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Originally published In Press as doi:10.1074/jbc.M405608200 on June 4, 2004

J. Biol. Chem., Vol. 279, Issue 33, 34302-34310, August 13, 2004
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Linking Receptor-mediated Endocytosis and Cell Signaling

EVIDENCE FOR REGULATED INTRAMEMBRANE PROTEOLYSIS OF MEGALIN IN PROXIMAL TUBULE*

Zhiying Zou{ddagger}, Brian Chung{ddagger}, Thao Nguyen{ddagger}, Sueann Mentone§, Brent Thomson{ddagger}, and Daniel Biemesderfer{ddagger}

From the Departments of {ddagger}Internal Medicine and §Cellular and Molecular Physiology, School of Medicine, Yale University, New Haven, Connecticut 06520-8029

Received for publication, May 19, 2004 , and in revised form, June 3, 2004.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
Megalin, a member of the low density lipoprotein receptor gene family, is required for efficient protein absorption in the proximal tubule. Recent studies have shown that the low density lipoprotein receptor-related protein, another member of this gene family, is proteolytically processed by {gamma}-secretase implying a role for low density lipoprotein receptor-related protein in a Notchlike signaling pathway. This pathway has been shown to involve: 1) metalloprotease-mediated ectodomain shedding and {gamma}-secretase-mediated intramembrane proteolysis of some receptors. Experiments were performed to determine whether megalin undergoes similar processing. By immunocytochemistry, immunoblotting, and a fluorogenic enzyme assay presenilin-1 (required for {gamma}-secretase activity) and {gamma}-secretase activity were found in the brush border of proximal kidney tubules where megalin is localized. Using a fluorogenic peptide containing an amyloid precursor protein {gamma}-secretase cleavage site and Compound E, a specific {gamma}-secretase inhibitor, we found high levels of {gamma}-secretase activity in renal brush border membrane vesicles. Immunoblotting analysis of renal microsomes and opossum kidney proximal tubule (OKP) cells using antibodies directed to the cytosolic domain of megalin showed a 35–40-kDa, membrane-associated, carboxyl-terminal fragment of megalin (MCTF). When cells were incubated with 200 nM phorbol 12-myristate 13-acetate, the appearance of the MCTF increased 2.5-fold and was blocked by metalloprotease inhibitors. When the cells were incubated with {gamma}-secretase inhibitor Compound E, it caused a 2-fold increase in MCTF. Finally, incubating the cells with 1 µM vitamin D-binding protein resulted in a 25% increase in the appearance of the MCTF. In summary, the MCTF is produced by protein kinase C regulated, metalloprotease-mediated ectodomain shedding and is the substrate for {gamma}-secretase. We postulate that the enzymatic processing of megalin represents part of a novel ligand-dependent signaling pathway in the proximal tubule that links receptor-mediated endocytosis with cell signaling.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
The proximal tubule reabsorbs low molecular weight protein (<70 kDa) by a mechanism that is largely dependent on the scavenger receptor megalin. In the healthy adult this amounts to as much as 600 mg of protein/day. However in renal disease, where the glomerular barrier is damaged, protein delivery to the proximal tubule increases dramatically and reaches levels that are eventually cytotoxic. Under these conditions the endocytic capacity of the proximal tubule becomes saturated and results in progressive nephropathy (1). In addition to proteinuria and sodium retention, the nephrotic syndrome is characterized by the up-regulation of several vasoactive and inflammatory genes (2). Although the pathologic course of the nephrotic syndrome has been fairly well characterized, the molecular mechanisms that link protein absorption with disease as well as with gene regulation in the proximal tubule are not understood.

Recently many studies have shown that a process known as regulated intramembrane proteolysis (RIP)1 acts on some membrane receptors and links receptor-mediated endocytosis and intracellular signaling events. RIP is an evolutionarily conserved mechanism involving the sequential, regulated proteolysis of membrane proteins culminating in the release of the intracellular domain of the protein from the membrane. The released intracellular domain in turn is either translocated to the nucleus where it acts as a transcription regulator (3) or it may activate other components of signaling pathways. First described for the sterol-regulatory element-binding protein (4), RIP has been shown to be involved in processing the Notch receptor (5), the amyloid precursor protein (APP) (6) as well as a growing list of other receptors including the ErbB-4, ATF6, and IRE1 (for a review, see Ref. 7).

For intramembrane cleavage to occur, RIP first requires ectodomain shedding of the receptor. Ectodomain shedding frequently occurs as multiple steps and is mediated either by members of the metalloprotease-disintegrin family of proteases, also called ADAMs (a disintegrin and metalloprotease domain), or by a matrix metalloprotease (MMP). The RIP of APP is clinically important since aberrant processing causes the accumulation of the A{beta} peptide fragments seen in the senile plaques that are pathogenic in Alzheimer's disease (8). In some instances, for example Notch and ErbB-4, ectodomain shedding has been shown to be ligand-dependent (5, 9). However, in the case of other receptors such as APP, the role of ligand binding is unclear, and constitutive processing seems to occur (10). In all cases studied to date, ectodomain shedding is regulated by the activity of protein kinase C (PKC) (for a review, see Ref. 11).

The shedding process produces characteristic membrane-associated carboxyl-terminal fragments, which in turn form the substrate for {gamma}-secretase. {gamma}-Secretase activity acts at a site within the transmembrane domain and is mediated by a protein complex that includes presenilin-1, presenilin-2, and nicastrin (12). Growing evidence supports the notion that the proteolytic activity of the {gamma}-secretase complex is mediated by the presenilins. Presenilin-1 and -2 are themselves proteolytically cleaved in the Golgi apparatus into amino-terminal and carboxyl-terminal fragments that remain associated in the active {gamma}-secretase complex (13).

Recent studies of the low density lipoprotein receptor-related protein (LRP), a member of the low density lipoprotein receptor gene family, showed that this receptor is also subjected to RIP (14). Since megalin is also a member of this gene family and closely related to LRP, we carried out studies to determine whether megalin is proteolytically processed by similar mechanisms. Here we report evidence for both {gamma}-secretase activity and cleaved forms of megalin in the brush border of the proximal tubule. We also show in opossum kidney proximal tubule (OKP) cells that megalin is processed by both matrix metalloprotease and {gamma}-secretase activities and that processing is both ligand-dependent and regulated by PKC.


   EXPERIMENTAL PROCEDURES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
RESULTS
 DISCUSSION
 REFERENCES
 
Materials—The fluorogenic {gamma}-secretase substrate (no. 565764), Compound E ({gamma}-secretase inhibitor XVIII, no. 565779), MMP inhibitor III (no. 444264), Gö6983 (no. 365251), and vitamin D-binding protein (Gcglobulin, no. 345802) were purchased from Calbiochem. The phorbol 12-myristate 13-acetate (PMA, no. P 8139), 4{alpha}PMA (no. P 148), and 5-(N-ethyl-N-isopropyl)amiloride (EIPA, no. A 3085) were obtained from Sigma.

Cloning the Carboxyl Terminus of Opossum (OKP Cell) Megalin— Opossum kidney mRNA was obtained from cultured OKP cells using TRIzolTM reagent (Invitrogen, no. 15596-018) followed by treatment with DNase (Invitrogen, no. 18068015) to remove contaminating genomic DNA. The cDNA was synthesized using oligo(dT)-primed total RNA (SuperScript First-Strand Synthesis System, Invitrogen, no. 11904-018). Using primers designed from the published rat megalin nucleotide sequence (15) we identified a 350-bp fragment of opossum megalin. A 3'-terminal 3.4-kb fragment of opossum kidney megalin mRNA was PCR-amplified by 3' rapid amplification of cDNA ends (Invitrogen, no. 18373012) using the gene-specific forward primer (5'-CAACTGTCCTTGTGTTCCAATCAGTC-3') and a universal amplification primer (5'-GGCCACGCGTCGACTAGTAC-3') supplied in the 3' rapid amplification of cDNA ends kit with Herculase Taq polymerase (Stratagene, no. 600310). The fragment was then ligated into pCR-Blunt vector (Invitrogen, no. K270020), and the sequence was confirmed by DNA sequencing. This opossum partial cDNA sequence was submitted to GenBankTM under the accession number AY627686 [GenBank] .

Constructing the GST-fused Opossum Kidney Cell Megalin Carboxyl Terminus as Antigen for Making Antibodies—A3'-end 660-bp fragment of the opossum megalin gene encoding the entire intracellular domain was PCR-amplified using forward primer 5'-GCTCCCTTCTGCCATCTCTTCCTAAGC-3' and reverse primer 5'-CAGTGGCACTAGTTATACCTCAGAGTC-3'. The PCR product was ligated into pDrive PCR vector (Qiagen) and digested out with BamHI and SalI. The GST-fused megalin carboxyl terminus was constructed by ligating the digested PCR product into pGEX-6p-2 vector (Amersham Biosciences). The GST-megalin fusion protein was expressed in bacteria and purified using Glutathione-SepharoseTM4B (Amersham Biosciences). The purified fusion protein was dialyzed into phosphate-buffered saline and stored at –70 °C.

{gamma}-Secretase Assay—{gamma}-Secretase activity was measured as described previously (16). Briefly solubilized brush border membrane vesicles were incubated with 8 µM fluorogenic substrate NMA (2-N-methylaminobenzoyl)-Gly-Gly-Val-Val-Ile-Ala-Thr-Val-Lys(DNP(dinitro-phenyl))-D-Arg-D-Arg-D-Arg-NH2 (Calbiochem) in 150 µl of assay buffer containing 50 mM Tris-HCl, pH 6.8, 2 mM EDTA, 0.25% CHAPSO (w/v) at 37 °C overnight with or without 30 nM specific {gamma}-secretase inhibitor Compound E (Calbiochem). After incubation, reactions were centrifuged at 16,100 x g for 15 min and chilled on ice. Supernatants were transferred to a 96-well plate, and fluorescence was measured using a plate reader (POLARSTAR Galaxy, BMG Lab Technologies, Ltd.) with excitation wavelength at 350 nm and emission wavelength at 440 nm. The data were analyzed and presented using Microsoft Excel.

Primary Antibodies—For anti-megalin antibodies, monoclonal and polyclonal antibodies were raised to the GST fusion protein representing the entire cytosolic domain of opossum megalin. For polyclonal antibodies, New Zealand White rabbits were immunized by the Immunization Service, Department of Comparative Medicine, Yale University School of Medicine. Immune sera were compared with preimmune sera by immunocytochemistry using fixed opossum kidney cryosections and by immunoblotting using specific and control fusion proteins as well as kidney microsomes as antigen. The serum was affinity-depleted of unwanted bacterial reactivity by passing over a CNBr-activated Sepharose column to which was attached control GST. This antibody is named anti-MC-220.

For monoclonal antibodies, BALB/c mice were immunized with the megalin carboxyl-terminal fusion protein using the pertussis/alum method (17). Splenocytes from an immunized mouse were fused with Ag8 cells, and hybridomas were prepared by routine methods using Cloning FactorTM (IGEN International, Inc., Gaithersburg, MD). Hybridomas secreting IgG with specificity for the megalin carboxyl-terminal domain were selected by screening by enzyme-linked immunosorbent assay using the GST-megalin carboxyl terminus and GST control fusion proteins as antigen. Hybridomas shown to be positive to the megalin fusion protein and not the control were screened by Western blot using fusion proteins and renal microsomes as antigen. mAb 6E10 was selected, and purified IgG was prepared by Protein G affinity chromatography. The specificity of the anti-megalin antibodies is shown in Fig. 1.



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  		FIG. 1.
Characterization of anti-megalin antibodies. Antibodies to native opossum megalin (mAb 6A6) or to the carboxyl-terminal cytosolic domain of opossum megalin (anti-MC-220 and mAb 6E10) were characterized by immunoblotting (panels A and B) and by immunocytochemistry (panel C). In panel A, purified GST (lanes 1, 3, and 5) and GST-megalin carboxyl terminus (lanes 2, 4, and 6) were probed with rabbit anti-GST antibody (lanes 1 and 2), mAb 6E10 (lanes 3 and 4), and anti-MC-220 (lanes 5 and 6). Molecular masses are shown on the left. In panel B, 100 µg of solubilized rat (lanes 2 and 3) or opossum (lanes 5, 6, and 7) renal microsomes were used for immunoprecipitation using anti-MC-220 (lane 2), mAb 6E10 (lane 5), and mAb 6A6 (lane 6). Unrelated antibodies, rabbit anti-Na+/Pi cotransporter (lane 3) or mAb 4E9 (anti-Na+/H+ exchanger isoform 1, lane 7), were used as control. As blotting controls, 10 µg of rat (lane 1) or opossum (lane 4) renal microsomal protein were used. Protein and immune complexes were analyzed by Western blot with previously characterized anti-megalin antibodies mAb DC6 (lanes 1–3) and rabbit anti-gp330CT (lanes 4–7). Panel C shows immunostaining of perfusion-fixed rat kidney with anti-MC-220 and mAb 6E10 and perfusion-fixed opossum kidney section with mAb 6A6.

 
A monoclonal antibody (mAb 6A6) was raised to native opossum kidney megalin. Advantage was taken of the antigenicity of the extracellular domain of megalin (18, 19). Mice were immunized with opossum kidney brush border membrane vesicles, and hybridomas were prepared as described above. Hybridomas were first screened by enzyme-linked immunosorbent assay using brush border membrane vesicles as antigen. Positive hybridomas were subsequently screened by immunocytochemistry using 5-µm cryosections of opossum kidney. Supernatants that stained the coated pit region of the brush border were then screened by Western blot using opossum microsomes as antigen. For comparison and as a positive control, a polyclonal antibody to the carboxyl terminus of rat megalin was used in the blotting screens. The specificity of mAb 6A6 is shown in Fig. 1. As seen in Fig. 1, all of the newly developed antibodies to opossum megalin (anti-MC220, 6E10, and 6A6) reacted with megalin by immunoprecipitation and immunoblotting and stained the brush border (coated pits) of the proximal tubule.

An additional mAb raised to megalin (mAb DC6) was a gift from Drs. Markus Exner and Dontscho Kerjaschki, Vienna, Austria. This mAb has been described previously and labels megalin in numerous mammalian species (20). These mAbs were used as purified IgG from hybridoma supernatants. Finally a polyclonal antibody to the megalin carboxyl terminus (anti-gp330CT) was a gift from Drs. Marilyn Farquhar and Robert Orlando at the University of California in San Diego. This antibody was raised to a fusion protein representing an 18-amino acid peptide within the carboxyl-terminal cytoplasmic domain of rat megalin (21).

A polyclonal antibody to the amino-terminal fragment of presenilin-1 was purchased from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA). As controls in some of the experiments described below, a polyclonal antibody to the Na+/Pi cotransporter (22) and the Na+/H+ exchanger isoform 1 (23) were used.

Antibody Conjugates—For indirect immunofluorescence microscopy, Alexa Fluor 594-conjugated goat anti-mouse or Alexa Fluor 488-conjugated goat anti-rabbit were purchased from Molecular Probes, Inc., Eugene, OR. For immunoblotting, horseradish peroxidase-conjugated rabbit anti-goat IgG (heavy and light chain-specific), goat anti-mouse ({gamma} chain-specific), or goat anti-rabbit (heavy and light chain-specific) were purchased from Zymed Laboratories Inc..

Experimental Animals—Sprague-Dawley rats (Charles River) were used to prepare microsomes (24) and brush border membrane vesicles using the divalent cation precipitation method (25). Animals were sacrificed by injection of sodium pentobarbital (Butler Co., Columbus, OH).

Two wild caught opossums were purchased from Northeastern Wild-life, South Plymouth, NY, and the kidneys were used for screening hybridomas. The animals were handled and anesthetized by veterinarians from the Section of Comparative Medicine, Yale University Medical School. The kidneys of one animal were used to prepare brush border membrane vesicles according to the divalent cation precipitation method (25). The kidneys of the other animal were perfusion-fixed and prepared for immunocytochemistry as described below.

Density Separation of Postmitochondrial Microsomes—The density separation of cellular membranes was accomplished by isopycnic centrifugation using OptiPrepTM (Nycomed Pharma, Oslo, Norway) density gradients. OptiPrep was diluted to appropriate concentrations from stocks using 20 mM Tricine (pH 7.8) and 8% sucrose according to the manufacturer's protocols. Preformed OptiPrep gradients were made using a Gradient MasterTM (Biocomp Inc., New Brunswick, Canada). 2 mg of postmitochondrial microsomes in 5% OptiPrep were layered on the top of 15–25% OptiPrep gradients. Gradients were centrifuged to equilibrium (at least 2 h) at 28,000 rpm using an SW 41 rotor in a Beckman ultracentrifuge. 1-ml fractions were manually collected from the top and stored at –70 °C. For analysis by immunoblotting equal volumes (10 µl) of each fraction were used.

SDS-PAGE and Immunoblotting—Protein samples were solubilized in SDS-PAGE sample buffer and separated by SDS-PAGE using 3.5–8% gradient or 7.5% polyacrylamide gels according to Laemmli (26). For immunoblotting, proteins were transferred to PVDF (Millipore Immobilon-P) at 400 mA for 4–6hat4 °C with a TransphorTM transfer electrophoresis unit (Hoefer Scientific Instruments, San Francisco, CA) and stained with Ponceau S in 0.5% trichloroacetic acid. Immunoblotting was performed as follows. PVDF membranes containing transferred protein from entire gels were incubated first in Blotto (5% nonfat dry milk in phosphate-buffered saline, pH 7.4) for 1–3 h to block non-specific binding of antibody followed by overnight incubation in primary antibody. Primary antibodies, diluted in Blotto, were used at dilutions ranging from 1:1000 to 1:5000. The membranes were then washed in Blotto and incubated for 1 h with an appropriate horseradish peroxidase-conjugated secondary antibody diluted 1:2000 in Blotto. After washing three times in Blotto, one time in phosphate-buffered saline (pH 7.4), and one time in distilled water, bound antibody was detected with the ECLTM chemiluminescence system (Amersham Biosciences) according to the manufacturer's protocols. In some experiments PVDF blots were reprobed with additional primary antibodies after stripping away the first antibody. Stripping was accomplished by incubating the PVDF membranes in 2% SDS, 100 mM {beta}-mercaptoethanol, 50 mM Tris (pH 6.9) for 60 min at 70 °C.

Tissue Preparation for Immunocytochemistry—Rats were anesthetized with sodium pentobarbital injected intravenously, and the kidneys were perfusion-fixed with paraformaldehyde-lysine-periodate fixative (27) as described previously (28). For immunofluorescence and immunogold labeling tissue was cryoprotected by incubating for 1 h in 2.3 M sucrose with 50% polyvinylpyrrolidone, mounted on aluminum nails, and stored in liquid nitrogen (28).

Indirect immunofluorescence microscopy was performed using either semithin cryosections of fixed tissue or Epon sections of the same tissue that was further subjected to antigen retrieval. Cryosections were prepared and stained exactly as described previously (17).

For antigen retrieval fixed tissue was embedded in Epon 812 as described previously (28). However, the tissue was not subjected to osmium tetroxide or uranyl acetate steps. After embedding 0.5-µm sections were cut with glass knives, and the sections were mounted on glass slides. The sections were then etched by incubating for 5 min in a solution containing 10 ml of 100% methanol, 5 ml of propylene oxide, and 2 g of KOH. The slides were then washed two times for 5 min each in 100% methanol and once in Tris-buffered saline (TBS) (50 mM Tris, 100 mM NaCl, pH 7.4). For antigen retrieval a 10 mM sodium citrate buffer (pH 6.0) was used. Briefly 500 ml of buffer in a 2-liter glass beaker were heated to boiling in a microwave oven. The slides were added to the hot buffer and heated in the oven for 20 min at ~40% power. After cooling the sections were washed three times for 15 min each in TBS, quenched for 15 min in 0.5 M ammonium chloride, and washed again in TBS. After an additional 5-min wash in 1% SDS the section were stained as described above.

For immunoelectron microscopy ultrathin cryosections were labeled using the colloidal gold technique. Briefly ultrathin cryosections were cut using a Reichert Ultracut E ultramicrotome fitted with an FC-4E cryoattachment and picked up on carbon-coated Formvar grids. Sections were labeled overnight either with anti-MC-220 or with mAb DC6 either diluted 1:50 in TBS containing 0.1% bovine serum albumin and 10% goat serum. After washing in TBS with 0.1% bovine serum albumin the sections were incubated for 1 h in goat anti-rabbit or goat anti-mouse gold conjugate (10 nm) diluted 1:20 in TBS containing 0.1% bovine serum albumin and 10% goat serum. After processing as described previously (28, 29) the sections were examined and photographed with a Zeiss 910 electron microscope. Digital images were prepared by scanning negatives with a ScanMaker 8700 (Microtek, Inc.) and were processed using Adobe Photoshop.

Analysis of Megalin Carboxyl-terminal Fragment—OKP cells were grown to confluence in 24-well culture plates (COSTAR, Inc.). Cells were serum-starved for 48 h prior to each experiment. Cells were treated in different conditions and then solubilized for 30 min on ice using 30 mM Tris, 20 mM MES (pH 7.4) containing 1% Triton X-100, 15 mM sodium pyrophosphate, 50 mM NaF, 0.7 mg/ml pepstatin A, 0.5 mg/ml leupeptin, 40 mg/ml phenylmethylsulfonyl fluoride, and Complete Protease InhibitorTM EDTA-free (Roche Applied Science) tablet (one tablet/25 ml of buffer). Insoluble material was removed by centrifugation for 10 min at 21,000 x g at 4 °C using a Micromax RF tabletop centrifuge (IEC, Needhan Heights, MA), and protein concentration from each extraction was estimated using the Lowry assay (30). 10 µg of total proteins from each treatment were separated by SDS-PAGE using 3.5–8% gradient gels and then transferred electrophoretically to PVDF membrane. Immunoblotting was performed as described previously using the anti-MC-220 antibody. The densitometry of the Western blot was measured using NIH Image 1.63 program.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
DISCUSSION
 REFERENCES
 
{gamma}-Secretase Expression in the Renal Brush Border—Regulated intramembrane proteolysis is mediated by {gamma}-secretase activity. Since presenilin is the active component of the {gamma}-secretase protein complex (31), we first sought to localize presenilin-1 protein in kidney and compare its renal distribution with that of megalin. As seen in Fig. 2, panel A, presenilin-1 localizes to the brush border of the proximal tubule where megalin is expressed (32).



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  		FIG. 2.
Localization of {gamma}-secretase activity and presenilin-1 protein in kidney. In panel A, paraformaldehyde-lysine-periodatefixed rat kidney cortex was immunostained with a polyclonal antibody to presenilin-1 amino-terminal fragment using an antigen retrieval protocol (see "Experimental Procedures"). Staining was concentrated to the brush border region of all proximal tubules (P) as well as in large vacuoles (arrows) of S1 proximal segments (S1). G, glomerulus. Panel B shows an immunoblot detecting presenilin-1 amino-terminal fragment (NTF) in rat kidney. 25 µg of either rat kidney cortical microsomes (M) or rat kidney brush border membrane vesicles (BBMV) were prepared for immunoblotting using a 7.5% SDS-polyacrylamide gel. The blot was probed with the anti-presenilin-1 amino-terminal fragment polyclonal antibody (1:1000). Molecular masses are presented on the left. Panel C shows {gamma}-secretase activity in brush border membrane vesicles. The enzyme activities were presented in fluorescence units as a function of protein concentration with or without the {gamma}-secretase inhibitor Compound E (30 nM).

 
To support our immunocytochemical data presented in Fig. 2 (panel A), we performed immunoblotting experiments using rat kidney microsomes and brush border membrane vesicles prepared from the renal cortex (Fig. 2, panel B). This figure shows that the anti-presenilin-1 antibody detected a protein of a size that is consistent with the presenilin-1 amino-terminal fragment and that it is enriched in microvillar membranes.

Finally we assayed {gamma}-secretase activity in renal membranes. As shown in Fig. 2, panel C, we were able to detect a significant level of {gamma}-secretase activity in brush border membrane vesicles that is inhibitable by the specific inhibitor Compound E. Taken together, these data show that presenilin-1 protein and {gamma}-secretase activity are present in the renal brush border where megalin is localized.

Evidence for Proteolysis of Megalin in Proximal Tubule— Since signaling pathways involving RIP require several proteolytic events (11), we looked for evidence of megalin processing in the proximal tubule. Previous studies by Bachinsky and co-workers (33) reported two forms of megalin in the brush border. In addition to megalin found in coated pits and endosomes, they found megalin on the microvillar surface that seemed to lack the cytoplasmic domain of the receptor suggesting that this region of the receptor was removed. To confirm this observation and localize the extracellular and cytosolic domains of megalin at high resolution we carried out immunoelectron microscopy with antibodies specific for either the cytoplasmic or extracellular domains of the receptor. As seen in Fig. 3, panel A, the extracellular domain of megalin was detected on the microvillar surface along coated pits and within dense apical tubules of proximal tubule cells. In contrast, while the cytosolic domain was found in the dense apical tubules and coated pits no staining was detected on the microvillar surface (panel B). We found the same staining pattern with another polyclonal antibody (anti-gp330CT from Dr. Farquhar) and our monoclonal (mAb 6E10) antibody to the cytosolic domain of megalin (data not shown). The inability to detect the cytosolic domain of megalin on microvilli indicates that either this region of the receptor has been proteolytically removed or that this region is occluded from antibody binding possibly through the interaction of megalin with other proteins. It should be noted that if this staining pattern is the result of proteolytic activity, such as ectodomain shedding, then the amino-terminal domain must remain associated with the microvillar membrane through interaction(s) with other components of the membrane.



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  		FIG. 3.
Subcellular localization of extracellular and intracellular domains of megalin in rat proximal tubule. Ultrathin cryosections of paraformaldehyde-lysine-periodate-fixed rat kidney cortex were immunolabeled with antibodies to megalin ectodomain (mAb DC6, panel A) and cytosolic domain (anti-MC-220, panel B). Specifically bound primary antibody was detected with secondary antibody conjugated to 10-nm colloidal gold. mv, microvilli; *, coated pits; M, mitochondria; arrows, dense apical tubules. Magnification bar, 100 nm.

 
To look for further evidence of proteolytic cleavage of megalin we biochemically analyzed renal microsomes using our anti-megalin carboxyl-terminal antibodies. We previously showed that 15–25% OptiPrep gradients separate dense membrane fractions enriched for markers of the intermicrovillar coated pit region of the brush border (Fig. 4, arrow) from other renal microsomes (34). Fig. 4 shows that when we examined microsomes separated on similar gradients we found much of the intact megalin localized to the dense membrane fractions as expected. However, we also detected a second major band of ~35–40 kDa along with several minor bands. The 35–40-kDa fragment of megalin will be referred to as the megalin carboxyl-terminal fragment (MCTF). These data suggest that a significant fraction of megalin is proteolytically cleaved in the brush border and provide further evidence for megalin processing. Moreover since the MCTF reached equilibrium in the density gradients this fragment must be membrane-associated.



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  		FIG. 4.
Immunoblot using anti-megalin carboxyl-terminal antibodies of rat renal microsomes separated on an OptiPrep density gradient. 2 mg of rat renal microsomal proteins were separated on a 15–25% OptiPrep density gradient. Equal volumes (10 µl) of each fraction were separated by SDS-PAGE using a 3.5–8% gradient gel and then analyzed by Western blot with polyclonal antibody anti-MC-220. Fraction 1 is the top fraction, and Fraction 15 is the bottom fraction. Intact megalin is seen at the top of the blot, while the 35–40-kDa MCTF is seen near the bottom. * denotes a minor carboxyl-terminal fragment of megalin that is frequently observed. The 118-kDa band seen in lane 1 represents nonspecific staining of the {beta}-galactosidase prestained molecular mass standard. Molecular masses are presented on the left.

 
Based on the data presented above and the fact that MCTF is the appropriate size to include the cytosolic and membrane domains of megalin, we postulate that this fragment is produced following ectodomain shedding. If megalin processing is part of a Notch-like signaling pathway, then we predict that the MCTF is the product of metalloprotease-mediated ectodomain shedding and forms the substrate for {gamma}-secretase activity. Therefore, the amount of MCTF seen at any time should reflect a state of equilibrium between these activities.

Evidence for Megalin Ectodomain Shedding in OKP Cells—To test this hypothesis, we studied the processing of megalin in more detail in the opossum proximal tubule cell line OKP. To look for evidence of megalin ectodomain shedding in OKP cells we biochemically analyzed cell culture supernatant and solubilized OKP cells using specific antibodies to either the ecto- or cytosolic domains of megalin. As shown in Fig. 5, mAb 6A6 (megalin extracellular domain) stained several protein bands in the cell supernatant, while mAb 6E10 (megalin cytosolic domain) detected nothing in the supernatant. However, when mAb 6E10 was used to probe blots prepared from whole cell lysates, it not only stained intact megalin but also an ~35–40-kDa megalin carboxyl-terminal fragment similar to that found in kidney. This fragment was not detected with mAb 6A6 (data not shown). Together these data suggest that megalin ectodomain shedding occurs constitutively in OKP cells.



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  		FIG. 5.
Proteolytic processing of megalin: evidence of megalin ectodomain shedding. OKP cells were grown to confluence and serum-starved for 48 h. Medium from the cell culture was clarified by centrifugation at 100,000 x g for 30 min and concentrated with Centricon® 10. Proteins from the concentrated medium were separated by SDS-PAGE and analyzed by Western blot with mAb 6E10 (cytoplasmic domain) (panel A) and mAb 6A6 (extracellular domain) (panel B). Whole cell lysate was probed with mAb 6E10 as control (panel C). The asterisk marks the major carboxyl-terminal fragment of megalin.

 
Megalin Is Processed by Matrix Metalloprotease and {gamma}-Secretase Activities in OKP Cells—Previous studies have shown that ectodomain shedding prior to RIP is metalloprotease-mediated and PKC-regulated. To test this part of our model, we studied the effect of PMA, a PKC activator, in conjunction with general matrix metalloprotease inhibitors on the appearance of the MCTF in serum-starved OKP cells. As seen in Fig. 6, PMA increased the amount of MCTF by ~2.5-fold. The PMA-induced increase in MCTF was completely blocked by the PKC inhibitor Gö6983. The PMA effect was also blocked by the matrix metalloprotease inhibitor MMP inhibitor III (Fig. 7) as well as by other metalloprotease inhibitors, tumour necrosis factor-{alpha} processing inhibitor-1 and -2 (data not shown). These data confirm that the formation of the MCTF is dependent on PKC-regulated, metalloprotease-mediated activity and suggests that the MCTF is produced following megalin ectodomain shedding.



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  		FIG. 6.
PKC activates megalin ectodomain shedding and increases the appearance of the MCTF. OKP cells were grown to confluence and serum-starved for 48 h. Serum-starved OKP cells were treated with 200 nM 4{alpha}PMA or PMA in the absence or presence of PKC inhibitor Gö6983 at 37 °C for 30 min. The cells were then solubilized in Tris-MES buffer containing 1% Triton X-100. 10 µg of total cell proteins were separated by SDS-PAGE using a 3.5–8% gradient gel and analyzed by Western blot using anti-MC-220. Panel A, representative Western blots. "Megalin" indicates the intact form of the protein; MCTF indicates the megalin carboxyl-terminal fragment. Panel B, densitometric analysis of the MCTF from six experiments.

 



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  		FIG. 7.
PKC-activated megalin ectodomain shedding depends on matrix metalloprotease activity. Serum-starved OKP cells were treated with 200 nM 4{alpha}PMA or PMA in the absence or presence of 20 µM matrix metalloprotease inhibitor MMP inhibitor III (MMPI) at 37 °C for 30 min. Panel A, representative blot showing the MCTF. Panel B, densitometric analysis of the MCTF from six experiments.

 
Our model also predicts that the MCTF is the substrate for {gamma}-secretase. Fig. 8 shows the effect of incubating OKP cells with the {gamma}-secretase inhibitor Compound E. As seen here, inhibiting {gamma}-secretase results in a significant increase in the appearance of the MCTF. These data are consistent with our hypothesis that {gamma}-secretase catalyzes the cleavage of the MCTF.



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  		FIG. 8.
The {gamma}-secretase inhibitor Compound E increases the accumulation of megalin carboxyl-terminal fragment. Serum-starved OKP cells were treated with or without 500 nM Compound E at 37 °C for 1 h. Panel A, representative blots showing MCTF. Panel B, densitometric analysis of the MCTF from six experiments.

 
EIPA Activates Megalin Ectodomain Shedding—In our studies of megalin processing we discovered that the amiloride analog EIPA dramatically increased the appearance of the MCTF in OKP cells. Figs. 9 and 10 show that 100 µM EIPA resulted in a 3–5-fold increase in the amount of MCTF. This effect was completely blocked by matrix metalloprotease inhibitors (Fig. 9) but not by PKC inhibitors (Fig. 10). These data indicate that EIPA activates matrix metalloprotease activity and increases megalin ectodomain shedding. Activation is not via a PKC pathway. In addition, neither amiloride nor other Na+/H+ exchange inhibitors (S3226 and HOE694) were able to mimic the effect of EIPA (data not shown).



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  		FIG. 9.
EIPA-activated megalin ectodomain shedding is blocked by matrix metalloprotease inhibitors. Serum-starved OKP cells were treated with 100 µM EIPA in the absence or presence of 20 µM matrix metalloprotease inhibitor MMP inhibitor III (MMPI) at 37 °C for 1 h. Cells without treatment served as control. Panel A, representative blots showing MCTF. Panel B, densitometric analysis of the MCTF from six experiments.

 



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  		FIG. 10.
EIPA activates megalin ectodomain shedding independently of PKC. Serum-starved OKP cells were treated with 100 µM EIPA in the absence or presence of PKC inhibitor 1 µM Gö6983 at 37 °C for 1 h. Panel A, representative blot showing MCTF. Panel B, densitometric analysis of the MCTF from five experiments.

 
Vitamin D-binding Protein Activates Megalin Ectodomain Shedding—Finally we examined the role of ligand binding on megalin processing. Since vitamin D-binding protein (DBP) is a known ligand of megalin we carried out experiments to determine whether DBP regulates megalin shedding. When we incubated serum-starved OKP cells in varying amounts of DBP (0.1–5.0 µM) we saw no detectable effects on the amount of the MCTF present in the cells (data not shown). We postulated that if DBP activates shedding at a low level then {gamma}-secretase might degrade the newly formed fragment as quickly as it is generated. To test this idea we looked at ligand binding while {gamma}-secretase activity was blocked with Compound E (Fig. 11). Under these conditions we found addition of DBP resulted in a modest but significant increase in the appearance of MCTF suggesting that ligand binding does activate megalin processing.



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  		FIG. 11.
DBP activates ectodomain shedding of megalin. Serum-starved OKP cells were treated with 500 nM Compound E in the absence or presence of 1 µM DBP at 37 °C for 1 h. Panel A, representative blot showing the MCTF. Panel B, densitometric analysis of the MCTF from nine experiments.

 

   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
REFERENCES
 
This study provides the first evidence for regulated intramembrane proteolysis of megalin and implies that megalin processing may link protein absorption and cell signaling in the proximal tubule. We show here that {gamma}-secretase activity as well as presenilin-1 protein are expressed in the renal brush border where megalin is localized. In addition, using newly developed antibodies to the cytosolic domain of megalin we provide morphological and biochemical evidence for regulated proteolytic processing of megalin in both kidney and in the proximal tubule cell line OKP.

Ectodomain Shedding of Megalin—Our data showing {gamma}-secretase-mediated RIP of megalin in the proximal tubule adds megalin to a growing list of receptors including Notch, APP, and LRP that utilize sequential proteolytic processing culminating in the release of the cytosolic domain of the receptor. In all cases studied RIP is preceded by, and dependent upon, ectodomain shedding of the receptor mediated by members of either the ADAM or the MMP families of proteases. Our data presented here are consistent with this model. Because of its role in the pathogenesis of Alzheimer's disease, the shedding process of APP has been studied in great detail. Ectodomain shedding of APP is mediated by at least two proteases, {alpha}- and {beta}-secretase (35). In the processing of the Notch receptor, the initial ectodomain cleavage occurs during biosynthesis in the Golgi apparatus by the enzyme furin. Once at the plasma membrane, {alpha}-secretase Kuzbanian (KUZ; ADAM 10)-mediated shedding may also be required prior to {gamma}-secretase cleavage of the receptor in the transmembrane domain (36). As with LRP (37), our knowledge of the enzymes that mediate megalin ectodomain shedding is incomplete. In our studies we showed that several broad spectrum inhibitors (MMP inhibitor III and tumor necrosis factor-{alpha} protease inhibitor-1 and -2) of metalloprotease activity were able to block constitutive shedding of megalin as well as ectodomain shedding activated by PKC and EIPA. At this point the molecular nature of the metalloprotease activity(ies) responsible for megalin ectodomain shedding is unknown.

The subcellular location of the metalloprotease-mediated ectodomain shedding and {gamma}-secretase cleavage of megalin is not known. Studies are underway to determine whether these events occur on the plasma membrane or within the endocytic pathway. Although we know nothing about the localization of the enzyme(s) responsible for megalin ectodomain shedding, it is important to note that much of the staining for presenilin-1 seems to be localized to the coated pit region of the brush border as well as in large cytoplasmic vacuoles in S1 proximal segments. These data suggest that at least part of megalin processing takes place intracellularly.

In cultured epithelial cells that express megalin both "soluble" and "insoluble" forms of the receptor have been reported (33, 38). We also found soluble amino-terminal fragments of megalin lacking the carboxyl terminus as well as membrane-associated carboxyl-terminal fragments of megalin in OKP cells (shown in Fig. 5). These findings are consistent with a model of regulated processing of the receptor. It is of interest to note that megalin ectodomain shedding plays a key role in the progression of glomerulonephritis (Heymann nephritis) in rats. In this model circulating anti-megalin antibodies have been shown to bind to megalin expressed on the base of the glomerular epithelial foot processes. Antibody binding results in ectodomain shedding and accumulation of antibody-antigen complexes (immune deposits) in the glomerular basement membrane leading to glomerulonephritis (39, 40). Although it is unclear whether antibody binding to megalin in other cell types leads to ectodomain shedding, the possibility that antibody-activated, metalloprotease-mediated ectodomain shedding of megalin occurs in the glomerular epithelium should be investigated.

We have confirmed previous findings (33) that megalin found on the microvillar membrane of the brush border cannot be labeled with antibodies to the cytosolic domain. It remains to be determined whether this staining pattern represents receptor that has been proteolytically cleaved. However, the fact that different antibodies (two polyclonal and one monoclonal antibody) to the cytosolic domain of megalin, and therefore presumably to different epitopes, failed to label the receptor on the microvilli suggests that this might be the case. If so, the ectodomain must be bound to the microvillus through interaction with other components of the membrane.

Although RIP is thought to link receptor-mediated endocytosis with cell signaling, the requirement for ligand-dependent activation of receptor processing has not always been well documented. Ligand binding of both the Notch receptor and ErbB-4 is known to activate processing of the receptors (9). However, in other instances, most notably APP, processing appears to be constitutive rather than ligand-dependent (10). In a similar vein, May and co-workers (14) failed to demonstrate ligand dependence for LRP processing. In our study, we found evidence for both constitutive and ligand-induced processing of megalin. We found that constitutive processing of megalin was regulated by PKC and activated by EIPA in the absence of ligand indicating that ligand binding is not a requirement for shedding. We were only able to detect significant effects on megalin processing using vitamin D-binding protein in the presence of the {gamma}-secretase inhibitor Compound E. This finding suggests that, when produced at low levels, {gamma}-secretase activity can degrade the MCTF as quickly as it is formed.

Our finding that EIPA activates megalin ectodomain shedding was accidental and unexpected. It is worth noting that 100 µM EIPA activated megalin shedding to a greater extent than either ligand (DBP) or PMA. It remains to be determined whether EIPA can activate shedding of other proteins in different cell types. The fact that other, more specific inhibitors of Na+/H+ exchange could not mimic the EIPA effect suggest that the activation of the protease did not result from inhibiting the Na+/H+ exchanger. Similarly other weak bases such as amiloride and HOE694 did not increase megalin shedding thus ruling out an effect of weak bases in general.

{gamma}-Secretase in Kidney—Presenilin-1 is expressed in many tissues during development (42) and in the adult (43). However, our finding that the adult kidney expresses high levels of presenilin-1 and {gamma}-secretase activity in the brush border is novel. Although details of its function in kidney are still limited, this observation provides evidence for a major role of {gamma}-secretase in proximal tubule physiology. It will be important to determine whether other renal proteins in addition to megalin are processed by RIP.

Our data suggest that the MCTF we observe in both kidney and OKP cells is in equilibrium between ectodomain shedding and the RIP mediated by {gamma}-secretase activity. However, we have not been able to identify the soluble megalin intracellular domain (which is distinct from the membrane-associated MCTF) predicted to be the product of {gamma}-secretase activity. This failure is not totally unexpected since other studies have shown that the {gamma}-secretase products of receptors such as Notch, APP, and ErbB-4 are very difficult to detect. Usually the {gamma}-secretase products can only be detected when the proteins are overexpressed in cultured cells (31, 44) or studied in a cell-free system (45). Together these data indicate that the half-life of the megalin intracellular domain is short. An important goal of future studies will be to understand the fate of the megalin intracellular domain in the proximal tubule.

Role of RIP of Megalin in Proximal Tubule—It is generally assumed that RIP is involved in signaling cascades that link extracellular events such as receptor-mediated endocytosis with transcriptional regulation (for a review, see Ref. 7). For example, the ligands Delta and Jagged bind the Notch receptor leading to RIP and activation of transcription factor CSL (CBF1, Su(H), and Lag-1). At this point possible cellular signaling events activated by megalin processing are not known. In a recent review on the role of lipoprotein receptors in signal transduction May and Herz (46) suggested that megalin might be involved in regulating genes of the vitamin D pathway. This conclusion was based on the fact 1) that uptake and activation of vitamin D in the proximal tubule is dependent on megalin (41) and 2) that a recently identified megalin-binding protein, MegBP, also associates with Ski-interacting protein (SKIP), a known regulator of genes involved in the vitamin D pathway. Our finding that DBP activates megalin processing supports this model. It will be important to assess the effects of other ligands. If all ligands activate megalin processing, and thus distal signaling events, it would seem unlikely that regulation would involve a specific set of genes such as those involved in the vitamin D metabolic pathway. This is obviously an important subject for future studies.


   FOOTNOTES
 
* This work was supported by National Institutes of Health Grant DK543933 (to D. B.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

The nucleotide sequence(s) reported in this paper has been submitted to the GenBankTM/EBI Data Bank with accession number(s) AY627686 [GenBank] . Back

To whom correspondence and reprint requests should be addressed: Section of Nephrology, Dept. of Internal Medicine, Yale University School of Medicine, 300 Cedar St., TAC S255, P. O. Box 208029, New Haven, CT 06520-8029. Tel.: 203-785-6739; Fax: 203-785-4904; E-mail: daniel.biemesderfer{at}yale.edu.

1 The abbreviations used are: RIP, regulated intramembrane proteolysis; APP, amyloid precursor protein; ADAM, a disintegrin and metalloprotease domain; MMP, matrix metalloprotease; PKC, protein kinase C; LRP, low density lipoprotein receptor-related protein; OKP, opossum kidney proximal tubule; GST, glutathione S-transferase; CHAPSO, 3-[(3-cholamidopropyl)dimethylammonio]-2-hydroxy-1-propanesulfonic acid; mAb, monoclonal antibody; Tricine, N-[2-hydroxy-1,1-bis(hydroxymethyl)ethyl]glycine; PVDF, polyvinylidene difluoride; TBS, Tris-buffered saline; MES, 4-morpholineethanesulfonic acid; MCTF, megalin carboxyl-terminal fragment; EIPA, 5-(N-ethyl-N-isopropyl)amiloride; DBP, vitamin D-binding protein. Back


   ACKNOWLEDGMENTS
 
We are grateful to Dr. Peter Aronson for critically reading this manuscript.



   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 

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