NHE3 serves as a molecular tool for cAMP-mediated regulation of receptor-mediated endocytosis

Gekle, Michael; Serrano, Oscar K.; Drumm, Karina; Mildenberger, Sigrid; Freudinger, Ruth; Gaßner, Birgit; Jansen, H W; Christensen, Erik

doi:10.1152/ajprenal.00206.2001

Cited by 33 publications

(30 citation statements)

References 39 publications

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“…This process occurs via a highly active and tightly regulated receptor-mediated pathway involving the albumin binding megalin/cubulin scavenger receptor (5,8,27). In addition to the receptor complex itself, the uptake of albumin appears to require the formation of a complex that includes the ion transporting proteins V-type H ϩ -ATPase, Na ϩ -H ϩ exchanger isoform 3 (NHE3), and the Cl Ϫ channel ClC-5 (11,20,27). The V-type H ϩ -ATPase plays a crucial role in the acidification of the endosomes/lysosomes required for the dissociation of albumin from the receptor (27), whereas NHE3 is thought to play a role in facilitating the initial acidification of the nascent endosome (11).…”

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confidence: 99%

PKC-α-mediated remodeling of the actin cytoskeleton is involved in constitutive albumin uptake by proximal tubule cells

Hryciw¹,

Pollock²,

Poronnik³

2005

American Journal of Physiology-Renal Physiology

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One key role of the renal proximal tubule is the reabsorption of proteins from the glomerular filtrate by constitutive receptor-mediated endocytosis. In the opossum kidney (OK) renal proximal tubule cell line, inhibition of protein kinase C (PKC) reduces albumin uptake, although the isoforms involved and mechanisms by which this occurs have not been identified. We used pharmacological and molecular approaches to investigate the role of PKC-α in albumin endocytosis. We found that albumin uptake in OK cells was inhibited by the pan-PKC blocker bisindolylmaleimide-1 and the isoform-specific PKC blockers Gö-6976 and 2′,3,3′,4,4′-hexahydroxy-1,1′-biphenyl-6,6′-dimethanol dimethyl ether, indicating a role for PKC-α. Overexpression of a kinase deficient PKC-α(K368R) but not wild-type PKC-α significantly reduced albumin endocytosis. Western blot analysis of fractionated cells showed an increased association of PKC-α-green fluorescent protein with the membrane fraction within 10–20 min of exposure to albumin. We used phalloidin to demonstrate that albumin induces the formation of clusters of actin at the apical surface of OK cells and that these clusters correspond to the location of albumin uptake. These clusters were not present in cells grown in the absence of albumin. In cells treated either with PKC inhibitors or overexpressing kinase-deficient PKC-α(K368R) this actin cluster formation was significantly reduced. This study identifies a role for PKC-α in constitutive albumin uptake in OK cells by mediating assembly of actin microfilaments at the apical membrane.

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confidence: 99%

PKC-α-mediated remodeling of the actin cytoskeleton is involved in constitutive albumin uptake by proximal tubule cells

Hryciw¹,

Pollock²,

Poronnik³

2005

American Journal of Physiology-Renal Physiology

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“…Basal microvillus NHE3 was increased by myosin VI KD, whereas the secondmessenger-induced (FSH and CCH) decrease in microvillus NHE3 activity and amount was blocked. Because these two agents have been shown to stimulate endocytosis without altering the rates of exocytosis (Gekle et al, 2002;Lee-Kwon et al, 2003;Musch et al, 2007;Zachos et al, 2013), we propose that both basal and stimulated endocytosis require myosin VI. Additionally, because EGF treatment of myosin VI KD cells still led to an increase in microvillar NHE3 activity, our results further indicate that myosin VI is not involved in the stimulated exocytosis of NHE3 in the Na + -absorptive cell.…”

Section: Discussionmentioning

confidence: 92%

Myosin VI mediates the movement of NHE3 down the microvillus in intestinal epithelial cells

Chen

Hubbard

Murtazina

et al. 2014

Journal of Cell Science

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The intestinal brush border Na + /H + exchanger NHE3 is tightly regulated through changes in its endocytosis and exocytosis. Myosin VI, a minus-end-directed actin motor, has been implicated in endocytosis at the inter-microvillar cleft and during vesicle remodeling in the terminal web. Here, we asked whether myosin VI also regulates NHE3 movement down the microvillus. The basal NHE3 activity and its surface amount, determined by fluorometry of the ratiometric pH indicator BCECF and biotinylation assays, respectively, were increased in myosin-VI-knockdown (KD) Caco-2/ Bbe cells. Carbachol (CCH) and forskolin (FSK) stimulated NHE3 endocytosis in control but not in myosin VI KD cells. Importantly, immunoelectron microscopy results showed that NHE3 was preferentially localized in the basal half of control microvilli but in the distal half in myosin VI KD cells. Treatment with dynasore duplicated some aspects of myosin VI KD: it increased basal surface NHE3 activity and prevented FSK-induced NHE3 endocytosis. However, NHE3 had an intermediate distribution along the microvillus (between that in myosin VI KD and untreated cells) in dynasore-treated cells. We conclude that myosin VI is required for basal and stimulated endocytosis of NHE3 in intestinal cells, and suggest that myosin VI also moves NHE3 down the microvillus.

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“…It has been suggested that increased levels of cAMP reduce albumin uptake by inhibiting NHE3 (11). cAMP inhibits NHE3 via an NHERF1-ezrin-protein kinase A complex (53) and basal level phosphorylation of NHE3 by protein kinase A is reported in proximal tubule cells (54).…”

Section: Discussionmentioning

confidence: 99%

“…The percent inhibition of total albumin uptake was 21.9 Ϯ 3% for the pSHAG-FF and 21.8 Ϯ 3% when NHERF1 was silenced (n ϭ 3). uptake by OK cells is inhibited by cAMP, an effect that has been attributed to inhibition of NHE3 (11). We therefore investigated whether 8-Br-cAMP could inhibit the increase in albumin uptake observed following silencing of NHERF1.…”

Section: Roles Of Nherf2 and Nherf1 In Albumin Endocytosismentioning

confidence: 99%

“…The functional basis for the obligate role of NHE3 in albumin uptake is less clear and may involve initiating the acidification of the nascent endosome by the electroneutral exchange of endosomal Na ϩ for cytosolic H ϩ (9,10) or inhibiting fusion of the early endosome (10). This is demonstrated by reduced albumin uptake in the OK cell line when NHE3 is inhibited and the abolition of albumin uptake in NHE3-deficient OK cells (9,11). These data are complemented by the finding of elevated levels of urinary protein in NHE3 knock-out mice (12).…”

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confidence: 99%

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Regulation of Albumin Endocytosis by PSD95/Dlg/ZO-1 (PDZ) Scaffolds

Hryciw

Ekberg

Ferguson

et al. 2006

Journal of Biological Chemistry

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The constitutive reuptake of albumin from the glomerular filtrate by receptor-mediated endocytosis is a key function of the renal proximal tubules. Both the Cl ؊ channel ClC-5 and the Na ؉ -H ؉ exchanger isoform 3 are critical components of the macromolecular endocytic complex that is required for albumin uptake, and therefore the cell-surface levels of these proteins may limit albumin endocytosis. This study was undertaken to investigate the potential roles of the epithelial PDZ scaffolds, Na ؉ -H ؉ exchange regulatory factors, NHERF1 and NHERF2, in albumin uptake by opossum kidney (OK) cells. We found that ClC-5 co-immunoprecipitates with NHERF2 but not NHERF1 from OK cell lysate. Experiments using fusion proteins demonstrated that this was a direct interaction between an internal binding site in the C terminus of ClC-5 and the PDZ2 module of NHERF2. In OK cells, NHERF2 is restricted to the intravillar region while NHERF1 is located in the microvilli. Silencing NHERF2 reduced both cell-surface levels of ClC-5 and albumin uptake. Conversely, silencing NHERF1 increased cell-surface levels of ClC-5 and albumin uptake, presumably by increasing the mobility of NHE3 in the membrane and its availability to the albumin uptake complex. Surface biotinylation experiments revealed that both NHERF1 and NHERF2 were associated with the plasma membrane and that NHERF2 was recruited to the membrane in the presence of albumin. The importance of the interaction between NHERF2 and the cytoskeleton was demonstrated by a significant reduction in albumin uptake in cells overexpressing an ezrin binding-deficient mutant of NHERF2. Thus NHERF1 and NHERF2 differentially regulate albumin uptake by mechanisms that ultimately alter the cell-surface levels of ClC-5.

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NHE3 serves as a molecular tool for cAMP-mediated regulation of receptor-mediated endocytosis

Cited by 33 publications

References 39 publications

PKC-α-mediated remodeling of the actin cytoskeleton is involved in constitutive albumin uptake by proximal tubule cells

PKC-α-mediated remodeling of the actin cytoskeleton is involved in constitutive albumin uptake by proximal tubule cells

Myosin VI mediates the movement of NHE3 down the microvillus in intestinal epithelial cells

Regulation of Albumin Endocytosis by PSD95/Dlg/ZO-1 (PDZ) Scaffolds

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