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,
*
Department of Internal Medicine, University of Michigan, Ann Arbor,
Michigan.
Department of Biological Chemistry, University of Michigan, Ann Arbor,
Michigan.
Howard Hughes Medical Institute, University of Michigan, Ann Arbor,
Michigan.
Correspondence to Dr. Ben Margolis, Howard Hughes Medical Institute, University of Michigan, 4570 MSRB II, Box 0650, 1150 W. Medical Center Drive, Ann Arbor, MI 48109. Phone: 734-764-3567; Fax: 734-763-9323; E-mail: bmargoli{at}umich.edu
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The LDLR family members contain NPXY (where N is arginine, P is proline, X is any amino acid, and Y is tyrosine) motifs in their cytoplasmic domains, which have the potential to bind to a modular protein-protein interaction domain known as the phosphotyrosine binding (PTB) domain (8). In addition, megalin and a Caenorhabditis elegans homolog are the only known LDLR family members to have an SXV (where S is serine, X is any amino acid, and V is valine) stretch of amino acids at their carboxyl-termini. This sequence of amino acids can serve as a binding motif for another protein-protein interaction domain, referred to as the PDZ domain (9). Furthermore, these binding motifs are conserved in all species from which megalin has been isolated to date.
The 80- to 90-amino acid PDZ domain was first identified in the postsynaptic protein PSD-95, the Drosophila dlg tumor suppressor gene, and the tight-junction protein ZO-1. PDZ domains are currently recognized as central organizers of protein complexes at the plasma membrane. These protein complexes mediate the adhesive properties of particular cells, the formation of the paracellular barrier (tight junctions), ion transport, and transmission of signals that regulate growth, differentiation, and homeostasis between adjacent cells. The asymmetrical localization of a number of transmembrane receptors and ion channels has recently been shown to be a consequence of their association with proteins containing PDZ domains (10,11). For example, in C. elegans, the proper localization of the receptor tyrosine kinase Let-23 to the basolateral surface of vulval precursor cells is dependent on a complex of three PDZ proteins, i.e., Lin-2, Lin-7, and Lin-10. The PDZ domain of Lin-7 binds directly to the carboxyl-terminus of Let-23, and mutations in lin-7, as well as in lin-2 or lin-10, result in mislocalization of Let-23 to the apical domain of the plasma membrane (12). The binding of class I PDZ domains is mediated by a (S/T)X(V/L) (where T is threonine and L is leucine) motif found at the carboxyl-termini of their cognate binding partners (9).
To isolate potential PTB or PDZ domain proteins that might bind megalin, we used the yeast two-hybrid system to screen an embryonic mouse brain cDNA library, using most of the cytoplasmic tail of megalin as bait. We report the isolation of a partial cDNA clone that contains a single PDZ domain; we named the protein GASP (glycoprotein 330/megalin-associated protein). GASP cDNA seems to be a truncated version of a larger mouse protein that shares a high degree of identity with human atrophin-1-interacting protein-1 (AIP-1) and rat synaptic scaffolding molecule (S-SCAM). The PDZ domain of GASP is also similar to the fifth PDZ domain of the mouse protein membrane-associated guanylate kinase 1 (MAGI-1) (13). We show, using biochemical techniques, that GASP cDNA and the fifth PDZ domain of MAGI-1 bind directly to the carboxyl-terminus of megalin. In addition, we show that MAGI-1 is expressed in a podocyte-specific manner within kidney glomeruli. We speculate that these associations with megalin are potentially important for the assembly of a multiprotein complex at subcellular locations in glomerular podocytes.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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V) was
generated by PCR, sequenced, and cloned into pAS2-1 in a manner similar to
that used for the wild-type (WT) megalin tail.
Glutathione-S-transferase (GST) fusion proteins were constructed
in pGSTag (14) using the
following inserts: the entire cDNA insert of mbp1b excised from pGAD1318 (see
below) (GST-GASP5), a mouse EST cDNA containing the carboxyl-terminal half of
the fourth PDZ domain and the entire fifth PDZ domain of MAGI-1 (GST-MAG15),
and the WT and mutant V cytoplasmic tails of megalin used in the yeast
two-hybrid screening (GST-megalin WT and GST-megalin V, respectively).
In addition, complementary sense and antisense oligonucleotides coding for the
last 19 amino acids of N-methyl-D-aspartate receptor 2B (NR2B) and
the activin type IIA receptor (ActRIIA) were annealed and cloned into pGSTag,
yielding GST-NR2B and GST-ActRIIA, respectively. Myc-tagged expression
constructs were prepared in pRK5-Myc
(15). An EcoRI
fragment containing the entire coding sequence of clone mpb1b was cloned into
pRK5-Myc to yield Myc-GASP5. The EST cDNA of MAGI-1 (see above) was cloned
into pRK5-Myc to yield Myc-MAGI5. MAGI-1 PCR products containing either a
variant of the fourth PDZ domain, the first three PDZ domains, or full-length
MAGI-1 were cloned into pRK5-Myc to generate Myc-MAGI4b, Myc-MAGI1-3, and
Myc-MAGI FL, respectively. The GASP cDNA and EST cDNA of MAGI-1 were subcloned
into the plasmid pET28c+ (Novagen, Madison, WI) for the creation of
His6-tagged proteins.
Yeast Two-Hybrid Screening
Yeast strain Y190 was transformed with the GAL4-megalin bait construct (see
above) and an oligo(dT)-primed embryonic mouse brain cDNA library that was
cloned into pGAD1318 (a kind gift from Dr. J. Camonis, Institut Curie, INSERM
U-248, Paris, France) downstream of the GAL4 activation domain. A total of 3
x 107 colonies were screened on Leu-Trp-His selection plates.
Positive clones that survived additional rounds of selection for histidine and
LacZ expression were isolated and grown in liquid selection medium
for plasmid DNA isolation. Plasmid DNA isolated from yeast was used to
transform DH5
bacteria for subsequent plasmid amplification,
purification, and sequencing. The megalin V mutant construct in pAS2-1
was used to confirm the interaction of the positive clones after their
nucleotide sequences were established.
Cell Culture and Transfections
Human embryonic kidney 293 (HEK293) cells and MDM cells (a rabbit kidney
epithelial cell line provided by Dr. David Kershaw, University of Michigan,
Ann Arbor, MI) were grown in Dulbecco's modified Eagle's medium (Life
Technologies, Grand Island, NY) supplemented with 10% fetal calf serum, 2 mM
L-glutamine, 100 U/ml penicillin G, and 100 µg/ml streptomycin.
HEK293 cells at approximately 50% confluence were transiently transfected with the Myc-tagged expression constructs, using the calcium phosphate precipitation method. Twenty hours after the addition of DNA, the cells were washed once with phosphate-buffered saline (PBS) and fresh medium was added. The cells were incubated for an additional 24 h before collection for lysate preparation.
Tissue and Cell Lysates
Tissue lysates were prepared as follows. Adult mouse tissues were placed in
a Dounce homogenizer with Triton lysis buffer [50 mM Hepes, pH 7.5, 150 mM
NaCl, 1.5 mM MgCl2, 1 mM ethylene glycol bis(ß-aminoethyl
ether)-N,N,N',N'-tetraacetic acid, 10% glycerol, 1%
Triton X-100] plus protease inhibitors (4 µg/ml phenylmethylsulfonyl
fluoride, 10 µg/ml aprotinin, and 10 µg/ml leupeptin) and were
homogenized with 25 to 30 strokes. The lysates were centrifuged at 20,000
x g for 30 min at 4°C. Supernatants were transferred to
fresh tubes, and the pellets were resuspended in a volume of lysis buffer
equal to the volume of supernatant.
Glomerular extracts were prepared by passing mouse kidneys that had been cut in half through a Nitex mesh with a pore size of 90 µm (Sefar America, Kansas City, MO), using ice-cold PBS. The flow-through fraction was then passed over Nitex mesh with a pore size of 46 µm, and the retained glomeruli were washed extensively with ice-cold PBS. Glomeruli were transferred to a 50-ml tube and centrifuged at 2000 x g for 3 min at 4°C. The pelleted glomeruli were resuspended in lysis buffer, sonicated with three 15-s bursts, and processed as described above.
Transiently transfected HEK293 cells in 60-mm dishes were washed once with PBS, scraped into 200 µl of Triton lysis buffer, transferred to microfuge tubes, briefly vortex-mixed, and incubated on ice for 10 min. The lysates were then processed as described above.
RNA (Northern) Blot Analysis
A 523-bp EcoRI-XbaI fragment of clone mbp1b was labeled
with [32P]dCTP (NEN, Boston, MA) using the Rediprime II random
prime labeling system (Amersham Pharmacia Biotech, Piscataway, NJ) and was
used to probe a mouse multiple-tissue RNA blot (Clontech, Palo Alto, CA). The
RNA blot was prehybridized in 10 ml of prehybridization/hybridization solution
[5x SSC, 100 µg/ml herring sperm DNA, 50% formamide, 10x
Denhardt's solution, 1.5% dextran sulfate, 0.1% sodium dodecyl sulfate (SDS)]
for 5 h at 42°C. A total of 1 x 107 cpm of probe was
added to the blot and hybridized overnight at 42°C. The blot was washed
once with 2x SSC/0.1% SDS at 42°C for 20 min and then twice with
0.1x SSC/0.1% SDS at 42°C for 20 min each. The blot was exposed to
x-ray film for 5 d at -70°C.
Reverse Transcription-PCR
Total RNA was isolated from a mouse glomerular preparation and used to
synthesize first-strand cDNA with a 3'-RACE kit (Life Technologies,
Rockville, MD). Specific primers for mouse MAGI-1 were used to PCR amplify
different regions, which were subsequently cloned into pGEM-T Easy vector
(Promega, Madison, WI) and sequenced. Individual PCR clones containing the
different regions of MAGI-1 were either subcloned into pRK5-Myc or used to
construct a full-length cDNA, using conventional DNA cloning techniques.
Antibodies
The polyclonal anti-GASP and anti-MAGI-1 antibodies used for immunoblotting
and immunostaining were prepared by injecting rabbits with purified GST fusion
proteins, i.e., GST-GASP5 and GST-MAG15, respectively. Anti-GASP
antibody UM200 and anti-MAGI-1 antibody UM209 were affinity-purified using
His6-tagged protein columns, as described previously
(16). Rabbit polyclonal
anti-glycoprotein 330 antibody was a gift from Dr. Joachim Herz (University of
Texas Southwestern Medical Center, Dallas, TX). The anti-Myc antibody was a
mouse monoclonal antibody (clone 9E10) obtained from mouse ascites fluid. The
anti-glomerular epithelial cell membrane protein-tyrosine phosphatase 1
(GLEPP1) antibody was a mouse monoclonal against rat GLEPP1 (1B4). Horseradish
peroxidase-conjugated sheep anti-mouse secondary antibodies were obtained from
Amersham Pharmacia Biotech.
GST Fusion Protein Precipitation
GST fusion proteins were produced in DH5 bacteria as described
previously (15). Tissue or
cell lysates were combined with 10 to 20 µg of GST fusion protein bound to
glutathione-agarose beads and were incubated on a rocker at room temperature
for 2 h. The beads were then washed twice with ice-cold PBS. Forty microliters
of 1x SDS sample buffer was added to the beads, and the mixture was
maintained at 100°C for 5 min. Samples were subjected to
SDS-polyacrylamide gel electrophoresis (PAGE), transferred to nitrocellulose
membranes, and blotted with the appropriate primary antibodies and horseradish
peroxidase-conjugated secondary antibodies. Blot were developed with
chemiluminescence reagents (NEN, Boston, MA) and exposed to x-ray film.
Immunofluorescence Microscopy
Cryostat sections (5 µm) of rat kidney were fixed in acetone and blocked
with 10% goat serum/10% rat serum in PBS. Sections were double-labeled by
incubation with a mixture of affinity-purified polyclonal anti-MAGI-1 (UM209)
and monoclonal anti-GLEPP1 primary antibodies for 1 h at room temperature. The
sections were washed twice with PBS and incubated for 1 h at room temperature
with a mixture of preabsorbed FITC-conjugated goat anti-rabbit IgG (1:50) and
rhodamine-conjugated goat anti-mouse IgG (1:100) secondary antibodies. The
sections were then washed with PBS, mounted, and photographed with a Nikon
807427 immunofluorescence microscope (Mager Scientific, Inc., Dexter, MI)
using the Metamorph imaging program (Version 3.51; Universal Imaging Corp.,
Downingtown, PA).
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The 5'-end of the cloned GASP cDNA lacks a Kozak consensus sequence for translation initiation, indicating that the cDNA probably represents a truncated version of a presumably larger transcript. This was verified by searching nucleotide and protein databases, because GASP exhibited a high degree of identity with human and rat proteins referred to as AIP-1 and S-SCAM, respectively (17,18). GASP appears to represent the carboxyl-terminal end of a mouse counterpart to these two proteins (Figure 1C). Although the PDZ domain of GASP is identical at the amino acid level to the fifth PDZ domain of AIP-1 and S-SCAM, the sequences outside the PDZ domain seem to diverge somewhat, especially in the sequences downstream from the PDZ domain (Figure 1D). The site at which all three proteins diverge after the PDZ domain is analogous to the site at which alternative splicing results in three different carboxyl-termini in the closely related protein MAGI-1 (13). The PDZ domain of GASP exhibits 76% identity, at the amino acid level, with the fifth PDZ domain of MAGI-1 and brain-specific angiogenesis inhibitor-associated protein 1 (BAP-1), the human counterpart of MAGI-1 (19), and 50% similarity with the last PDZ domain in a C. elegans clone, K01A6.1.
The tissue distribution of GASP was investigated by Northern analysis of a mouse multiple-tissue blot. A 523-bp fragment from the 3'-untranslated region of GASP was used as the probe. GASP was found to be expressed predominantly in brain, with major transcript sizes of approximately 7.4, 6.5, and 3.0 kb (Figure 2). A low level of expression was detected in testis, and no appreciable expression was observed in the other tissues examined. This pattern of expression of GASP is similar to the reported expression of AIP-1 and S-SCAM (17,18).
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To further examine the interaction of GASP with megalin, GST fusion protein
precipitation assays were performed. A GST fusion protein containing the GASP
cDNA was produced in bacteria, bound to glutathione-agarose beads, and added
to mouse kidney lysates to precipitate endogenous megalin. The GASP fusion
protein was successful in precipitating megalin from kidney lysates, whereas
GST alone was not (Figure 3A).
In a reciprocal experiment, GST fusion proteins were produced with the WT and
V megalin tails, which had been used in the yeast two-hybrid assay, and
were added to mouse brain lysates. The megalin WT fusion protein was capable
of precipitating endogenous GASP from brain tissue
(Figure 3B). In addition, the
megalin WT fusion protein was able to precipitate GASP cDNA expressed as a
Myc-tagged protein in HEK293 cells (Figure
3C). The megalin V mutant was unable precipitate GASP from
either the brain lysate or the lysate of HEK293 cells expressing Myc-tagged
GASP cDNA. These data confirm that the PDZ domain within GASP mediates its
interaction with megalin and appears to be specific for the fifth PDZ domain
in endogenous full-length GASP.
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The interaction of rat S-SCAM with proteins present at postsynaptic densities within neurons was recently demonstrated (18). One of the proteins found to interact with S-SCAM was NR2B, and the interaction was mediated by the fifth PDZ domain of S-SCAM. In addition, a recently identified mouse protein, which appears to be identical to GASP (except at the extreme carboxyl-terminus), was cloned by means of its interaction with ActRIIA (20). This interaction was reported to occur through the fifth PDZ domain of the mouse protein (referred to as ARIP-1). We tested both NR2B and ActRIIA for their abilities to bind to GASP. GST fusion proteins were constructed with the last 19 amino acids of NR2B, ActRIIA, and megalin and were used in precipitation assays with Myc-tagged GASP. The shorter version of the megalin carboxyl-terminal tail was able to bind to GASP, as was NR2B (Figure 3D). However, ActRIIA was not able to bind to GASP in this assay.
The interaction of megalin with GASP may have a significant function in the brain (see Discussion); however, our interest in the interaction of megalin with other proteins is focused primarily on the kidney and on how these interactions could potentially affect the localization and/or function of megalin. MAGI-1, a close homolog of GASP, is expressed in a more ubiquitous manner, including in kidney. In addition, the domain structure of MAGI-1 is identical to that of the AIP-1/S-SCAM proteins, and the fifth PDZ domain of MAGI-1 displays a high degree of similarity to the fifth PDZ domain in GASP/AIP-1/S-SCAM. Therefore, we wanted to examine a potential interaction between megalin and MAGI-1. To investigate this possibility, an EST cDNA that contained the carboxyl-terminal half of the fourth PDZ domain and the entire fifth PDZ domain of MAGI-1 was fused to GST and used in a precipitation assay with kidney lysates. This GST fusion protein, GST-MAG15, was able to precipitate endogenous megalin from kidney lysates in a manner similar to that of GASP (Figure 3A). This result prompted us to focus on the function of MAGI-1 with respect to its interaction with megalin and to more precisely determine the localization of MAGI-1 within the kidney.
The specificity of the carboxyl-terminus of megalin for the individual PDZ
domains of MAGI-1 was investigated by expressing Myc-tagged regions of MAGI-1
in HEK293 cells and using the resulting cell lysates in GST fusion protein
precipitation assays. Myc-tagged, PCR-generated regions of MAGI-1 containing
either PDZ domains 1 to 3 (Myc-MAGI1-3) or PDZ domain 4 (Myc-MAGI4b) and a
Myc-tagged version of the MAGI-1 EST cDNA (Myc-MAG15)
(Figure 4A) were combined with
GST fusion proteins with megalin WT or mutant V tails. The fusion
protein with the megalin WT tail was capable of precipitating Myc-MAG15,
whereas the V mutant was not (Figure
4B), illustrating that the interaction of megalin with MAGI-1,
like that with GASP, was mediated via a PDZ domain. This interaction is
apparently specific for the fifth PDZ domain of MAGI-1, inasmuch as neither
Myc-MAGI1-3 nor Myc-MAGI4b was precipitated by the megalin WT tail
(Figure 4B).
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Although MAGI-1 is apparently expressed in the kidney at the RNA level (13), it has not been further localized. To determine where MAGI-1 is expressed within the kidney, we performed immunofluorescence assays with adult rat kidney sections, using an affinity-purified polyclonal antibody directed against GST-MAGI5. MAGI-1 expression was observed almost exclusively in glomeruli of adult kidneys, where it paralleled the expression pattern of GLEPP1 (Figure 5). GLEPP1 expression in the kidney was previously observed specifically in podocytes (21), suggesting that MAGI-1 is also confined to podocytes.
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The nature of the glomerulus-expressed MAGI-1 was examined by Western analysis of glomerular preparations. Isolated glomeruli obtained from adult mouse kidneys were lysed in one of three different extraction buffers, and samples of the resulting supernatant and pellet fractions were analyzed for the presence of MAGI-1. Endogenous MAGI-1 from glomerular preparations was found to be insoluble in Triton lysis buffer; all of the protein was observed in the pellet fraction (Figure 6A). MAGI-1 was still partially observed in the pellet fraction with RIPA buffer, although most of the protein was soluble. As expected, MAGI-1 was completely soluble in 6 M urea lysis buffer. These results suggest that MAGI-1 is tightly associated in a complex with the cytoskeleton. It was also observed that MAGI-1 existed in three different forms in the glomerular preparations (Figure 6A), with the largest form migrating at approximately 185 to 190 kD. These glomerulus-derived forms of MAGI-1 were larger than the forms observed previously in lysates from MDCK cells (13). We also examined endogenous MAGI-1 in MDM cells (a rabbit kidney epithelial cell line). MDM cells form monolayers and express junctional proteins (e.g., ZO-1) in a manner similar to that of MDCK cells (Patrie KM, Margolis B, unpublished observations). We found that MDM cells contained three forms of MAGI-1, which migrated with sizes comparable to those observed in MDCK cells (Figure 6B). However, the forms of MAGI-1 observed in MDM cells migrated with apparent molecular sizes smaller than those of the forms observed in the mouse glomerular preparations. In addition, MAGI-1 from this cell line was soluble in Triton lysis buffer. It appears, therefore, that the expression of MAGI-1 differs not only in size but also in subcellular association, depending on the source examined. Although we have not precisely defined the subcellular localization of MAGI-1 within podocytes, MAGI-1 has been observed to colocalize with ZO-1 at apparent tight-junctional sites in MDM cells (Patrie KM, Margolis B, unpublished observations) and MDCK cells (Patrie KM, Margolis B, unpublished observations) (22).
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The appearance of larger forms of MAGI-1 in glomerular preparations suggests that podocytes may express forms of MAGI-1 that are not found in cell lines or other tissues. We therefore wanted to clone a glomerulus-specific MAGI-1 cDNA. Using total RNA isolated from glomerular preparations, single-stranded cDNA was produced with an oligo(dT) primer, and different regions of MAGI-1 were then cloned by PCR. The individual regions of MAGI-1 were ligated together to produce a full-length MAGI-1 cDNA. During the course of this PCR-based cloning, we found that inserts from eight individual PCR clones derived from the carboxyl-terminus of MAGI-1 were the same size; when three of these clones were sequenced, they were found to be identical. The sequences of these clones coded for the variant c tail of MAGI-1 (13). The retrieval of only one of the three known splice variants previously observed for this region of MAGI-1 indicated to us that glomerular podocytes preferentially express this variant, compared with the other two known splice variants. Furthermore, the 3'-untranslated region of this glomerular variant c was not only shorter but also different in nucleotide sequence, compared with the published sequence. The significance of this difference is unknown at this time.
We also identified a number of splice variants in other regions of MAGI-1 that had not been previously reported. Figure 7 illustrates the three different clones obtained with primers in and around the fourth PDZ domain. A plausible explanation for alternative splicing within this PDZ domain may be to create PDZ domains with binding specificities for different target proteins. Other insertions in MAGI-1 were routinely observed and included an 84-bp insertion immediately preceding the third PDZ domain and a 36-bp insertion between the two WW domains (data not shown). The significance of these latter two insertions is not known at this time.
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Because the size of MAGI-1 observed in glomerular extracts was larger than
those observed in cell lines, the full-length MAGI-1 cDNA that was constructed
from the glomerular RNA consisted of clones containing the insertions
mentioned above (together with a clone containing the PDZ4c splice variant),
as well as the longer c version of the carboxyl-terminus. Lysates from HEK293
cells expressing the full-length MAGI-1 cDNA and glomerular extracts were
analyzed by Western blotting. MAGI-1 cloned from glomerular RNA migrated at
the same relative size as the largest form of MAGI-1 observed in glomerular
extracts (Figure 8A). In
addition, the full-length MAGI-1 was precipitated from HEK293 lysates by
GST-megalin WT but not by the GST-megalin 
V mutant
(Figure 8B).
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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GASP also shares a significant degree of homology with the mouse protein MAGI-1 and its human counterpart BAP-1. Together, these two groups of proteins, i.e., GASP/AIP-1/S-SCAM and MAGI-1/BAP-1, comprise a subfamily of membrane-associated guanylate kinase (MAGUK) proteins. The prototypical MAGUK proteins, for example PSD-95, are involved in the clustering of transmembrane receptor proteins and ion channels at precise locations in the plasma membranes of cells. Unlike the restricted pattern of expression exhibited by GASP, however, MAGI-1 is expressed in a more ubiquitous manner (13). Its reported expression in the kidney coincides with our finding that the fifth PDZ domain of MAGI-1 could bind megalin, which is itself abundantly expressed in the kidney. The binding of megalin to MAGI-1 was specifically mediated by the fifth PDZ domain of MAGI-1, inasmuch as the other PDZ domains of MAGI-1 were unable to bind to megalin and a megalin mutant lacking the carboxyl-terminal valine abolished the interaction with the fifth PDZ domain of MAGI-1. By using immunofluorescence assays in mouse kidney sections, we found that the expression of MAGI-1 was predominantly restricted to glomeruli. This pattern of MAGI-1 expression directly overlapped with the expression of the podocyte-specific protein GLEPP1. We are therefore confident that MAGI-1 is also expressed in a podocyte-specific manner. Although megalin is predominantly expressed in kidney proximal tubules, it has also been observed in the sole plates of podocyte foot processes, albeit at a much lower level of expression. The lack of expression of MAGI-1 in proximal tubules may have some significance with respect to megalin function. Megalin is viewed as an endocytic receptor that plays a scavenging role in the proximal tubules, clearing the glomerular filtrate of any remaining proteins that have progressed through the glomerulus. An interaction between the cytoplasmic tail of megalin and MAGI-1 might potentially interfere with the endocytic apparatus responsible for the function of megalin in proximal tubules. In podocytes, however, this interaction would presumably prevent or greatly reduce the endocytosis of megalin. Although endocytosis of megalin in podocytes, either in complex with MAGI-1 or not, cannot be entirely ruled out at this point, a nonendocytic function of megalin can be hypothesized by comparison with other members of the LDLR family that have been demonstrated to interact with potential signaling molecules. Both LDLR and LRP interact with the neuronal adaptor proteins mammalian Disabled (mDab) and FE65 (25). These interactions occur through binding of the PTB domains of mDab and FE65 to the NPXY motifs located in the cytoplasmic tails of LDLR and LRP. Interestingly, one of the three NPXY motifs in megalin is nearly identical (in the core NPXY motif and the flanking amino acids) to the NPXY motif in LDLR and LRP that binds to mDab, which suggests that megalin may also bind to mDab or a closely related protein. Indeed, megalin was recently shown to bind to Dab2, a protein that is highly homologous to mDab, and this binding was mediated through one of the NPXY motifs in the cytoplasmic tail of megalin (26). It is plausible that megalin may also serve in some signaling capacity that could be facilitated by its interaction with MAGI-1. A potential signaling complex could be assembled, with MAGI-1 serving as a docking site for a number of proteins that bind to the other PDZ or WW domains of MAGI-1. Whether MAGI-1 binds to the same proteins as reported for the homologous S-SCAM protein or to other proteins confined to the kidney remains to be investigated.
MAGI-1 was observed in the insoluble fraction of glomerular preparations
extracted with Triton X-100. Insolubility of proteins in this detergent is
often attributed to tight association with the cytoskeleton. In contrast,
MAGI-1 in a rabbit kidney epithelial cell line was found to be Triton-soluble
and migrated faster during SDS-PAGE analysis. The bulk of the difference in
the sizes of MAGI-1 from these two sources is most likely attributable to the
differential expression of splicing variants in the carboxyl-terminus of this
protein. We have found that the splicing variant for this region that is
preferentially expressed in glomeruli is variant c. This variant is much
larger than the other two splice variants found in this region and also
contains a number of tracts of basic amino acids. Although these polybasic
regions are thought to function as nuclear localization signals
(13), small polybasic regions
in the MAGUK protein hDLG (a human homologue to the Drosophila dlg
protein) are responsible for its interaction with the amino-terminal FERM
domain in protein 4.1 and ezrin
(27). This interaction would
likely result in hDLG associating with actin microfilaments through the
F-actin-binding domains of protein 4.1 and ezrin. Whether a similar mechanism
occurs with MAGI-1 or whether an interaction with an alternative protein is
responsible for the potential cytoskeletal association of MAGI-1 is not known
at this time. The fact that MAGI-1 could be bound to an actin-based
cytoskeleton fits well with the concept of MAGI-1 anchoring transmembrane
proteins at defined cellular locations. Indeed, the morphologic features of
the podocyte foot processes are dependent on a microfilament-based contractile
apparatus composed of actin, myosin II, -actinin, talin, and vinculin
(28). Recently, other proteins
were found to bind to MAGI-1, including ß-catenin
(29) and the tumor suppressor
PTEN (phosphatase and tensin homologue deleted on chromosome 10)
(30). Although the binding and
localization of these two proteins with MAGI-1 were analyzed in cell lines, it
has yet to be determined whether ß-catenin and PTEN are expressed in the
kidney with patterns similar to that we demonstrated for MAGI-1. Because
MAGI-1 has the potential to interact with a number of proteins and form a
multiprotein complex within podocytes, it will be of interest to determine
whether MAGI-1 plays a role in the overall targeting or anchoring of these
proteins and to evaluate the normal function of these complexes. In addition,
determination of how these interactions are affected in disease conditions
that result in the effacement of podocyte foot processes may lead to a better
understanding of the overall changes that occur at the molecular level within
podocytes.
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Acknowledgments |
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References |
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-catenin. Biochem Biophys Res Commun256
: 456-461,1999[Medline]
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