Journal of the American Society of Nephrology
2007 JASN IMPACT FACTOR 7.111 HOME   AUTHOR INFO   EDITORIAL BOARD   SUBSCRIBE   FEEDBACK   ALERTS   HELP 
    advanced
CURRENT ISSUE ARCHIVES JASN Express ONLINE SUBMISSION


This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow reprints & permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Malhas, A. N.
Right arrow Articles by Price, R. G.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Malhas, A. N.
Right arrow Articles by Price, R. G.
J Am Soc Nephrol 13:19-26, 2002
© 2002 American Society of Nephrology

Interaction of the Leucine-Rich Repeats of Polycystin-1 with Extracellular Matrix Proteins: Possible Role in Cell Proliferation

Ashraf N. Malhas, Ramadan A. Abuknesha and Robert G. Price

Division of Life Sciences, King’s College London, London, United Kingdom.

Correspondence to Professor R.G. Price, Division of Life Sciences, King’s College London, 150 Stamford Street, London SE1 9NN, UK. Phone: +44-0-2078484451; Fax: +44-0-2078484500; E-mail: robert.price{at}kcl.ac.uk


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
ABSTRACT. Polycystin-1, the product of the PKD1 gene, is a membrane-bound multidomain protein with a unique structure and a molecular weight of {approx}460 kD. The purpose of this study is to investigate the binding of the cystein-flanked leucine-rich repeats (LRR) of polycystin-1 to extracellular matrix (ECM) components. These interactions may play a role in normal renal development as well as the pathogenesis of autosomal-dominant polycystic kidney disease (ADPKD). In vitro assays were used to assess the binding of a fusion protein containing the LRR of polycystin-1 and that of affinity purified polycystin-1 to a number of ECM components. The results showed that the LRR modulate the binding of polycystin-1 to collagen I, fibronectin, laminin, and cyst fluid–derived laminin fragments. The addition of the LRR fusion protein to cells in culture resulted in a significant dose-dependant reduction in the rate of proliferation. Cyst fluid–derived laminin fragments had a stimulatory effect on cell proliferation, which was reversed by the LRR fusion protein. These results suggest that the LRR of polycystin-1 act as mediators of the polycystin-1 interaction with the ECM. The observed suppression effect of the LRR on cell proliferation suggests a functional role of the LRR-mediated polycystin-1 involvement in cell-matrix and cell-cell interactions. These interactions may result in the enhanced cell proliferation that is a characteristic feature of ADPKD.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
Autosomal-dominant polycystic kidney disease (ADPKD) is one of the most common genetic diseases in humans, with an incidence rate of {approx}1:1000 (1). The predominantly affected organ in ADPKD is the kidney, which is characterized by enlargement of tubule segments to form epithelial-lined fluid-filled cysts (2). There are different forms of ADPKD, which are caused by mutations in at least three different alleles. Approximately 85% of ADPKD cases are caused by mutations in the PKD1 locus. The rest of the ADPKD cases are accounted for by mutations in the PKD2 and possibly the PKD3 loci. PKD2 has a late onset and is less severe, so some cases may be undiagnosed (3). The PKD1 product, polycystin-1, is predicted to be a plasma membrane–associated glycoprotein that contains 4302 amino acids and has a molecular weight of {approx}460 kD (4,5). Polycystin-1 has a unique structure unlike that of any known protein and may be representative of a new family. The extracellular part of polycystin-1 has two complete and one incomplete leucine-rich repeats flanked by cysteine-rich domains, a cell-wall and stress-response component domain, a C-type lectin domain, a LDL domain, 16 "Ig-like" PKD repeats, and a domain that shows high homology to the sea urchin receptor for egg jelly. The intracellular part contains several potential phosphorylation sites, a proline-rich sequence, a heterotrimeric G protein activation sequence, and a coiled-coil domain (reviewed in reference (6). Polycystin-1 may be involved in cell-cell and cell-matrix interactions, which are important in renal development, but the nature of its involvement in the pathogenesis of ADPKD is not currently understood.

Significant evidence is now available to suggest that polycystin-1 plays an important role in signal transduction. Immunohistochemical studies in our laboratory have shown that polycystin-1 has a wide tissue distribution, with the highest expression in ADPKD and normal embryonic kidneys. Its expression is downregulated in the normal adult kidney (7). A recent study (8) has shown that, after normal cells attach to collagen I in a subconfluent density, polycystin-1 is part of a complex that includes vinculin, talin, tensin, {alpha}-actinin, paxillin, p130cas, c-src, and focal adhesion kinase. In confluent normal cells, polycystin-1 localizes at cell-cell junctions where it interacts with E-cadherin and {alpha}-, ß-, and {gamma}-catenin. ß-catenin has a structural role in cell adhesion as well as being part of the wnt signaling pathway, which plays an important role in cell development (9). Polycystin-1 also activates heterotrimeric G proteins (10), inhibits the degradation of the regulator of G protein signaling protein, RGS7 (11), and can stimulate AP-1–dependent transcription through the c-Jun terminal kinase pathway (12). All this evidence suggests that polycystin-1 is part of a signal transduction pathway that plays a role in normal cell development and is perturbed in ADPKD. Previous studies in our laboratory have established that the extracellular component laminin has the ability to enhance the proliferation of tubular epithelial cells in vitro (13,14), and we have more recently demonstrated that the polycystin-1 C-type lectin domain binds carbohydrate structures present in type IV collagen (15).

The LRR of polycystin-1 are among the extracellular domains that may be involved in extracellular matrix (ECM) interactions and signal transduction. Most members of the leucine-rich repeat (LRR) superfamily of proteins are considered to be adhesive proteins, e.g., toll, slit, and the small leucine-rich proteoglycans, biglycan, fibromodulin, decorin, and lumican (16,17). Some members of the superfamily such as CD14 and nerve growth factor receptor (trk) are involved in signal transduction (18). The ability of the members of the LRR superfamily to interact with other proteins is aided by the LRR nonglobular shape and exposed surface of parallel ß-sheets, resulting in a large surface area available for interaction and thus high affinity (19). As in the case of polycystin-1, LRR are usually flanked by cysteine-rich domains (20). Specificity of the protein-protein interaction depends on nonconsensus residues, length of repeats, and flanking domains (16,21).

The purpose of this study is to further investigate the role of the extracellular domains of polycystin-1 in cell-cell and cell-matrix interactions as well as their role in cell proliferation. The binding of the LRR of polycystin-1 to ECM components and their effect on cell proliferation were investigated by use of affinity-purified polycystin-1 and a fusion protein that contains the LRR of polycystin-1.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Sequence Alignment of the LRR of Polycystin-1 with Related Protein Domains
The amino acid sequence of the LRR and the two cysteine-rich flanking domains of polycystin-1 were compared with those of decorin, trkA, and slit. The latter proteins were chosen from a Fasta3 search (http://www.ebi.ac.uk) by use of the appropriate domains of polycystin-1 as the input sequences.

Cell Cultures
Human brain astrocytoma cells were obtained from the European Cell Culture Collection, and embryonic kidney epithelial (293) cells were provided by Dr. M. Slade (MRC Oncology Laboratory, Imperial College London School of Medicine). Cell monolayers were maintained in Dulbecco’s Modified Eagle’s Medium supplemented with 10% fetal bovine serum, 2000 U/ml penicillin, 2000 µg/ml streptomycin, and 20 mM L-glutamine (Sigma, Poole, UK). Both cell lines used express polycystin-1. Kidneys were obtained from patients with bilateral cystic disease with a familial history of ADPKD. Two kidneys (from one male and one female patient) were maintained at 4°C, and fluid was aspirated from superficial cysts. Cyst fluid was snap frozen and stored at -70°C. Kidneys were obtained from UK hospitals, and all procedures were carried out within the guidelines of the King’s College Ethical Committee regulations.

Production of a Fusion Protein Containing the LRR of Polycystin-1
Total RNA was isolated from 293 cells ({approx}1.4 x 105) by use of TRI-reagent (Sigma). Reverse transcription was performed with the use of random hexamer primers and Moloney Murine Leukemia Virus reverse transcriptase (Promega, Southampton, UK). Nucleotides 258 to 1014 of the PKD1 cDNA (accession number L33243) were amplified by PCR by use of 5'-GGCCTGTGGCTCGGATCCCTGGCGGGG-3' as the sense primer and 5'-AAAGAATTCCGTGGAGGAGGGTGGGGCC-3' as the antisense primer, which incorporate the BamHI and EcoRI restriction sites, respectively. PCR was performed by use of Taq polymerase for 35 cycles (95°C for 1 min, 42°C for 1 min, and 74°C for 2 min), followed by a final extension at 74°C for 10 min. The 756-bp PCR product was purified by use of the Wizard PCR Preps DNA Purification System (Promega, UK), cloned into the pGEX-2T expression vector (Amersham Pharmacia Biotech, Amersham, UK) in frame with the glutathione S-transferase (GST) coding region by use of T4 DNA ligase, and transformed into Escherichia coli strain BL21. Transformed E. coli cultures were grown in 100 ml of LB medium (1% tryptone, 0.5% yeast extract, and 1% NaCl) that contained ampicilin (100 µg/ml) and induced for 2 h at 37°C with isopropyl-ß-D-thiogalactopyranoside at a final concentration of 1 mM. Cells were harvested by centrifugation at 5000 x g for 10 min at 4°C, washed with phosphate-buffered saline, reharvested, and resuspended in phosphate-buffered saline that contained 5 mM dithiothreitol. The cells were then sonicated on ice before the addition of Triton X-100 to a final concentration of 1% followed by centrifugation at 20,000 x g for 10 min at 4°C. The GST-LRR fusion protein was purified from the cell lysate by use of the Bulk Purification Module (Amersham Pharmacia Biotech). Unpurified, purified, and control E. coli cell lysates were analyzed by sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE) in a BioRad Mini-Protean II system and by Western blot analysis that used goat anti-GST (1:1000) and rabbit anti-goat IgG–horseradish peroxidase (HRP) conjugate (1:5000), according to standard procedures (22,23).

Characterization of Polycystin-1 Present in 293 Cell Lysates
Two-Dimensional Gel Electrophoresis (2-D GE).
A total of 293 cell monolayers (1.5 x 106 cells) were trypsinated, washed with 50 mM Tris and 50 mM sorbitol, and lysed on ice for 1 h with the use of lysis buffer (50 mM Tris, 8 M urea, 1% Triton X-100, 50 mM NaF, 0.1 mM Na3VO4, 1 mM PMSF, 5 µg/ml pepstatin, 25 µg/ml aprotonin, and 25 µg/ml leupeptin). The cell lysate (100 µl) was centrifuged at 10,000 x g for 30 min at 4°C, and the supernatant analyzed by 2-D GE. Isoelectric focusing (first dimension) was performed by use of Bio-Rad’s Mini-Protean II 2-D system according to the method of O’Farrell (24), and the second dimension (SDS-PAGE) was performed with the use of Bio-Rad’s Mini-Protean II slab cell (22). The proteins were then either visualized by silver staining (25) or analyzed by Western blotting that used rabbit anti-polycystin-1 antibodies (1:500) (7), goat anti-rabbit IgG-HRP (1:20000), and enhanced chemiluminescence (Amersham Pharmacia Biotech).

Analysis of Isolated Polycystin-1 by Mass Spectrometry.
After silver staining and Western blotting, the spot corresponding to polycystin-1 was excised from the stained gel, destained by use of 30 mM potassium ferricyanide and 100 mM sodium thiosulfate, and then digested by trypsin as described in reference 26. The digested peptide fragments were extracted from the gel and analyzed by matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrometry with the use of a LASERMAT 2000 mass spectrometer (Thermo Bioanalysis Ltd., Hemel Hempstead, UK), as described elsewhere (27). The monoisotopic mass values of the tryptic digest fragments were used to search the NCBI nonredundant database by use of the program ProFound, and analysis of the tryptic cleavage of polycystin-1 was performed by use of the program ProteinInfo (http://www.proteometrics.com).

Isolation and Purification of Polycystin-1.
Polycystin-1 was immunoaffinity purified from 293 cells, as described elsewhere (27). The identity of the purified material as intact polycystin-1 was confirmed by SDS-PAGE and Western blotting, followed by tryptic digestion of the visualized band and MALDI-TOF mass spectrographic analysis of the tryptic fragments.

In Vitro Dot Blot Overlay Binding Assays
Laminin fragments (N3L, N3U, and N5) were extracted from cyst fluid as described elsewhere (13). Collagens I, II, III, and IV, whole engleberth holm-swarm laminin, fibronectin (Sigma), cyst fluid, and the laminin fragments (2 µl) were dot blotted on nitrocellulose membranes at the following concentrations (in mg/ml): 1, 0.5, 0.25, 0.125, and 0.01. The concentration of proteins in cyst fluid represents total proteins rather than a specific protein component. The membranes were blocked with 2% milk in Tris-buffered saline that contained 0.5 mM MgCl2 and 0.9 mM CaCl2 for 1 h at room temperature, washed three times with Tris-buffered saline for a total of 15 min, and incubated with the GST-LRR fusion protein (10 µg) for 2 h at 37°C with gentle agitation. The membranes were washed as before, incubated with goat anti-GST (1:1000) for 1 h at room temperature, washed, and then incubated with anti-goat IgG–HRP conjugate (1:2000) for 1 h at room temperature. After a final wash, the membranes were developed with 3,3'-diamino-benzidine-tetrahydrochloride (Merck, Poole, UK) for 5 min before being washed with distilled water and scanned at 530 nm (Joyce-Loebl Cromoscan 3 plate scanner). The above binding assay was repeated by use of purified polycystin-1 (1 µg) as the binding protein both in the absence and presence of the GST-LRR fusion protein (2 µg) as well as free GST and bovine serum albumin. In this case, the antibodies used were rabbit anti-polycystin-1 (1:500) and goat anti-rabbit IgG-HRP (1:5000).

Cell Proliferation Assays
Human brain astrocytoma and 293 cells were seeded into 24-well plates at a density of 5 x 104 cells/ml and grown as described above. Cells were harvested by trypsinization at 24, 48, 72, 96, and 120 h, and viable cells were counted by use of an improved Neubauer hemocytometer. Cells were grown in the presence of 100 ng/ml cyst fluid, 100 ng/ml of the LRR, or both. Different LRR concentrations of 20 and 40 ng/ml were also tested. Proliferation assays where the cyst fluid and LRR were omitted, as well as those where GST was added instead, were also performed for control purposes. All proliferation assays were carried out in triplicate and repeated three times.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Sequence Alignment
The comparison of the sequence alignment of the LRR and cysteine flanking domains of polycystin-1 with those of selected proteins and the consensus sequences according to Kobe and Diesenhofer (20) are shown in (Figure 1). LRR are most commonly 24 residues long and contain a leucine or an aliphatic amino acid at positions 2, 5, 7, 12, 16, 21, and 24. The LRR of polycystin-1 are of the B-type repeats because of the presence of an asparagine residue at position 10.



View larger version (38K):
[in this window]
[in a new window]
[as a PowerPoint slide]
  		Figure 1. Multiple sequence alignment of (A) leucine-rich repeats (LRR), (B) the amino-flanking cystein-rich domains, and (C) the carboxy-flanking cystein-rich domains of human polycystin-1 (P98161), nerve growth factor receptor, trkA (P04629), decorin (P07585), and the Drosophila slit protein (P24014). The consensus sequences shown are those according to Kobe and Deisenhofer (16). A dot denotes any amino acid, "a" denotes an aliphatic amino acid, and a dash denotes that an amino acid may be present or absent at the specified position. The carboxy-flanking cystein-rich domain of decorin is not shown because it has a consensus sequence that is different from the proteins shown here.

 
Production and Characterization of the GST-LRR Fusion Protein
A GST-fusion protein containing the LRR and the two cysteine-rich flanking domains of polycystin-1 (residues 22 to 268) was expressed in Escherichia coli. The identity of the GST-LRR fusion protein ({approx}58 kD) was confirmed by SDS-PAGE and Western blot analysis (Figure 2).



View larger version (48K):
[in this window]
[in a new window]
[as a PowerPoint slide]
  		Figure 2. Sodium dodecyl sulfate–polyacrylamide gel electrophoresis and immunoblot analyses of the glutathione S-transferase (GST)–LRR fusion protein. Lane 1, Escherichia coli cells transformed with pGEX-2T showing the expression of a 29-kD protein corresponding to the molecular weight of free GST. Lane 2, Escherichia coli cells transformed with pGEX-2T-LRR showing the expression of a 58-kD protein corresponding to the predicted molecular weight of the GST-LRR fusion protein. Lane 3, Amersham Rainbow Molecular Weight Markers. Lane 4, immunoblot of purified GST-LRR fusion protein ({approx}58 kD).

 
Proteomic Analysis of Polycystin-1 from 293 Cells
After 2-D GE of 293 cell lysates, proteins were visualized by silver staining, and polycystin-1 was detected by Western blot analysis (Figure 3). Polycystin-1 was visualized as a single spot with a molecular weight of {approx}460 kD and an isoelectric point (PI) value of {approx}6.3. The spot corresponding to polycystin-1 on the stained gel was excised, subjected to tryptic digestion, and the resulting tryptic fragments were detected by MALDI-TOF mass spectometry. The molecular weights obtained for the fragments were used to search the NCBInr database by use of the program ProFound. The search parameters were as follows: database, NCBInr (4/17/2001); taxonomy category, Homo sapiens; protein mass range, 300 to 500 kD; protein PI range, 0 to 14; search for, single protein; digest chemistry, trypsin; maximum missed cuts, 1; modification, by acrylamide (Cys); charge state, MH+ and peptide mass tolerance (MON), ± 0.1 Da. The identity of the protein was found to match the computer-predicted structure of the human polycystic kidney disease 1 protein, polycystin-1 (accession numbers AAC37576, A38971, and NP_000287) with a Z score of 2.43 and sequence coverage of 24%.



View larger version (50K):
[in this window]
[in a new window]
[as a PowerPoint slide]
  		Figure 3. 293 cell lysate was analyzed by two-dimensional gel electrophoresis followed by immunoblotting with the use of anti-polycystin-1 antibodies. The visualized polycystin-1 has a molecular weight ({approx}460 kD) and isoelectric point (PI) value ({approx}6.3) corresponding to those predicted from its gene sequence.

 
In Vitro Binding Blot Overlay Assays
Blot overlay assays were used to assess the binding of the LRR of polycystin-1 to collagen I, II, III, and IV, laminin, fibronectin, cyst fluid, and the laminin fragments N3L, N3U, and N5. Both the GST-LRR fusion protein and the purified whole polycystin-1 were shown to bind to collagen I, laminin, fibronectin, cyst fluid, and the laminin fragments N3L and N3U (Figure 4). No binding to collagens II, III, and IV, the N5 laminin fragment, or milk protein used in the blocking was detected. Binding to the cyst fluid generated the highest signal, whereas no significant difference in signal intensity generated by the binding to the other ligands was observed. The binding of the purified polycystin-1 to the above ligands was almost identical to that of the LRR fusion protein. Decreasing the ligand concentration resulted in decreased signal intensity (Figure 5).



View larger version (26K):
[in this window]
[in a new window]
[as a PowerPoint slide]
  		Figure 4. Blot overlay assays of (A) GST-LRR fusion protein and (B) purified polycystin-1 binding to cyst fluid (CF), laminin (Lam), N3L, N3U, N5, fibronectin (Fn), collagen IV (CIV), collagen III (CIII), collagen II (CII), and collagen I (CI). All ligands were used at a concentration of 1 mg/ml. The plots show the integral reflectance at 530 nm.

 


View larger version (41K):
[in this window]
[in a new window]
[as a PowerPoint slide]
  		Figure 5. Effect of varying the ligand concentration on the binding of the GST-LRR fusion protein to laminin, the laminin fragments N3L and N3U, fibronectin, and collagen I.

 
When excess GST-LRR fusion protein was added to the polycystin-1 assay, significant inhibition of binding to the ECM components and the cyst fluid resulted (Figure 6). No inhibition of binding was observed when GST or bovine serum albumin were added. This indicates that the LRR domains mediate the binding of polycystin-1 to the above ligands.



View larger version (24K):
[in this window]
[in a new window]
[as a PowerPoint slide]
  		Figure 6. The binding of the affinity purified polycystin-1 (1 µg) to cyst fluid, laminin, the laminin fragments N3L and N3U, fibronectin, and collagen I in the absence (-LRR) and presence (+LRR) of excess GST-LRR fusion protein (2 µg). The addition of excess GST-LRR fusion protein competes with the binding of the LRR of the purified polycystin-1 to the extracellular membrane proteins. The results for the other ligands used were identical to those shown for laminin and collagen I. The addition of excess GST or bovine serum albumin did not have any effect on the binding of polycystin-1 to the ligands.

 
Effect of the LRR on Cell Proliferation
To determine the effect of the polycystin-1 LRR on the proliferation of human brain astrocytoma and 293 cells, the LRR fusion protein was added to cells in culture at a concentration of 20 ng/ml and the cells counted at fixed time intervals over a period of 5 d. The results show a significant decrease in the rate of cell proliferation over the 5-d period. Increasing the LRR fusion protein concentration from 20 to 40 ng/ml resulted in further suppression of cell proliferation (Figure 7).



View larger version (18K):
[in this window]
[in a new window]
[as a PowerPoint slide]
  		Figure 7. The effect of the LRR fusion protein on the proliferation of (A) 293 cells and (B) human brain astrocytoma (MOG-G-CCM) cells. The LRR fusion protein results in a significant dose-dependent suppression of cell proliferation. Growth conditions are described in the text. The values shown are from the means ± SD. In some cases, the SD values are too small and therefore are not demonstrable.

 
The addition of cyst fluid at a concentration of 100 ng/ml to both cell lines resulted in enhanced proliferation over the 5-d period. Adding the LRR fusion protein at a concentration of 100 ng/ml together with the cyst fluid resulted in an inhibition of the cyst fluid effect—i.e., the stimulatory effect of cyst fluid was reversed by the LRR fusion protein (Figure 8). The addition of 40 ng/ml of cyst fluid resulted in a decrease in the rate of proliferation to the baseline (control) levels (results not shown).



View larger version (16K):
[in this window]
[in a new window]
[as a PowerPoint slide]
  		Figure 8. The effect of cyst fluid and the LRR fusion protein on (A) 293 cells and (B) MOG-G-CCM cells. Each of the cyst fluid and the LRR fusion protein was added at a concentration of 100 ng/ml. The values shown are from the means ± SD. In some cases, the SD values are too small and therefore are not demonstrable.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
This study shows that the LRR of polycystin-1 have the ability to interact with the ECM components collagen I, laminin, and fibronectin as well as cyst fluid–derived laminin fragments. Blot overlay assays that were used in this study were first described by Glenny and Weber (28) and are considered to be a powerful tool for studying protein-protein interactions and identifying possible ligands (29). We have also shown that the addition of the LRR of polycystin-1 to cells in culture has a suppressive effect on proliferation.

It is known that several changes in the composition of the ECM occur during the course of normal tissue development. Fibronectin, laminin, and collagens are among the ECM components that play important roles in this process (30). Fibronectin and laminin affect cell adhesion, morphology, migration, and differentiation. A shift in the collagen isotypes, including type I collagen, is observed during differentiation (31). The binding of the LRR of polycystin-1 to the above ligands may therefore play a role in tissue development. The composition of the ECM is altered as ADPKD progresses, which suggests that polycystin-1-ECM interactions have a functional role.

Recent studies have shown that polycystin-1 is associated with a focal adhesion complex that binds to collagen I in a subconfluent density that resembles the state of uteric bud cells during development (8). Herein we show that this collagen I binding may be mediated by the three incomplete LRR of polycystin-1 in a way that is similar to the binding of collagen I to decorin, an extracellular small leucine-rich proteoglycans that has 10 LRR forming an arch shape, which allows interaction with a single triple helix of collagen I (32). Collagen I forms ionic and polar interactions with the residues of 3 of the 10 LRR of decorin (32–34).

The growth suppression effect observed upon the addition of the polycystin-1 LRR to cells in culture in this study was found to be very similar to that of decorin (35). The ectopic expression as well as the exogenous addition of recombinant decorin induces growth suppression in a number of cell lines (35). Growth suppression is associated with the induction of p21 (36), a protein that controls transition from G1-S and inhibits cyclin-dependent kinases and proliferating cell nuclear antigen, arresting the cells in G1. Transfection with the small leucine-rich proteoglycans biglycan and fibromodulin does not have the same effect (36), which indicates that the effect is not caused by the presence of any LRR. Hence, it is possible that polycystin-1 may be involved in a signaling pathway that is affected in ADPKD, causing abnormal cell proliferation.

The addition of the LRR fusion protein demonstrated in this study is therefore suppressing baseline proliferation. The LRR fusion protein added to cells in culture may be competing with the binding of the LRR of polycystin-1 to a ligand, which might be an autocrine laminin-like growth factor in the medium that possibly triggers a signal transduction pathway enhancing cell proliferation. Alternatively, the LRR may have a suppressant effect on proliferation by binding to a putative cell surface receptor (see below).

Cyst fluid–derived laminin fragments as well as epidermal growth factor present in cyst fluid have the ability to enhance cell proliferation (13), and herein we show that this effect can be reversed by the addition of the polycystin-1 LRR to cells in culture. One possible explanation of the observed effect is that the cyst fluid components may cause cell proliferation through a cell-signaling pathway of which polycystin-1 and, more specifically, the cystein-flanked LRR domains are components. The addition of the LRR fusion protein to cells in culture competes with the binding of the cyst fluid component to polycystin-1, producing the observed reduction in proliferation. Another possible explanation is that the added LRR bind to a cell surface receptor (see below), causing the observed reduction in cell proliferation. The observation that the addition of 40 ng/ml of LRR to cells in the presence of cyst fluid reduces proliferation to the baseline (control) levels and that the addition of 100 ng/ml of LRR reduces proliferation to lower than baseline levels may indicate that polycystin-1 regulates cell proliferation through two mechanisms—one by binding to extracellular ligands to enhance proliferation and the other by binding to a cell surface receptor to reduce proliferation, hence maintaining a balance.

The exact role of polycystin-1 in tubule development and cyst formation is still not fully understood. Although many would presume that a loss of function resulting from mutations in the PKD1 gene is the primary cystogenic force, the increased expression of normal polycystin-1 in mice has been found to induce cyst formation (37), which suggests a possible alternative mechanism in which the protein level is important for maintaining a balance in the rate of proliferation. The latter is also supported by the observation that polycystin-1 expression is upregulated in the fetal stage and in patients with ADPKD and downregulated in normal adults (7). Our results can therefore suggest two possible models for the polycystin-1 involvement in development and cystogenesis (Figure 9). The first model assumes that polycytsin-1 is part of a pathway that induces cell proliferation in response to binding to ECM proteins and therefore the addition of the LRR fusion protein to cells in culture competes with the LRR in polycystin-1, resulting in the observed decrease in cell proliferation. The second model assumes that the LRR of polycystin-1 bind to a cell surface receptor on adjacent cells to reduce the rate of proliferation. In this model, a mutation in the PKD1 gene would result in an abnormal polycystin-1 that is unable to bind to its receptor, hence resulting in the uncontrolled cell proliferation observed in ADPKD.



View larger version (14K):
[in this window]
[in a new window]
[as a PowerPoint slide]
  		Figure 9. Possible mechanisms by which polycystin-1 (P) may control cell proliferation. The first mechanism is by binding to an extracellular factor (A), e.g., laminin or a cyst fluid component, which enhances cell proliferation. The second mechanism is by binding to a putative cell surface receptor (R), resulting in decreased cell proliferation. A balance between both processes is needed to maintain normal levels of proliferation, which are altered in autosomal dominant polycystic kidney disease.

 
Polycystin-1 isolated by immunoaffinity chromatography had the same structure as that predicted from the gene sequence (27). In this study, we identified a single polycystin-1 spot among the proteins present in a 293-cell lysate using 2-D GE. The PI of the isolated polycystin-1 and its amino acid structure were both consistent with those predicted from the gene sequence. In addition, we have shown that polycystin-1 has the ability to interact with a number of ECM components through the LRR domains and that these interactions may be part of a signal transduction pathway which is essential for normal cell development. Mutations in the PKD1 gene encoding polycystin-1 would interfere with the normal function of polycystin-1 in the pathway, resulting in the abnormal cell proliferation that is a characteristic feature of ADPKD.


   Acknowledgments
 
We would like to thank Dr. Andrew Kicman and Hendrik Neubert (Drug Control Center, King’s College London) for their assistance with the MALDI-TOF MS. The work was partially funded by an ORS studentship.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

  1. Pirson Y, Chauvneau D, Van Gijn J: Subarachnoid haemorrhage in ADPKD patients: How to recognize and how to manage. Nephrol Dial Transplant 11: 1236–1238, 1996[Free Full Text]
  2. Gabow P: Autosomal-dominant polycystic kidney-disease. N Engl J Med 329: 332–342, 1993[Free Full Text]
  3. The International Polycystic Kidney Disease Consortium: Polycystic kidney-disease—the complete structure of the pkd1 gene and its protein. Cell 81: 289–298, 1995[CrossRef][Medline]
  4. The European Polycystic Kidney Disease Consortium: Breakpoint break for consortium studying adult polycystic kidney-disease. Cell 77: 881–894, 1994[CrossRef][Medline]
  5. Geng L, Segal Y, Piessel B, Deng N, Pei Y, Rennke H, Glucksmann-Kuis A, Schneider M, Ericsson M, Reeders S, Zhou J: Identification and localization of polycystin, the PKD1 gene product. J Clin Invest 98: 2674–2682, 1996[Medline]
  6. Wilson P: Polycystin-1: New aspects of structure, function and regulation. J Am Soc Nephrol 12: 834–845, 2001[Abstract/Free Full Text]
  7. Weston B, Jeffery S, Jeffrey I, Sharaf S, Carter N, Saggar-Malik A, Price RG: Polycystin expression during embryonic development of human kidney in adult tissues and ADPKD tissue. Histochem J 29: 847–856, 1997[CrossRef][Medline]
  8. Geng L, Burrow C, Li H, Wilson P: Modification of the composition of polycystin-1 multiprotein complexes by calcium and tyrosine phosphorylation. Biochim Biophys Acta 1535: 21–35, 2000[Medline]
  9. Novak A, Dedhar S: Signalling through ß-catenin and Lef/Tcf. Cell Mol Life Sci 56: 523–537, 1999[CrossRef][Medline]
  10. Parnell S, Magenheimer B, Maser R, Rankin C, Smine A, Okamoto T, Calvet J: The polycystic kidney disease-1 protein, polycystin-1, binds and activates heterotrimeric G-proteins in vitro. Biochem Biophys Res Comm 251: 625–631, 1998[CrossRef][Medline]
  11. Kim E, Arnould T, Sellin L, Benzing T, Fan M, Gruning W, Sokol S, Drummond I, Walz G: The polycystic kidney disease 1 gene product modulates Wnt signalling. J Biol Chem 274: 4947–4953, 1999[Abstract/Free Full Text]
  12. Arnould T, Kim E, Tsiokas L, Jochimsen F, Gruning W, Chang J, Walz G: The polycystic kidney disease 1 gene product mediates protein kinase C alpha-dependent and c-Jun N-terminal kinase-dependent activation of the transcription factor AP-1. J Biol Chem 273: 6013–6018, 1998[Abstract/Free Full Text]
  13. Slade M, Kirby R, Pocsi I, Jones J, Price R: Presence of laminin fragments in cyst fluid from patients with autosomal dominant polycystic kidney disease ADPKD: Role in proliferation of tubular epithelial cells. Biochim Biophys Acta 1401: 203–210, 1998[Medline]
  14. Slade M, Pocsi I, Kirby R, Kay Jones J, Ganz S, Taylor S, Price R: Isolation of pepsin-resistant laminin fragments from human placenta: Effect on epithelial cells cultured from kidneys of patients with autosomal dominant polycystic kidney disease (ADPKD). Biochim Biobys Acta 1310: 25–31, 1996
  15. Weston B, Bagneris C, Stirling J, Price RG: The polycystin-1 C-type lectin domain binds carbohydrate in a calcium-dependent manner, and interacts with extracellular matrix proteins in vitro. Biocim Biophys Acta 1536: 161–176, 2001[Medline]
  16. Kobe B, Deisenhofer J: The leucine-rich repeat: A versatile binding motif. Trends Biochem Sci 19: 415–421, 1994[CrossRef][Medline]
  17. Iozzo R: The biology of the small leucine-rich proteoglycans. J Biol Chem 274: 18843–18846, 1999[Free Full Text]
  18. Windisch J, Marsteine R, Schneider R: Nerve growth factor binding sites on trkA mapped to single 24-amino acid leucine-rich motif. J Biol Chem 270: 28133–28138, 1995[Abstract/Free Full Text]
  19. Kobe B, Deisenhofer J: A structural basis of the interaction between leucine-rich repeats and protein ligands. Nature 374: 183–186, 1995[CrossRef][Medline]
  20. Fisher L, Heegeard A, Vetter U, Vogel W, Just W, Termir J, Young M: Human biglycan gene. J Biol Chem 266: 14371–14377, 1991[Abstract/Free Full Text]
  21. Svensson L, Heingard D, Oldberg A: Decorin-binding sites for collagen type I are mainly located in leucine-rich repeats 4–5. J Biol Chem 270: 20712–20716, 1995[Abstract/Free Full Text]
  22. Lamelli U: Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 259: 680–685, 1970
  23. Gershoni J, Palade G: Electrophoretic transfer of proteins from sodium dodecyl-sulfate polyacrylamide gels to a positively charged membrane-filter. Anal Biochem 124: 396–405, 1982[CrossRef][Medline]
  24. O’Farell P: High resolution two dimensional electrophoresis of proteins. J Biol Chem 250: 4007–4021, 1975[Abstract/Free Full Text]
  25. Ansorge W: Fast and sensitive detection of protein and dna bands by treatment with potassium-permanganate. J Biochem Biophys Methods 11: 13–20, 1985[CrossRef][Medline]
  26. Helmann U, Wernstedt C, Gonez J, Heldin C: Improvement of an in-gel digestion procedure for the micropreparation of internal protein-fragments for amino-acid sequencing. Anal Biochem 224: 451–455, 1995[CrossRef][Medline]
  27. Malhas AN, Abuknesha RA, Price RG: Polycystin-1: Immunoaffinity purification and characterization by mass spectrometry. FEBS Lett 505: 313–316, 2001[CrossRef][Medline]
  28. Glenny J, Weber K: Calmodulin-binding proteins of the microfilaments present in isolated brush borders and microvilli of interstitial epithelial cells. J Biol Chem 255: 10551–10554, 1980[Abstract/Free Full Text]
  29. Crawford A, Beckerle M: Blot overlay assays.In: Cell Biology, a Laboratory Manual, 3rd ed. edited by Cellis J, San Diego, Academic Press, 1997,pp 301–308
  30. Ekblom P: Formation of basement membranes in the embryonic kidney: An immunohistological study. J Cell Biol 91: 1–10, 1981[Abstract/Free Full Text]
  31. Ekblom P, Lehtonen E, Saxen L, Timpl R: Shift in collagen type as an early response to induction of the metanephric mesenchyme. J Cell Biol 89: 276–283, 1981[Abstract/Free Full Text]
  32. Weber I, Harrison R, Iozzo R: Model structure of decorin and implications for collagen fibrillogenesis. J Biol Chem 271: 31767–31770, 1996[Abstract/Free Full Text]
  33. Ruoslahti E, Yamaguchi Y: Proteoglycans as modulators of growth factor activities. Cell 64: 867–869, 1991[CrossRef][Medline]
  34. Keene D, San Antonio J, Mayne R, McQuillan D, Sarris G, Santoro S, Iozzo R: Decorin binds near the C terminus of type I collagen. J Biol Chem 275: 21801–21804, 2000[Abstract/Free Full Text]
  35. De Luca A, Santra M, Baldi A, Giordano A, Iozzo R: Decorin-induced growth suppression is associated with up-regulation of p21, an inhibitor of cyclin-dependent kinases. J Biol Chem 271: 18961–18965, 1996[Abstract/Free Full Text]
  36. Santra M, Mann D, Mercer E, Skorski T, Calabretta B, Iozzo R: Ectopic expression of decorin protein core causes a generalised growth suppression in neoplastic cells of various histogenetic origins and requires endogenous p21, an inhibitor of cyclin-dependent kinases. J Clin Invest 100: 149–157, 1997[Medline]
  37. Pritchard L, Sloane-Stanley JA, Sharpe JA, Aspinwall R, Lu WN, Buckle V, Strmecki L, Walker D, Ward CJ, Alpers CE, Zhou J, William WG, Harris PC: A human PKD1 transgene generates functional polycystin-1 in mice and is associated with a cystic phenotype. Hum Mol Genet 9: 2617–2627, 2000[Abstract/Free Full Text]
Received for publication July 27, 2001. Accepted for publication September 23, 2001.




This article has been cited by other articles:


Home page
 Am. J. Physiol. Renal Physiol.Home page
T. Weimbs
Polycystic kidney disease and renal injury repair: common pathways, fluid flow, and the function of polycystin-1
Am J Physiol Renal Physiol, November 1, 2007; 293(5): F1423 - F1432.
[Abstract] [Full Text] [PDF]

Home page
 J. Am. Soc. Nephrol.Home page
M. B. Shannon, B. L. Patton, S. J. Harvey, and J. H. Miner
A Hypomorphic Mutation in the Mouse Laminin {alpha}5 Gene Causes Polycystic Kidney Disease
J. Am. Soc. Nephrol., July 1, 2006; 17(7): 1913 - 1922.
[Abstract] [Full Text] [PDF]

Home page
 J. Biol. Chem.Home page
F. Qian, W. Wei, G. Germino, and A. Oberhauser
The Nanomechanics of Polycystin-1 Extracellular Region
J. Biol. Chem., December 9, 2005; 280(49): 40723 - 40730.
[Abstract] [Full Text] [PDF]

Home page
 Hum Mol GenetHome page
Q. Li, N. Montalbetti, P. Y. Shen, X.-Q. Dai, C. I. Cheeseman, E. Karpinski, G. Wu, H. F. Cantiello, and X.-Z. Chen
Alpha-actinin associates with polycystin-2 and regulates its channel activity
Hum. Mol. Genet., June 15, 2005; 14(12): 1587 - 1603.
[Abstract] [Full Text] [PDF]

Home page
 Am. J. Physiol. Renal Physiol.Home page
M. Silberberg, A. J. Charron, R. Bacallao, and A. Wandinger-Ness
Mispolarization of desmosomal proteins and altered intercellular adhesion in autosomal dominant polycystic kidney disease
Am J Physiol Renal Physiol, June 1, 2005; 288(6): F1153 - F1163.
[Abstract] [Full Text] [PDF]

Home page
 J. Am. Soc. Nephrol.Home page
H. Kim, W. Jeong, K. Ahn, C. Ahn, and S. Kang
Siah-1 Interacts with the Intracellular Region of Polycystin-1 and Affects Its Stability via the Ubiquitin-Proteasome Pathway
J. Am. Soc. Nephrol., August 1, 2004; 15(8): 2042 - 2049.
[Abstract] [Full Text] [PDF]

Home page
 Am. J. Physiol. Renal Physiol.Home page
H. F. Cantiello
Regulation of calcium signaling by polycystin-2
Am J Physiol Renal Physiol, June 1, 2004; 286(6): F1012 - F1029.
[Abstract] [Full Text] [PDF]

Home page
 NEJMHome page
P. D. Wilson
Polycystic Kidney Disease
N. Engl. J. Med., January 8, 2004; 350(2): 151 - 164.
[Full Text] [PDF]

Home page
 J. Am. Soc. Nephrol.Home page
A. J. Streets, L. J. Newby, M. J. O'Hare, N. O. Bukanov, O. Ibraghimov-Beskrovnaya, and A. C.M. Ong
Functional Analysis of PKD1 Transgenic Lines Reveals a Direct Role for Polycystin-1 in Mediating Cell-Cell Adhesion
J. Am. Soc. Nephrol., July 1, 2003; 14(7): 1804 - 1815.
[Abstract] [Full Text] [PDF]

This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow reprints & permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Malhas, A. N.
Right arrow Articles by Price, R. G.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Malhas, A. N.
Right arrow Articles by Price, R. G.

HOME CURRENT ISSUE ARCHIVES JASN Express ONLINE SUBMISSION AUTHOR INFO
EDITORIAL BOARD SUBSCRIBE FEEDBACK ALERTS HELP