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1
Lou, Xiaojing, Tammie McQuistan, Robert A. Orlando, and Marilyn Gist Farquhar. "GAIP, GIPC and Gαi3 are Concentrated in Endocytic Compartments of Proximal Tubule Cells: Putative Role in Regulating Megalin’s Function." Journal of the American Society of Nephrology 13, no. 4 (April 2002): 918–27. http://dx.doi.org/10.1681/asn.v134918.
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ABSTRACT. Megalin is the most abundant endocytic receptor in the proximal tubule epithelium (PTE), where it is concentrated in clathrin-coated pits (CCPs) and vesicles in the brush border region. The heterotrimeric G protein alpha subunit, Gαi3, has also been localized to the brush border region of PTE. By immunofluorescence GIPC and GAIP, components of G protein-mediated signaling pathways, are also concentrated in the brush border region of PTE and are present in megalin-expressing cell lines. By cell fractionation, these signaling molecules cosediment with megalin in brush border and microvillar fractions. GAIP is found by immunoelectron microscopy in CCPs, and GIPC is found in CCPs and apical tubules of endocytic compartments in the renal brush border. In precipitation assays, GST-GIPC specifically binds megalin. The concentration of Gαi3, GIPC, and GAIP with megalin in endocytic compartments of the proximal tubule, where extensive endocytosis occurs, and the interaction between GIPC and the cytoplasmic tail of megalin suggest a model whereby G protein-mediated signaling may regulate megalin’s endocytic function and/or trafficking.
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RADER, KATHERINE, ROBERT A. ORLANDO, XIAOJING LOU, and MARILYN GIST FARQUHAR. "Characterization of ANKRA, a Novel Ankyrin Repeat Protein that Interacts with the Cytoplasmic Domain of Megalin." Journal of the American Society of Nephrology 11, no. 12 (December 2000): 2167–78. http://dx.doi.org/10.1681/asn.v11122167.
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Abstract. Ankyrin-repeat family A protein (ANKRA) is a novel protein that interacts directly and specifically with the cytoplasmic tail of megalin in the yeast two-hybrid system and glutathione-S-transferase pull-down assays. ANKRA has three ankyrin repeats and shows 61% overall homology to regulatory factor X, ankyrin repeat-containing protein. Mapping studies show that the three ankyrin repeats and C-terminus of ANKRA are required for binding to a unique juxtamembrane, 19-amino acid sequence on the megalin tail. Point mutational analysis reveals that a proline-rich motif (PXXPXXP) within this region is the site of ANKRA binding. ANKRA interacts with megalin but not with low-density lipoprotein receptor related protein, in keeping with the fact that the sequence of the megalin tail is unique. By cell fractionation, ANKRA is found both in the cytosol and associated with membranes enriched in megalin in L2 cells and proximal tubule cells. By immunofluorescence, ANKRA is concentrated near megalin along the plasma membrane of L2 cells and in the kidney cortex is expressed in glomerular and proximal tubule epithelia which also express megalin. These observations suggest that ANKRA may play a unique role in megalin's function as a clearance receptor in the kidney and L2 cells. In addition, ANKRA may have other partners because northern blot analysis reveals that ANKRA is more broadly expressed than megalin, and by immunofluorescence ANKRA is also expressed in connecting tubule cells and principal cells of collecting ducts.
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Takeda, Tetsuro, Hajime Yamazaki, and Marilyn G. Farquhar. "Identification of an apical sorting determinant in the cytoplasmic tail of megalin." American Journal of Physiology-Cell Physiology 284, no. 5 (May 1, 2003): C1105—C1113. http://dx.doi.org/10.1152/ajpcell.00514.2002.
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Megalin is the main endocytic receptor of the proximal tubule and is responsible for reabsorption of many filtered proteins. In contrast to other members of the low-density lipoprotein (LDL) receptor gene family, it is expressed on the apical plasma membrane (PM) of polarized epithelial cells. To identify megalin's apical sorting signal, we generated deletion mutants and chimeric minireceptors composed of complementary regions of megalin and LDL receptor-related protein (LRP) and assessed the distribution of the mutants in Madin-Darby canine kidney (MDCK) cells by immunofluorescence and cell surface biotinylation. Megalin and LRP minireceptors are correctly targeted to the apical and basolateral PM, respectively, of MDCK cells. We found that the information that directs apical sorting is present in the cytoplasmic tail (CT) of megalin, which contains three NPXY motifs, YXXØ, SH3, and dileucine motifs, and a PDZ-binding motif at its COOH terminus. Deletion analysis established that amino acids 107–136 of the megalin-CT containing the second NPXY-like motif are critical for apical sorting and targeting, whereas the regions containing the first and third NPXY motifs are required for efficient endocytosis. We conclude that the megalin-CT contains a novel apical sorting determinant and that cytoplasmic sorting machinery exists in MDCK cells for some apical transmembrane proteins.
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Hosojima, Michihiro, Hiroyoshi Sato, Keiko Yamamoto, Ryohei Kaseda, Taeko Soma, Asako Kobayashi, Akiyo Suzuki, et al. "Regulation of Megalin Expression in Cultured Proximal Tubule Cells by Angiotensin II Type 1A Receptor- and Insulin-Mediated Signaling Cross Talk." Endocrinology 150, no. 2 (February 1, 2009): 871–78. http://dx.doi.org/10.1210/en.2008-0886.
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Impairment of proximal tubular endocytosis of glomerular-filtered proteins including albumin results in the development of proteinuria/albuminuria in patients with chronic kidney disease. However, the mechanisms regulating the proximal tubular function are largely unknown. This study aimed to investigate the role of angiotensin II type 1A receptor (AT1AR)- and insulin-mediated signaling pathways in regulating the expression of megalin, a multiligand endocytic receptor in proximal tubule cells (PTCs). Opossum kidney PTC-derived OK cells that stably express rat AT1AR but are deficient in endogenous angiotensin II receptors (AT1AR-OK cells) were used for this study. Treatment of the cells with angiotensin II suppressed mRNA and protein expression of megalin at 3- and 24-h incubation time points, respectively. Cellular uptake and degradation of albumin and receptor-associated protein, megalin’s endocytic ligands were suppressed 24 h after angiotensin II treatment. The AT1AR-mediated decrease in megalin expression was partially prevented by ERK inhibitors. Insulin competed with the AT1AR-mediated ERK activation and decrease in megalin expression. Inhibitors of phosphatidylinositol 3-kinase (PI3K), a major component of insulin signaling, also suppressed megalin expression, and activation of the insulin receptor substrate (IRS)/PI3K system was prevented by angiotensin II. Collectively the AT1AR-mediated ERK signaling is involved in suppressing megalin expression in the OK cell line, and insulin competes with this pathway. Conversely, the insulin-IRS/PI3K signaling, with which angiotensin II competes, tends to stimulate megalin expression. In conclusion, there is AT1AR- and insulin-mediated competitive signaling cross talk to regulate megalin expression in cultured PTCs. Angiotensin II type 1A receptor- and insulin-mediated competitive signaling cross-talk regulates the expression of megalin, a multi-ligand endocytic receptor, in cultured proximal tubule cells.
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Gravotta, Diego, Andres Perez Bay, Caspar T. H. Jonker, Patrick J. Zager, Ignacio Benedicto, Ryan Schreiner, Paulo S. Caceres, and Enrique Rodriguez-Boulan. "Clathrin and clathrin adaptor AP-1 control apical trafficking of megalin in the biosynthetic and recycling routes." Molecular Biology of the Cell 30, no. 14 (July 2019): 1716–28. http://dx.doi.org/10.1091/mbc.e18-12-0811.
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Megalin (gp330, LRP-2) is a protein structurally related to the low-density lipoprotein receptor family that displays a large luminal domain with multiligand binding properties. Megalin localizes to the apical surface of multiple epithelia, where it participates in endocytosis of a variety of ligands performing roles important for development or homeostasis. We recently described the apical recycling pathway of megalin in Madin–Darby canine kidney (MDCK) cells and found that it is a long-lived, fast recycling receptor with a recycling turnover of 15 min and a half-life of 4.8 h. Previous work implicated clathrin and clathrin adaptors in the polarized trafficking of fast recycling basolateral receptors. Hence, here we study the role of clathrin and clathrin adaptors in megalin’s apical localization and trafficking. Targeted silencing of clathrin or the γ1 subunit of clathrin adaptor AP-1 by RNA interference in MDCK cells disrupted apical localization of megalin, causing its redistribution to the basolateral membrane. In contrast, silencing of the γ2 subunit of AP-1 had no effect on megalin polarity. Trafficking assays we developed using FM4-HA-miniMegalin-GFP, a reversible conditional endoplasmic reticulum–retained chimera, revealed that clathrin and AP-1 silencing disrupted apical sorting of megalin in both biosynthetic and recycling routes. Our experiments demonstrate that clathrin and AP-1 control the sorting of an apical transmembrane protein.
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Sun, Yuan, Alexandre Goes Martini, Manoe J. Janssen, Ingrid M. Garrelds, Rosalinde Masereeuw, Xifeng Lu, and A. H. Jan Danser. "Megalin." Hypertension 75, no. 5 (May 2020): 1242–50. http://dx.doi.org/10.1161/hypertensionaha.120.14845.
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Megalin is an endocytic receptor contributing to protein reabsorption. Impaired expression or trafficking of megalin increases urinary renin and allowed the detection of prorenin, which normally is absent in urine. Here, we investigated (pro)renin uptake by megalin, using both conditionally immortalized proximal tubule epithelial cells and Brown Norway Rat yolk sac cells (BN16). To distinguish binding and internalization, cells were incubated with recombinant human (pro)renin at 4°C and 37°C, respectively. (Pro)renin levels were assessed by immunoradiometric assay. At 4°C, BN16 cells bound 3× more prorenin than renin, suggestive for a higher affinity of prorenin. Similarly, at 37°C, prorenin accumulated at 3- to 4-fold higher levels than renin in BN16 cells. Consequently, depletion of medium prorenin (but not renin) content occurred after 24 hours. No such differences were observed in conditionally immortalized proximal tubule epithelial cells, and M6P (mannose-6-phosphate) greatly reduced conditionally immortalized proximal tubule epithelial cells (pro)renin uptake, suggesting that these cells accumulate (pro)renin largely via M6P receptors. M6P did not affect (pro)renin uptake in BN16 cells. Yet, inhibiting megalin expression with siRNA greatly reduced (pro)renin binding and internalization by BN16 cells. Furthermore, treating BN16 cells with albumin, an endogenous ligand of megalin, also decreased binding and internalization of (pro)renin, while deleting the (pro)renin receptor affected the latter only. Exposing prorenin’s prosegment with the renin inhibitor aliskiren dramatically increased prorenin binding, while after prosegment cleavage with trypsin prorenin binding was identical to that of renin. In conclusion, megalin might function as an endocytic receptor for (pro)renin and displays a preference for prorenin. Megalin-mediated endocytosis requires the (pro)renin receptor.
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Sun, Y., I. M. Garrelds, X. Lu, and A. Danser. "MEGALIN." Journal of Hypertension 37 (July 2019): e134. http://dx.doi.org/10.1097/01.hjh.0000571732.76261.b9.
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Klassen, R. Bryan S., Patricia L. Allen, Vecihi Batuman, Kimberly Crenshaw, and Timothy G. Hammond. "Light chains are a ligand for megalin." Journal of Applied Physiology 98, no. 1 (January 2005): 257–63. http://dx.doi.org/10.1152/japplphysiol.01090.2003.
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Cubilin and megalin are giant glycoprotein receptors abundant on the luminal surface of proximal tubular cells of the kidney. We showed previously that light chains are a ligand for cubilin. As cubilin and megalin share a number of common ligands, we further investigated the ligand specificity of these receptors. Three lines of evidence suggest that light chains can also bind megalin: 1) anti-megalin antiserum largely displaces brush-border light chain binding and megalin-expressing BN-16 cell uptake more than anti-cubilin antiserum, 2) direct binding studies on isolated proteins using surface plasmon resonance techniques confirm that megalin binds light chains, and 3) light chains compete with known megalin ligands for brush-border membrane binding and BN-16 cell uptake. The megalin-light chain interaction is divalent ion dependent and similar for both κ- and λ-light chains. A fit of the data on light chain binding to megalin over a concentration range 0.078–2.5 mg/ml leads to an estimated dissociation constant of 6 × 10−5 M, corresponding approximately to one light chain-binding site per megalin and in the same range for dissociation constants for cubilin binding. These data suggest that light chains bind the tandem megalin-cubilin complex. Megalin is the major mediator of light chain entry into megalin-expressing membrane such as the apical surface of proximal tubular epithelial cells.
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Wen, Lu, Pia K. Andersen, Dina M. U. Husum, Rikke Nørregaard, Zhanzheng Zhao, Zhangsuo Liu, and Henrik Birn. "MicroRNA-148b regulates megalin expression and is associated with receptor downregulation in mice with unilateral ureteral obstruction." American Journal of Physiology-Renal Physiology 313, no. 2 (August 1, 2017): F210—F217. http://dx.doi.org/10.1152/ajprenal.00585.2016.
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Megalin is a multiligand, endocytic receptor that is important for the normal, proximal tubule reabsorption of filtered proteins, hormones, enzymes, essential nutrients, and nephrotoxins. Megalin dysfunction has been associated with acute, as well as chronic kidney diseases. Tubular proteinuria has been observed following unilateral ureteral obstruction (UUO), suggesting megalin dysfunction; however, the pathophysiological mechanism has not been determined. To identify potential regulators of megalin expression, we examined renal microRNAs (miRNAs) expression and observed an upregulation of microRNA-148b (miR-148b) in obstructed mouse kidneys 7 days after UUO, which was associated with a significant reduction in proximal tubule megalin expression and accumulation of megalin ligands. By in silico miRNA target prediction analysis, we identified megalin messenger RNA (mRNA) as a potential target of miR-148b and confirmed using a dual-luciferase reporter assay that miR-148b targeted the 3′-untranslated region of the megalin gene. Transfection of LLC-PK1 cells with miR-148b mimic reduced endogenous megalin mRNA and protein levels in a concentration-dependent manner, while transfection with miR-148b inhibitor resulted in an increase. Our findings suggest that miR-148b directly downregulates megalin expression and that miR-148b negatively regulates megalin expression in UUO-induced kidney injury. Furthermore, the identification of a miRNA regulating megalin expression may allow for targeted interventions to modulate megalin function and proximal tubule uptake of proteins, as well as other ligands.
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Nagai, Junya, Hiroaki Tanaka, Naoki Nakanishi, Teruo Murakami, and Mikihisa Takano. "Role of megalin in renal handling of aminoglycosides." American Journal of Physiology-Renal Physiology 281, no. 2 (August 1, 2001): F337—F344. http://dx.doi.org/10.1152/ajprenal.2001.281.2.f337.
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The role of megalin in tissue distribution of aminoglycosides was examined in normal rats and maleate-treated rats that shed megalin from the renal brush-border membrane. In normal rats, amikacin administered intravenously accumulated most abundantly in the renal cortex, followed by the renal medulla. No amikacin was detected in other tissues. Tissue distributions of amikacin were well correlated with megalin levels in each tissue. Bolus administration of gentamicin increased urinary excretion of megalin ligands (vitamin D binding protein and calcium), suggesting the competition between gentamicin and these megalin ligands in renal tubules. Ligand blotting showed that binding of45Ca2+ to megalin was inhibited by aminoglycosides. Both megalin levels and amikacin accumulation in renal cortex were decreased by maleate injection. Then, amikacin accumulation recovered proportionate to megalin levels. These findings suggest that megalin is involved in the renal cortical accumulation of aminoglycosides in vivo. In addition, the interaction between aminoglycosides and calcium in the kidney may be due to the competition among these compounds to bind to megalin.
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Storm, Tina, Erik I. Christensen, Julie Nelly Christensen, Tine Kjaergaard, Niels Uldbjerg, Agnete Larsen, Bent Honoré, and Mette Madsen. "Megalin Is Predominantly Observed in Vesicular Structures in First and Third Trimester Cytotrophoblasts of the Human Placenta." Journal of Histochemistry & Cytochemistry 64, no. 12 (October 23, 2016): 769–84. http://dx.doi.org/10.1369/0022155416672210.
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The membrane receptor megalin is crucial for normal fetal development. Besides its expression in the developing fetus, megalin is also expressed in the human placenta. Similar to its established function in the kidney proximal tubules, placental megalin has been proposed to mediate uptake of vital nutrients. However, details of megalin expression, subcellular localization, and function in the human placenta remain to be established. By immunohistochemical analyses of first trimester and term human placenta, we showed that megalin is predominantly expressed in cytotrophoblasts, the highly proliferative cells in placenta. Only limited amounts of megalin could be detected in syncytiotrophoblasts and least in term placenta syncytiotrophoblasts. Immunocytochemical analyses furthermore showed that placental megalin associates with structures of the endolysosomal apparatus. Combined, our results clearly place placental megalin in the context of endocytosis and trafficking of ligands. However, due to the limited expression of megalin in syncytiotrophoblasts, especially in term placenta, it appears that the main role for placental megalin is not to mediate uptake of nutrients from the maternal bloodstream, as previously proposed. In contrast, our results point toward novel and complex functions for megalin in the cytotrophoblasts. Thus, we propose that the perception of placental megalin localization and function should be revised.
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Nagai, Masaaki, Timo Meerloo, Tetsuro Takeda, and Marilyn Gist Farquhar. "The Adaptor Protein ARH Escorts Megalin to and through Endosomes." Molecular Biology of the Cell 14, no. 12 (December 2003): 4984–96. http://dx.doi.org/10.1091/mbc.e03-06-0385.
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Megalin is an endocytic receptor that binds multiple ligands and is essential for many physiological processes such as brain development and uptake of proteins by the kidney tubule, yolk sac, and thyroid. The cytoplasmic tail of megalin contains two FXNPXY motifs. Autosomal recessive hypercholesterolemia (ARH) is an adaptor protein that binds to the FXNPXY motif of the low-density lipoprotein receptor as well as clathrin and AP-2. We found that ARH also binds to the first FXNPXY motif of megalin in two-hybrid, pull-down and coimmunoprecipitation assays. ARH colocalizes with megalin in clathrin coated pits and in recycling endosomes in the Golgi region. When cells are treated with nocodazole, the recycling endosomes containing megalin and ARH disperse. On internalization of megalin, ARH and megalin are first seen in clathrin coated pits followed by sequential localization in early endosomes and tubular recycling endosomes in the pericentriolar region followed by their reappearance at the cell surface. Expression of ARH in Madin-Darby canine kidney cells expressing megalin mini-receptors enhances megalin-mediated uptake of125I-lactoferrin, a megalin ligand. These results show that ARH facilitates endocytosis of megalin, escorts megalin along its endocytic route and raise the possibility that transport through the endosomal system is selective and requires interaction with specific adaptor proteins.
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Gonzalez-Villalobos, Romer, R. Bryan Klassen, Patricia L. Allen, L. G. Navar, and Timothy G. Hammond. "Megalin binds and internalizes angiotensin II." American Journal of Physiology-Renal Physiology 288, no. 2 (February 2005): F420—F427. http://dx.doi.org/10.1152/ajprenal.00243.2004.
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Megalin is an abundant membrane protein heavily involved in receptor-mediated endocytosis. The major functions of megalin in vivo remain incompletely defined as megalin typically faces specialized milieus such as glomerular filtrate, airways, epididymal fluid, thyroid colloid, and yolk sac fluid, which lack many of its known ligands. In the course of studies on ANG II internalization, we were surprised when only part of the uptake of labeled ANG II into immortalized yolk sac cells (BN-16 cells) was blocked by specific peptide inhibitors and direct competitors of the angiotensin type 1 receptor. This led us to test if megalin was a receptor for ANG II. Four lines of direct evidence demonstrate that megalin and, to a lesser extent, its chaperone protein cubilin are receptors for ANG II. First, in BN-16 cells anti-megalin and anti-cubilin antisera interfere with ANG II uptake. Second, also in BN-16 cells, pure ANG II competes for uptake of a known megalin ligand. Third, in proximal tubule cell brush-border membrane vesicles extracted from mice, anti-megalin antisera interfere with ANG II binding. Fourth, purified megalin binds ANG II directly in surface plasmon resonance experiments. The finding that megalin is a receptor for ANG II suggests a major new function for the megalin pathway in vivo. These results also indicate that ANG II internalization in some tissues is megalin dependent and that megalin may play a role in regulating proximal tubule ANG II levels.
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Zheng, Gang, Michele Marino, Jun Zhao, and Robert T. McCluskey. "Megalin (gp330): A Putative Endocytic Receptor for Thyroglobulin (Tg)." Endocrinology 139, no. 3 (March 1, 1998): 1462–65. http://dx.doi.org/10.1210/endo.139.3.5978.
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Megalin (gp330) is a large glycoprotein receptor found mainly on a group of absorptive epithelial cells, including renal proximal tubule, epididymal and thyroid cells. Megalin has been shown to bind multiple, unrelated ligands, mainly in vitro, and to mediate endocytosis of ligands in cultured cells. However, physiologic ligands of megalin are largely unknown. In the present study we have demonstrated that purified rat megalin binds rat thyroglobulin (Tg) in solid phase assays, with an estimated Kd of 9.2±0.6 nM. Binding was calcium dependent and was almost completely inhibited by excess Tg, by three megalin ligands—lactoferrin, lipoprotein lipase and apolipoprotein J- and by the receptor associated protein (RAP), which inhibits binding of all megalin ligands. Three anti-megalin antibodies partially inhibited Tg binding to megalin. 125I labeled Tg bound to megalin was released by EDTA and heparin; the released product was shown by SDS-PAGE and autoradiography to be 660 kD (dimeric) Tg. However, an immunoblotting experiment showed binding of megalin both to monomeric (330 kD) and dimeric Tg. We propose that megalin, which is known to mediate ligand endocytosis and is found on the apical surface of thyrocytes, may participate in the endocytosis of Tg from the colloid, a process that is required for hormone release from Tg.
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Nagai, J., E. I. Christensen, S. M. Morris, T. E. Willnow, J. A. Cooper, and R. Nielsen. "Mutually dependent localization of megalin and Dab2 in the renal proximal tubule." American Journal of Physiology-Renal Physiology 289, no. 3 (September 2005): F569—F576. http://dx.doi.org/10.1152/ajprenal.00292.2004.
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Disabled-2 (Dab2) is a cytoplasmic adaptor protein that binds to the cytoplasmic tail of the multiligand endocytic receptor megalin, abundantly expressed in renal proximal tubules. Deletion of Dab2 induces a urinary increase in specific plasma proteins such as vitamin D binding protein and retinol binding protein (Morris SM, Tallquist MD, Rock CO, and Cooper JA. EMBO J 21: 1555–1564, 2002). However, the subcellular localization of Dab2 in the renal proximal tubule and its function have not been fully elucidated yet. Here, we report the characterization of Dab2 in the renal proximal tubule. Immunohistocytochemistry revealed colocalization with megalin in coated pits and vesicles but not in dense apical tubules and the brush border. Kidney-specific megalin knockout almost abolished Dab2 staining, indicating that Dab2 subcellular localization requires megalin in the proximal tubule. Reciprocally, knockout of Dab2 led to a redistribution of megalin from endosomes to microvilli. In addition, there was an overall decrease in levels of megalin protein observed by immunoblotting but no decrease in clathrin or α-adaptin protein levels or in megalin mRNA. In rat yolk sac epithelial BN16 cells, Dab2 was present apically and colocalized with megalin. Introduction of anti-Dab2 antibody into BN16 cells decreased the internalization of 125I-labeled receptor-associated protein, substantiating the role of Dab2 in megalin-mediated endocytosis. The present study shows that Dab2 is localized in the apical endocytic apparatus of the renal proximal tubule and that this localization requires megalin. Furthermore, the study suggests that the urinary loss of megalin ligands observed in Dab2 knockout mice is caused by suboptimal trafficking of megalin, leading to decreased megalin levels.
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Klassen, R. Bryan, Kimberly Crenshaw, Renata Kozyraki, Pierre J. Verroust, Laura Tio, Sílvia Atrian, Patricia L. Allen, and Timothy G. Hammond. "Megalin mediates renal uptake of heavy metal metallothionein complexes." American Journal of Physiology-Renal Physiology 287, no. 3 (September 2004): F393—F403. http://dx.doi.org/10.1152/ajprenal.00233.2003.
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Although several heavy metal toxins are delivered to the kidney on the carrier protein metallothionein (MT), uncertainty as to how MT enters proximal tubular cells limits treatment strategies. Prompted by reports that MT-I interferes with renal uptake of the megalin ligand β2-microglobulin in conscious rats, we tested the hypothesis that megalin binds MT and mediates its uptake. Three lines of evidence suggest that binding of MT to megalin is critical in renal proximal tubular uptake of MT-bound heavy metals. First, MT binds megalin, but not cubilin, in direct surface plasmon resonance studies. Binding of MT occurs at a single site with a Kd ∼10−4 and, as with other megalin ligands, depends on divalent cations. Second, antisera and various known megalin ligands inhibit the uptake of fluorescently labeled MT in model cell systems. Anti-megalin antisera, but not control sera, displace >90% bound MT from rat renal brush-border membranes. Megalin ligands including β2-microglobulin and also recombinant MT fragments compete for uptake by megalin-expressing rat yolk sac BN-16 cells. Third, megalin and fluorescently labeled MT colocalize in BN-16 cells, as shown by fluorescent microscopic techniques. Follow-up surface plasmon resonance and flow cytometry studies using overlapping MT peptides and recombinant MT fragments identify the hinge SCKKSCC region of MT as a critical site for megalin binding. These findings suggest that disruption of the SCKKSCC motif can inhibit proximal tubular MT uptake and thereby eliminate much of the renal accumulation and toxicity of heavy metals such as cadmium, gold, copper, and cisplatinum.
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Suzuki, Takahiro, Hiroaki Yamaguchi, Jiro Ogura, Masaki Kobayashi, Takehiro Yamada, and Ken Iseki. "Megalin Contributes to Kidney Accumulation and Nephrotoxicity of Colistin." Antimicrobial Agents and Chemotherapy 57, no. 12 (October 7, 2013): 6319–24. http://dx.doi.org/10.1128/aac.00254-13.
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ABSTRACTInterest has recently been shown again in colistin because of the increased prevalence of infections caused by multidrug-resistant Gram-negative bacteria. Although the potential for nephrotoxicity is a major dose-limiting factor in colistin use, little is known about the mechanisms that underlie colistin-induced nephrotoxicity. In this study, we focused on an endocytosis receptor, megalin, that is expressed in renal proximal tubules, with the aim of clarifying the role of megalin in the kidney accumulation and nephrotoxicity of colistin. We examined the binding of colistin to megalin by using a vesicle assay. The kidney accumulation, urinary excretion, and concentrations in plasma of colistin in megalin-shedding rats were also evaluated. Furthermore, we examined the effect of megalin ligands and a microtubule-depolymerizing agent on colistin-induced nephrotoxicity. We found that cytochromec, a typical megalin ligand, inhibited the binding of colistin to megalin competitively. In megalin-shedding rats, renal proximal tubule colistin accumulation was decreased (13.5 ± 1.6 and 21.3 ± 2.6 μg in megalin-shedding and control rats, respectively). Coadministration of colistin and cytochromecor albumin fragments resulted in a significant decrease in urinaryN-acetyl-β-d-glucosaminidase (NAG) excretion, a marker of renal tubular damage (717.1 ± 183.9 mU/day for colistin alone, 500.8 ± 102.4 mU/day for cytochromecwith colistin, and 406.7 ± 156.7 mU/day for albumin fragments with colistin). Moreover, coadministration of colistin and colchicine, a microtubule-depolymerizing agent, resulted in a significant decrease in urinary NAG excretion. In conclusion, our results indicate that colistin acts as a megalin ligand and that megalin plays a key role in the accumulation in the kidney and nephrotoxicity of colistin. Megalin ligands may be new targets for the prevention of colistin-induced nephrotoxicity.
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Gamat, M., G. Shaw, and M. B. Renfree. "170. THE ROLE OF MEGALIN IN PROSTATE DEVELOPMENT OF THE MOUSE." Reproduction, Fertility and Development 22, no. 9 (2010): 88. http://dx.doi.org/10.1071/srb10abs170.
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Prostatic development is dependent on androgens; but the precise mechanism by which androgens mediate their effect is still unclear. Megalin, a cell membrane transporter, may shuttle sex steroids into cells to regulate androgen-responsive genes responsible for prostatic bud induction in the urogenital sinus (UGS). In megalin knockout mice, testicular descent fails and the vagina fails to open in females, both of which are dependent on sex steroid signalling (Hammes et al. 2005) . In this megalin-mediated pathway, SHBG-bound sex steroids bind to megalin, which is internalised. The SHBG-sex steroid complex is released, and the sex steroid is released from SHBG where it can bind to the androgen receptor to regulate androgen responsive genes. Receptor-Associated Protein (RAP) is a molecular chaperone protein that protects newly synthesised megalin from binding to potential ligands in the cytoplasm prior to insertion into the cell membrane. We hypothesised that megalin may shuttle SHBG-bound androgens across the cell membrane. This study characterised the expression and evaluated a possible role for megalin in the development of the mouse prostate. Megalin, SHBG and RAP transcripts were detected in the developing male and female UGS of the mouse from day E14.5 to day E18.5 (when prostatic buds start to form) and in the adult prostate. Megalin, SHBG and RAP protein were localised in the urogenital epithelium. To assess the role of megalin in prostatic development, UGS tissues were incubated with androgens in the presence and absence of RAP. Incubating UGS tissues with RAP did NOT inhibit prostatic bud initiation. Furthermore, in the UGS of megalin knockout mice, prostatic bud formation appeared to be identical to those of wild-type littermates. These results demonstrate that megalin is not involved in prostatic bud initiation. However, the ubiquitous expression of megalin suggests that its role is redundant in the prostate. (1) Hammes A et al. (2005) Role of endocytosis in cellular uptake of sex steroids. Cell 122(5), 751–62.
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ZIAK, MARTIN, DONTSCHO KERJASCHKI, MARILYN G. FARQUHAR, and JÜRGEN ROTH. "Identification of Megalin as the Sole Rat Kidney Sialoglycoprotein Containing Poly α2,8 Deaminoneuraminic Acid." Journal of the American Society of Nephrology 10, no. 2 (February 1999): 203–9. http://dx.doi.org/10.1681/asn.v102203.
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Abstract. Recently, poly α2,8 deaminoneuraminic acid (poly α2,8 KDN) was demonstrated in various embryonic and adult mammalian tissues. This study reports the purification and characterization of the single poly α2,8 KDN-bearing glycoprotein from rat kidney. Amino acid sequences of proteolytic fragments shared homology with megalin, a member of the LDL receptor family. Immunochemical analysis supported this finding, since immunoprecipitated poly α2,8 KDN-bearing glycoprotein was immunoreactive with anti-megalin antibodies in Western blotting and conversely immunoprecipitated megalin was immunoreactive with the monoclonal anti-poly α2,8 KDN antibody. Furthermore, receptor-associated protein affinity-purified megalin reacted with the anti-poly α2,8 KDN antibody. By immunoelectron microscopy, labeling for both poly α2,8 KDN and megalin coincided in the brush border, endocytic invaginations and vesicles, and apical dense tubules of proximal convoluted tubules. Immunoreactivity for poly α2,8 KDN on purified megalin was abolished by β-elimination reaction but not by N-glycosidase F treatment. These data identified megalin as the sole glycoprotein of rat kidney, which contains poly α2,8 KDN present on O-glycosidically linked oligosaccharides. Furthermore, this study shows that megalin carries N-glycosidically linked hybrid and complex-type oligosaccharides terminating with sialic acid. Both poly α2,8 KDN and sialic acids on megalin may contribute to the binding of Ca2+ and cationic ligands.
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Khan, Sono S., Martin Petkovich, Rachel M. Holden, and Michael A. Adams. "Megalin and Vitamin D Metabolism—Implications in Non-Renal Tissues and Kidney Disease." Nutrients 14, no. 18 (September 7, 2022): 3690. http://dx.doi.org/10.3390/nu14183690.
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Megalin is an endocytic receptor abundantly expressed in proximal tubular epithelial cells and other calciotropic extrarenal cells expressing vitamin D metabolizing enzymes, such as bone and parathyroid cells. The receptor functions in the uptake of the vitamin D-binding protein (DBP) complexed to 25 hydroxyvitamin D3 (25(OH)D3), facilitating the intracellular conversion of precursor 25(OH)D3 to the active 1,25 dihydroxyvitamin D3 (1,25(OH)2D3). The significance of renal megalin-mediated reabsorption of 25(OH)D3 and 1,25(OH)2D3 has been well established experimentally, and other studies have demonstrated relevant roles of extrarenal megalin in regulating vitamin D homeostasis in mammary cells, fat, muscle, bone, and mesenchymal stem cells. Parathyroid gland megalin may regulate calcium signaling, suggesting intriguing possibilities for megalin-mediated cross-talk between calcium and vitamin D regulation in the parathyroid; however, parathyroid megalin functionality has not been assessed in the context of vitamin D. Within various models of chronic kidney disease (CKD), megalin expression appears to be downregulated; however, contradictory results have been observed between human and rodent models. This review aims to provide an overview of the current knowledge of megalin function in the context of vitamin D metabolism, with an emphasis on extrarenal megalin, an area that clearly requires further investigation.
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Dahlke, Eileen, Yaman Anan, Lea Maximiliane Klie, Ariane Elisabeth Hartkopf, and Franziska Theilig. "Megalin Orchestrates FcRn Endocytosis and Trafficking." Cells 12, no. 1 (December 22, 2022): 53. http://dx.doi.org/10.3390/cells12010053.
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The neonatal Fc receptor (FcRn) is highly expressed in the renal proximal tubule and is important for the reclamation of albumin by cellular transcytosis to prevent its loss in the urine. The initial event of this transcellular transport mechanism is the endocytosis of albumin by the apical scavenger receptors megalin and cubilin. An interaction of megalin and FcRn was postulated, however, evidence is still missing. Similarly, the intracellular trafficking of FcRn remains unknown and shall be identified in our study. Using a Venus-based bimolecular fluorescence complementation system, we detected an interaction between megalin and FcRn in the endosomal compartment, which significantly increased with the induction of endocytosis using albumin or lactoglobulin as a ligand. The interaction between megalin and FcRn occurred at a neutral and acidic pH between the extracellular domains of both proteins. Amnionless, another transmembrane acceptor of cubilin, revealed no interaction with FcRn. With the induction of endocytosis by albumin or lactoglobulin, super resolution microscopy demonstrated a redistribution of megalin and FcRn into clathrin vesicles and early endosomes. This trafficking into clathrin vesicles was impaired in megalin-deficient cells upon albumin-induced endocytosis, supporting the role of megalin in FcRn redistribution. Our results indicate that megalin and FcRn specifically bind and interact within their extracellular domains. The availability of megalin is necessary for the redistribution of FcRn. Megalin, therefore, orchestrates FcRn endocytosis and intracellular trafficking as an early event intranscytosis.
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Czekay, R. P., R. A. Orlando, L. Woodward, E. D. Adamson, and M. G. Farquhar. "The expression of megalin (gp330) and LRP diverges during F9 cell differentiation." Journal of Cell Science 108, no. 4 (April 1, 1995): 1433–41. http://dx.doi.org/10.1242/jcs.108.4.1433.
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The receptor-associated protein, RAP, is a chaperonin-like molecule that binds to two members of the low density lipoprotein receptor (LDLR) superfamily-megalin (gp330) and the LDL receptor-related protein (LRP). In F9 embryonal carcinoma cells, expression of RAP mRNA increases when differentiation is induced with retinoic acid and dibutyryl-cyclic AMP. We have investigated the expression of megalin and LRP and their interaction with RAP in F9 cells using biochemical and immunocytochemical methods. Both receptors are expressed in uninduced F9 cells, but only megalin co-precipitates with RAP. When F9 cells were induced to differentiate into parietal endoderm, the expression of megalin was dramatically increased. The expression of megalin exceeded that of LRP and RAP by an order of magnitude and both receptors co-precipitated with RAP. By immunoelectron microscopy, megalin and LRP were localized to clathrin-coated pits at the cell surface in both undifferentiated and differentiated F9 cells, whereas RAP was found mainly in the ER. A sizeable pool of LRP was also detected in the ER. When F9 cells were grown in suspension in the presence of RA and induced to develop into embryoid bodies, the expression of megalin and LRP segregated into different cell types: megalin was found in the outer epithelial layer and LRP in the stem cells of the inner core. Our results demonstrate that F9 cells induced to differentiate in monolayer culture express megalin, LRP and RAP, and RAP is capable of interacting simultaneously with both receptors. In embryoid bodies the expression of megalin and LRP diverges, and only megalin is expressed in the outer epithelial layer.(ABSTRACT TRUNCATED AT 250 WORDS)
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Kurosaki, Yoshifumi, Akemi Imoto, Fumitaka Kawakami, Masanori Yokoba, Tsuneo Takenaka, Takafumi Ichikawa, Masato Katagiri, and Naohito Ishii. "Oxidative stress increases megalin expression in the renal proximal tubules during the normoalbuminuric stage of diabetes mellitus." American Journal of Physiology-Renal Physiology 314, no. 3 (March 1, 2018): F462—F470. http://dx.doi.org/10.1152/ajprenal.00108.2017.
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Megalin, an endocytic receptor expressed in proximal tubule cells, plays a critical role in renal tubular protein reabsorption and is associated with the albuminuria observed in diabetic nephropathy. We have previously reported increased oxidant production in the renal cortex during the normoalbuminuric stage of diabetes mellitus (DM); however, the relationship between oxidative stress and renal megalin expression during the normoalbuminuric stage of DM remains unclear. In the present study, we evaluated whether oxidative stress affects megalin expression in the normoalbuminuric stage of DM in a streptozotocin-induced diabetic rat model and in immortalized human proximal tubular cells (HK-2). We demonstrated that increased expression of renal megalin accompanies oxidative stress during the early stage of DM, before albuminuria development. Telmisartan treatment prevented the diabetes-induced elevation in megalin level, possibly through an oxidative stress-dependent mechanism. In HK-2 cells, hydrogen peroxide significantly increased megalin levels in a dose- and time-dependent manner; however, the elevation in megalin expression was decreased following prolonged exposure to severe oxidative stress induced by 0.4 mmol/l hydrogen peroxide. High-glucose treatment also significantly increased megalin expression in HK-2 cells. Concurrent administration of the antioxidant N-acetyl-cysteine blocked the effects of high glucose on megalin expression. Furthermore, the hydrogen peroxide-induced increase in megalin expression was blocked by treatment with phosphatidylinositol 3-kinase and Akt inhibitors. Increase of phosphorylated Akt expression was also seen in the renal cortex of diabetic rats. Taken together, our results indicate that mild oxidative stress increases renal megalin expression through the phosphatidylinositol 3-kinase-Akt pathway in the normoalbuminuric stage of DM.
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Faber, Kirsten, Vibeke Hvidberg, Søren K. Moestrup, Björn Dahlbäck, and Lars Bo Nielsen. "Megalin Is a Receptor for Apolipoprotein M, and Kidney-Specific Megalin-Deficiency Confers Urinary Excretion of Apolipoprotein M." Molecular Endocrinology 20, no. 1 (January 1, 2006): 212–18. http://dx.doi.org/10.1210/me.2005-0209.
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Abstract Apolipoprotein (apo) M is a novel apolipoprotein belonging to the lipocalin protein superfamily, i.e. proteins binding small lipophilic compounds. Like other apolipoproteins, it is expressed in hepatocytes and secreted into plasma where it associates with high-density lipoprotein particles. In addition, apoM is expressed at high levels in the kidney tubule cells. In this study, we show that the multiligand receptor megalin, which is expressed in kidney proximal tubule cells, is a receptor for apoM and mediates its uptake in the kidney. To examine apoM binding to megalin, a recombinant apoM was expressed in Escherichia coli and used in surface plasmon resonance and cell culture studies. The results showed apoM binding to immobilized megalin [dissociation constant (Kd) ∼ 0.3–1 μm] and that the apoM was endocytosed by cultured rat yolk sac cells in a megalin-dependent manner. To examine the importance of apoM binding by megalin in vivo, we analyzed mice with a tissue-specific deficiency of megalin in the kidney. Megalin deficiency was associated with pronounced urinary excretion of apoM, whereas apoM was not detected in normal mouse, human, or rat urine. Gel filtration analysis showed that the urinary apoM-containing particles were small and devoid of apoA-I. The results suggest that apoM binds to megalin and that megalin-mediated endocytosis in kidney proximal tubules prevents apoM excretion in the urine.
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Gonzalez-Villalobos, Romer, R. Bryan Klassen, Patricia L. Allen, Kelly Johanson, Chasity B. Baker, Hiroyuki Kobori, L. G. Navar, and Timothy G. Hammond. "Megalin binds and internalizes angiotensin-(1–7)." American Journal of Physiology-Renal Physiology 290, no. 5 (May 2006): F1270—F1275. http://dx.doi.org/10.1152/ajprenal.00164.2005.
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Megalin is a multiligand receptor heavily involved in protein endocytosis. We recently demonstrated that megalin binds and mediates internalization of ANG II. Although there is a strong structural resemblance between ANG II and ANG-(1–7), their physiological actions and their affinity for the angiotensin type 1 receptor (AT1R) are dissimilar. Therefore, the hypothesis of the present work was to test whether megalin binds and internalizes ANG-(1–7). The uptake of ANG-(1–7) was determined by exposure of confluent monolayers of BN/MSV cells (a model representative of the yolk sac epithelium) to fluorescently labeled ANG-(1–7) (100 nM) and measurement of the amount of cell-associated fluorescence after 4 h by flow cytometry. Anti-megalin antisera and an AT1R blocker (olmesartan) were used to interfere with uptake via megalin and the AT1R, respectively. ANG-(1–7) uptake was prevented by anti-megalin antisera (63%) to a higher degree than olmesartan (13%) ( P < 0.001). In analysis by flow cytometry of binding experiments performed in brush-border membrane vesicles isolated from kidneys of CD-1 mice, anti-megalin antisera interfered with ANG-(1–7) binding more strongly than olmesartan ( P < 0.05 against positive control). Interactions of megalin with ANG-(1–7) at a molecular level were studied by surface plasmon resonance, demonstrating that ANG-(1–7) binds megalin dose and time dependently and with an affinity similar to ANG II. These results show that the scavenger receptor megalin binds and internalizes ANG-(1–7).
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Gamat, M., M. B. Renfree, A. J. Pask, and G. Shaw. "230. Megalin, RAP and Nkx3.1 expression in the developing reproductive tract of a marsupial, the tammar wallaby." Reproduction, Fertility and Development 20, no. 9 (2008): 30. http://dx.doi.org/10.1071/srb08abs230.
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Androgens induce the differentiation of the urogenital sinus (UGS) to form a prostate. An early marker of this response is upregulation of the transcription factor Nkx3.1 in the urogenital epithelium in the precursors of prostatic buds. In tammars, prostate differentiation begins ~3 weeks after birth and after the time the testis starts to secrete androgens, and 2 weeks after androgen stimulated Wolffian duct differentiation. The reason for this delay in prostate differentiation is unexplained. Androgen receptors are present in the UGS, and the potent androgen, androstanediol, induces prostatic development in females. Whilst androgens may diffuse into cells by across the cell membrane, there is increasing evidence that steroids are also internalised actively via the cell-surface transport molecule Megalin. We are exploring the possibility that the delay may be related to the establishment of a Megalin-mediated pathway. Megalin is a cell surface receptor expressed on epithelia and mediates the endocytosis of a wide range of ligands, including SHBG-bound sex steroids. Megalin action is regulated by Receptor Associated Protein (RAP), which acts as an antagonist to Megalin action. This study cloned partial sequences of Megalin, RAP and Nkx3.1 and examined their expression in the developing urogenital sinus of the tammar wallaby using RT–PCR. The cellular distribution of Megalin protein in the developing UGS was examined using immunohistochemistry. Megalin, RAP and Nkx3.1 in the tammar were all highly conserved with eutherian orthologueues. Megalin and Nkx3.1 transcripts were detected in the liver, kidney, ovary, testis and developing urogenital sinus of male and female tammars. In the developing UGS of the tammar, there was strong staining for Megalin protein in the urogenital epithelium with some diffuse staining in the surrounding mesenchyme. Together, these results suggest that Megalin could be a key gene in the mediation of androgen action in prostatic development in the tammar wallaby.
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OLEINIKOV, Andrew V., Jun ZHAO, and Sudesh P. MAKKER. "Cytosolic adaptor protein Dab2 is an intracellular ligand of endocytic receptor gp600/megalin." Biochemical Journal 347, no. 3 (April 25, 2000): 613–21. http://dx.doi.org/10.1042/bj3470613.
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gp600/megalin, an endocytic receptor, belongs to the low-density lipoprotein receptor family. It is most abundant in the renal proximal tubular cells, where it is implicated in the reabsorption of a number of molecules filtered through the glomerulus. The cytoplasmic tail (CT) of gp600/megalin contains a number of sequence similarities, which indicate that gp600/megalin might be involved in signal transduction. To find intracellular proteins that would interact with the gp600/megalin CT, a human kidney cDNA library was screened by using the yeast two-hybrid system. The phosphotyrosine interaction domain (PID) of the Disabled protein 2 (Dab2), a mammalian structural analogue of Drosophila Disabled, was found to bind to the gp600/megalin CT in this system. The interaction between these two proteins was confirmed by a binding assay in vitro and by the co-immunoprecipitation of both proteins from renal cell lysates. The gp600/megalin CT contains three ΨXNPXY motifs (in which Ψ represents a hydrophobic residue) that are potentially able to interact with PID. Analysis of the CT deletion and point-mutation variants of gp600/megalin by the two-hybrid system revealed that the third ΨXNPXY motif is most probably involved in this interaction. Dab2 is a mitogen-responsive phosphoprotein thought to be an adaptor molecule involved in signal transduction, and a suggested negative regulator of cell growth. Dab2 is the first intracellular ligand identified for gp600/megalin; gp600/megalin is the first known transmembrane receptor that interacts with the cytosolic protein Dab2. We speculate that their interaction might involve gp600/megalin in signal transduction pathways or might mediate the intracellular trafficking of this receptor.
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Mazzocchi, Luciana C., Christine U. Vohwinkel, Konstantin Mayer, Susanne Herold, Rory E. Morty, Werner Seeger, and István Vadász. "TGF-β inhibits alveolar protein transport by promoting shedding, regulated intramembrane proteolysis, and transcriptional downregulation of megalin." American Journal of Physiology-Lung Cellular and Molecular Physiology 313, no. 5 (November 1, 2017): L807—L824. http://dx.doi.org/10.1152/ajplung.00569.2016.
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Disruption of the alveolar-capillary barrier is a hallmark of acute respiratory distress syndrome (ARDS) that leads to the accumulation of protein-rich edema in the alveolar space, often resulting in comparable protein concentrations in alveolar edema and plasma and causing deleterious remodeling. Patients who survive ARDS have approximately three times lower protein concentrations in the alveolar edema than nonsurvivors; thus the ability to remove excess protein from the alveolar space may be critical for a positive outcome. We have recently shown that clearance of albumin from the alveolar space is mediated by megalin, a 600-kDa transmembrane endocytic receptor and member of the low-density lipoprotein receptor superfamily. In the currents study, we investigate the molecular mechanisms by which transforming growth factor-β (TGF-β), a key molecule of ARDS pathogenesis, drives downregulation of megalin expression and function. TGF-β treatment led to shedding and regulated intramembrane proteolysis of megalin at the cell surface and to a subsequent increase in intracellular megalin COOH-terminal fragment abundance resulting in transcriptional downregulation of megalin. Activity of classical protein kinase C enzymes and γ-secretase was required for the TGF-β-induced megalin downregulation. Furthermore, TGF-β-induced shedding of megalin was mediated by matrix metalloproteinases (MMPs)-2, -9, and -14. Silencing of either of these MMPs stabilized megalin at the cell surface after TGF-β treatment and restored normal albumin transport. Moreover, a direct interaction of megalin with MMP-2 and -14 was demonstrated, suggesting that these MMPs may function as novel sheddases of megalin. Further understanding of these mechanisms may lead to novel therapeutic approaches for the treatment of ARDS.
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BIRN, HENRIK, HENRIK VORUM, PIERRE J. VERROUST, SØREN K. MOESTRUP, and ERIK ILSØ CHRISTENSEN. "Receptor-Associated Protein Is Important for Normal Processing of Megalin in Kidney Proximal Tubules." Journal of the American Society of Nephrology 11, no. 2 (February 2000): 191–202. http://dx.doi.org/10.1681/asn.v112191.
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The receptor-associated protein (RAP) has been identified as a chaperone regulating the expression and processing of the LDL receptor-related protein. RAP also binds to the related 600-kD multiligand endocytic receptor megalin expressed in many absorptive epithelia including renal proximal tubule. The present study examines the effect of RAP gene disruption on megalin expression and subcellular distribution in the proximal tubule as well as the effect on tubular protein reabsorption. It is shown that RAP is important for the normal expression and function of megalin. Megalin expression was reduced to approximately 23% estimated by immunoblotting and supported by immunocytochemistry and by the amount of megalin recovered by RAP affinity chromatography. Light-and electron microscope immunocytochemistry as well as analyses on separated membrane fractions showed significant changes in the subcellular distribution of megalin. A significant reduction in the normal brush border labeling was observed in association with increased labeling of rough endoplasmic reticulum and the smooth paramembranous endoplasmic reticulum along the basolateral membranes. RAP deficiency was associated with changes in urinary protein composition, enabling the identification of α-amylase as a new ligand for megalin. In addition, an increased excretion of vitamin D-binding protein, a recently identified ligand to megalin, was observed supporting changes in tubular protein reabsorption. The present data show that RAP is of crucial importance for normal processing and function of megalin, suggesting a chaperone-like function of this protein in the kidney proximal tubule.
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Ahuja, Rajiv, Raghunatha Yammani, Joseph A. Bauer, Seema Kalra, Shakuntla Seetharam, and Bellur Seetharam. "Interactions of cubilin with megalin and the product of the amnionless gene (AMN): effect on its stability." Biochemical Journal 410, no. 2 (February 12, 2008): 301–8. http://dx.doi.org/10.1042/bj20070919.
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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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Mahadevappa, Ravikiran, Rikke Nielsen, Erik Ilsø Christensen, and Henrik Birn. "Megalin in acute kidney injury: foe and friend." American Journal of Physiology-Renal Physiology 306, no. 2 (January 15, 2014): F147—F154. http://dx.doi.org/10.1152/ajprenal.00378.2013.
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The kidney proximal tubule is a key target in many forms of acute kidney injury (AKI). The multiligand receptor megalin is responsible for the normal proximal tubule uptake of filtered molecules, including nephrotoxins, cytokines, and markers of AKI. By mediating the uptake of nephrotoxins, megalin plays an essential role in the development of some types of AKI. However, megalin also mediates the tubular uptake of molecules implicated in the protection against AKI, and changes in megalin expression have been demonstrated in AKI in animal models. Thus, modulation of megalin expression in response to AKI may be an important part of the tubule cell adaption to cellular protection and regeneration and should be further investigated as a potential target of intervention. This review explores current evidence linking megalin expression and function to the development, diagnosis, and progression of AKI as well as renal protection against AKI.
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Gburek, Jakub, Henrik Birn, Pierre J. Verroust, Boguslawa Goj, Christian Jacobsen, Søren K. Moestrup, Thomas E. Willnow, and Erik I. Christensen. "Renal uptake of myoglobin is mediated by the endocytic receptors megalin and cubilin." American Journal of Physiology-Renal Physiology 285, no. 3 (September 2003): F451—F458. http://dx.doi.org/10.1152/ajprenal.00062.2003.
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Nephrotoxicity of myoglobin is well recognized as playing a part in the development of acute renal failure in settings of myoglobinuria. However, the molecular mechanism of myoglobin uptake in renal proximal tubules has not been clarified. Here, we report that the endocytic receptors megalin and cubilin are involved in renal reabsorption of myoglobin. Both receptors were captured from solubilized renal brush-border membranes by affinity chromatography using myoglobin-Sepharose. Myoglobin bound to purified megalin and cubilin with Kdvalues of 2.0 and 3 μM, respectively, as evaluated by surface plasmon resonance analysis. Apomyoglobin bound to megalin with the same affinity, and the affinity of apomyoglobin to cubilin was reduced ( Kd= 5 μM). Radioiodinated myoglobin could be displaced by apomyoglobin in inhibition studies using isolated renal brush-border membranes ( Ki∼ 2 μM). Receptor-associated protein as well as antibodies directed against megalin and cubilin markedly inhibited the uptake of fluorescent-labeled myoglobin by cultured yolk sac BN-16 cells. The significance of megalin- and cubilin-mediated endocytosis for myoglobin uptake in vivo was demonstrated by use of kidney-specific megalin knockout mice. Injected myoglobin was extensively reabsorbed by megalin-expressing proximal tubular cells, whereas there was very little uptake in the megalin-deficient cells. In conclusion, this study establishes the molecular mechanism of myoglobin uptake in the renal proximal tubule involving the endocytic receptors megalin and cubilin. Identification of the receptors for tubular uptake of myoglobin may be essential for development of new therapeutic strategies for myoglobinuric acute renal failure.
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Shah, Mehul, Oscar Y. Baterina, Vanessa Taupin, and Marilyn G. Farquhar. "ARH directs megalin to the endocytic recycling compartment to regulate its proteolysis and gene expression." Journal of Cell Biology 202, no. 1 (July 8, 2013): 113–27. http://dx.doi.org/10.1083/jcb.201211110.
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Receptors internalized by endocytosis can return to the plasma membrane (PM) directly from early endosomes (EE; fast recycling) or they can traffic from EE to the endocytic recycling compartment (ERC) and recycle from there (slow recycling). How receptors are sorted for trafficking along these two pathways remains unclear. Here we show that autosomal recessive hypercholesterolemia (ARH) is required for trafficking of megalin, a member of the LDL receptor family, from EE to the ERC by coupling it to dynein; in the absence of ARH, megalin returns directly to the PM from EE via the connecdenn2/Rab35 fast recycling pathway. Binding of ARH to the endocytic adaptor AP-2 prevents fast recycling of megalin. ARH-mediated trafficking of megalin to the ERC is necessary for γ-secretase mediated cleavage of megalin and release of a tail fragment that mediates transcriptional repression. These results identify a novel mechanism for sorting receptors for trafficking to the ERC and link ERC trafficking to regulated intramembrane proteolysis (RIP) and expression of megalin.
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Li, Yuanli, Rong Cong, and Daniel Biemesderfer. "The COOH terminus of megalin regulates gene expression in opossum kidney proximal tubule cells." American Journal of Physiology-Cell Physiology 295, no. 2 (August 2008): C529—C537. http://dx.doi.org/10.1152/ajpcell.00037.2008.
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We recently reported that megalin is subjected to regulated intramembrane proteolysis (RIP) and includes 1) protein kinase C (PKC)-regulated, metalloprotease-mediated ectodomain shedding producing a membrane-bound megalin COOH-terminal fragment (MCTF) and 2) γ-secretase-mediated cleavage of the MCTF producing a soluble megalin intracellular domain (MICD). Based on studies of RIP of other receptors, the MICD is predicted to target to the nucleus and regulate gene expression. To determine whether RIP of megalin regulates proximal tubule gene expression, we stably expressed the transfected MCTF (tMCTF) or transfected MICD (tMICD) in opossum kidney proximal tubule (OKP) cells and examined the resulting phenotype. Immunoblotting and immunocytochemical analysis of tMCTF cells showed the tMCTF was expressed and constitutively processed by γ-secretase. Analysis of specific protein expression in tMCTF- and tMICD-transfected cells using Western blot showed endogenous megalin and Na+/H+ exchanger 3 (NHE3) protein expression to be dramatically lower than that of control cells. Expression of other proteins including myosin VI, β-adaptin, and the Na-K-ATPase appeared unchanged. Analysis of specific mRNA expression using quantitative real-time PCR showed megalin and NHE3 mRNA levels were significantly lower in tMCTF- and tMICD-transfected cells compared with controls. Inhibition of γ-secretase activity in tMCTF cells resulted in an 8- to 10-fold recovery of megalin mRNA within 4 h. These data show that the COOH-terminal domain of megalin regulates expression of specific proteins in OKP cells and provides the first evidence that RIP of megalin may be part of a signaling pathway linking protein absorption and gene expression in proximal tubule.
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Yammani, Raghunatha R., Mukut Sharma, Shakuntla Seetharam, John E. Moulder, Nancy M. Dahms, and Bellur Seetharam. "Loss of albumin and megalin binding to renal cubilin in rats results in albuminuria after total body irradiation." American Journal of Physiology-Regulatory, Integrative and Comparative Physiology 283, no. 2 (August 1, 2002): R339—R346. http://dx.doi.org/10.1152/ajpregu.00752.2001.
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The role of the renal apical brush-border membrane (BBM) endocytic receptors cubilin and megalin in the onset of albuminuria in rats exposed to a single dose of total body irradiation (TBI) has been investigated. Albuminuria was evident as immunoblot (IB) analysis of the urine samples from TBI rats revealed excretion of large amounts of albumin. IB analysis of the BBM proteins did not reveal any significant changes in cubilin or megalin levels, but 125I-albumin binding to BBM from TBI rats declined by 80% with a fivefold decrease (from 0.5 to 2.5 μM) in the affinity for albumin. IB analysis of cubilin from the BBM demonstrated a 75% loss when purified using albumin, but not intrinsic factor (IF)-cobalamin (Cbl) ligand affinity chromatography. Immunoprecipitation (IP) of Triton X-100 extract of the BBM with antiserum to cubilin followed by IB of the immune complex with an antiserum to megalin revealed a 75% loss of association between megalin and cubilin. IP studies with antiserum to cubilin or megalin and IB with antiserum to the cation-independent mannose 6-phosphate/insulin-like growth factor II-receptor (CIMPR) revealed that CIMPR interacted with both cubilin and megalin. In addition, TBI did not disrupt the association of CIMPR with either cubilin or megalin in BBM. These results suggest that albuminuria noted in TBI rats is due to selective loss of albumin and megalin, but not CIMPR or IF-Cbl binding by cubilin. Furthermore, these results also suggest that albumin and IF-Cbl binding to cubilin occur at distinct sites and that in the rat renal BBM, CIMPR interacts with both cubilin and megalin.
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Kasabri, Violet, Amal Akour, Nailya Bulatova, Maysa Suyagh, Al Motassem Yousef, Hussein Al Hawari, Sameeha Al Shelleh, Jameel Bzour, and Hiba Fahmawi. "A Pre-Post Study of Vitamin D Supplement Effects on Urinary Megalin: The Emerging Predictive Role of Megalin in Diabetic Nephropathy Progression." Endocrine, Metabolic & Immune Disorders - Drug Targets 20, no. 9 (November 5, 2020): 1552–57. http://dx.doi.org/10.2174/1871530320666200525012811.
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Introduction: Megalin is a renal proximal tubular protein that reabsorbs vitamin D from glomerular filtrates. Previous studies found significantly higher levels of urinary megalin in chronic microvascular complications of diabetes with associated metabolic derangements. This study aimed at testing the effect of vitamin D supplements on urinary megalin levels in diabetic nephropathy (DN) patients with vitamin D hypovitaminosis. Methods: Sixty-three participants with vitamin D deficiency and diabetic nephropathy (DN) were enrolled in the pre-post study; urinary megalin levels with various clinical parameters and serum levels of vitamin D3 were measured and compared to the baseline at 3- and 6-month intervals. Results: Interestingly, a supplementation related increase in serum vitamin D3 levels at 3- and 6- month interventions affected a constellation of ameliorations in the DN progression of clinical and metabolic factors. There was a decrease in ACR with a concomitant decrease in urinary megalin and a decrease in blood pressure, fasting plasma glucose (FPG), and low-density lipoprotein – cholesterol (LDL-C) – but an increase in glomerular filtration rate (GFR). Principally, pellet urinary megalin associated positively (p < 0.05) with vitamin D hypovitaminosis and the albumin-to-creatinine ratio (ACR) but negatively (p < 0.05) with Ca2+ and body mass index (BMI). Conclusion: Vitamin D supplementation could elucidate underlying pathophysiological mechanisms and a prognostic significance of urinary megalin association with DN, obesity/MetS-related dyslipidemia, and hyperglycemia modification. Megalin is a putative sensitive and precise predictive marker and an emerging therapeutic target of renal anomalies.
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PATRIE, KEVIN M., ANDREW J. DRESCHER, MEERA GOYAL, ROGER C. WIGGINS, and BEN MARGOLIS. "The Membrane-Associated Guanylate Kinase Protein MAGI-1 Binds Megalin and Is Present in Glomerular Podocytes." Journal of the American Society of Nephrology 12, no. 4 (April 2001): 667–77. http://dx.doi.org/10.1681/asn.v124667.
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Abstract. The transmembrane endocytic receptor glycoprotein 330/megalin (hereafter referred to as megalin) is localized to the apical membrane domain of epithelial cells, where it is involved in the uptake of proteins from extracellular sources. The cytoplasmic domain of megalin contains amino acid motifs that have the potential to bind to other proteins, which may influence its localization or function. The yeast two-hybrid system was used to search for proteins that bind to the cytoplasmic tail of megalin, and a protein fragment from a mouse embryonic cDNA library that contained a single PDZ domain was identified. This protein, which was namedglycoprotein 330-associatedprotein (GASP), appears to be a truncated mouse counterpart of the human and rat proteins atrophin-1-interacting protein-1 and synaptic scaffolding molecule, respectively. The interaction of GASP with megalin is mediated by the PDZ domain of GASP binding to the DSDV motif found at the carboxyl-terminus of megalin. A mutant version of megalin that lacks the terminal valine is unable to bind to GASP, illustrating the PDZ domain-dependent interaction between these two proteins. A close homolog of GASP,i.e., membrane-associated guanylate kinase with inverted orientation-1 (MAGI-1), is more ubiquitous in its tissue distribution (including kidney) and is also able to specifically bind to megalin via its fifth PDZ domain. Immunofluorescence studies of adult kidney revealed that MAGI-1 is expressed in the glomerulus of the kidney, in a manner that parallels the expression of the podocyte-specific protein glomerular epithelial protein 1. Western analysis of endogenous MAGI-1 from glomerular preparations suggests that it is associated with the cytoskeleton and seems to be expressed in a different form, compared with cell line-derived endogenous MAGI-1. The association of megalin with MAGI-1 may allow the assembly of a multiprotein complex, in which megalin may serve a nonendocytic function in glomerular podocytes.
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CHRISTENSEN, ERIK ILSØ, JAN ØIVIND MOSKAUG, HENRIK VORUM, CHRISTIAN JACOBSEN, THOMAS E. GUNDERSEN, ANDERS NYKJÆR, RUNE BLOMHOFF, THOMAS E. WILLNOW, and SØREN K. MOESTRUP. "Evidence for an Essential Role of Megalin in Transepithelial Transport of Retinol." Journal of the American Society of Nephrology 10, no. 4 (April 1999): 685–95. http://dx.doi.org/10.1681/asn.v104685.
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Abstract. Transepithelial transport of retinol is linked to retinol-binding protein (RBP), which is taken up and also synthesized in a number of epithelia. By immunocytochemistry of human, rat, and mouse renal proximal tubules, a strong staining in apical endocytic vacuoles, lysosomes, endoplasmic reticulum, Golgi, and basal vesicles was observed, in accordance with luminal endocytic uptake as well as a constitutive synthesis and basal secretion of RBP. Analysis of mice with target disruption of the gene for the major endocytic receptor of proximal tubules, megalin, revealed no RBP in proximal tubules of these mice. Western blotting and HPLC of the urine of the megalin-deficient mice instead revealed a highly increased urinary excretion of RBP and retinol, demonstrating that glomerular filtered RBP-retinol of megalin-deficient mice escapes uptake by proximal tubules. A direct megalin-mediated uptake of purified RBP-retinol was indicated by surface plasmon resonance analysis and uptake in immortalized rat yolk sac cells. Uptake was partially inhibited by a polyclonal megalin antibody and the receptor-associated protein. The present data show that the absence of RBP-binding megalin causes a significantly increased loss of RBP and retinol in the urine, demonstrating a crucial role of megalin in vitamin A homeostasis.
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Kapetanaki, Stefania, Ashok Kumar Kumawat, Katarina Persson, and Isak Demirel. "TMAO Suppresses Megalin Expression and Albumin Uptake in Human Proximal Tubular Cells Via PI3K and ERK Signaling." International Journal of Molecular Sciences 23, no. 16 (August 9, 2022): 8856. http://dx.doi.org/10.3390/ijms23168856.
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Trimethylamine-N-oxide (TMAO) is a uremic toxin, which has been associated with chronic kidney disease (CKD). Renal tubular epithelial cells play a central role in the pathophysiology of CKD. Megalin is an albumin-binding surface receptor on tubular epithelial cells, which is indispensable for urine protein reabsorption. To date, no studies have investigated the effect of TMAO on megalin expression and the functional properties of human tubular epithelial cells. The aim of this study was first to identify the functional effect of TMAO on human renal proximal tubular cells and second, to unravel the effects of TMAO on megalin-cubilin receptor expression. We found through global gene expression analysis that TMAO was associated with kidney disease. The microarray analysis also showed that megalin expression was suppressed by TMAO, which was also validated at the gene and protein level. High glucose and TMAO was shown to downregulate megalin expression and albumin uptake similarly. We also found that TMAO suppressed megalin expression via PI3K and ERK signaling. Furthermore, we showed that candesartan, dapagliflozin and enalaprilat counteracted the suppressive effect of TMAO on megalin expression. Our results may further help us unravel the role of TMAO in CKD development and to identify new therapeutic targets to counteract TMAOs effects.
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LARSSON, Mårten, Göran HJÄLM, Amos M. SAKWE, Åke ENGSTRÖM, Anna-Stina HÖGLUND, Erik LARSSON, Robert C. ROBINSON, Christian SUNDBERG, and Lars RASK. "Selective interaction of megalin with postsynaptic density-95 (PSD-95)-like membrane-associated guanylate kinase (MAGUK) proteins." Biochemical Journal 373, no. 2 (July 15, 2003): 381–91. http://dx.doi.org/10.1042/bj20021958.
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Megalin is an integral membrane receptor belonging to the low-density lipoprotein receptor family. In addition to its role as an endocytotic receptor, megalin has also been proposed to have signalling functions. Using interaction cloning in yeast, we identified the membrane-associated guanylate kinase family member postsynaptic density-95 (PSD-95) as an interaction partner for megalin. PSD-95 and a truncated version of megalin were co-immunoprecipitated from HEK-293 cell lysates overexpressing the two proteins, which confirmed the interaction. The two proteins were found to be co-localized in these cells by confocal microscopy. Immunocytochemical studies showed that cells in the parathyroid, proximal tubuli of the kidney and placenta express both megalin and PSD-95. We found that the interaction between the two proteins is mediated by the binding of the C-terminus of megalin, which has a type I PSD-95/Drosophila discs-large/zona occludens 1 (PDZ)-binding motif, to the PDZ2 domain of PSD-95. The PSD-95-like membrane-associated guanylate kinase (‘MAGUK’) family contains three additional members: PSD-93, synapse-associated protein 97 (SAP97) and SAP102. We detected these proteins, apart from SAP102, in parathyroid chief cells, a cell type having a marked expression of megalin. The PDZ2 domains of PSD-93 and SAP102 were also shown to interact with megalin, whereas no interaction was detected for SAP97. The SAP97 PDZ2 domain differed at four positions from the other members of the PSD-95 subfamily. One of these residues was Thr389, located in the αB-helix and part of the hydrophobic pocket of the PDZ2 domain. Surface plasmon resonance experiments revealed that mutation of SAP97 Thr389 to alanine, as with the other PSD-95-like membrane-associated guanylate kinases, induced binding to megalin.
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Birn, Henrik, Thomas E. Willnow, Rikke Nielsen, Anthony G. W. Norden, Christian Bönsch, Søren K. Moestrup, Ebba Nexø, and Erik I. Christensen. "Megalin is essential for renal proximal tubule reabsorption and accumulation of transcobalamin-B12." American Journal of Physiology-Renal Physiology 282, no. 3 (March 1, 2002): F408—F416. http://dx.doi.org/10.1152/ajprenal.00206.2000.
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Megalin has previously been shown to bind and mediate endocytosis of transcobalamin (TC)-B12. However, the physiological significance of this has not been established, and other TC-B12binding proteins have been suggested to mediate renal uptake of this vitamin complex. The present study demonstrates by the use of megalin-deficient mice that megalin is, in fact, essential for the normal renal reabsorption of TC-vitamin B12and for renal accumulation of this highly conserved vitamin. Megalin-deficient mice excrete increased amounts of TC and B12in the urine, revealing a defective renal tubular uptake of TC-B12. The urinary B12excretion is increased ∼4-fold, resulting in an ∼28-fold higher renal B12clearance. This is associated with an ∼4-fold decrease in B12content in megalin-deficient kidney cortex. Thus megalin is important to prevent urinary loss of vitamin B12. In addition, light- and electron-microscopic immunocytochemistry demonstrate lysosomal accumulation of B12in rat and mouse proximal tubules. In rats this accumulation is correlated with vitamin intake. Thus renal lysosomal B12accumulation is dependent on vitamin status, indicating a possible reserve function of this organelle in the rat kidney.
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Bryniarski, Mark A., Benjamin M. Yee, Irum Jaffri, Lee D. Chaves, Jin Ah Yu, Xiaowen Guan, Nazanin Ghavam, Rabi Yacoub, and Marilyn E. Morris. "Increased megalin expression in early type 2 diabetes: role of insulin-signaling pathways." American Journal of Physiology-Renal Physiology 315, no. 5 (November 1, 2018): F1191—F1207. http://dx.doi.org/10.1152/ajprenal.00210.2018.
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The megalin/cubilin complex is responsible for the majority of serum protein reclamation in the proximal tubules. The current study examined if decreases in their renal expression, along with the albumin recycling protein neonatal Fc receptor (FcRn) could account for proteinuria/albuminuria in the Zucker diabetic fatty rat model of type 2 diabetes. Immunoblots of renal cortex samples obtained at worsening disease stages demonstrated no loss in megalin, cubilin, or FcRn, even when proteinuria was measured. Additionally, early diabetic rats exhibited significantly increased renal megalin expression when compared with controls (adjusted P < 0.01). Based on these results, the ability of insulin to increase megalin was examined in a clonal subpopulation of the opossum kidney proximal tubule cell line. Insulin treatments (24 h, 100 nM) under high glucose conditions significantly increased megalin protein ( P < 0.0001), mRNA ( P < 0.0001), and albumin endocytosis. The effect on megalin expression was prevented with inhibitors against key effectors of insulin intracellular signaling, phosphatidylinositide 3-kinase and Akt. Studies using rapamycin to inhibit the mechanistic target of rapamycin complex 1 (mTORC1) resulted in a loss of insulin-induced megalin expression. However, subsequent evaluation demonstrated these effects were independent of initial mTORC1 suppression. The presented results provide insight into the expression of megalin, cubilin, and FcRn in type 2 diabetes, which may be impacted by elevated insulin and glucose. Furthermore, proximal tubule endocytic activity in early diabetics may be enhanced, a process that could have a significant role in proteinuria-induced renal damage.
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Czekay, R. P., R. A. Orlando, L. Woodward, M. Lundstrom, and M. G. Farquhar. "Endocytic trafficking of megalin/RAP complexes: dissociation of the complexes in late endosomes." Molecular Biology of the Cell 8, no. 3 (March 1997): 517–32. http://dx.doi.org/10.1091/mbc.8.3.517.
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Megalin (gp330) is a member of the low-density lipoprotein receptor gene family. Like other members of the family, it is an endocytic receptor that binds a number of specific ligands. Megalin also binds the receptor-associated protein (RAP) that serves as an exocytic traffic chaperone and inhibits ligand binding to the receptor. To investigate the fate of megalin/RAP complexes, we bound RAP glutathione-S-transferase fusion protein (RAP-GST) to megalin at the surface of L2 yolk sac carcinoma cells and followed the trafficking of the complexes by immunofluorescence and immunogold labeling and by their distribution on Percoll gradients. We show that megalin/RAP-GST complexes, which are internalized via clathrin-coated pits, are delivered to early endosomes where they accumulate during an 18 degrees C temperature block and colocalize with transferrin and transferrin receptor. Upon release from the temperature block, the complexes travel to late endosomes where they colocalize with rab7 and can be coprecipitated with anti-RAP-GST antibodies. Dissociation of the complex occurs in late endosomes and is most likely triggered by the low pH (approximately 5.5) of this compartment. RAP is then rapidly delivered to lysosomes and degraded whereas megalin is recycled to the cell surface. When the ligand, lipoprotein lipase, was bound to megalin, the receptor was found to recycle through early endosomes. We conclude that in contrast to receptor/ligand complexes, megalin/RAP complexes traffic through late endosomes, which is a novelty for members of the low-density lipoprotein receptor gene family.
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Li, Xiao C., and Jia L. Zhuo. "Mechanisms of AT1a receptor-mediated uptake of angiotensin II by proximal tubule cells: a novel role of the multiligand endocytic receptor megalin." American Journal of Physiology-Renal Physiology 307, no. 2 (July 15, 2014): F222—F233. http://dx.doi.org/10.1152/ajprenal.00693.2013.
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The present study tested the hypothesis that the multiligand endocytic receptor megalin is partially involved in the uptake of ANG II and downstream signaling responses in mouse proximal tubule cells (mPCT) by interacting with AT1a receptors. mPCT cells of wild-type (WT) and AT1a receptor-deficient (AT1a-KO) mice were treated with vehicle, the AT1 receptor blocker losartan (10 μM), or a selective megalin small interfering (si) RNA for 48 h. The uptake of fluorescein (FITC)-labeled ANG II (10 nM, 37°C) and downstream signaling responses were analyzed by fluorescence imaging and Western blotting. AT1a receptors and megalin were abundantly expressed in mPCT cells, whereas AT1a receptors were absent in AT1a-KO mPCT cells ( P < 0.01). In WT mPCT cells, FITC-ANG II uptake was visualized at 30 min in the cytoplasm and in the nuclei 1 h after exposure. Losartan alone completely blocked the uptake of FITC-ANG II, whereas megalin siRNA inhibited only 30% of the response ( P < 0.01). The remaining FITC-ANG II uptake in the presence of megalin siRNA was completely abolished by losartan. ANG II induced threefold increases in phosphorylated MAP kinases ERK1/2 and a onefold increase in phosphorylated sodium and hydrogen exchanger 3 (NHE3) proteins, which were also blocked by losartan and megalin-siRNA. By contrast, losartan and megalin siRNA had no effects on these signaling proteins in AT1a-KO mPCT cells. We conclude that the uptake of ANG II and downstream MAP kinases ERK1/2 and NHE3 signaling responses in mPCT cells are mediated primarily by AT1a receptors. However, megalin may also play a partial role in these responses to ANG II.
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Chiu-Ugalde, Jazmin, Franziska Theilig, Thomas Behrends, Julia Drebes, Carolin Sieland, Prema Subbarayal, Josef Köhrle, Annette Hammes, Lutz Schomburg, and Ulrich Schweizer. "Mutation of megalin leads to urinary loss of selenoprotein P and selenium deficiency in serum, liver, kidneys and brain." Biochemical Journal 431, no. 1 (September 14, 2010): 103–11. http://dx.doi.org/10.1042/bj20100779.
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Distribution of selenium (Se) within the mammalian body is mediated by SePP (selenoprotein P), an Se-rich glycoprotein secreted by hepatocytes. Genetic and biochemical evidence indicate that the endocytic receptors ApoER2 (apolipoprotein E receptor 2) and megalin mediate tissue-specific SePP uptake. In the present study megalin-mutant mice were fed on diets containing adequate (0.15 p.p.m.) or low (0.08 p.p.m.) Se content and were analysed for tissue and plasma Se levels, cellular GPx (glutathione peroxidase) activities and protein expression patterns. Megalin-mutant mice displayed increased urinary Se loss, which correlated with SePP excretion in their urine. Accordingly, serum Se and SePP levels were significantly reduced in megalin-mutant mice, reaching marginal levels on the low-Se diet. Moreover, kidney Se content and expression of renal selenoproteins were accordingly reduced, as was SePP internalization along the proximal tubule epithelium. Although GPx4 expression was not altered in testis, Se and GPx activity in liver and brain were significantly reduced. When fed on a low-Se diet, megalin-mutant mice developed impaired movement co-ordination, but no astrogliosis. These findings suggest that megalin prevents urinary SePP loss and participates in brain Se/SePP uptake.
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Saito, Akihiko, Hiroyoshi Sato, Noriaki Iino, and Tetsuro Takeda. "Molecular Mechanisms of Receptor-Mediated Endocytosis in the Renal Proximal Tubular Epithelium." Journal of Biomedicine and Biotechnology 2010 (2010): 1–7. http://dx.doi.org/10.1155/2010/403272.
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Receptor-mediated endocytosis is a pivotal function of renal proximal tubule epithelial cells (PTECs) to reabsorb and metabolize substantial amounts of proteins and other substances in glomerular filtrates. The function accounts for the conservation of nutrients, including carrier-bound vitamins and trace elements, filtered by glomeruli. Impairment of the process results in a loss of such substances and development of proteinuria, an important clinical sign of kidney disease and a risk marker for cardiovascular disease. Megalin is a multiligand endocytic receptor expressed at clathrin-coated pits of PTEC, playing a central role in the process. Megalin cooperates with various membrane molecules and interacts with many intracellular adaptor proteins for endocytic trafficking. Megalin is also involved in signaling pathways in the cells. Megalin-mediated endocytic overload leads to damage of PTEC. Further studies are needed to elucidate the mechanism of megalin-mediated endocytosis and develop strategies for preventing the damage of PTEC.
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Marinò, Michele, Luca Chiovato, Nicholas Mitsiades, Francesco Latrofa, David Andrews, Sophia Tseleni-Balafouta, A. Bernard Collins, Aldo Pinchera, and Robert T. McCluskey. "Circulating Thyroglobulin Transcytosed by Thyroid Cells Is Complexed with Secretory Components of Its Endocytic Receptor Megalin*." Journal of Clinical Endocrinology & Metabolism 85, no. 9 (September 1, 2000): 3458–67. http://dx.doi.org/10.1210/jcem.85.9.6804.
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Abstract After its endocytosis from the colloid, some thyroglobulin (Tg) is transcytosed intact across thyrocytes, accounting in part for its presence in the circulation. We previously showed that megalin (gp330), an endocytic Tg receptor, mediates apical to basolateral Tg transcytosis. Here we investigated whether a portion of megalin remains combined with Tg after its transcytosis, using studies with cultured thyroid cells and in vivo observations. FRTL-5 cells, a rat thyroid cell line, cultured on filters in dual chambers form tight junctions and exhibit features of polarity, with expression of megalin exclusively on the upper (apical) surface. After the addition of unlabeled Tg to the upper chamber and incubation at 37 C, some Tg was transcytosed intact across FRTL-5 cells into the lower chamber. Two antimegalin ectodomain antibodies precipitated transcytosed Tg in fluids collected from the lower chamber. After the addition of Tg to surface-biotinylated FRTL-5 cells, an anti-Tg antibody and the two antimegalin ectodomain antibodies precipitated high molecular mass biotinylated material in fluids collected from the lower chamber, corresponding to much of the megalin ectodomain, as well as smaller amounts of lower molecular mass material. The results indicate that Tg transcytosed across FRTL-5 cells remains complexed with megalin ectodomain components, which we refer to as megalin secretory components. In aminotriazole-treated rats, which develop increased megalin-mediated Tg transcytosis, antimegalin antibodies precipitated some of the Tg in the serum. Tg was also precipitated by antimegalin antibodies in sera from patients with Graves’ disease, in which we found increased megalin expression on the apical surface of thyrocytes. In contrast, in thyroidectomized patients with metastatic papillary thyroid carcinoma, in whom Tg is directly secreted by neoplastic thyroid cells into the circulation rather than transcytosed, serum Tg was not precipitated by antimegalin antibodies. The detection of Tg-megalin complexes may help identify the source of serum Tg in patients with thyroid diseases.
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Orlando, R. A., D. Kerjaschki, and M. G. Farquhar. "Megalin (gp330) possesses an antigenic epitope capable of inducing passive Heymann nephritis independent of the nephritogenic epitope in receptor-associated protein." Journal of the American Society of Nephrology 6, no. 1 (July 1995): 61–67. http://dx.doi.org/10.1681/asn.v6161.
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The Heymann nephritis antigenic complex (HNAC) consists of two glycoproteins, megalin (gp330), and the receptor-associated protein (RAP). HNAC is expressed on the surface of the glomerular epithelium where it plays a primary role in the pathogenesis of Heymann nephritis (HN). Several models were previously proposed describing how antibody binding epitopes in HNAC may contribute to the initiation and progression of HN. Although these models suggest that nephritogenic epitopes capable of initiating HN are present in both megalin and RAP, the structural relationship between these epitopes has not been established. Previously a nephritogenic epitope was identified and characterized in RAP that initiates immune complex formation in HN. In this report, the immunologic relationship between nephritogenic epitopes in megalin and RAP were examined to determine whether these epitopes are immunologically distinct or antigenically related. To this end, a polyclonal antibody to megalin was generated that does not recognize RAP by immunoblotting or immunoprecipitation and whether this antibody is capable of inducing passive HN was determined. It was found that antimegalin antibodies devoid of RAP cross-reactivity induced the formation of subepithelial immune deposits (passive HN) when injected into rats. Antibodies eluted from glomeruli of the injected rats recognized only megalin by immunoblotting a cortical extract and did not recognize a RAP fusion protein or any other renal protein. In addition, the eluted antibodies immunoprecipitated two proteolytic fragments of megalin (140 and 75 kd) identifying a pathogenic epitope within a smaller fragment of megalin.(ABSTRACT TRUNCATED AT 250 WORDS)
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Norden, Anthony G. W., Marta Lapsley, Takashi Igarashi, Catherine L. Kelleher, Philip J. Lee, Takeshi Matsuyama, Steven J. Scheinman, et al. "Urinary Megalin Deficiency Implicates Abnormal Tubular Endocytic Function in Fanconi Syndrome." Journal of the American Society of Nephrology 13, no. 1 (January 2002): 125–33. http://dx.doi.org/10.1681/asn.v131125.
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ABSTRACT. Normal reabsorption of glomerular filtrate proteins probably requires recycling of the endocytic receptors megalin (gp330) and cubilin. Both receptors are located on the luminal surface of the renal proximal tubule epithelium. Whether abnormal amounts of receptor are present in the urine of patients with Dent’s disease, Lowe’s syndrome, or autosomal dominant idiopathic Fanconi syndrome was explored. They are all forms of the renal Fanconi syndrome and are associated with tubular proteinuria. Urine samples of equal creatinine contents were dialyzed, lyophilized, and subjected to electrophoresis on nonreducing sodium dodecyl sulfate-5% polyacrylamide gels. Proteins were blotted and probed with anti-megalin IgG, anti-cubilin IgG, or receptor-associated protein. Megalin and cubilin levels detected by immunochemiluminescence were measured as integrated pixels and expressed as percentages of the normal mean values. A striking deficiency of urinary megalin, compared with normal individuals (n = 42), was observed for eight of nine families with Dent’s disease (n = 10) and for the two families with Lowe’s syndrome (n = 3). The family with autosomal dominant idiopathic Fanconi syndrome (n = 2) exhibited megalin levels within the normal range. The measured levels of cubilin were normal for all patients. These results are consistent with defective recycling of megalin to the apical cell surface of the proximal tubules and thus decreased loss into urine in Dent’s disease and Lowe’s syndrome. This defect would interfere with the normal endocytic function of megalin, result in losses of potential ligands into the urine, and produce tubular proteinuria.
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Fowlkes, John L., R. Clay Bunn, Gael E. Cockrell, Lindsey M. Clark, Elizabeth C. Wahl, Charles K. Lumpkin, and Kathryn M. Thrailkill. "Dysregulation of the Intrarenal Vitamin D Endocytic Pathway in a Nephropathy-Prone Mouse Model of Type 1 Diabetes." Experimental Diabetes Research 2011 (2011): 1–7. http://dx.doi.org/10.1155/2011/269378.
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Microalbuminuria in humans with Type 1 diabetes (T1D) is associated with increased urinary excretion of megalin, as well as many megalin ligands, including vitamin-D-binding protein (VDBP). We examined the DBA/2J diabetic mouse, nephropathy prone model, to determine if megalin and VDBP excretion coincide with the development of diabetic nephropathy. Megalin, VDBP, and 25-hydroxy-vitamin D (25-OHD) were measured in urine, and genes involved in vitamin D metabolism were assessed in renal tissues from diabetic and control mice at 10, 15, and 18 weeks following the onset of diabetes. Megalin, VDBP, and 25-OHD were increased in the urine of diabetic mice. 1-α hydroxylase (CYP27B1) mRNA in the kidney was persistently increased in diabetic mice, as were several vitamin D-target genes. These studies show that intrarenal vitamin D handling is altered in the diabetic kidney, and they suggest that in T1D, urinary losses of VDBP may portend risk for intrarenal and extrarenal vitamin D deficiencies.