Specific Association of Megalin and the Na+/H+ Exchanger Isoform NHE3 in the Proximal Tubule

Biemesderfer, Daniel; Nagy, Tamás; DeGray, Brenda; Aronson, Peter S.

doi:10.1074/jbc.274.25.17518

Cited by 111 publications

(88 citation statements)

References 47 publications

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“…These transporters are presumably located on microvilli (Fig. 2) and likely correspond to the fraction of exchangers that Biemesderfer et al (27) found not to associate with megalin. As such, these exchangers are most likely to function actively in ion transport.…”

Section: Discussionmentioning

confidence: 99%

Rho GTPases dictate the mobility of the Na/H exchanger NHE3 in epithelia: Role in apical retention and targeting

Alexander

Furuya

Szászi

et al. 2005

Proc. Natl. Acad. Sci. U.S.A.

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Proximal tubular reabsorption of filtered sodium by the sodium͞ hydrogen exchanger isoform 3 (NHE3), located on the apical membrane, is fundamental to the maintenance of systemic volume and pH homeostasis. NHE3 is finely regulated by a variety of hormones and by changes in ionic composition and volume, likely requiring redistribution of the exchangers. We analyzed the subcellular distribution and dynamics of the exchangers by generating an epithelial line expressing NHE3 tagged with an exofacial epitope, which enabled us to monitor exchanger mobility and traffic in intact cells. Using determinations of fluorescence recovery after photobleaching in combination with dynamic measurements of subcellular distribution, we found that, in renal epithelial cells, NHE3 exists in four distinct subcompartments: a virtually immobile subpopulation that is retained on the apical membrane by interaction with the actin cytoskeleton in a manner that depends on the sustained activity of Rho GTPases; a mobile subpopulation on the apical membrane, which can be readily internalized; and two intracellular compartments that can be differentiated by their rate of exchange with the apical pool of NHE3. We provide evidence that detachment of the immobile fraction from its cytoskeletal anchorage leads to rapid internalization. These observations suggest that modulation of the mobile fraction of NHE3 on the apical membrane can alter the number of functional exchangers on the cell surface and, consequently, the rate of transepithelial ion transport. Regulation of the interaction of NHE3 with the actin cytoskeleton can therefore provide a new mode of regulation of sodium and hydrogen transport. fluorescence recovery after photobleaching ͉ pH regulation ͉ sodium proton exchange ͉ hypertension ͉ Madin-Darby canine kidney cells

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Section: Discussionmentioning

confidence: 99%

Rho GTPases dictate the mobility of the Na/H exchanger NHE3 in epithelia: Role in apical retention and targeting

Alexander

Furuya

Szászi

et al. 2005

Proc. Natl. Acad. Sci. U.S.A.

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“…We have recently reported an association between NHE3 and the putative scavenger receptor megalin in renal brush border membranes (1). Moreover, we found that renal brush border NHE3 exists in two states with distinct sedimentation coefficients, a 9.6 S megalin-free form and a 21 S megalin-bound form (1).…”

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confidence: 93%

“…Antibodies to NHE3-In a previous paper, we described in detail the development and characterization of isoform-specific monoclonal antibodies (mAbs) 1 to a restricted region of the carboxyl terminus of NHE3 (10). mAbs 2B9, 4F5, and 19F5 were raised to a fusion protein (fpNHE3-702-832) that reproduced the C-terminal 131 amino acids of rabbit NHE3 (11).…”

Section: Methodsmentioning

confidence: 99%

“…Moreover, we found that renal brush border NHE3 exists in two states with distinct sedimentation coefficients, a 9.6 S megalin-free form and a 21 S megalin-bound form (1). The purpose of the present study was to use membrane fractionation methods to determine whether these two oligomeric forms of NHE3 are expressed in distinct microdomains of the renal brush border and, if so, to compare the functional activity of NHE3 in these microdomains.…”

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confidence: 99%

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Active (9.6 S) and Inactive (21 S) Oligomers of NHE3 in Microdomains of the Renal Brush Border

Biemesderfer

DeGray

Aronson

2001

Journal of Biological Chemistry

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We have previously shown that Na ؉ -H ؉ exchanger isoform NHE3 exists as both 9.6 and 21 S (megalin-associated) oligomers in the renal brush border (1). To characterize the oligomeric forms of the renal brush border Na ؉ -H ؉ exchanger in more detail, we performed membrane fractionation studies. We found that similar amounts of NHE3 were present in microvilli and a nonmicrovillar membrane domain of high density (dense vesicles). Horseradish peroxidase-labeled endosomes were not prevalent in the dense membrane fraction. However, megalin, which localizes primarily to the intermicrovillar microdomain of the brush border, was enriched in the dense vesicles, implicating this microdomain as the likely source of these membranes. Immunolocalization of NHE3 confirmed that a major fraction of the transporter colocalized with megalin in the intermicrovillar region of the brush border. Immunoprecipitation studies demonstrated that in microvilli the majority of NHE3 was not bound to megalin, while in the dense vesicles most of the NHE3 coprecipitated with megalin. Moreover, sucrose velocity gradient centrifugation experiments revealed that most NHE3 in microvilli sedimented with an S value of 9.6, while the S value of NHE3 in dense vesicles was 21. Finally, we examined the functional state of NHE3 in both membrane fractions. As assayed by changes in acridine orange fluorescence, imposing an outwardly directed Na ؉ gradient caused generation of an inside acid pH gradient in the microvilli, indicating Na ؉ -H ؉ exchange activity, but not in the dense vesicles. Taken together, these data demonstrate that renal brush border NHE3 exists in two oligomeric states: a 9.6 S active form present in microvilli and a 21 S, megalin-associated, inactive form in the intermicrovillar microdomain of the apical plasma membrane. Thus, regulation of renal brush border Na ؉ -H ؉ exchange activity may be mediated by shifting the distribution between these forms of NHE3.In the kidney, the activity of the Na ϩ -H ϩ exchanger isoform NHE3, located on the apical microvillar membrane of the proximal tubule, plays a major role in mediating transepithelial bicarbonate and NaCl reabsorption (2-4). Numerous physiologic studies of brush border Na ϩ -H ϩ exchange have shown that this activity is regulated by such hormones as angiotensin II (5) and parathyroid hormone (6) as well as by systemic alterations in acid-base balance (2, 3). Several laboratories have presented evidence suggesting that NHE3 is regulated by posttranslational mechanisms that may include membrane trafficking between an intracellular compartment and the plasma membrane (7-9). Such models predict that NHE3 must be localized in a nonmicrovillar membrane compartment, which functions as a store of transporter, as well as on the microvillar membrane where NHE3 is active.We have recently reported an association between NHE3 and the putative scavenger receptor megalin in renal brush border membranes (1). Moreover, we found that renal brush border NHE3 exists in two states with distinct sediment...

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“…Other molecules might be involved in the alteration of the subcellular distribution of NaPi-II. The mechanism for the internalization of NaPi-II might be distinct from the case of the Na ϩ /H ϩ exchanger, whereby specific association and co-internalization with megalin have been previously reported in the proximal tubules (28). Acute impairment of megalin function is often observed in renal tubulopathy caused by aminoglycosides and anticancer drugs (19,20).…”

Section: Discussionmentioning

confidence: 99%

Intraperitoneal Administration of Recombinant Receptor-Associated Protein Causes Phosphaturia via an Alteration in Subcellular Distribution of the Renal Sodium Phosphate Co-Transporter

Yamagata

Ozono

Hashimoto

et al. 2005

Journal of the American Society of Nephrology

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Megalin is a multifunctional endocytic receptor that is expressed in renal proximal tubules and plays critical roles in the renal uptake of various proteins. It was hypothesized that megalin-dependent endocytosis might play a role in renal phosphate reabsorption. For addressing the short-term effects of altered megalin function, a recombinant protein for the soluble form of 39-kD receptor-associated protein (RAP) was administered intraperitoneally to 7-wk-old mice. Histidine (His)-tagged soluble RAP (amino acids 39 to 356) lacking the amino-terminal signal peptide and the carboxy-terminal endoplasmic reticulum retention signal was prepared by bacterial expression (designated His-sRAP). After the direct interaction between His-sRAP and megalin was confirmed, mice were given a single intraperitoneal administration of His-sRAP (3.5 mg/dose). Immunostaining and Western blot analyses demonstrated the uptake of His-sRAP and the accelerated internalization of megalin in proximal tubular cells 1 h after administration. In addition, internalization of the type II sodium/phosphate co-transporter (NaPi-II) was observed. The effects of three sequential administrations of His-sRAP (3.5 mg/dose, three doses at 4-h intervals) then were examined, and increased urinary excretion of low molecular weight proteins, including vitamin D-binding protein, was found, which is consistent with findings reported for megalin-deficient mice. It is interesting that urinary excretion of phosphate was also increased, and the protein level of NaPi-II in the brush border membrane was decreased. Serum concentration of 25-hydroxyvitamin D was decreased, whereas the plasma level of intact parathyroid hormone was not altered by the administration of His-sRAP. The results suggest that the His-sRAP-induced acceleration of megalin-mediated endocytosis caused phosphaturia via altered subcellular distribution of NaPi-II.

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Specific Association of Megalin and the Na+/H+ Exchanger Isoform NHE3 in the Proximal Tubule

Cited by 111 publications

References 47 publications

Rho GTPases dictate the mobility of the Na/H exchanger NHE3 in epithelia: Role in apical retention and targeting

Rho GTPases dictate the mobility of the Na/H exchanger NHE3 in epithelia: Role in apical retention and targeting

Active (9.6 S) and Inactive (21 S) Oligomers of NHE3 in Microdomains of the Renal Brush Border

Intraperitoneal Administration of Recombinant Receptor-Associated Protein Causes Phosphaturia via an Alteration in Subcellular Distribution of the Renal Sodium Phosphate Co-Transporter

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