Podocalyxin Activates RhoA and Induces Actin Reorganization through NHERF1 and Ezrin in MDCK Cells
Sandra Schmieder,
Masaaki Nagai,
Robert A. Orlando,
Tetsuro Takeda and
Marilyn G. Farquhar
Department of Cellular and Molecular Medicine, University of California, San Diego, San Diego, California
Correspondence to Dr. Marilyn G. Farquhar, Department of Cellular and Molecular Medicine, George Palade Laboratories Room 210, 9500 Gilman Drive, La Jolla, CA 92093-0651. Phone: 858-534-7711; Fax: 858-534-8549; E-mail: mfarquhar{at}ucsd.edu
ABSTRACT. Podocalyxin (PC) is the major sialoglycoprotein expressedon the apical membrane of the podocyte. Previously it was shownthat PC is connected to actin through the PC/NHERF2/ezrin complex,and this connection is disrupted in the nephrotic syndrome.For assessing whether expression of PC affects the organizationof the actin cytoskeleton, MDCK cell lines stably expressingeither full-length PC or a PC mutant lacking the NHERF bindingsite was established. It was found that full-length PC but notthe PC mutant is connected to actin, induces redistributionof actin toward the apical membrane, and leads to increasedRhoA activity. By immunofluorescence redistribution of RhoAand RhoGDI was observed in the presence of both full-lengthPC and the PC mutant. With the use of pulldown assays, PC andezrin were found to interact directly and the ezrin bindingsite was mapped to the juxtamembrane region of PC’s cytoplasmictail. It is concluded that PC binds to ezrin both directly andindirectly. PC activates RhoA through NHERF and ezrin, leadingto redistribution of actin filaments. These results suggestthat in podocytes, PC may also regulate foot process architecturethrough RhoA.
The unique foot process and filtration slit architecture ofpodocytes is essential for maintaining glomerular filtration.Disruption of this organization is characteristically seen inglomerular diseases associated with the nephrotic syndrome.Although the molecular mechanisms that lead to these derangementsare not yet fully understood, it has been shown that the denseactin network present in the foot processes is normally anchoredto apical and basal membrane proteins as well as to proteinsin the slit diaphragm region (1) and undergoes substantial changesin the nephrotic syndrome (2–6).
We have previously shown that podocalyxin (PC), the major apicalsialoglycoprotein of the podocyte, serves as an anti-adhesinthat maintains the filtration slits between the foot processesopen (7) and is connected to the actin cytoskeleton indirectlythrough NHERF2 (8), a PDZ protein, and ezrin, a member of theERM (ezrin-radixin-moesin) family of actin-binding proteins(9). Moreover, we found that the connection of PC to the actincytoskeleton is disrupted in puromycin aminonucleoside nephrosis(8), a rat model in which there is a dramatic reorganizationof the actin cytoskeleton (5,6).
In this article, we investigate how the association of PC withactin is regulated, focusing on the small GTPase RhoA, becauseRhoA has been shown to activate ezrin (10) and ezrin connectsvarious plasma membrane proteins, including PC, to actin filamentsin its open or active conformation (11). Activated ERM proteinsalso bind and sequester RhoGDI (12–15), a negative regulatorof Rho GTPases, thereby initiating the activation of RhoA (13)and maintaining ERM activation (16). Moreover, it is clear thatproper regulation of Rho GTPase is required for maintainingthe differentiation of podocytes, as RhoGDI –/–mice, lacking RhoGDI, show massive proteinuria and loss of footprocess architecture (17).
In this study, we used MDCK cells to investigate further theconnection of PC to actin, to assess the effects of expressionof PC on actin organization, and to determine the involvementof RhoA in these effects. We show here that expression of PCleads to redistribution of actin and RhoGDI toward the apicalmembrane and activation of RhoA. Our findings suggest that inthe podocyte, PC participates in the organization of the actincytoskeleton that maintains the foot process architecture throughactivation of RhoA.
Antibodies
PC mAb 5A (18) and rabbit polyclonal 0601 which recognizes thecytoplasmic tail of PC (7) were described previously. Anti-EBP50(NHERF1) was provided by Dr. Anthony Bretscher (Cornell University,Ithaca, NY), and polyclonal anti-E3KARP (NHERF2) was providedby Dr. Chris Yun (Johns Hopkins University, Baltimore, MD).Anti-ezrin (3C12) was purchased from NeoMarkers (Fremont, CA),and anti-RhoA (26C4) and anti-RhoGDI (A-20) were purchased fromSanta Cruz Biotechnology (Santa Cruz, CA).
Cell Culture and Retroviral Infection
MDCK cells and HEK293 cells were maintained as described previously(19). MDCK cells stably expressing PC or mutant PC(DTHL), lackingthe last four amino acids of the cytoplasmic tail that constitutethe NHERF binding site, and those expressing empty vector weregenerated using a retrovirus system as described in detail elsewhere(19).
Immunofluorescence Microscopy
MDCK cells that were grown on glass coverslips or Costar Transwellfilters (Corning, Palo Alto, CA) for 3 d after confluence werefixed in 2% paraformaldehyde, permeabilized, and incubated sequentiallywith primary antibodies (7) followed by cross-absorbed Alexa488 or Alexa 594 goat anti-rabbit or anti-mouse F(ab')2 (MolecularProbes, Eugene, OR). Actin was detected with Texas Red-phalloidin.Cells were examined with a Zeiss Axiophot equipped for epifluorescenceor a BioRad MRC1024 confocal microscope equipped with Lasersharp3.1 software. Vertical sections were generated in 0.3-µmsteps. Axiophot images were acquired with a Hamamatsu ORCA-ERcamera and processed with Scion Image software. Confocal imageswere processed with Adobe Photoshop.
SDS-PAGE and Immunoblotting
Cells were lysed in 0.5% Triton X-100, 20 mM Tris-HCl (pH 7.4),and 150 mM NaCl that contained Complete protease inhibitor mix(Roche, Indianapolis, IN). Proteins were quantified using theBradford assay (BioRad, Hercules, CA), heated 5 min at 95°Cin Laemmli sample buffer (LSB), separated by SDS-PAGE, and transferredonto polyvinylidene difluoride membranes. Membranes were incubated(2 h) with primary antibodies followed by horseradish peroxidase-coupledsecondary antibodies and detection by chemiluminescence. Proteinbands were quantified by densitometry using Scan Analysis Software(BioSoft, Cambridge, England).
Alkaline Phosphatase Treatment
MDCK cell lysates (20 µg) were suspended in dephosphorylationbuffer (50 mM Tris [pH 9], 1 mM MgCl2, 0.1 mM ZnCl2, and 1 mMspermidine), mixed with 15 U of alkaline phosphatase (Roche),incubated at 30°C for 30 min, and immunoblotted for NHERF1.In controls, alkaline phosphatase was omitted and 200 nM okadaicacid was added to inhibit serine/threonine phosphatases.
Differential Detergent Extraction
Differential detergent extraction of cells was carried out asdescribed previously (8). Briefly, cells were lysed in 0.5%Triton X-100, the lysate was centrifuged, the pellet was reextractedwith RIPA buffer (0.1% SDS, 0.5% sodium deoxycholate, 1% TritonX-100, 150 mM NaCl, 20 mM HEPES, pH 7.4, and 1 mM EDTA), andthe lysate was again centrifuged. LSB was added to the Triton-soluble(TS), RIPA-soluble (RS), and RIPA-insoluble pellet (RI), andequal volumes of each fraction were analyzed by immunoblotting.
Assay for Active RhoA
A pulldown assay for active RhoA was carried out with a GSTfusion protein containing the Rho-binding domain (GST-RBD) ofRhotekin (provided by Dr. Martin Schwartz, University of Virginia,Charlottesville, VA) (20). MDCK cells were lysed in 1% TritonX-100, 0.5% sodium deoxycholate, 0.1% SDS, 50 mM Tris-HCl (pH7.2), 500 mM NaCl, and 10 mM MgCl2 that contained Complete.After centrifugation, lysates were incubated (90 min at 4°C)with 10 µg of GST-RBD immobilized on glutathione-agarosebeads. Bound proteins were eluted in LSB, and RhoA was detectedby immunoblotting.
Pulldown Assay Using In Vitro Translated Proteins
cDNA encoding the cytoplasmic tail of PC (PCT) or the PCT(DTHL)mutant were amplified by PCR and inserted into pGEX-KG (AmershamPharmacia Biotech, Piscataway, NJ), and GST fusion proteinswere produced. 35S-labeled ezrin or a N-terminal fragment ofezrin (amino acids 1 to 310) were produced by TNT T7 Quick CoupledTranscription/Translation system (Promega, Madison, WI) in thepresence of [35S]methionine and pCR3 constructs (provided byDr. Heinz Furthmayr, Stanford University, Stanford, CA). Invitro translation products were incubated (2 h at 4°C) withGST or GST fusion proteins immobilized on glutathione-Sepharosebeads in 200 mM NaCl, 20 mM Tris-HCl (pH 8), and 0.2% TritonX-100 that contained Complete. Bound proteins were analyzedby autoradiography.
Pulldown Assay Using Purified Recombinant Proteins
cDNA encoding PCT and a truncated PCT (PCT-N) lacking the 12N-terminal amino acids of the cytoplasmic tail (HQRISQRKDQQR)were amplified by PCR and inserted into pET28 (Novagen, Madison,WI). The QuickChange site-directed mutagenesis kit (Stratagene,La Jolla, CA) was used to introduce point mutations in the PCTchanging residues H411, R413, and S415 to alanines. His-taggedproteins (His-PCT, His-PCT-N, His-PCT-S/A, His-PCT-HR/AA, andHis-PCT-HRS/AAA) were produced according to the manufacturer’sinstructions. Ezrin and N'-ezrin were amplified by PCR and insertedinto pGEX-KG, and GST fusion proteins were produced. His-taggedPCT or PCT mutants were incubated for 2 h at 4°C with GSTalone, GST-ezrin, or GST-N'-ezrin in 500 mM NaCl, 20 mM Tris-HCl(pH 8), and 0.2% Triton X-100 that contained Complete, and boundproteins were detected by immunoblotting.
Characterization of MDCK Cell Lines
MDCK cell lines were generated that stably express PC (MDCK-PC)or a mutant (MDCK-PC[DTHL]), lacking the four C-terminal aminoacids that constitute the PDZ binding motif through which PCbinds the PDZ proteins NHERF1 and NHERF2 (8). A control cellline (MDCK-mock) was infected with empty retrovirus. PC wasdetected at the expected size, 140 kD, in MDCK-PC and MDCK-PC(DTHL)cells but not in controls that lack endogenous PC (Figure 1A).Ezrin migrated at 81 kD, and NHERF1 migrated as several bands,48 to 52 kD, in all cell lines. By densitometry, the upper NHERF1bands represent 60% of the total NHERF1 signal in MDCK-PC cellsand only 40% in the other cell lines. After alkaline phosphatasedigestion (Figure 1B), only the 48-kD band was seen, indicatingthat the upper bands represent phosphorylated forms of NHERF1.Increased phosphorylation of NHERF1 was not seen in the PC mutantlacking the NHERF1 binding site, suggesting that direct bindingof PC to NHERF1 is required for enhanced phosphorylation ofNHERF1. NHERF2 was not detected in any of the MDCK cells.
Figure 1. Expression of podocalyxin (PC), ezrin, NHERF1, and NHERF2 in MDCK cells. (A) Exogenous PC is present in MDCK-PC and MDCK-PC(DTHL) cell lysates but not in MDCK-WT and MDCK-mock cells. Endogenous ezrin and NHERF1 are present in all cell lines at similar levels, whereas NHERF2 is absent. NHERF1 migrates as several bands, 48 to 52 kD. In MDCK-PC cells, the 48-kD band is decreased, and the upper, 52-kD band is increased. Cell lysates (20 µg) were separated by 7.5% SDS-PAGE and immunoblotted with anti-podocalyxin 5A (1:1000), anti-ezrin 3C12 (1:500), anti-NHERF (1:1000), and anti-NHERF2 (1:1000). (B) Identification of phosphorylated forms of NHERF1. After alkaline phosphatase treatment of MDCK-PC cell lysates, the higher molecular weight forms of NHERF1 disappear and the 48-kD form is correspondingly increased. MDCK-PC cell lysate (20 µg) was incubated without (–; lane 1) or with (+; lane 2) alkaline phosphatase for 30 min at 30°C, separated by 7.5% SDS-PAGE, and immunoblotted with anti-NHERF1 (1:1000).
Actin Is Redistributed in MDCK-PC Cells
We next assessed the effect of expressing PC on the distributionof actin in MDCK cells by immunofluorescence. PC was seen exclusivelyalong the apical membrane (Figure 2C) as previously reported(7). The apical targeting of PC in MDCK cells corresponds tothe apical localization of PC in podocytes along the tops andsides of the foot processes above the level of the slit membranes(21). The PC(DTHL) mutant was also localized mainly on the apicalmembrane, but some staining was also seen inside the cell (Figure 2F).Similar findings were recently reported for the same truncationmutant of rabbit PC expressed in MDCK cells (22).
Figure 2. Distribution of actin and PC in MDCK cells as seen by confocal microscopy (vertical or X-Z sections). In MDCK-mock cells (A), actin is uniformly distributed along the apical, lateral, and basal domains of the cell membrane. In MDCK-PC cells (B through D), actin staining is increased at the apical domain (B) and overlaps with PC located along the apical domain (C). The increase in apical actin correlates with the expression level of PC (*). In MDCK-PC(DTHL) cells (E through G), overlap of PC with actin at the apical domain (F and G) is not striking as in MDCK-PC cells, and some of the PC is found intracellularly (F, arrows). Cells were grown on filters, stained for actin with phalloidin-Texas Red (1:200) and for PC with mAb 5A (1:50), and analyzed by confocal microscopy. Bar = 15 µm.
In MDCK-mock (Figure 2A) and MDCK-PC(DTHL) (Figure 2E) cells,actin was distributed along the apical, lateral, and basal cellmembranes with similar staining intensities. By contrast, inMDCK-PC cells, actin staining was increased along the apicaland decreased along the basal domain (Figure 2B). The extentof the redistribution correlated with the expression level ofPC (Figure 2C, D, and G). Horizontal views at the apical levelshowed that actin overlapped with PC (Figure 3, A through C)but not with the PC(DTHL) mutant (Figure 3, D through F), andthis overlap was greater in cells with high expression levelsof PC. Taken together, these observations indicate that expressionof PC in MDCK cells leads to redistribution of actin towardthe apical membrane and that the interaction between PC andNHERF1 is required for this to occur.
Figure 3. Horizontal sections at the apical level showing staining for actin and PC in MDCK cells. In MDCK-PC cells (A through C), there is striking overlap between actin and PC staining (yellow in C), which correlates with the expression level of PC. In MDCK-PC(DTHL) cells (D through F), there is little overlap in the distribution of actin and PC. Cells were prepared and analyzed as in Figure 2. Bar = 15 µm
NHERF1 and Ezrin Connect PC to the Actin Cytoskeleton
Next, we examined the association of PC and ezrin with actinusing a differential detergent extraction method (8) that preservescytoskeletal integrity and the interactions of noncytoskeletalproteins with the cytoskeleton (23,24). Cell components wereseparated into TS, RS, and RI fractions (Figure 4). After extractionof MDCK-PC cells, PC was found in all three fractions: mostof it was found in the TS fraction, but significant amountswere detected in the RS and RI fractions. As previously shown,some of the PC co-sediments with actin (9). In contrast, PC(DTHL)was detected only in the TS fraction, indicating dissociationfrom actin filaments. Ezrin distributed in all three fractions,but the amount found in the RI fraction was increased twofoldin MDCK-PC cells over controls, indicating that expression ofPC leads to increased activation of ezrin and its associationwith actin. However, the amount of ezrin found in the RI fractionof MDCK-PC(DTHL) cells was decreased twofold over controls.These results indicate that when full-length PC is expressedin MDCK cells, some of the protein is connected to the actincytoskeleton. The increased solubility of the PC mutant indicatesthat ezrin alone is not sufficient to connect PC to the actincytoskeleton. Rather, full engagement of PC with the cytoskeletonrequires NHERF1 interactions with PC’s C-terminal PDZbinding domain.
Figure 4. Distribution of PC and ezrin in fractions obtained by selective detergent extraction. In MDCK-PC cells, full-length PC is present in the Triton X-100-soluble (TS), RIPA-soluble (RS), and RIPA-insoluble (RI) fractions (lanes 7, 8, and 9, respectively), whereas in MDCK-PC(DTHL) cells, the PC mutant is present exclusively in the TS fraction (lane 10). Ezrin is distributed in all three fractions in all cell types; however, in MDCK-PC cells, the amount of ezrin in the RI fractions (lane 9) is increased compared with MDCK-WT and MDCK-mock cells (compare with lanes 3 and 6). By contrast, in MDCK-PC(DTHL) cells, the amount of ezrin found in the RI fraction was decreased (lane 12). MDCK cells were extracted sequentially as described in Materials and Methods. Equal volumes of the different fractions were immunoblotted for PC and ezrin as in Figure 1.
RhoGDI and RhoA Are Redistributed in MDCK-PC Cells
Increased actin-bound ezrin indicates an increase in activeezrin in MDCK-PC cells. Because activated ezrin binds RhoGDI(12–15), we investigated the distribution of RhoGDI inMDCK-PC cells versus controls. In vertical sections of MDCK-mockcells (Figure 5A) and MDCK-WT (not shown), RhoGDI was distributedthroughout the cytoplasm, whereas in both MDCK-PC (Figure 5, B through D)and MDCK-PC(DTHL) cells (Figure 5, E and F), thedistribution of RhoGDI had shifted and was more concentratedat the apical membrane, where PC and the PC(DTHL) mutant werefound. The redistribution of RhoGDI in cells expressing thePC mutant lacking the NHERF binding site indicates that RhoGDI’srecruitment to the vicinity of PC does not involve binding toNHERF1.
Figure 5. Distribution of RhoGDI and PC in MDCK cell lines. In MDCK-mock cells (A), RhoGDI is distributed throughout the cytoplasm, whereas in MDCK-PC (B) and MDCK-PC(DTHL) (E) cells, RhoGDI is enriched along the apical membrane in cells expressing full-length and mutant PC. Apical RhoGDI enrichment is strongest in cells with the highest expression levels of full-length and mutant PC (*). Co-localization of RhoGDI and PC correlates with the expression level of PC (B through D). Cells were grown on filters and stained with anti-RhoGDI (1:200) and anti-PC 5A mAb (1:50) and analyzed by confocal microscopy. Bar = 15 µm.
Active RhoA is known to translocate to the plasma membrane,where it interacts with various effector proteins to mediatedownstream signaling and to affect the distribution and organizationof actin. We reasoned that RhoA might also be redistributedin cells expressing PC, as binding of RhoGDI to activated ezrindissociates the RhoGDI/RhoA complex, allowing subsequent activationand redistribution of RhoA (25). We found that in controls,RhoA was distributed throughout the cytoplasm and was most concentratedin the juxtanuclear region (Figure 6, A and B). In MDCK-PC (Figure 6C)and PC(DTHL) (Figure 6D) cells, there was a partial shiftin RhoA staining to the cell membranes. Thus, expression ofPC or the PC mutant in MDCK cells results in redistributionof both RhoGDI and RhoA independent of NHERF binding to PC.
Figure 6. Distribution of RhoA in MDCK cells. In MDCK-WT (A) and MDCK-mock (B) cells, RhoA is distributed throughout the cytoplasm, but is most concentrated in the juxtanuclear region. In MDCK-PC (C) and MDCK-PC(DTHL) (D) cells, RhoA is distributed along the cell membrane (arrows), and cytoplasmic staining is typically decreased. MDCK cells were grown on coverslips, labeled with anti-RhoA (1:400), and examined by immunofluorescence microscopy. Bar = 5 µm.
RhoA Is Activated in MDCK-PC Cells
To determine whether RhoA activity is increased in cells expressingPC, we carried out a pulldown assay for active RhoA (20). AllMDCK cell lines showed similar levels of total RhoA, but theratio of GTP-bound RhoA to total RhoA was threefold higher inMDCK-PC cells compared with the other cell lines, includingMDCK-PC(DTHL) cells (Figure 7). The absence of enhanced RhoAactivity in the mutant indicates that expression of PC leadsto increased RhoA activity through NHERF1. From these results,we conclude that interaction between PC and NHERF1 is not requiredfor the redistribution of RhoGDI and RhoA, but it is requiredfor activation of RhoA, the connection of PC to the actin cytoskeletonand redistribution of actin.
Figure 7. Determination of RhoA activity in MDCK cells. (A) The amount of GTP-bound RhoA (bottom panel) in MDCK-PC cell lysates (lane 3) is higher than in the other cell lines (lanes 1, 2, and 4). The amounts of total RhoA in the lysates (top panel) are similar in all MDCK cell lines (lanes 1 through 4). A pulldown assay for GTP-bound RhoA was carried out as described in Materials and Methods. Total RhoA (5 µl of lysate) and GTP-bound RhoA (700 µl of lysate) were detected by immunoblotting with anti-RhoA mAb (1:400). (B) Graphic representation of the relative RhoA activity in MDCK cells. In MDCK-PC cells (lane 3), RhoA activity increased approximately threefold compared with MDCK-WT (lane 1), MDCK-mock (lane 2), and MDCK-PC(DTHL) cells (lane 4). The amounts of total and GTP-bound RhoA were quantified by densitometry. The relative RhoA activity was calculated from the ratios of GTP-bound RhoA/total RhoA and normalized to the ratio obtained for MDCK-WT cells (lane 1).
PC Can Bind Directly to Ezrin
The recruitment of RhoGDI to the plasma membrane in cells expressingthe PC(DTHL) mutant missing the NHERF binding site was surprising,because we have previously shown that PC associates with ezrinthrough NHERF (8). It raised the possibility that PCT can binddirectly to ezrin independent of NHERF. To test this possibility,we carried out a pulldown assay with GST-PCT or GST-PCT(DTHL)and 35S-labeled, in vitro-translated N-terminal fragment ofezrin (N'-ezrin). With N'-ezrin, we observed binding to bothGST-PCT and GST-PCT(DTHL) (Figure 8A). Binding to GST-PCT(DTHL)was weaker than to GST-PCT but was above that observed withGST alone. Full-length ezrin did not bind to any of the fusionproteins (not shown), most likely because it assumes an inactivehead-to-tail conformation that partially masks the PC bindingsite (11).
Figure 8. The cytoplasmic tail of PC (PCT) interacts directly with the N-terminal domain of ezrin. (A) In vitro-translated, N-terminal fragment of ezrin (N'ezrin) interacts with GST-PCT (lane 3) but not with GST alone (lane 2). Binding of N'ezrin to GST-PCT(DTHL) (lane 4) is reduced compared with GST-PCT. Equimolar amounts of GST (5 µg), GST-PCT (7 µg), and GST-PCT(DTHL) (7 µg) bound to glutathione-Sepharose beads were incubated with in vitro-translated, radiolabeled N'ezrin. Proteins bound to the beads were separated by 12% SDS-PAGE and analyzed by autoradiography. Lane 1 shows 100% of input. (B) Purified His-tagged PCT directly interacts with purified GST-N'ezrin (lane 3) and GST-ezrin (lane 4) but not with GST alone (lane 2). His-PCT (100 µg) was incubated with GST, GST-N'ezrin, or GST-ezrin (50 µg) bound to glutathione-agarose beads. Precipitates were separated by 12% SDS-PAGE and analyzed by immunoblotting using anti-PC 0601 (1:5000). Lane 1: 1% of His-PCT used in the assay. (C) Amino acid sequence alignment of the intracellular juxtamembrane regions of rat PC (accession no. NP 620203) and human intercellular adhesion molecule-3 (ICAM-3; accession no. NP 002153). Amino acids present in both sequences are indicated in bold. (D) Mapping the ezrin binding site in PCT with purified, recombinant proteins. Direct interaction between GST-N'ezrin and His-tagged PCT is reduced by 22, 46, 95, and 100%, respectively, in the following PCT mutants: His-PCT-S/A (lane 2), His-PCT-HR/AA (lane 3), His-PCT-HRS/AAA (lane 4), and His-PCT-N (lane 5). Purified GST-N'ezrin (50 µg) was immobilized on glutathione-agarose beads and incubated with His-PCT, His-PCT-S/A, PCT-HR/AA, His-PCT-HRS/AAA, or His-PCT-N (100 µg). Precipitates were analyzed by 12% SDS-PAGE and immunoblotted for PC as in B.
Next, we carried out a pulldown assay using purified His-taggedPCT and GST-ezrin or GST-N'-ezrin. We found that His-taggedPCT bound to GST-ezrin as well as to GST-N'-ezrin but not toGST alone (Figure 8B). As above, binding was stronger for GST-N'-ezrinthan for GST-ezrin. We conclude that PCT can bind directly tothe N-terminus of ezrin.
Identification of the Ezrin-Binding Site within the PCT
Sequence alignment (Figure 8C) revealed that the PCT possessesa sequence, HQRIS, in the juxtamembrane region that is similarto the HQRS sequence found in intercellular adhesion molecule-3(ICAM-3) (26). The basic residues H and R within this sequenceand several serines in the cytoplasmic tail are required forinteraction of ICAM-3 with ezrin (26). We tested the abilityof His-tagged PCT containing either a single mutation of S415to A (His-PCT-S/A), a double mutation of H411 and R413 residuesto alanines (His-PCT-HR/AA), a triple mutation of all threeresidues (His-PCT-HRS/AAA), or a 12 residue, N-terminal truncation(His-PCT-N), to interact with GST-N'-ezrin. We found that theinteraction of these mutants with GST-N'-ezrin was reduced by22, 46, 95, and 100%, respectively (Figure 8D). We concludethat the ezrin binding site is within the 12 juxtamembrane aminoacids of PCT and that residues H411, R413, and S415 are essentialfor binding of PC to ezrin.
The actin cytoskeleton is anchored to membrane proteins thatprovide structural support to the cell membrane, and its organizationdetermines cell shape. In several glomerular diseases, especiallyminimal-change nephrotic syndrome, reorganization of the actincytoskeleton accompanies changes in foot process organizationof podocytes, but the molecular events involved remain unknown(2). We have shown that PC is connected to actin through NHERFand ezrin under normal conditions, forming the PC/NHERF/ezrin/actincomplex, and is disconnected from actin in experimental modelsof nephrosis (8). Here we show that expression of PC in MDCKcells leads to phosphorylation of NHERF1, activation of ezrin,activation of RhoA, and redistribution of actin and that theseeffects are dependent on the interaction of PC with NHERF.
Our findings support the following model (Figure 9) for theeffects of PC on activation of RhoA and the assembly of thePC/NHERF1/ezrin/actin complex: PC binds to NHERF, which in turnbinds activated ezrin. Activated ezrin initiates the activationof RhoA through its ability to bind and sequester RhoGDI, releasingGDP-RhoA (13). The presence of NHERF in the complex is requiredfor the activation of RhoA. Once RhoA is activated, it signalsdownstream to maintain activation of ezrin, allowing connectionof PC to actin at the apical cell membrane. This model is basedon our observations and previous work by others as discussedbelow.
Figure 9. Proposed model for the PC-induced reorganization of actin in MDCK cells through the RhoA pathway. PC interacts with ezrin both directly and indirectly through NHERF1. The juxtamembrane region of PC interacts directly with the N-terminus of ezrin. The C-terminal PDZ-binding motif of PC (blue arrow) interacts with the second PDZ domain of NHERF1. PC-associated ezrin recruits RhoGDI, releasing GDP-RhoA. In the presence of NHERF1, RhoA-GDP is converted to RhoA-GTP, possibly because NHERF1 recruits a guanine nucleotide exchange factor (GEF) for RhoA. Activated RhoA-GTP interacts with effector proteins that lead to activation of ezrin, connecting PC to actin and concentrating actin at the apical domain. Activated RhoA also can modulate tight junction structure and function (not shown).
We found that RhoA activity is increased in cells expressingPC but not in those expressing the PC mutant lacking the NHERFbinding site, indicating that interaction with NHERF1 is essentialfor activation of RhoA. NHERF proteins bind and organize variousfunctional macromolecular complexes through their two PDZ domains,allowing, for example, the 2-adrenergic receptor (2-AR)-mediatedregulation of the Na+/H+ exchanger NHE-3 (27) or of cystic fibrosistransmembrane conductance regulator (CFTR) (28) and the reciprocalPKA-mediated regulation between CFTR and NHE3 (29). It is interestingthat stimulation of 2-AR (30) and the P2Y1 purinergic receptor(31,32)—both of which are connected to actin through NHERF1and ezrin—have also been reported to activate RhoA. Thus,it seems likely that NHERF recruits a factor responsible forRhoA activation, possibly a RhoGEF.
RhoA is known to activate ezrin (10) and to regulate the connectionof ezrin-binding membrane proteins to actin (33,34). The findingthat expression of the PC(DTHL) mutant does not activate RhoAand does not result in the redistribution of actin suggeststhat activation of RhoA is required for connection of PC toactin, probably because activated RhoA provides positive feedbackthat maintains ezrin in an active state capable of binding actin.
In addition to the ability of PC to bind ezrin indirectly throughNHERF, we found using pulldown assays that PC can bind directlyto ezrin. PC is the first membrane protein found to interactwith ezrin both directly and indirectly via NHERF. These twomechanisms—direct and indirect—have been describedfor the connection of membrane proteins to actin filaments throughezrin (11). One set of membrane proteins, e.g., CD44 (12), ICAM-1(35), ICAM-2 (35), ICAM-3 (26), syndecan 2 (34,36), and NHE1(36), bind directly to ezrin, whereas the other, e.g., NHE-3(37), 2-AR, and CFTR (27), interacts with ezrin through NHERFproteins.
Using alanine mutagenesis and purified, recombinant proteins,we identified the binding site for ezrin in the cytoplasmictail of PC. Sequence analysis had revealed that PC’s cytoplasmictail contains an amino acid sequence HQRIS in the juxtamembraneregion that resembles a sequence in ICAM-3 HQRS shown to beinvolved in ezrin binding (26). Mutational analysis confirmedthat, as in ICAM-3, two positively charged residues histineand arginine (H411 and R413) and a serine (S415) are involvedin the direct binding of PC to ezrin. Replacement of these residueswith alanines reduced ezrin binding by 95%, indicating thatthese residues, which are conserved in rabbit and human PC,are crucial for the interaction. That binding of ezrin was totallyabolished when the 12 juxtamembrane residues were removed providesfurther evidence that this region is essential for ezrin binding.The ezrin binding sites of PC, ICAM-3, and syndecan 2 seem tobe distinct from the juxtamembrane clusters of three consecutivepositively charged residues responsible for ezrin binding inCD44 (38), CD43, ICAM-1 (35), ICAM-2 (39), and NHE1 (36). Ezrinbinding sites on membrane proteins are often located in thejuxtamembrane region, which may allow for regulation of ezrinbinding by phosphoinositides. Ezrin binds phosphatidylinositol4,5-bisphosphate (PIP2) (43), and the interaction between ezrinand membrane proteins is facilitated by PIP2 (12,26,39).
The functional significance of the direct interaction betweenPC and ezrin is not clear and requires further investigation.One possibility is that a direct interaction between PC andezrin might allow transient disruption of the PC-NHERF interactionassociated with regulatory events such as phosphorylation thatmight induce conformational changes of NHERF1 and the cytoplasmictail of PC, both of which are known to be phosphorylated (41,42,43).
We have previously shown that expression of PC in MDCK cellsmodifies the distribution of junctional proteins and decreasestransepithelial resistance (7). RhoA and Rac1 are known to beinvolved in regulation of tight junction structure and function(44). Therefore, the available evidence suggests that PC participatesin the regulation of tight junction structure and function throughactivation of RhoA, which is known to promote actin reorganizationat the junctional level in MDCK cells (44,45).
During glomerular development, expression of PC correlates withthe establishment of foot process architecture, opening of intercellularspaces, disappearance of tight junctions, and appearance ofthe slit diaphragms (46). In a previous report, we showed thatPC’s ectodomain presents anti-adhesion properties thatcontribute to regulation of the maintenance of the filtrationpathway between neighboring podocytes (7). Our finding thatthe cytoplasmic tail of PC is connected to the actin cytoskeleton(9) and that loss of glomerular foot processes is associatedwith disruption of the PC-actin connection (8) suggested thatPC may contribute to regulation of podocyte cell shape throughits cytoplasmic tail. Our present results in MDCK cells demonstratethat PC activates the RhoA pathway and induces actin reorganization,providing further support for this conclusion.
Acknowledgments
This work was supported by National Institutes of Health GrantDK17724 (to M.G.F.) and by a German Academic Exchange ServiceFellowship (to S.S.).
We thank Dr. Larry Goldstein (University of California San Diego)for the use of his Bio-Rad MRC 1024 confocal microscope.
Footnotes
S.S.’s current affiliation is Laboratoire de Physiologiedes Membranes Cellulaires, Université de Nice-SophiaAntipolis/CNRS, La Darse, Villefranche sur mer, France.
Mundel P, Shankland SJ: Podocyte biology and response to injury. J Am Soc Nephrol 13: 3005–3015, 2002[Free Full Text]
Oh J, Reiser J, Mundel P: Dynamic (re)organization of the podocyte actin cytoskeleton in the nephrotic syndrome. Pediatr Nephrol 19: 130–137, 2004[CrossRef][Medline]
Pavenstadt H, Kriz W, Kretzler M: Cell biology of the glomerular podocyte. Physiol Rev 83: 253–307, 2003[Abstract/Free Full Text]
Lachapelle M, Bendayan M: Contractile proteins in podocytes: Immunocytochemical localization of actin and alpha-actinin in normal and nephrotic rat kidneys. Virchows Arch B Cell Pathol Incl Mol Pathol 60: 105–111, 1991[Medline]
Whiteside CI, Cameron R, Munk S, Levy J: Podocytic cytoskeletal disaggregation and basement-membrane detachment in puromycin aminonucleoside nephrosis. Am J Pathol 142: 1641–1653, 1993[Abstract]
Kubosawa H, Kondo Y: Modulation of cytoskeletal organization of podocytes during the course of aminonucleoside nephrosis in rats. Pathol Int 44: 578–586, 1994[Medline]
Takeda T, Go WY, Orlando RA, Farquhar MG: Expression of podocalyxin inhibits cell-cell adhesion and modifies junctional properties in Madin-Darby canine kidney cells. Mol Biol Cell 11: 3219–3232, 2000[Abstract/Free Full Text]
Takeda T, McQuistan T, Orlando RA, Farquhar MG: Loss of glomerular foot processes is associated with uncoupling of podocalyxin from the actin cytoskeleton. J Clin Invest 108: 289–301, 2001[CrossRef][Medline]
Orlando RA, Takeda T, Zak B, Schmieder S, Benoit VM, McQuistan T, Furthmayr H, Farquhar MG: The glomerular epithelial cell anti-adhesin podocalyxin associates with the actin cytoskeleton through interactions with ezrin. J Am Soc Nephrol 12: 1589–1598, 2001[Abstract/Free Full Text]
Matsui T, Yonemura S, Tsukita S: Activation of ERM proteins in vivo by Rho involves phosphatidyl-inositol 4-phosphate 5-kinase and not ROCK kinases. Curr Biol 9: 1259–1262, 1999[CrossRef][Medline]
Bretscher A, Edwards K, Fehon RG: ERM proteins and merlin: Integrators at the cell cortex. Nat Rev Mol Cell Biol 3: 586–599, 2002[CrossRef][Medline]
Hirao M, Sato N, Kondo T, Yonemura S, Monden M, Sasaki T, Takai Y, Tsukita S: Regulation mechanism of ERM (ezrin/radixin/moesin) protein/plasma membrane association: Possible involvement of phosphatidylinositol turnover and Rho-dependent signaling pathway. J Cell Biol 135: 37–51, 1996[Abstract/Free Full Text]
Takahashi K, Sasaki T, Mammoto A, Takaishi K, Kameyama T, Tsukita S, Takai Y: Direct interaction of the Rho GDP dissociation inhibitor with ezrin/radixin/moesin initiates the activation of the Rho small G protein. J Biol Chem 272: 23371–23375, 1997[Abstract/Free Full Text]
Maeda M, Matsui T, Imamura M, Tsukita S: Expression level, subcellular distribution and rho-GDI binding affinity of merlin in comparison with ezrin/radixin/moesin proteins. Oncogene 18: 4788–4797, 1999[CrossRef][Medline]
Hamada K, Seto A, Shimizu T, Matsui T, Takai Y, Tsukita S, Hakoshima T: Crystallization and preliminary crystallographic studies of RhoGDI in complex with the radixin FERM domain. Acta Crystallogr D Biol Crystallogr 57: 889–890, 2001[CrossRef][Medline]
Bretscher A, Chambers D, Nguyen R, Reczek D: ERM-Merlin and EBP50 protein families in plasma membrane organization and function. Annu Rev Cell Dev Biol 16: 113–143, 2000[CrossRef][Medline]
Togawa A, Miyoshi J, Ishizaki H, Tanaka M, Takakura A, Nishioka H, Yoshida H, Doi T, Mizoguchi A, Matsuura N, Niho Y, Nishimune Y, Nishikawa S, Takai Y: Progressive impairment of kidneys and reproductive organs in mice lacking Rho GDI. Oncogene 18: 5373–5380, 1999[CrossRef][Medline]
Miettinen A, Dekan G, Farquhar MG: Monoclonal antibodies against membrane proteins of the rat glomerulus. Immunochemical specificity and immunofluorescence distribution of the antigens. Am J Pathol 137: 929–944, 1990[Abstract]
Nagai M, Meerloo T, Takeda T, Farquhar MG: The adaptor protein ARH escorts megalin to and through endosomes. Mol Biol Cell 14: 4984–4996, 2003[Abstract/Free Full Text]
Ren XD, Kiosses WB, Schwartz MA: Regulation of the small GTP-binding protein Rho by cell adhesion and the cytoskeleton. EMBO J 18: 578–585, 1999[CrossRef][Medline]
Kerjaschki D, Sharkey DJ, Farquhar MG: Identification and characterization of podocalyxin—The major sialoprotein of the renal glomerular epithelial cell. J Cell Biol 98: 1591–1596, 1984[Abstract/Free Full Text]
Li Y, Li J, Straight SW, Kershaw DB: PDZ domain-mediated interaction of rabbit podocalyxin and Na(+)/H(+) exchange regulatory factor-2. Am J Physiol Renal Physiol 282: F1129–F1139, 2002[Abstract/Free Full Text]
Lenk R, Ransom L, Kaufmann Y, Penman S: A cytoskeletal structure with associated polyribosomes obtained from HeLa cells. Cell 10: 67–78, 1977[CrossRef][Medline]
Fey EG, Capco DG, Krochmalnic G, Penman S: Epithelial structure revealed by chemical dissection and unembedded electron microscopy. J Cell Biol 99: 203s–208s, 1984[Abstract/Free Full Text]
Takaishi K, Sasaki T, Kameyama T, Tsukita S, Takai Y: Translocation of activated Rho from the cytoplasm to membrane ruffling area, cell-cell adhesion sites and cleavage furrows. Oncogene 11: 39–48, 1995[Medline]
Serrador JM, Vicente-Manzanares M, Calvo J, Barreiro O, Montoya MC, Schwartz-Albiez R, Furthmayr H, Lozano F, Sanchez-Madrid F: A novel serine-rich motif in the intercellular adhesion molecule 3 is critical for its ezrin/radixin/moesin-directed subcellular targeting. J Biol Chem 277: 10400–10409, 2002[Abstract/Free Full Text]
Hall RA, Premont RT, Chow CW, Blitzer JT, Pitcher JA, Claing A, Stoffel RH, Barak LS, Shenolikar S, Weinman EJ, Grinstein S, Lefkowitz RJ: The 2-adrenergic receptor interacts with the Na+/H+-exchanger regulatory factor to control Na+/H+ exchange. Nature 392: 626–630, 1998[CrossRef][Medline]
Naren AP, Cobb B, Li C, Roy K, Nelson D, Heda GD, Liao J, Kirk KL, Sorscher EJ, Hanrahan J, Clancy JP: A macromolecular complex of beta 2 adrenergic receptor, CFTR, and ezrin/radixin/moesin-binding phosphoprotein 50 is regulated by PKA. Proc Natl Acad Sci U S A 100: 342–346, 2003[Abstract/Free Full Text]
Bagorda A, Guerra L, Di Sole F, Hemle-Kolb C, Cardone RA, Fanelli T, Reshkin SJ, Gisler SM, Murer H, Casavola V: Reciprocal protein kinase A regulatory interactions between cystic fibrosis transmembrane conductance regulator and Na+/H+ exchanger isoform 3 in a renal polarized epithelial cell model. J Biol Chem 277: 21480–21488, 2002[Abstract/Free Full Text]
Yamauchi J, Hirasawa A, Miyamoto Y, Itoh H, Tsujimoto G: 2-adrenergic receptor/cyclic adenosine monophosphate (cAMP) leads to JNK activation through Rho family small GTPases. Biochem Biophys Res Commun 284: 1199–1203, 2001[CrossRef][Medline]
Sauzeau V, Le Jeune H, Cario-Toumaniantz C, Vaillant N, Gadeau AP, Desgranges C, Scalbert E, Chardin P, Pacaud P, Loirand G: P2Y(1), P2Y(2), P2Y(4), and P2Y(6) receptors are coupled to Rho and Rho kinase activation in vascular myocytes. Am J Physiol Heart Circ Physiol 278: H1751–H1761, 2000[Abstract/Free Full Text]
Wilde JI, Retzer M, Siess W, Watson SP: ADP-induced platelet shape change: An investigation of the signalling pathways involved and their dependence on the method of platelet preparation. Platelets 11: 286–295, 2000[CrossRef][Medline]
Wojciak-Stothard B, Williams L, Ridley AJ: Monocyte adhesion and spreading on human endothelial cells is dependent on Rho-regulated receptor clustering. J Cell Biol 145: 1293–1307, 1999[Abstract/Free Full Text]
Granes F, Urena JM, Rocamora N, Vilaro S: Ezrin links syndecan-2 to the cytoskeleton. J Cell Sci 113 [Suppl]: 1267–1276, 2000[Abstract]
Heiska L, Alfthan K, Gronholm M, Vilja P, Vaheri A, Carpen O: Association of ezrin with intercellular adhesion molecule-1 and -2 (ICAM-1 and ICAM-2). Regulation by phosphatidylinositol 4, 5-bisphosphate. J Biol Chem 273: 21893–21900, 1998[Abstract/Free Full Text]
Denker SP, Huang DC, Orlowski J, Furthmayr H, Barber DL: Direct binding of the Na-H exchanger NHE1 to ERM proteins regulates the cortical cytoskeleton and cell shape independently of H(+) translocation. Mol Cell 6: 1425–1436, 2000[CrossRef][Medline]
Yun CH, Lamprecht G, Forster DV, Sidor A: NHE3 kinase A regulatory protein E3KARP binds the epithelial brush border Na+/H+ exchanger NHE3 and the cytoskeletal protein ezrin. J Biol Chem 273: 25856–25863, 1998[Abstract/Free Full Text]
Legg JW, Isacke CM: Identification and functional analysis of the ezrin-binding site in the hyaluronan receptor, CD44. Curr Biol 8: 705–708, 1998[CrossRef][Medline]
Yonemura S, Hirao M, Doi Y, Takahashi N, Kondo T, Tsukita S: Ezrin/radixin/moesin (ERM) proteins bind to a positively charged amino acid cluster in the juxta-membrane cytoplasmic domain of CD44, CD43, and ICAM-2. J Cell Biol 140: 885–895, 1998[Abstract/Free Full Text]
Barret C, Roy C, Montcourrier P, Mangeat P, Niggli V: Mutagenesis of the phosphatidylinositol 4,5-bisphosphate (PIP(2)) binding site in the NH(2)-terminal domain of ezrin correlates with its altered cellular distribution. J Cell Biol 151: 1067–1080, 2000[Abstract/Free Full Text]
Hall RA, Spurney RF, Premont RT, Rahman N, Blitzer JT, Pitcher JA, Lefkowitz RJ: G protein-coupled receptor kinase 6A phosphorylates the Na+/H+ exchanger regulatory factor via a PDZ domain-mediated interaction. J Biol Chem 274: 24328–24334, 1999[Abstract/Free Full Text]
He J, Lau AG, Yaffe MB, Hall RA: Phosphorylation and cell cycle-dependent regulation of Na+/H+ exchanger regulatory factor-1 by Cdc2 kinase. J Biol Chem 276: 41559–41565, 2001[Abstract/Free Full Text]
Schmieder S, Takeda T, Farquhar MG: Increased phosphorylation of podocalyxin in glomeruli from PAN nephrotic and protamine sulfate treated rats [Abstract]. J Am Soc Nephrol 12: A3603, 2001
Jou TS, Schneeberger EE, Nelson WJ: Structural and functional regulation of tight junctions by RhoA and Rac1 small GTPases. J Cell Biol 142: 101–115, 1998[Abstract/Free Full Text]
Takaishi K, Sasaki T, Kotani H, Nishioka H, Takai Y: Regulation of cell-cell adhesion by rac and rho small G proteins in MDCK cells. J Cell Biol 139: 1047–1059, 1997[Abstract/Free Full Text]
Schnabel E, Dekan G, Miettinen A, Farquhar MG: Biogenesis of podocalyxin—the major glomerular sialoglycoprotein—in the newborn rat kidney. Eur J Cell Biol 48: 313–326, 1989[Medline]
Received for publication January 15, 2004.
Accepted for publication May 28, 2004.
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Top
Abstract
Introduction
–/– mice, lacking RhoGDI
DTHL), lacking the last four amino acids of the cytoplasmic tail that constitute the NHERF binding site, and those expressing empty vector were generated using a retrovirus system as described in detail elsewhere (19). 
81 kD, and NHERF1 migrated as several bands, 48 to 52 kD, in all cell lines. By densitometry, the upper NHERF1 bands represent 
2-adrenergic receptor (
