scholarly journals Interaction of a farnesylated protein with renal type IIa Na/Pi co-transporter in response to parathyroid hormone and dietary phosphate

2004 ◽  
Vol 377 (3) ◽  
pp. 607-616 ◽  
Author(s):  
Mikiko ITO ◽  
Sachi IIDAWA ◽  
Michiyo IZUKA ◽  
Sakiko HAITO ◽  
Hiroko SEGAWA ◽  
...  
Keyword(s):  
Parathyroid Hormone ◽  
Rat Kidney ◽  
Intracellular Loop ◽  
Interacting Protein ◽  
Opossum Kidney ◽  
Opossum Kidney Cells ◽  
Dietary Phosphate ◽  
Dibasic Amino Acid ◽  
Farnesylated Protein ◽  
Hybrid Screening

Treatment with PTH (parathyroid hormone) or a high-Pi diet causes internalization of the type IIa sodium-dependent phosphate (Na/Pi IIa) co-transporter from the apical membrane and its degradation in the lysosome. A dibasic amino acid motif (KR) in the third intracellular loop of the co-transporter is essential for protein's PTH-induced retrieval. To elucidate the mechanism of internalization of Na/Pi IIa, we identified the interacting protein for the endocytic motif by yeast two-hybrid screening. We found a strong interaction of the Na/Pi IIa co-transporter with a small protein known as the PEX19 (human peroxisomal farnesylated protein; PxF, Pex19p). PEX19 can bind to the KR motif, but not to a mutant with this motif replaced with NI residues. PEX19 is highly expressed in mouse and rat kidney. Western blot analysis indicates that PEX19 is located in the cytosolic and brush-border membrane fractions (microvilli and the subapical component). Overexpression of PEX19 stimulated the endocytosis of the Na/Pi IIa co-transporter in opossum kidney cells in the absence of PTH. In conclusion, the present study indicates that PEX19 may be actively involved in controlling the internalization and trafficking of the Na/Pi IIa co-transporter.

Different effects of arsenate and phosphonoformate on Pi transport adaptation in opossum kidney cells

2009 ◽  
Vol 297 (3) ◽  
pp. C516-C525 ◽  
Author(s):  
Ricardo Villa-Bellosta ◽  
Víctor Sorribas
Keyword(s):  
Rat Kidney ◽  
Opossum Kidney ◽  
Ok Cells ◽  
Opossum Kidney Cells ◽  
Competitive Inhibitors ◽  
Renal Adaptation ◽  
Proximal Tubular ◽  
Dietary Phosphate ◽  
Definition Of

The main nonhormonal mechanism for controlling inorganic phosphate (Pi) homeostasis is renal adaptation of the proximal tubular Pi transport rate to changes in dietary phosphate content. Opossum kidney (OK) cell line is an in vitro renal model that maintains the ability of renal adaptation to the extracellular Pi concentration. We have studied how two competitive inhibitors of Pi transport, arsenate [As(V)] and phosphonoformate (PFA), affect adaptation to low and high Pi concentrations. OK cells show very high affinity for As(V) (inhibitory constant, Ki 0.12 mM) when compared with the rat kidney. As(V) very efficiently reversed the adaptation of OK cells to low Pi (0.1 mM), whereas PFA induced adaptation similar to 0.1 mM Pi. Adaptation with 2 mM Pi or As(V) was characterized by decreases in the maximal velociy ( Vmax) of Pi transport and an abundance of the NaPi-IIa Pi transporter in the plasma membrane, shown by the protein biotinylation. Conversely, PFA and 0.1 mM Pi increased the Vmax and transporter abundance. Changes in the Vmax were limited to a 50% variation, which was not paralleled by changes in the concentration of Pi or of the inhibitor. OK cells are very sensitive to As(V), but the effects are reversible and noncytotoxic. These effects can be interpreted as As(V) being transported into the cell, thereby mimicking a high Pi concentration. PFA blocks the uptake of Pi but is not transported, and it therefore simulates a low Pi concentration inside the cell. To conclude, a mathematical definition of the adaptation process is reported, thereby explaining the limited changes in Pi transport Vmax.

scholarly journals Human munc13 Is a Diacylglycerol Receptor that Induces Apoptosis and May Contribute to Renal Cell Injury in Hyperglycemia

1999 ◽  
Vol 10 (5) ◽  
pp. 1609-1619 ◽  
Author(s):  
Yong Song ◽  
Menachem Ailenberg ◽  
Mel Silverman
Keyword(s):  
Phorbol Ester ◽  
Renal Cell ◽  
Cell Injury ◽  
Rat Kidney ◽  
Diabetic Rat ◽  
Opossum Kidney ◽  
Opossum Kidney Cells ◽  
Renal Cell Line ◽  
Diabetic Rat Model

We have previously shown that human munc13 (hmunc13) is up-regulated by hyperglycemia under in vitro conditions in human mesangial cell cultures. The purpose of the present study was to determine the cellular function of hmunc13. To do this, we have investigated the subcellular localization of hmunc13 in a transiently transfected renal cell line, opossum kidney cells. We have found that hmunc13 is a cytoplasmic protein and is translocated to the Golgi apparatus after phorbol ester stimulation. In addition, cells transfected with hmunc13 demonstrate apoptosis after treatment with phorbol ester, but cells transfected with an hmunc13 deletion mutant in which the diacylglycerol (C1) binding domain is absent exhibit no change in intracellular distribution and no induction of apoptosis in the presence of phorbol ester stimulation. We conclude that both the diacylglycerol-induced translocation and the apoptosis represent functional activity of hmunc13. We have also demonstrated that munc13-1 and munc13-2 are localized mainly to cortical epithelial cells in rat kidney and both are overexpressed under conditions of hyperglycemia in a streptozotocin-treated diabetic rat model. Taken together, our data suggest that hmunc13 serves as a diacylglycerol-activated, PKC-independent signaling pathway capable of inducing apoptosis and that this pathway may contribute to the renal cell complications of hyperglycemia.

Regulation of Na+/H+ exchange in opossum kidney cells by parathyroid hormone, cyclic AMP and phorbol esters

1990 ◽  
Vol 415 (4) ◽  
pp. 461-470 ◽  
Author(s):  
Corinna Helmle-Kolb ◽  
Marshall H. Montrose ◽  
Gerti Stange ◽  
Heini Murer
Keyword(s):  
Parathyroid Hormone ◽  
Cyclic Amp ◽  
Phorbol Esters ◽  
Kidney Cells ◽  
Opossum Kidney ◽  
Opossum Kidney Cells

Parathyroid hormone degradation by opossum kidney cells via receptor-mediated endocytosis and lysosomal hydrolysis

1992 ◽  
Vol 127 (3) ◽  
pp. 267-270 ◽  
Author(s):  
Toru Yamaguchi ◽  
Makoto Arao ◽  
Masaaki Fukase
Keyword(s):  
Parathyroid Hormone ◽  
Biologically Active ◽  
Main Role ◽  
Kidney Cells ◽  
Binding Studies ◽  
Opossum Kidney ◽  
Receptor Mediated Endocytosis ◽  
Opossum Kidney Cell ◽  
Opossum Kidney Cells

The mechanisms involved in parathyroid hormone (PTH) degradation by proximal renal tubule cells were studied using an opossum kidney cell line possessing PTH receptors as an in vitro model system. One hour incubation of 5 nmol/l human (h) PTH-(1-84) with intact opossum kidney cells (4.0× 106 cells) resulted in about 70% degradation and disappearance of hPTH-(1-84) from the medium, as determined by a two-site immunoradiometric assay. Preincubation with 100 nmol/l h[Nle8, Nle18, Tyr34]PTH-(1-34)amide for 6, 24, 48 and 72 h caused a 26, 47, 62 and 73% decrease, respectively, in PTH degradation by opossum kidney cells. Binding studies with 125I-labeled h[Nle8, Nle18, Tyr34]PTH-(1-34)amide as a radioligand showed that PTH receptor binding decreased with the time of pretreatment with the agonist. Pretreatments of the cells with monensin, an inhibitor of endocytosis, and the lysosomotropic agents such as chloroquine, ammonium chloride and leupeptin, inhibited degradation of hPTH-(1-84) by 87, 71, 76 and 72%, respectively. Concentrations of 5 nmol/l hPTH-(39-84) and hPTH-(39-68), which are known not to bind to PTH receptors appreciably, were not degraded by opossum kidney cells during 1 h incubations. Thus intact, biologically active PTH, but not its inactive fragments, is degraded by opossum kidney cells, by receptor-mediated endocytosis and lysosomal hydrolysis. A mechanism resembling the peritubular uptake of intact PTH by perfused kidneys reported previously appears to play a main role in PTH metabolism by cultured renal cells.

Interactions of cubilin with megalin and the product of the amnionless gene (AMN): effect on its stability

2008 ◽  
Vol 410 (2) ◽  
pp. 301-308 ◽  
Author(s):  
Rajiv Ahuja ◽  
Raghunatha Yammani ◽  
Joseph A. Bauer ◽  
Seema Kalra ◽  
Shakuntla Seetharam ◽  
...  
Keyword(s):  
Rat Kidney ◽  
Intrinsic Factor ◽  
Transmembrane Proteins ◽  
Immunohistochemical Analysis ◽  
Protein Product ◽  
Kidney Cells ◽  
Opossum Kidney ◽  
Opossum Kidney Cells ◽  
Polarized Epithelia

Cubilin, a 456 kDa multipurpose receptor lacking in both transmembrane and cytoplasmic domains is expressed in the apical BBMs (brush border membranes) of polarized epithelia. Cubilin interacts with two transmembrane proteins, AMN, a 45–50 kDa protein product of the amnionless gene, and megalin, a 600 kDa giant endocytic receptor. In vitro, three fragments of cubilin, the 113-residue N-terminus and CUB domains 12–17 and 22–27, demonstrated Ca2+-dependent binding to megalin. Immunoprecipitation and immunoblotting studies using detergent extracts of rat kidney BBMs revealed that cubilin interacts with both megalin and AMN. Ligand (intrinsic factor–cobalamin)-affinity chromatography showed that in renal BBMs, functional cubilin exists as a complex with both AMN and megalin. Cubilin and AMN levels were reduced by 80% and 55–60% respectively in total membranes and BBMs obtained from kidney of megalin antibody-producing rabbits. Immunohistochemical analysis and turnover studies for cubilin in megalin or AMN gene-silenced opossum kidney cells showed a significant reduction (85–90%) in cubilin staining and a 2-fold decrease in its half-life. Taken together, these results indicate that three distinct regions of cubilin bind to megalin and its interactions with both megalin and AMN are essential for its intracellular stability.

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