. To gain more insight into the contributions of the three residues to establishing the selectivity of these pumps for Na(+) ions versus protons, we generated Na,K-ATPase constructs in which these residues are replaced by their H,K-ATPase counterparts either singly or in combinations. Surprisingly, none of the point mutants nor even the triple mutant was able to hydrolyze ATP at pH 6.0 at a rate greater than 20% of their respective V(max)s. For the point mutants L319F and N326Y, protons seem to competitively inhibit ATP hydrolysis at pH 6.0, based on the low apparent affinity for Na(+) ions at pH 6.0 compared to pH 7.5. It would appear, therefore, that the cation selectivity of Na,K- and H,K-ATPase is generated through a cooperative effort between residues of transmembrane segments and the flanking loops that connect these transmembrane domains. This view is further supported by homology modeling of the Na,K-ATPase based on the crystal structure of the SERCA pump.
Assuntos
ATPase Trocadora de Hidrogênio-Potássio/metabolismo , ATPase Trocadora de Sódio-Potássio/metabolismo , Estômago/enzimologia , Animais , Sequência de Bases , Cátions , Primers do DNA , ATPase Trocadora de Hidrogênio-Potássio/química , ATPase Trocadora de Hidrogênio-Potássio/genética , Concentração de Íons de Hidrogênio , Células LLC-PK1 , Mutação Puntual , ATPase Trocadora de Sódio-Potássio/química , ATPase Trocadora de Sódio-Potássio/genética , Suínos , Vanadatos/metabolismoRESUMO
Cystic fibrosis is caused by mutations in the gene encoding the cystic fibrosis transmembrane conductance regulator (CFTR). The most common mutation, DeltaF508, results in the production of a misfolded CFTR protein that is retained in the endoplasmic reticulum and targeted for degradation. Curcumin is a nontoxic Ca-adenosine triphosphatase pump inhibitor that can be administered to humans safely. Oral administration of curcumin to homozygous DeltaF508 CFTR mice in doses comparable, on a weight-per-weight basis, to those well tolerated by humans corrected these animals' characteristic nasal potential difference defect. These effects were not observed in mice homozygous for a complete knockout of the CFTR gene. Curcumin also induced the functional appearance of DeltaF508 CFTR protein in the plasma membranes of transfected baby hamster kidney cells. Thus, curcumin treatment may be able to correct defects associated with the homozygous expression of DeltaF508 CFTR.
Assuntos
Membrana Celular/metabolismo , Curcumina/farmacologia , Regulador de Condutância Transmembrana em Fibrose Cística/metabolismo , Fibrose Cística/tratamento farmacológico , Retículo Endoplasmático/metabolismo , Mucosa Nasal/efeitos dos fármacos , Animais , Cálcio/metabolismo , Calnexina/metabolismo , Linhagem Celular , Cricetinae , Curcumina/administração & dosagem , Curcumina/uso terapêutico , Fibrose Cística/genética , Fibrose Cística/fisiopatologia , Regulador de Condutância Transmembrana em Fibrose Cística/química , Regulador de Condutância Transmembrana em Fibrose Cística/genética , Eletrólitos/farmacologia , Marcação de Genes , Glicosilação , Humanos , Mucosa Intestinal/efeitos dos fármacos , Mucosa Intestinal/fisiologia , Obstrução Intestinal/prevenção & controle , Isoproterenol/farmacologia , Potenciais da Membrana/efeitos dos fármacos , Camundongos , Camundongos Knockout , Mutação , Mucosa Nasal/fisiologia , Polietilenoglicóis/farmacologia , Dobramento de Proteína , Reto , TransfecçãoRESUMO
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.
Assuntos
Proteínas de Membrana/biossíntese , Biossíntese de Proteínas , Proteínas , Animais , Western Blotting , Células COS , Linhagem Celular , Membrana Celular/metabolismo , Células Cultivadas , DNA Complementar/metabolismo , Retículo Endoplasmático/metabolismo , Regulação da Expressão Gênica , Camundongos , Camundongos Transgênicos , Microscopia de Fluorescência , Modelos Biológicos , Mutação , Testes de Precipitina , Ligação Proteica , RNA Mensageiro/metabolismo , Proteínas Recombinantes de Fusão/metabolismo , Canais de Cátion TRPP , TransfecçãoRESUMO
Renal regulation of mammalian water homeostasis is mediated by the aquaporin-1 (AQP1) water channel, which is expressed in the apical and basolateral membranes of proximal tubules and descending limbs of Henle, and aquaporin-2 (AQP2), which is redistributed from intracellular vesicles to the apical membrane (AM) of collecting duct cells with vasopressin. In transfected Madin-Darby canine kidney cells, AQP1 and AQP2 are regulated similarly, which indicates that routing elements reside in their primary sequences. We studied the role of the AQP2 COOH terminus in apical routing and AQP2 shuttling. An AQP1 chimera (AQP1 with an AQP2 tail: AQP1/2-N220) was located only in the AM independent of forskolin treatment. Forskolin increased the apical expression of AQP1 and AQP1/2-N220 less than twofold; that of AQP2 increased more than fourfold with concomitant changes in osmotic water permeabilities. The dimeric AQP2 tail coupled to placental alkaline phosphatase (AQP2-Plap) was retained in intracellular vesicles different from those of homotetrameric wild-type AQP2; the same protein without the AQP2 tail (TMR-Plap) was only expressed in the AM. The study shows that the AQP2 COOH tail is necessary but not sufficient for routing to the AM and suggests that other parts of AQP2 are needed for AQP2 accumulation in intracellular vesicles.