Human Molecular Genetics

Human Molecular Genetics Advance Access originally published online on September 28, 2006
Human Molecular Genetics 2006 15(22):3280-3292; doi:10.1093/hmg/ddl404

This Article Abstract FREE Full Text (PDF) Supplementary Data All Versions of this Article: 15/22/3280    most recent ddl404v1 Alert me when this article is cited Alert me if a correction is posted Services Email this article to a friend Similar articles in this journal Similar articles in ISI Web of Science Similar articles in PubMed Alert me to new issues of the journal Add to My Personal Archive Download to citation manager Search for citing articles in: ISI Web of Science (24) Request Permissions Google Scholar Articles by Wu, Y. Articles by Chen, X.-Z. Search for Related Content PubMed PubMed Citation Articles by Wu, Y. Articles by Chen, X.-Z. Social Bookmarking          What's this?

© The Author 2006. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oxfordjournals.org

Kinesin-2 mediates physical and functional interactions between polycystin-2 and fibrocystin

Yuliang Wu^1,, Xiao-Qing Dai^1,, Qiang Li^1,, Carl X. Chen^1,3, Weiyi Mai⁴, Zahir Hussain¹, Wentong Long¹, Nicolás Montalbetti⁵, Guochun Li⁶, Richard Glynne⁶, Shaohua Wang², Horacio F. Cantiello^5,7, Guanqing Wu⁴ and Xing-Zhen Chen^1,*

¹ Membrane Protein Research Group, Department of Physiology and ² Department of Surgery, University of Alberta, Edmonton, Alberta, Canada, ³ School of Mechanical Engineering, Jimei University, Xiamen, Fujian, China, ⁴ Department of Medicine, Vanderbilt University, Nashville, TN, USA, ⁵ Laboratorio de Canales Iónicos, Facultad de Farmacia y Bioquímica, Buenos Aires, Argentina, ⁶ Genomics Institute of the Novartis Research Foundation, San Diego, CA, USA and ⁷ Renal Unit, Massachusetts General Hospital and Harvard Medical School, Charlestown, MA, USA

^* To whom correspondence should be addressed at: Department of Physiology, University of Alberta, 7-29 Medical Sciences Building, Edmonton, Alberta, Canada T6G 2H7. Tel: +1 7804922294; Fax: +1 7804928915; Email: xzchen{at}ualberta.ca

Received July 25, 2006; Accepted September 21, 2006

	ABSTRACT

TOP ABSTRACT INTRODUCTION RESULTS DISCUSSION MATERIALS AND METHODS SUPPLEMENTARY MATERIAL REFERENCES

 
Autosomal dominant polycystic kidney disease (ADPKD) is caused by mutations in PKD1, encoding polycystin-1 (PC1), or PKD2 (polycystin-2, PC2). Autosomal recessive PKD (ARPKD) is caused by mutations in PKHD1, encoding fibrocystin/polyductin (FPC). No molecular link between ADPKD and ARPKD has been determined. Here, we demonstrated, by yeast two-hybrid and biochemical assays, that KIF3B, a motor subunit of kinesin-2, associates with PC2 and FPC. Co-immunoprecipitation experiments using Madin-Darby canine kidney (MDCK) and inner medullary collecting duct (IMCD) cells and human kidney revealed that PC2 and KIF3B, FPC and KIF3B and, furthermore, PC2 and FPC are endogenously in the same complex(es), though no direct association between the PC2 and FPC intracellular termini was detected. In vitro binding and Far Western blot experiments demonstrated that PC2 and FPC are in the same complex only if KIF3B is present, presumably by forming a PC2–KIF3B–FPC complex. This was supported by our observation that altering KIF3B level in IMCD cells by over-expression or siRNA significantly affected complexing between PC2 and FPC. Immunofluorescence experiments showed that PC2, FPC and KIF3B partially co-localized in primary cilia of over-confluent and perinuclear regions of sub-confluent cells. Furthermore, KIF3B mediated functional modulation of purified PC2 channels by FPC in a planer lipid bilayer electrophysiology system. The FPC C-terminus substantially stimulated PC2 channel activity in the presence of KIF3B, whereas FPC or KIF3B alone had no effect. Taken together, we discovered that kinesin-2 is a linker between PC2 and FPC and mediates the regulation of PC2 channel function by FPC. Our study may be important for elucidating common molecular pathways for PKD of different genotypes.

	INTRODUCTION

TOP ABSTRACT INTRODUCTION RESULTS DISCUSSION MATERIALS AND METHODS SUPPLEMENTARY MATERIAL REFERENCES

 
The formation of kidney cysts is a pathological entity common to a number of inherited and acquired human diseases. Of these disorders, autosomal dominant polycystic kidney disease (ADPKD) is the most prominent, affects up to one in 500 individuals, and is caused by mutations in the PKD1 or PKD2 gene, encoding, respectively, the transmembrane proteins polycystin-1 (PC1) or polycystin-2 (PC2) (1–3). Autosomal recessive PKD (ARPKD) is less common than ADPKD, occurring in 1/20 000 infants and children (4), and is caused by mutations in the PKHD1 (polycystic kidney and hepatic disease 1) gene, encoding fibrocystin (also named polyductin and tigmin, abbreviated as FPC) (5–7).

PC2 is a 968 amino-acid protein with a molecular mass of 110 kDa, and has six putative transmembrane domains and intracellular N- and C-termini. PC2 is a member of the transient receptor potential (TRP) superfamily of channels and is indeed a non-selective cation channel permeable to Ca, K and Na (8,9). PC2 is known to co-assemble with the following proteins: PC1 (10), PC2 (11), TRPC1 (12), Golgi- and endoplasmic reticulum-associated protein PIGEA-14 (13), actin filament-associated proteins Hax-1 and CD2AP (14,15), actin filament proteins tropomyosin-1, troponin I and -actinin (16–18) and RhoA GTPase-binding protein mDia1 (19). PC2 is present in a variety of tissues, including kidney, testis, cardiac, skeletal and smooth muscles (2), but not all organs display known phenotypes associated with PKD2 mutations.

FPC is a 4074 amino-acid protein with a single transmembrane domain near its intracellular carboxyl tail and contains IPT/TIG (immunoglobin-like fold shared by plexins and transcription factors) and parallel beta-helix 1 (PbH1) repeats in its large extracellular N-terminus (5). This suggests that FPC may be a cell surface receptor implicated in protein–protein interactions. FPC shares a modest sequence similarity (23% identity) with two proteins of unknown function D86, a putative lymphocyte-secreted protein and TMEM2 (type II transmembrane protein) (5,6). FPC also shares structural similarity with hepatocyte growth factor receptor and plexins, which are involved in the regulation of cell adhesion and proliferation. FPC is highly expressed in kidney, but is also found in liver, pancreas, lung and testis (5–7,20). In kidney, FPC is expressed in various segments of a nephron, including collecting ducts, proximal convoluted tubules and thick ascending limbs of the loop of Henle (20–23). Interestingly, like other cystoproteins, such as polycystins (PC1 and PC2), polaris (homologous to Chlamydomonas intraflagellar transport protein 88, or IFT88) and inversin, FPC is also present in renal primary cilia (21–24).

Kinesins represent a diverse group of microtubule-associated motor proteins that drive a number of cellular transport events (25). Kinesin-2, first identified from sea urchin eggs (26), transports cargo to the plus end of a cilium (or flagellum), whereas dynein transports materials from tip to the cell body (27). Unlike the conventional heterotetrameric kinesin (kinesin-1), kinesin-2 is a heterotrimeric complex formed by a pair of homologous motor subunits, KIF3A and KIF3B, and a non-motor subunit KAP3 (kinesin-associated polypeptide 3). KIF3A and KIF3B are composed of three domains, an N-terminal motor domain, which contains ATP hydrolysis and microtubule binding sites, a central stalk domain, where two motor subunits form a -helical coiled-coil structure, and a globular C-terminus (28,29) (Fig. 1A). In neurons, kinesin-2 is also responsible for sorting and transporting materials that are synthesized within the cell body into and along extensive dendritic and axonal processes (30).

View larger version (23K): [in this window] [in a new window] [Download PowerPoint slide]   Figure 1. Interactions between PC2, FPC and kinesin-2 identified by yeast two-hybrid experiments. (A) Illustration of FPC, PC2, KIF3B and KIF3A proteins and their fragments. Numbers refer to amino acid positions; TM, transmembrane. (B) Quantification of interactions between PC1, PC2, FPC and KIF3B in a yeast liquid ß-galactosidase assay. Constructs were co-expressed in yeast strain Y187 as fusion proteins with either the GAL4 activation or the DNA-binding domain. P53+T-antigen serves as a positive control, whereas empty vectors (pGBKT7+pGADT7) as a negative control. Quantitative data are plotted with error bars corresponding to the SE based on three or more independent experiments. (C) Summary of interactions between PC2, FPC and kinesin-2 by a yeast colony lift assay. ‘+’ and ‘–’ indicate the presence and absence of interaction, respectively.

 
In the present study, we demonstrated that both PC2 and FPC associate with the motor subunit KIF3B. We then utilized various in vitro and in vivo approaches to show that PC2, KIF3B and FPC form a complex. Using a planner lipid bilayer electrophysiology system, we investigated the functional modulation of PC2 channel by FPC and the mediator role of KIF3B.

	RESULTS

TOP ABSTRACT INTRODUCTION RESULTS DISCUSSION MATERIALS AND METHODS SUPPLEMENTARY MATERIAL REFERENCES

 
Interaction of PC2 and FPC with KIF3B revealed by yeast two-hybrid system
A yeast two-hybrid system was used to screen proteins that associate with the C-terminus of PC2 (PC2C, amino-acid 682–968; Fig. 1A). A bait construct, pGBKT7–PC2C, was used to screen a human heart cDNA library (Clontech, Polo Alto, CA, USA). One plasmid isolated from the library represented a C-terminal fragment of human KIF3B protein (called thereafter KIF3BC, amino-acid 407–747; Fig. 1A), containing part of the coiled-coil region and the whole globular tail. Interestingly, the FPC intracellular C-terminus (FPCC, amino-acid 3882–4074) and the PC2 intracellular N-terminus (PC2N, amino-acid 1–215; Fig. 1A) also strongly associated with KIF3BC. We further found that a smaller fragment within KIF3BC (KIF3BC1, amino-acid 592–670) associates with FPCC. On the other hand, our liquid ß-galactosidase quantitative and colony lift assays revealed no binding between FPCC and the following fragments: PC2C, PC2N and the C-terminus of PC1 (PC1C, amino-acid 4088–4302), between KIF3BC and PC1C and between the KIF3B N-terminus (KIF3BN, amino-acid 1–406) and PC2C (or FPCC) (Fig. 1B and C). No ß-galactosidase activity was detected between empty vectors pGBKT7 and pGADT7 (Fig. 1B), which served as a negative control.

We also tested the KIF3A and KAP3 subunits utilizing the yeast colony lift assay. PC2C associated with KIF3A (full-length and the C-terminus, KIF3AC, amino-acid 403–702, but not the N-terminus, KIF3AN, amino-acid 1–402), whereas FPCC exhibited no association with these KIF3A fragments (Fig. 1C). Neither PC2C nor FPCC interacted with KAP3 (full length and truncated fragments KAP3N, amino-acid 1–448, and KAP3C, amino-acid 449–792). We also found no association between KIF3B and three extracellular fragments of FPC: FPCN1 (amino-acid 30–396), FPCN2 (amino-acid 1313–1852) and FPCN3 (amino-acid 3465–3849) (Fig. 1A and C). Of note, we confirmed the previously reported association between KIF3B and IFT20 (31), but found no binding between IFT20 and PC2 (or FPC) (Fig. 1C). Thus, KIF3B is the only identified intermediate protein in these assays that directly binds both PC2 and FPC.

In vitro and in vivo association of PC2 or FPC with KIF3B
We first employed immunoprecipitation (IP) and peptide blocking to validate the specificity of antibodies against FPC (hAR-C2m3C10 or C10), PC2 (1A11), both being previously reported (18,23), and KIF3B (Catalog no. 611535, mouse monoclonal, BD Biosciences). The specificity of C10 was verified in combination with the use of a FLAG antibody in Madin–Darby canine kidney (MDCK) cells expressing FLAG-FPCC (Fig. 2A) and the antibody hAR-Nm3G12 (G12) against the FPC N-terminus in native mouse inner medulla collecting duct (IMCD) cells (Fig. 2B). The presence of a small band of ~135 kDa, recognized by C10 but not G12, was previously reported and presumably corresponds to an N-terminal truncated variant of FPC (23). The specificity of 1A11 (Fig. 2C), no. 611535 (Fig. 2D), G-20 (PC2, data not shown) and C-18 (KIF3B, data not shown) was verified in native IMCD cells as well. We then utilized in vitro biochemical methods to further characterize the interaction between PC2/FPC and KIF3B. We first employed a glutathione S-transferase (GST) fusion protein affinity-binding method. For this purpose poly-histidine-tagged (His-) KIF3AC and KIF3BC, and GST-tagged (GST-) PC2C and FPCC were expressed in bacteria BL21 (DE3) in the presence of 1 mM IPTG (Fig. 3A, panels 1 and 2). Cell extracts containing PC2C or FPCC were incubated with purified KIF3AC or KIF3BC, and detected with a specific antibody against KIF3A or KIF3B. GST-PC2C co-precipitated with KIF3BC and KIF3AC, whereas GST-FPCC co-precipitated with KIF3BC, but not with KIF3AC (Fig. 3A, panels 3 and 4). GST alone exhibited no binding with KIF3AC or KIF3BC.

View larger version (55K): [in this window] [in a new window] [Download PowerPoint slide]   Figure 2. Validation of antibody specificity. (A) MDCK cells were transfected with FLAG-tagged FPCC and cells lysates were detected with anti-FLAG antibody (M2, left panel) or anti-FPC antibody hAR-C2m3C10 (C10, right panel). (B) IMCD cell lysates were immunoprecipitated (IP) with C10, immunoblotted (IB) with the same antibody (left panel) in the presence of FPCC protein as blocking peptide (BP, center panel), and IB with FPC antibody hAR-Nm3G12 (G12, right panel). +, with antibody; –, with non-immune mouse IgG. (C) IMCD cell lysates were IP with PC2 antibody 1A11, IB with the same antibody (left panel) in the presence of blocking peptide PC2C (right panel). (D) IMCD cell lysates were IP with KIF3B antibody (no. 611535), IB with the same antibody (left panel), and blocked with blocking peptide KIF3BC (right panel).

 

View larger version (37K): [in this window] [in a new window] [Download PowerPoint slide]   Figure 3. Association of KIF3B with PC2 and FPC revealed by GST pull-down, dot blot and co-IP. (A) Purified His-tagged KIF3BC and KIF3AC proteins were stained by Coomassie Brilliant Blue (CBB). E. coli extract expressing GST alone, GST-PC2C or GST-FPCC was incubated with purified His-KIF3BC or His-KIF3AC. GST-agarose was used to precipitate GST epitope-binding proteins. The resultant protein samples were IB with an antibody against GST, KIF3B (no. 611535) or KIF3A, as indicated. (B) Purified GST-KIF3BC, -PC2 N, -PC2C, -FPCC and GST protein alone were spotted on nitrocellulose membranes, incubated with IMCD cell lysates in a blocking buffer and then detected by the KIF3B antibody (no. 611535). GST-KIF3BC acts as a positive control, whereas BSA and PBS binding buffer are negative controls. (C) MDCK and IMCD cell lysates and total proteins from human kidney tissues were IP with an antibody against KIF3B (C-18), PC2 (G-20) or FPC (C2p). The precipitates were immunoblotted with an antibody against PC2 (1A11), FPC (C10) or KIF3B (no. 611535), as indicated. +, with antibody; –, with non-immune goat or rabbit IgG.

 
Because KIF3A was able to bind PC2 but not FPC, in the next experiments we focused on using KIF3B. We also performed dot blot overlay experiments to examine the PC2–KIF3B and FPC–KIF3B interactions. GST-PC2N, GST-PC2C, GST-FPCC and GST proteins purified from E. coli were spotted onto a nitrocellulose membrane, followed by incubation with lysates of IMCD or MDCK cells (data not shown). The membrane was then washed and probed with the mouse monoclonal antibody against KIF3B. Immunoreactive spots were observed with GST-PC2N, GST-PC2C and GST-FPCC, but not with GST, bovine serum albumin (BSA) or binding buffer alone (Fig. 3B), further demonstrating that PC2 and FPC associate with KIF3B.

To determine whether PC2 and FPC interact with kinesin-2 in vivo, we performed co-IP experiments using native IMCD cells, MDCK cells and human kidney tissues. Using antibodies against KIF3B, PC2 or FPC for precipitation, we detected associated proteins via immunoblotting. PC2 and FPC were detected in the immunoprecipitates using a KIF3B antibody, but not in the control immunoprecipitates using non-immune serum (Fig. 3C). Reciprocal co-IP using an antibody against PC2 or FPC also precipitated KIF3B from these cells or tissue lysates. These results demonstrate that the protein complexes in the forms of PC2–KIF3B and FPC–KIF3B are present in vivo in these cells and tissues. Note that the fact that KIF3B associates with both PC2 and FPC does not guarantee that these three proteins are present in a common complex. In fact, even without the existence of the triplex PC2–KIF3B–FPC, the simultaneous presence of the duplexes KIF3B–PC2 and KIF3B–FPC in vivo is sufficient to account for all our observed data. Therefore, whether PC2 and FPC are in a same protein complex requires further verification.

Interaction between PC2 and FPC
We performed reciprocal co-IP experiments using native cells and tissues, and revealed that the two proteins are in the same complex (Fig. 4A). Furthermore, we used siRNA to decrease the expression of FPC or PC2 in IMCD cells and found that FPC was more efficiently diminished than PC2 (Supplementary Material, Fig. S1). Thus, we chose native IMCD cells and those with either pkhd1 siRNA or FPCC over-expression for further co-IP assays. As expected, we found that the amount of immunoprecipitated PC2 in siRNA cells was smaller than the one in native cells, which itself was smaller than the one in over-expressed cells (right panel, Fig. 4B). Note that, compared with native IMCD cells, there was no appreciable change in the PC2 level in cells over-expressing FPCC (106±10%, from three Western blot (WB) data after normalization by the actin expression) or in those with pkhd1 siRNA (114±6%, N=3) (left panels, Fig. 4B).

View larger version (56K): [in this window] [in a new window] [Download PowerPoint slide]   Figure 4. Interaction between PC2 and FPC revealed by co-IP and pull-down. (A) IMCD cell lysates and total proteins from human kidney and heart tissues were IP with FPC (C10, upper panels) or PC2 (1A11, lower panels) antibody and detected by 1A11 or C10. +, with antibody; –, with non-immune mouse IgG. (B) IMCD cell lysates were IP with an FPC antibody (C2p) and IB with PC2 antibody (1A11, right panel). The expressions of FPC and PC2 were detected with C2p and 1A11, respectively, in cells over-expressing FLAG-FPCC, native cells (Ctrl) and pkhd1 siRNA cells. Actin was used as a loading control (left panels). (C) Pull-down experiments using MDCK cells over-expressing FLAG-FPCTMC (upper panels) or TAP–PC2 (lower panels). Proteins eluted from FLAG-M2 beads or protein A agarose were detected with indicated antibodies.

 
To further substantiate physical interaction between PC2 and FPC, we transfected MDCK cells with the FLAG-tagged FPC transmembrane domain plus FPCC (FPCTMC, amino-acids 3850–4074) or TAP–PC2, a PC2 construct containing two IgG and one calmodulin-binding domains used in our modified tandem affinity purification (TAP) (32). Analysis of derived cell lysates by FLAG-M2 beads chromatography and immunoblotting revealed retention of PC2, KIF3A and KIF3B along with FLAG-FPCTMC. Reciprocally, using protein A pull-down and immunoblotting, we observed retention of FPC, KIF3A and KIF3B along with TAP–PC2 (Fig. 4C). Similar results were found when FPCC was used in place of FPCTMC (data not shown). These results provide further support that PC2 and FPC are in a common complex, either via direct binding or via intermediate proteins that could include KIF3B. Note that no direct binding was revealed by yeast two-hybrid assays performed by Zhou's group (personal communication) and the present study (Fig. 1), which is in favor of the involvement of a mediator protein for the PC2–FPC interaction. However, owing to limitations associated with the yeast two-hybrid method, we cannot exclude the possibility that PC2 and FPC directly bind, e.g. via transmembrane domains or other soluble fragments not tested. Because no direct binding between FPC and KIF3A was found so far (Figs 1 and 3), the positive interaction between FPCTMC and KIF3A revealed here is presumably indirect because KIF3A binds KIF3B (abundantly present in native cells, see Fig. 5B and Supplementary Material, Fig. S1) which binds FPC. We consider KIF3B a candidate for the mediator protein, as it binds both PC2 and FPC. However, for KIF3B to act as an intermediate protein, a single KIF3B molecule must be able to simultaneously bind PC2 and FPC, which has so far not been proved.

View larger version (36K): [in this window] [in a new window] [Download PowerPoint slide]   Figure 5. Roles of KIF3B in the PC2xFPC interaction revealed by in vitro binding and co-IP. (A) GST pull-down assay. Purified GST-PC2C (left panel), GST-FPCC (right panel) or GST alone (both panels) was incubated with purified His-FPCC (left panel) or His-PC2C (right panel) with or without His-KIF3BC, His-KIF3BN proteins, as indicated. After extensive wash, the resultant protein samples were immunoblotted with an antibody against FPC (C10) or PC2 (1A11). (B) IMCD cell lysates were precipitated with FPC antibody (C2p) and detected by PC2 antibody (1A11, right panel). The expressions of KIF3B, FPC and PC2 were detected with KIF3B antibody (C-18), FPC (C2p) and PC2 (1A11), respectively, in cells over-expressing HA-KIF3BC, native cells (Ctrl) and kif3b siRNA cells.

 
Complexing between PC2 and FPC, and roles of KIF3B
We first examined the direct binding between PC2 and FPC by in vitro binding experiments. We found that GST-PC2C associated with His-FPCC and that His-PC2C associated with GST-FPCC in the presence of KIF3BC, but not it its absence or in the presence of KIF3BN (Fig. 5A). This result is in agreement with the finding by yeast two-hybrid, indicating that there is no direct association between PC2 and FPC, and demonstrates that KIF3B acts as a mediator of the interaction between PC2 and FPC. Thus, this in vitro binding assay indicates the existence of a PC2–KIF3B–FPC complex.

To verify the mediator role of KIF3B in the complexing between FPC and PC2, we used siRNA to decrease the KIF3B expression in IMCD cells and found that KIF3B was effectively diminished (Supplementary Material, Fig. S1). We then compared the PC2–FPC complexing between native IMCD cells and those with diminished KIF3B or over-expressed of KIF3BC by use of co-IP. Indeed, we found that the complexing strength between PC2 and FPC was significantly decreased in kif3b siRNA cells and increased in over-expressed cells (Fig. 5B), which confirms a bridging role of KIF3B. Of note, compared with native IMCD cells, there was no appreciable change in the PC2 and FPC expression levels in cells over-expressing KIF3BC (PC2, 109±12%; FPC, 96±12%; N=3) or in those with kif3b siRNA (PC2, 108±3%; FPC, 103±2%; N=3) (Fig. 5B).

We next created conditions under which native mediator proteins in MDCK cells, such as KIF3B, are separated from PC2 and FPC. For this, we performed Far WB experiments using transfected MDCK cell lines stably expressing GFP-tagged PC2, PC2C or FPCC (Fig. 6A), and purified GST-PC2C, GST-FPCC, His-KIF3AC or His-KIF3BC proteins from E. coli. To separate individual proteins, including mediator proteins, from any complexes present in the cell lysates, we ran SDS–PAGE of cell lysates. The samples were transferred to nitrocellulose membrane where proteins were denatured and then renatured, followed by incubation with purified partner proteins for 3 h to allow potential associations. This was followed by wash and regular WB to detect binding of incubated proteins. Incubation of FPCC alone (Fig. 6B, left panel) or of FPCC plus KIF3AC (Fig. 6C, left panel) did not result in the detection of FPCC on the sites where over-expressed GFP-PC2C (~60 kDa) or GFP-PC2 (~140 kDa) is found, indicating that FPCC is not bound directly to PC2C or full-length PC2, and that KIF3AC did not promote their binding. In contrast, incubation of FPCC plus KIF3BC resulted in the detection of FPCC on the sites where over-expressed GFP-PC2C or GFP-PC2 is found (Fig. 6D, left panel, lanes 3 and 4, indicated by arrows), demonstrating that KIF3BC mediates the complexing between PC2 and FPCC. Reciprocally, purified PC2C bound to the over-expressed GFP-FPCC (~50 kDa) in MDCK cells in the presence of KIF3BC (Fig. 6D, right panel, lane 3, indicated by arrow), but not in its absence or in the presence of KIF3AC (Fig. 6B and C, right panels). Thus, because in these assays any native mediator proteins were separated from PC2 and FPC, KIF3BC acted as a necessary and sufficient linker for complexing between PC2 and FPC, presumably by forming a PC2–KIF3B–FPC triplex.

View larger version (44K): [in this window] [in a new window] [Download PowerPoint slide]   Figure 6. Roles of KIF3B in the PC2xFPC interaction revealed by Far WB. (A) GFP, GFP-PC2C, GFP-PC2 or GFP-FPCC was stably expressed in MDCK cells. WB data for GFP-PC2C, GFP-PC2 (left panel), GFP-FPCC (right panel) and GFP (both panels) are shown. (B–D) Cell lysates were separated by SDS–PAGE and transferred onto nitrocellulose membranes. Proteins were denatured, renatured and then incubated with purified FPCC (left panels) or PC2C (right panels) proteins together with none (B, control), KIF3AC (C) or KIF3BC (D). Bound proteins were detected by FPC (C10, left panels) or PC2 antibody (1A11, right panels). BSA and GFP vector (lanes 1 and 2) serve as negative controls. In (D), arrows point to three novel bands [compared with (B) and (C)] corresponding to the binding of FPCC to the sites of PC2C (left panel, lane 3) and PC2 (left panel, lane 4) and to the binding of PC2C to the site of FPCC (right panel, lane 3), respectively.

 
To lend further support to the existence of a complex containing PC2, KIF3B and FPC, we conducted an iodixanol gradient fractionation experiment using native IMCD cells. KIF3B, FPC and KIF3A were found in light and middle fractions, whereas PC2 was distributed in middle and dense fractions (Supplementary Material, Fig. S2). All four proteins co-existed in some fractions, especially in fraction no. 8, indicative of the presence of a large protein complex containing these four proteins in IMCD cells.

Subcellular co-localization of PC2 and FPC with KIF3B
To examine where these three proteins are co-localized in cells, we performed indirect immunofluorescence assays in cultured over-confluent (ciliated) and sub-confluent IMCD cells. Consistent with reported ciliary localization of these proteins (22,23,33,34), we found that PC2, FPC and KIF3B were indeed present and partially co-localized in primary cilia of IMCD cells (Fig. 7A). When cells were sub-confluent, we found that PC2 and FPC were mostly intracellularly localized, and the three proteins seemed to co-localize to a perinuclear region (Fig. 7B).

View larger version (43K): [in this window] [in a new window] [Download PowerPoint slide]   Figure 7. Immunofluorescence staining for PC2, FPC and KIF3B in IMCD cells. (A) Ciliated IMCD cells were co-stained for PC2 (G-20), KIF3B (no. 611535) and FPC (C2p). (B) Sub-confluent cells were co-stained for PC2, KIF3B and FPC. Horizontal bars=20 µm.

 
Functional modulation of PC2 channel by FPC through KIF3B
We next wanted to determine whether functional interaction occurs between the three members of the complex PC2–KIF3B–FPC. Because PC2 is a cation channel, we examined whether/how FPC and/or KIF3B modulate PC2 channel activity. For this, we employed the lipid bilayer electrophysiology assay. We prepared full-length PC2 from MDCK stable cell line by a modified TAP method and purified His-KIF3BC and His-FPCC from E. coli. In the presence of PC2 alone, PC2 opened at three different conductance states (Fig. 8A and B). The main state opened only occasionally with an averaged open probability (NPo) value of 0.018±0.003 and an averaged single-channel conductance (G) value of 141±13 pS (N=5) (–20 to +160 mV) (Fig. 8B). The intermediate state opened with an NPo value of 0.07±0.01 and a G-value of 68.6±7.5 pS (N=14) (0 to +160 mV). The small (conductance) state opened most often, with an NPo value of 0.18±0.02 and a G-value of 50.6±5.4 pS (N=25) (+80 to +160 mV). Addition of KIF3BC and FPCC to the cis chamber substantially augmented the likelihood of PC2 channel opening at the main state, with a G-value of 150±7 pS (N=14) (Fig. 8C and D), not significantly different from the value obtained in the absence of KIF3BC and FPCC (P=0.51). The corresponding NPo value increased to 0.48±0.04 pS (N=14) (P=0.0001) (0 to +140 mV) (Fig. 9A). Under this condition, the intermediate and small conductance states of PC2 were also observed, with G-values of 80.3±11.6 (N=9) and 32.0±5.5 pS (N=19), respectively (Fig. 8C and D). The increased activity of PC2 at the main state by KIF3BC plus FPCC was also demonstrated by current density plotting of recordings at +20 mV (Fig. 9B). The small (conductance) state was observed less often than in the absence of both KIF3BC and FPCC. When FPCC or KIF3BC alone was added, multiple conductance states of PC2 were also present (Fig. 9C). However, addition of FPCC or KIF3BC alone was unable to increase the likelihood of PC2 opening at the main state (Fig. 9A). With only FPCC added, the G-values were 157±26 pS (N=5) (main state, –40 to +60 mV), 76.3±10.8 pS (N=16) (intermediate state, 0 to +160 mV) and 53.6±7.9 pS (N=27) (small state, +80 to +160 mV). With only KIF3BC added, the G-values were correspondingly 144±27 pS (N=3) (–40 to +60 mV), 62.7±10.2 pS (N=8) (0 to +160 mV) and 45.6±8.0 pS (N=20) (+80 to +160 mV). We also employed mean currents (without discerning various conductance states) to assess PC2 channel activity. No significant difference in the mean currents was observed among the three conditions where PC2 alone, PC2+FPCC or PC2+KIF3BC was present. In contrast, in average, the presence of KIF3BC and FPCC together increased the mean single-channel currents of PC2 by about 5-fold (Fig. 9D), consistent with the results presented by NPo and histogram plotting (Fig. 9B). We utilized the KIF3B truncation mutant, KIF3BN, as a negative control of KIF3B as it does not bind PC2 or FPC (Fig. 1) nor mediate the link between PC2 and FPC (Fig. 5A). From five paired experiments, we found that neither of FPCC+KIF3BN nor KIF3BN exhibited significant modulation effect on PC2 channel. Further, when we added FPCC or KIF3BC protein alone to the trans side, we did not observe significant functional effects on PC2 channel activity (N=10). Of note, KIF3A is not suitable candidate as a negative control in this regard because it exhibits functional modulation of PC2 channel (35). Thus, although KIF3B directly associates with PC2, it did not exert a significant effect on PC2; the stimulatory effect on PC2 was by the indirect partner, FPC, when KIF3B was present. In summary, together with the presence of the PC2–KIF3B–FPC triplex supported by our physical interaction data (Fig. 1–6), our functional data demonstrated that KIF3B mediates the stimulation of PC2 channel activity by FPC.

View larger version (29K): [in this window] [in a new window] [Download PowerPoint slide]   Figure 8. Multiple conductance states of PC2 channel and effects of FPCC and KIF3BC. TAP–PC2 purified from MDCK cells together with KIF3BC and FPCC proteins purified from E. coli were used in lipid bilayer experiments under asymmetrical solution condition (150 mM cis KCl and 15 mM trans KCl). Shown trace recordings are not intended to reflect the actual open probability (NPo) values. (A) Representative tracings of PC2 channel activity in the absence of FPCC and KIF3BC, illustrating multiple conductance states at indicated voltages. M, main state; I, intermediate state; S, small state. Closed levels are indicated by horizontal dashed lines. Inserts are shown with a different time and current scale. Traces were Gaussian filtered at 200 Hz in Clampfit 9. (B) Averaged I–V relationships, corresponding to the three conductance states, in the absence of FPCC and KIF3BC. (C) Representative tracings of PC2 channel activity in the presence of both FPCC and KIF3BC, illustrating multiple conductance states. (D) Averaged I–V curves, corresponding to the three states, in the presence of both FPCC and KIF3BC.

 

View larger version (23K): [in this window] [in a new window] [Download PowerPoint slide]   Figure 9. Effects of FPCC and KIF3BC on PC2 channel determined by NPo, current density and mean current. Experiment conditions are the same as in Figure 8. (A) Averaged open probability for PC2 channel opening at the main state in the presence or absence of FPCC and/or KIF3BC, as indicated. Double asterisk means P<0.01. Data were obtained for voltages between 0 and +140 mV. (B) Current density plots obtained from the all-point histogram analysis of 11 independent recordings in the presence of PC2 alone (left panel) and of seven independent recordings in the presence of PC2+KIF3BC+FPCC (right panel). Each recording is of 10 s long and was obtained at +20 mV. The three peaks in each panel (from left to right) correspond to the close, intermediate and main states, respectively. (C) Representative tracings of PC2 channel activity in the presence of FPCC or KIF3BC only, illustrating multiple conductance states at indicated voltages. (D) Mean PC2 single-channel current was calculated using the Clampfit 9 program from each tracing of 60 s long obtained at –100 or +20 mV in the presence of PC2, PC2+KIF3BC, PC2+FPCC or PC2+KIF3BC+FPCC, as indicated. Several (numbers are indicated in the figure) mean current values calculated by this way were averaged to produce an averaged value as presented.

 

	DISCUSSION

TOP ABSTRACT INTRODUCTION RESULTS DISCUSSION MATERIALS AND METHODS SUPPLEMENTARY MATERIAL REFERENCES

 
Human ADPKD and ARPKD share similar clinical manifestations despite of different genetic backgrounds. Moreover, mutations in a diverse number of proteins, including kinesin-2, polaris, cystin and inversin, which have no sequence similarity with either polycystins or fibrocystin, also result in renal cyst development (36–39). This raises the possibility that common molecular mechanisms of cystogenesis exist, namely the possibility that these cystoproteins are present in same complexes. Indeed, our present studies demonstrate that PC2 and FPC are part of the same protein complex. Using co-IP, two other groups recently found that FPC and PC2 are present in the same complex (personal communications). However, co-IP assays do not tell whether two proteins directly bind to each other. Data from our current study using several approaches and those by Zhou's group using yeast two-hybrid method (personal communication) show that FPC and PC2 do not bind directly to each other. Thus, the protein(s) mediating the PC2–FPC complexing remained to be identified. Using a combination of in vitro and in vivo approaches, including yeast two-hybrid, GST pull-down and Far WB, we have demonstrated that the kinesin-2 motor subunit KIF3B acts as a linker protein, which enables the formation of a triplex, presumably in the form of PC2–KIF3B–FPC. Thus, KIF3B represents the first molecular linker between ADPKD and ARPKD proteins, suggesting that the protein complex PC2–KIF3B–FPC is part of a common molecular pathway implicated in renal cystic diseases.

Kinesin-2, as a heterotrimer formed by KIF3A, KIF3B and KAP3, is important for numerous cell functions, in particular, cilium growth and cell cycle (25,28,33,40) and has profound pathological implications. Disruption of KIF3B in nodal cilia damages ciliary growth, which alters leftward nodal flow and consequently results in abnormal embryonic left–right asymmetry development (41). Similarly, mice with KIF3A knockout fail to synthesize cilia in the embryonic node and exhibits randomization of the left–right asymmetry and structural abnormalities of the neural tube, pericardium, brachial arches and somites (42,43). Kidney-specific inactivation of KIF3A resulted in cyst formation in renal tubular epithelial cells at postnatal day 5 and caused renal failure by postnatal day 21 (36). KAP3 is associated with adenomatous polyposis coli and responsible for its transportation along microtubules (44) and is required for axoneme growth and maintenance of the cilia in Drosophila type 1 sensory neurons (45).

Given the existence of a triplex PC2–KIF3B–FPC, it is important to determine where the three proteins co-localize, as this would provide hints as to their possible physiological roles. In ciliated IMCD cells, PC2 partially co-localized with KIF3B and FPC in primary cilia. Together with a recent report that PC2 and PC1 in renal primary cilia constitute part of a shear stress sensor responsive to tubular flow (34), our data suggest the possibility that the flow sensor is composed of a larger complex containing not only PC1 and PC2, but also KIF3B, FPC and possibly other proteins. Under this scenario, PC2 as a Ca-permeable channel in the primary cilium would be regulated by several proteins, including PC1, FPC and others. Interestingly, the single-channel activity of PC2 on the membrane of isolated primary cilia was recently detected (46).

Using the lipid bilayer electrophysiology assay, we demonstrated that FPC is capable of increasing PC2 channel function in the presence, but not in the absence of KIF3B. Thus, KIF3B likely links PC2 and FPC together not only in a structural complex, but also for the regulation of PC2 channel function, which establishes both physical and functional links between key proteins responsible for the pathogenesis of ADPKD and ARPKD. PC2 was previously reported to be functionally regulated by partner proteins, including PC1 (34) and -actinin (18). Our study shows that fibrocystin is another partner of PC2, which functionally up-regulates its channel function through KIF3B. Interestingly, although KIF3B directly binds PC2, it has no significant effect on PC2 channel function, indicating that it primarily serves as a linker protein.

Our lipid bilayer system to investigate the modulation of PC2 in the membrane by KIF3B and FPC introduced from cis (intracellular) side of the membrane is a highly simplified model in which microtubule and kinesin-2 motor are not present. However, the beauty and novelty here are that functional modulation occurs in such a simple setting. In a more physiological setting, such as primary cilium membrane that envelops a microtubule plus motor machineries, modulation should be more fine tuned and may simply be different, for example, where the associations of KIF3B with the two cystoproteins may be dynamic and under cellular regulations. It is also possible that the roles that KIF3B plays in the PC2–FPC complexing and in the modulation of PC2 by FPC are different from its role as a kinesin-2 motor subunit. Studies on their interaction using more physiological models will further help understanding crosstalk between ADPKD and ARPKD.

The pathways involved in renal and hepatic cyst formation and non-cystic manifestations in other organs for ADPKD and ARPKD remain illusive. In both C. elegans and Chlamydomonas, the kinesin-2 complex appears to interact with a large proteinaceous ‘raft’ complex that is composed of 15 different proteins (33). Our current study found that the ADPKD-related protein PC2, the ARPKD-related protein FPC and the kinesin-2 motor subunit KIF3B are in part together in primary cilia and the perinuclear cytoplasm of renal epithelial cells. Among the previously reported binding partners of PC2, FPC and KIF3B, such as PC1, -actinin, TRPC1, KIF3A (which has a direct association with PC2, see Fig. 1 and 3) and KAP3, etc., some partners, e.g. PC1, may also be in the same complex with PC2–KIF3B–FPC. Thus, PC2–KIF3B–FPC may potentially be part of a large protein complex which itself is part of a common pathway for dominant and recessive PKDs. Thus, one possible scenario would be that a triggering to the complex, e.g. a ligand binding to FPC or PC1, induces conformational changes to partners in the complex, which leads to modulated PC2 channel activity, which will then affect downstream processes.

	MATERIALS AND METHODS

TOP ABSTRACT INTRODUCTION RESULTS DISCUSSION MATERIALS AND METHODS SUPPLEMENTARY MATERIAL REFERENCES

 
Yeast two-hybrid analysis
A yeast two-hybrid screen was performed in the yeast strain AH109 containing Ade2, His3 and LacZ reporter genes under the control of the GAL4 upstream activating sequences as described recently (18), and the whole kit was purchased from Clontech. Briefly, the cDNA fragments encoding human PC2C or FPCC were subcloned in frame into the GAL4 DNA binding domain of the vector pGBKT7 by a PCR-based approach. PC2C was used as bait to screen a human heart cDNA library constructed in the vector pACT2 containing the GAL4 activation domain. Transformants were grown on the minimal synthetic dropout medium lacking leucine, tryptophan, adenine and histidine. Colonies survived were further screened for activation of a LacZ reporter gene by a filter lift assay. Plasmid cDNAs were isolated from the positive colonies and individually tested against the bait pGBKT7–PC2C and empty vector pGBKT7. Constructs of FPC, PC2 and kinesin-2 (Fig. 1A) in the pGBKT7 or pGADT7 vector were transformed into yeast strain Y187 containing LacZ reporter gene for pair-wise interaction assay. Furthermore, a liquid culture assay was used to quantify ß-galactosidase activity using ONPG as substrate according to the manufacture's instructions.

Cell culture and transfection
MDCK, IMCD and human embryonic kidney (HEK) 293 cells were cultured in Dulbecco's modified Eagle's medium supplemented with 10% fetal bovine serum. Cells of less than 25 cycles were cultured to full confluence before collection. Transient transfection of PC2, FPC and KIF3B was performed on MDCK cells cultured to 90–95% confluence using lipofectamine 2000 (Invitrogen, Carlsbad, CA, USA) according to the manufacturer's recommendation. Stable cell line was selected by applying G418 (0.5 mg/ml) and FACSAria cell sorting (BD Biosciences, San Jose, CA, USA).

Plasmids and antibodies
The cDNAs of KIF3A and KIF3B were generous gifts of Dr Tetsu Akiyama (University of Tokyo, Tokyo, Japan). The KAP3 and IFT20 cDNAs were obtained by RT–PCR from HEK 293 cells. pEGFPC2 (Clontech) was used to construct individual genes with an EGFP tag. These constructs were then used as templates to make cDNA constructs with an HA or a FLAG tag. All plasmids were verified by sequencing. Mouse monoclonal (Catalog no. 611535) and goat polyclonal (C-18) antibodies against KIF3B were purchased from BD Biosciences (Mississauga, ON, Canada) and Santa Cruz (Santa Cruz, CA, USA), respectively. Rabbit polyclonal antibody against KIF3A (Catalog no. K3513) and actin (Catalog no. A2066) were from Sigma-Aldrich (Oakville, ON, Canada). Mouse monoclonal anti-PC2 antibody 1A11 was raised against human GST-PC2C fusion protein (amino-acid 682–968), and goat polyclonal anti-PC2 antibody G-20 was purchased from Santa Cruz and characterized previously (18). Mouse monoclonal anti-FPC antibody hAR-C2m3C10 and rabbit polyclonal anti-FPC antibody hAR-C2p were raised against its cytoplamic domain (amino-acid 3872–4074), and mouse monoclonal anti-FPC antibody hAR-Nm3G12 was raised against its N-terminal domain (amino-acid 481–700), as described previously (23). Rabbit polyclonal anti-GFP was from BD Biosciences. The secondary antibodies, both fluorescein-labeled and peroxidase-conjugated IgGs, were from Chemicon International (Temecula, CA, USA).

GST pull-down
Precleared bacterial protein extracts (250 µl) containing GST-PC2C, GST-FPCC or GST alone was incubated with 2 µg of purified His-KIF3AC, His-KIF3BC or His-KIF3BN protein in the binding buffer (50 mM Tris, pH 7.5, 150 mM NaCl, 1 mM CaCl₂). The mixture was incubated at room temperature (RT) for 1 h with gentle shaking, followed by another hour of incubation after addition of 100 µl glutathione-agarose beads (Sigma-Aldrich). The beads were then washed several times with 140 mM NaCl, 10 mM Na₂HPO₄ and 1.8 mM KH₂PO₄ at pH 7.5, and the remaining proteins were eluted using 10 mM glutathione and 50 mM Tris at pH 8.0. The protein samples were resolved by SDS–PAGE (10%) and transferred onto nitrocellulose membranes (Bio-Rad, Hercules, CA, USA). The filters were then blocked with 3% skimmed milk powder, immunoblotted with KIF3A or KIF3B antibodies, respectively, and visualized with enhanced chemiluminescence (Amersham, Baie d'Urfe, Canada). Similar procedure was used for GST-pull down analysis between PC2 and FPC, except that all proteins were purified and that extensive washing was applied.

Dot blot overlay
Proteins of GST-PC2N, GST-PC2C and GST were prepared and described previously (18), and GST-FPCC and GST-KIF3BC were prepared similarly. All these proteins were spotted on dry nitrocellulose membrane strips that were then allowed to air dry for 10 min and saturated with phosphate-buffered saline (PBS) containing 3% BSA for 1 h at RT. The strips were subsequently incubated at 4°C overnight with native IMCD or MDCK cell lysates in 50 mM Tris, pH 7.5, 150 mM NaCl, 1 mM CaCl₂ or 1 mM EGTA, 1% BSA, washed with PBS twice (10 min each) and immunoblotted with KIF3B antibody. Purified GST-KIF3BC fusion protein was used as positive control.

Far Western blot
Equal amounts of BSA and cell lysates were run on SDS–PAGE and transferred to nitrocellulose membrane. Proteins were denatured and renatured in AC buffer (100 mM NaCl, 20 mM Tris, pH 7.6, 0.5 mM EDTA, 10% glycerol, 0.1% Tween-20, 2% milk powder, 1 mM DTT) by reducing guanidine–HCl. Briefly, it was denatured with 6 M guanidine–HCl in AC buffer for 30 min at RT. Then, it was washed in 3 M guanidine–HCl for 30 min at RT, then 1 M guanidine–HCl in AC buffer for 30 min at 4°C and then in 0.1 M for 30 min at 4°C. Finally, it was further renatured in AC buffer (without guanidine–HCl) overnight at 4°C. Then, membranes were incubated with bacterially purified proteins at 4°C for 3 h in AC buffer, followed by regular WB.

Co-immunoprecipitation
Reciprocal co-IP was performed using lysates of native IMCD, MDCK cells (2x10⁷ cells), human kidney and heart tissues (10 mg total proteins). Cell monolayers in 100 mm dishes were washed twice with PBS and solubilized in ice-cold CellLytic-M lysis buffer and proteinase inhibitor cocktail (Sigma-Aldrich). Fresh kidney and heart tissues were flash-frozen in liquid nitrogen, macerated to fine powder and solubilized in the lysis buffer. Supernatant was collected following centrifugation at 16 000g for 20 min. Equal amounts of total protein from postnuclear supernatants were precleared for 1 h with protein G-/A-Sepharose (Sigma-Aldrich) and then incubated for another hour on ice with antibody against PC2, FPC or KIF3B. After the addition of 150 µl of 50% protein G-/A-Sepharose, the mixtures were incubated for another hour with gentle shaking. The immune complexes absorbed to protein G-/A-Sepharose were washed three times with the lysis buffer. The precipitated proteins were analyzed by WB using antibodies against kinesin-2, FPC or PC2. Co-IP experiments were conducted reciprocally.

Immunofluorescence
Cells were grown on coverslips, fixed with 4% freshly prepared paraformaldehyde for 10 min at RT, washed twice with PBS, and permeabilized in 0.2% Triton X-100 at RT for 10 min. Cells were blocked by 5% non-fat milk in PBST (PBS plus 0.05% Tween-20) at RT for 1 h and then incubated at RT for 1 h with the PC2, FPC or KIF3B primary antibodies, followed by another hour of incubation with secondary antibodies. Cells were finally washed with PBS. Vectashield mounting medium (Vector Laboratories, Burlingame, CA, USA) was used to protect immunofluorescence signals from fading. Fluorescent images were captured on a motorized Olympus IX81 microscopy installed with a CCD cooling RT SE6 monochrome camera (Diagnostic Instruments, Sterling Heights, MI, USA). Final composite images were made using Image-Pro Plus 5.0 (Media Cybernetics Inc., Silver Spring, MD, USA).

Small interfering RNA
Three double-stranded RNA were synthesized for each of the mouse pkd2 and kif3b genes by Invitrogen (Supplementary data Table). For mouse FPC, the reported sequence (5'-AAAGTCAGGAGCTCCACCTAC-3') (47) was chosen. The altered expression of the target proteins was evaluated by immunoblotting, and mixed siRNAs were utilized to maximize the knock-down effect. A double-stranded RNA against human mitogen-activated protein kinase p38 -isoform was used as a control. IMCD cells at 50–60% confluency were transiently transfected using lipofectamine 2000 according to the manufacturer's instructions. Cell lysates were collected 48 h after transfection for WB and Co-IP assays.

Protein preparation and planer lipid bilayer electrophysiology
His-KIF3BC, His-KIF3BN and His-FPCC proteins were purified from E. coli in pET28a(+) vector (Novagen, La Jolla, CA, USA). TAP–PC2 was prepared and reconstituted in a lipid bilayer system as previously described (32) to assess the channel activity. Briefly, a lipid bilayer was formed with a mixture of 1-palmitoyl-2-oleoyl phosphatydil-choline and phosphatydil-ethanolamine (Avanti Polar Lipids, Birmingham, AL, USA) at a 3:7 ratio in a Delrin cup inserted in an acrylic chamber (Harvard Apparatus, Montreal, QC, Canada). The cis (or trans) compartment contained 150 mM KCl (or 15 mM), 15 µM Ca (by 1 mM EGTA and 1.01 mM CaCl₂), pH 7.4 (adjusted by MOPS-KOH). TAP–PC2 protein was added to the cis chamber in proximity of the membrane or used to directly ‘paint’ the membrane. Alternatively, TAP–PC2-containing proteoliposome was directly painted to form the membrane with PC2 channel proteins expectedly inserted. KIF3BC/KIF3BN and/or FPCC proteins were added to the cis chamber when studying their effects on PC2 channel. The cis compartment was clamped to a range of voltages using a Gapfree protocol generated by Clampex 8 (Axon Instruments, Union City, CA, USA) and the trans compartment was held at ground (0 mV). Currents and voltages were recorded at 200 µs per sample and Bessel filtered at 1 or 3 kHz with the amplifier PC-ONE (Dagan Corporation, Minneapolis, MN, USA), the AD/DA converter Digidata 1320A and the software Clampex 8 (Axon Instruments).

Data analysis
Data obtained from lipid bilayer experiments were analyzed using Clampfit 9. The all-point histogram was used to calculate single-channel amplitudes and to obtain data for current density plotting. The multiplication of open probability and the number of channels in the bilayer membrane (NPo, briefly designated ‘open probability') was obtained from currents generated by gap-free recordings of 20 s long. Analyzed data were plotted using Sigmaplot 9 (Jandel Scientific Software, San Rafael, CA, USA) and expressed in the form of mean±SE (N), where SE is the standard error and N the number of independent measurements.

	SUPPLEMENTARY MATERIAL

TOP ABSTRACT INTRODUCTION RESULTS DISCUSSION MATERIALS AND METHODS SUPPLEMENTARY MATERIAL REFERENCES

 
Supplementary Material is available at HMG Online.

	ACKNOWLEDGEMENTS

 
This work was supported by the Canadian Institutes of Health Research, the Canada Foundation for Innovation, the Kidney Foundation of Canada (X.Z.C.) and the Heart and Stroke Foundation of Canada (X.Z.C. and S.W.). X.Z.C. is a recipient of the Alberta Heritage Foundation for Medical Research Senior Scholarship. X.Q.D. is a recipient of AHFMR studentship. Q.L. is a recipient of the Kidney Foundation of Canada biomedical fellowship.

Conflict of Interest statement. None declared.

	FOOTNOTES

 
The authors wish it to be known that, in their opinion, the first three authors should be regarded as joint first authors.

	REFERENCES

TOP ABSTRACT INTRODUCTION RESULTS DISCUSSION MATERIALS AND METHODS SUPPLEMENTARY MATERIAL REFERENCES

 

1. Igarashi P. and Somlo S. (2002) Genetics and pathogenesis of polycystic kidney disease. J. Am. Soc. Nephrol. 13:2384–2398.[Free Full Text]

2. Mochizuki T., Wu G., Hayashi T., Xenophontos S.L., Veldhuisen B., Saris J.J., Reynolds D.M., Cai Y., Gabow P.A., Pierides A., et al. (1996) PKD2, a gene for polycystic kidney disease that encodes an integral membrane protein. Science 272:1339–1342.[Abstract]

3. The European Polycystic Kidney Consortium Disease. (1994) The polycystic kidney disease 1 gene encodes a 147 kb transcript and lies within a duplicated region on chromosome 16. Cell 77:881–894.[CrossRef][Web of Science][Medline]

4. Zerres K., Rudnik-Schoneborn S., Steinkamm C., Becker J., Mucher G. (1998) Autosomal recessive polycystic kidney disease. J. Mol. Med. 76:303–309.[CrossRef][Web of Science][Medline]

5. Ward C.J., Hogan M.C., Rossetti S., Walker D., Sneddon T., Wang X., Kubly V., Cunningham J.M., Bacallao R., Ishibashi M., et al. (2002) The gene mutated in autosomal recessive polycystic kidney disease encodes a large, receptor-like protein. Nat. Genet. 30:259–269.[CrossRef][Web of Science][Medline]

6. Onuchic L.F., Furu L., Nagasawa Y., Hou X., Eggermann T., Ren Z., Bergmann C., Senderek J., Esquivel E., Zeltner R., et al. (2002) PKHD1, the polycystic kidney and hepatic disease 1 gene, encodes a novel large protein containing multiple immunoglobulin-like plexin-transcription-factor domains and parallel beta-helix 1 repeats. Am. J. Hum. Genet. 70:1305–1317.[CrossRef][Web of Science][Medline]

7. Xiong H., Chen Y., Yi Y., Tsuchiya K., Moeckel G., Cheung J., Liang D., Tham K., Xu X., Chen X.Z., et al. (2002) A novel gene encoding a TIG multiple domain protein is a positional candidate for autosomal recessive polycystic kidney disease. Genomics 80:96–104.[CrossRef][Web of Science][Medline]

8. Hanaoka K., Qian F., Boletta A., Bhunia A.K., Piontek K., Tsiokas L., Sukhatme V.P., Guggino W.B., Germino G.G. (2000) Co-assembly of polycystin-1 and -2 produces unique cation-permeable currents. Nature 408:990–994.[CrossRef][Medline]

9. Gonzalez-Perrett S., Kim K., Ibarra C., Damiano A.E., Zotta E., Batelli M., Harris P.C., Reisin I.L., Arnaout M.A., Cantiello H.F. (2001) Polycystin-2, the protein mutated in autosomal dominant polycystic kidney disease (ADPKD), is a Ca2+-permeable nonselective cation channel. Proc. Natl Acad. Sci. USA 98:1182–1187.[Abstract/Free Full Text]

10. Qian F., Germino F.J., Cai Y., Zhang X., Somlo S., Germino G.G. (1997) PKD1 interacts with PKD2 through a probable coiled-coil domain. Nat. Genet. 16:179–183.[CrossRef][Web of Science][Medline]

11. Tsiokas L., Kim E., Arnould T., Sukhatme V.P., Walz G. (1997) Homo- and heterodimeric interactions between the gene products of PKD1 and PKD2. Proc. Natl Acad. Sci. USA 94:6965–6970.[Abstract/Free Full Text]

12. Tsiokas L., Arnould T., Zhu C., Kim E., Walz G., Sukhatme V.P. (1999) Specific association of the gene product of PKD2 with the TRPC1 channel. Proc. Natl Acad. Sci. USA 96:3934–3939.[Abstract/Free Full Text]

13. Hidaka S., Konecke V., Osten L., Witzgall R. (2004) PIGEA-14, a novel coiled-coil protein affecting the intracellular distribution of polycystin-2. J. Biol. Chem. 279:35009–35016.[Abstract/Free Full Text]

14. Gallagher A.R., Cedzich A., Gretz N., Somlo S., Witzgall R. (2000) The polycystic kidney disease protein PKD2 interacts with Hax-1, a protein associated with the actin cytoskeleton. Proc. Natl Acad. Sci. USA 97:4017–4022.[Abstract/Free Full Text]

15. Lehtonen S., Ora A., Olkkonen V.M., Geng L., Zerial M., Somlo S., Lehtonen E. (2000) In vivo interaction of the adapter protein CD2-associated protein with the type 2 polycystic kidney disease protein, polycystin-2. J. Biol. Chem. 275:32888–32893.[Abstract/Free Full Text]

16. Li Q., Dai Y., Guo L., Liu Y., Hao C., Wu G., Basora N., Michalak M., Chen X.Z. (2003) Polycystin-2 associates with tropomyosin-1, an actin microfilament component. J. Mol. Biol. 325:949–962.[CrossRef][Web of Science][Medline]

17. Li Q., Shen P.Y., Wu G., Chen X.Z. (2003) Polycystin-2 interacts with troponin I, an angiogenesis inhibitor. Biochemistry 42:450–457.[CrossRef][Medline]

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

19. Rundle D.R., Gorbsky G., Tsiokas L. (2004) PKD2 interacts and co-localizes with mDia1 to mitotic spindles of dividing cells: role of mDia1 in PKD2 localization to mitotic spindles. J. Biol. Chem. 279:29728–29739.[Abstract/Free Full Text]

20. Nagasawa Y., Matthiesen S., Onuchic L.F., Hou X., Bergmann C., Esquivel E., Senderek J., Ren Z., Zeltner R., Furu L., et al. (2002) Identification and characterization of Pkhd1, the mouse orthologue of the human ARPKD gene. J. Am. Soc. Nephrol. 13:2246–2258.[Abstract/Free Full Text]

21. Menezes L.F., Cai Y., Nagasawa Y., Silva A.M., Watkins M.L., Da Silva A.M., Somlo S., Guay-Woodford L.M., Germino G.G., Onuchic L.F. (2004) Polyductin, the PKHD1 gene product, comprises isoforms expressed in plasma membrane, primary cilium, and cytoplasm. Kidney Int. 66:1345–1355.[CrossRef][Web of Science][Medline]

22. Ward C.J., Yuan D., Masyuk T.V., Wang X., Punyashthiti R., Whelan S., Bacallao R., Torra R., LaRusso N.F., Torres V.E., et al. (2003) Cellular and subcellular localization of the ARPKD protein; fibrocystin is expressed on primary cilia. Hum. Mol. Genet. 12:2703–2710.[Abstract/Free Full Text]

23. Zhang M.Z., Mai W., Li C., Cho S.Y., Hao C., Moeckel G., Zhao R., Kim I., Wang J., Xiong H., et al. (2004) PKHD1 protein encoded by the gene for autosomal recessive polycystic kidney disease associates with basal bodies and primary cilia in renal epithelial cells. Proc. Natl Acad. Sci. USA 101:2311–2316.[Abstract/Free Full Text]

24. Wang S., Luo Y., Wilson P.D., Witman G.B., Zhou J. (2004) The autosomal recessive polycystic kidney disease protein is localized to primary cilia, with concentration in the basal body area. J. Am. Soc. Nephrol. 15:592–602.[Abstract/Free Full Text]

25. Miki H., Setou M., Kaneshiro K., Hirokawa N. (2001) All kinesin superfamily protein, KIF, genes in mouse and human. Proc. Natl Acad. Sci. USA 98:7004–7011.[Abstract/Free Full Text]

26. Cole D.G., Chinn S.W., Wedaman K.P., Hall K., Vuong T., Scholey J.M. (1993) Novel heterotrimeric kinesin-related protein purified from sea urchin eggs. Nature 366:268–270.[CrossRef][Medline]

27. Vale R.D. (2003) The molecular motor toolbox for intracellular transport. Cell 112:467–480.[CrossRef][Web of Science][Medline]

28. Cole D.G. (1999) Kinesin-II, the heteromeric kinesin. Cell Mol. Life Sci. 56:217–226.[CrossRef][Web of Science][Medline]

29. Scholey J.M. (2003) Intraflagellar transport. Annu. Rev. Cell Dev. Biol. 19:423–443.[CrossRef][Web of Science][Medline]

30. Goldstein L.S. and Yang Z. (2000) Microtubule-based transport systems in neurons: the roles of kinesins and dyneins. Annu. Rev. Neurosci. 23:39–71.[CrossRef][Web of Science][Medline]

31. Baker S.A., Freeman K., Luby-Phelps K., Pazour G.J., Besharse J.C. (2003) IFT20 links kinesin II with a mammalian intraflagellar transport complex that is conserved in motile flagella and sensory cilia. J. Biol. Chem. 278:34211–34218.[Abstract/Free Full Text]

32. Li Q., Dai X.Q., Shen P.Y., Cantiello H.F., Karpinski E., Chen X.Z. (2004) A modified mammalian tandem affinity purification procedure to prepare functional polycystin-2 channel. FEBS Lett. 576:231–236.[CrossRef][Web of Science][Medline]

33. Cole D.G., Diener D.R., Himelblau A.L., Beech P.L., Fuster J.C., Rosenbaum J.L. (1998) Chlamydomonas kinesin-II-dependent intraflagellar transport (IFT): IFT particles contain proteins required for ciliary assembly in Caenorhabditis elegans sensory neurons. J. Cell Biol. 141:993–1008.[Abstract/Free Full Text]

34. Nauli S.M., Alenghat F.J., Luo Y., Williams E., Vassilev P., Li X., Elia A.E., Lu W., Brown E.M., Quinn S.J., et al. (2003) Polycystins 1 and 2 mediate mechanosensation in the primary cilium of kidney cells. Nat. Genet. 33:129–137.[CrossRef][Web of Science][Medline]

35. Li Q., Montalbetti N., Wu Y., Ramos A.J., Raychowdhury M.K., Chen X.Z., Cantiello H.F. (2006) Polycystin-2 cation channel function is under the control of microtubular structures in primary cilia of renal epithelial cells. J. Biol. Chem. in press.

36. Lin F., Hiesberger T., Cordes K., Sinclair A.M., Goldstein L.S., Somlo S., Igarashi P. (2003) Kidney-specific inactivation of the KIF3A subunit of kinesin-II inhibits renal ciliogenesis and produces polycystic kidney disease. Proc. Natl Acad. Sci. USA 100:5286–5291.[Abstract/Free Full Text]

37. Otto E.A., Schermer B., Obara T., O'Toole J.F., Hiller K.S., Mueller A.M., Ruf R.G., Hoefele J., Beekmann F., Landau D., et al. (2003) Mutations in INVS encoding inversin cause nephronophthisis type 2, linking renal cystic disease to the function of primary cilia and left-right axis determination. Nat. Genet. 34:413–420.[CrossRef][Web of Science][Medline]

38. Pazour G.J., Dickert B.L., Vucica Y., Seeley E.S., Rosenbaum J.L., Witman G.B., Cole D.G. (2000) Chlamydomonas IFT88 and its mouse homologue, polycystic kidney disease gene tg737, are required for assembly of cilia and flagella. J. Cell Biol. 151:709–718.[Abstract/Free Full Text]

39. Avner E.D., Studnicki F.E., Young M.C., Sweeney W.E., Jr W.E., Piesco N.P., Ellis D., Fettermann G.H. (1987) Congenital murine polycystic kidney disease. I. The ontogeny of tubular cyst formation. Pediatr. Nephrol. 1:587–596.[CrossRef][Web of Science][Medline]

40. Haraguchi K., Hayashi T., Jimbo T., Yamamoto T., Akiyama T. (2005) Role of the kinesin-2 family protein, KIF3, during mitosis. J. Biol. Chem. 281:4094–4099.[Medline]

41. Nonaka S., Tanaka Y., Okada Y., Takeda S., Harada A., Kanai Y., Kido M., Hirokawa N. (1998) Randomization of left-right asymmetry due to loss of nodal cilia generating leftward flow of extraembryonic fluid in mice lacking KIF3B motor protein. Cell 95:829–837.[CrossRef][Web of Science][Medline]

42. Marszalek J.R., Ruiz-Lozano P., Roberts E., Chien K.R., Goldstein L.S. (1999) Situs inversus and embryonic ciliary morphogenesis defects in mouse mutants lacking the KIF3A subunit of kinesin-II. Proc. Natl Acad. Sci. USA 96:5043–5048.[Abstract/Free Full Text]

43. Takeda S., Yonekawa Y., Tanaka Y., Okada Y., Nonaka S., Hirokawa N. (1999) Left-right asymmetry and kinesin superfamily protein KIF3A: new insights in determination of laterality and mesoderm induction by kif3A–/– mice analysis. J. Cell Biol. 145:825–836.[Abstract/Free Full Text]

44. Jimbo T., Kawasaki Y., Koyama R., Sato R., Takada S., Haraguchi K., Akiyama T. (2002) Identification of a link between the tumour suppressor APC and the kinesin superfamily. Nat. Cell Biol. 4:323–327.[CrossRef][Web of Science][Medline]

45. Sarpal R., Todi S.V., Sivan-Loukianova E., Shirolikar S., Subramanian N., Raff E.C., Erickson J.W., Ray K., Eberl D.F. (2003) Drosophila KAP interacts with the kinesin II motor subunit KLP64D to assemble chordotonal sensory cilia, but not sperm tails. Curr. Biol. 13:1687–1696.[CrossRef][Web of Science][Medline]

46. Raychowdhury M.K., McLaughlin M., Ramos A.J., Montalbetti N., Bouley R., Ausiello D.A., Cantiello H.F. (2005) Characterization of single channel currents from primary cilia of renal epithelial cells. J. Biol. Chem. 280:34718–34722.[Abstract/Free Full Text]

47. Mai W., Chen D., Ding T., Kim I., Park S., Cho S.Y., Chu J.S., Liang D., Wang N., Wu D., et al. (2005) Inhibition of Pkhd1 impairs tubulomorphogenesis of cultured IMCD cells. Mol Biol. Cell 16:4398–4409.[Abstract/Free Full Text]

CiteULike    Connotea    Del.icio.us    What's this?

This article has been cited by other articles:

				L. Tsiokas Function and regulation of TRPP2 at the plasma membrane Am J Physiol Renal Physiol, July 1, 2009; 297(1): F1 - F9. [Abstract] [Full Text] [PDF]

				D.-C. Fischer, U. Jacoby, L. Pape, C. J. Ward, E. Kuwertz-Broeking, C. Renken, H. Nizze, U. Querfeld, B. Rudolph, D. E. Mueller-Wiefel, et al. Activation of the AKT/mTOR pathway in autosomal recessive polycystic kidney disease (ARPKD) Nephrol. Dial. Transplant., June 1, 2009; 24(6): 1819 - 1827. [Abstract] [Full Text] [PDF]

				P. C. Harris 2008 Homer W. Smith Award: Insights into the Pathogenesis of Polycystic Kidney Disease from Gene Discovery J. Am. Soc. Nephrol., June 1, 2009; 20(6): 1188 - 1198. [Abstract] [Full Text] [PDF]

				P. Zhang, Y. Luo, B. Chasan, S. Gonzalez-Perrett, N. Montalbetti, G. A. Timpanaro, M. d. R. Cantero, A. J. Ramos, W. H. Goldmann, J. Zhou, et al. The multimeric structure of polycystin-2 (TRPP2): structural-functional correlates of homo- and hetero-multimers with TRPC1 Hum. Mol. Genet., April 1, 2009; 18(7): 1238 - 1251. [Abstract] [Full Text] [PDF]

				M. C. Hogan, L. Manganelli, J. R. Woollard, A. I. Masyuk, T. V. Masyuk, R. Tammachote, B. Q. Huang, A. A. Leontovich, T. G. Beito, B. J. Madden, et al. Characterization of PKD Protein-Positive Exosome-Like Vesicles J. Am. Soc. Nephrol., February 1, 2009; 20(2): 278 - 288. [Abstract] [Full Text] [PDF]

				I. Kim, C. Li, D. Liang, X.-Z. Chen, R. J. Coffy, J. Ma, P. Zhao, and G. Wu Polycystin-2 Expression Is Regulated by a PC2-binding Domain in the Intracellular Portion of Fibrocystin J. Biol. Chem., November 14, 2008; 283(46): 31559 - 31566. [Abstract] [Full Text] [PDF]

				G. Liang, J. Yang, Z. Wang, Q. Li, Y. Tang, and X.-Z. Chen Polycystin-2 down-regulates cell proliferation via promoting PERK-dependent phosphorylation of eIF2{alpha} Hum. Mol. Genet., October 15, 2008; 17(20): 3254 - 3262. [Abstract] [Full Text] [PDF]

				L. Geng, W. Boehmerle, Y. Maeda, D. Y. Okuhara, X. Tian, Z. Yu, C.-u. Choe, G. I. Anyatonwu, B. E. Ehrlich, and S. Somlo Syntaxin 5 regulates the endoplasmic reticulum channel-release properties of polycystin-2 PNAS, October 14, 2008; 105(41): 15920 - 15925. [Abstract] [Full Text] [PDF]

				V. E. Torres Role of Vasopressin Antagonists Clin. J. Am. Soc. Nephrol., July 1, 2008; 3(4): 1212 - 1218. [Abstract] [Full Text] [PDF]

				M Adams, U M Smith, C V Logan, and C A Johnson Recent advances in the molecular pathology, cell biology and genetics of ciliopathies J. Med. Genet., May 1, 2008; 45(5): 257 - 267. [Abstract] [Full Text] [PDF]

				G. Liang, Q. Li, Y. Tang, K. Kokame, T. Kikuchi, G. Wu, and X.-Z. Chen Polycystin-2 is regulated by endoplasmic reticulum-associated degradation Hum. Mol. Genet., April 15, 2008; 17(8): 1109 - 1119. [Abstract] [Full Text] [PDF]

				R. Rohatgi, L. Battini, P. Kim, S. Israeli, P. D. Wilson, G. L. Gusella, and L. M. Satlin Mechanoregulation of intracellular Ca2+ in human autosomal recessive polycystic kidney disease cyst-lining renal epithelial cells Am J Physiol Renal Physiol, April 1, 2008; 294(4): F890 - F899. [Abstract] [Full Text] [PDF]

				I. Kim, Y. Fu, K. Hui, G. Moeckel, W. Mai, C. Li, D. Liang, P. Zhao, J. Ma, X.-Z. Chen, et al. Fibrocystin/Polyductin Modulates Renal Tubular Formation by Regulating Polycystin-2 Expression and Function J. Am. Soc. Nephrol., March 1, 2008; 19(3): 455 - 468. [Abstract] [Full Text] [PDF]

				J.-y. Kaimori and G. G. Germino ARPKD and ADPKD: First Cousins or More Distant Relatives? J. Am. Soc. Nephrol., March 1, 2008; 19(3): 416 - 418. [Full Text] [PDF]

				A.-R. Gallagher, E. L. Esquivel, T. S. Briere, X. Tian, M. Mitobe, L. F. Menezes, G. S. Markowitz, D. Jain, L. F. Onuchic, and S. Somlo Biliary and Pancreatic Dysgenesis in Mice Harboring a Mutation in Pkhd1 Am. J. Pathol., February 1, 2008; 172(2): 417 - 429. [Abstract] [Full Text] [PDF]

				K. Huang, D. R. Diener, A. Mitchell, G. J. Pazour, G. B. Witman, and J. L. Rosenbaum Function and dynamics of PKD2 in Chlamydomonas reinhardtii flagella J. Cell Biol., November 5, 2007; 179(3): 501 - 514. [Abstract] [Full Text] [PDF]

				M. A. Garcia-Gonzalez, L. F. Menezes, K. B. Piontek, J. Kaimori, D. L. Huso, T. Watnick, L. F. Onuchic, L. M. Guay-Woodford, and G. G. Germino Genetic interaction studies link autosomal dominant and recessive polycystic kidney disease in a common pathway Hum. Mol. Genet., August 15, 2007; 16(16): 1940 - 1950. [Abstract] [Full Text] [PDF]

				S. Wang, J. Zhang, S. M. Nauli, X. Li, P. G. Starremans, Y. Luo, K. A. Roberts, and J. Zhou Fibrocystin/Polyductin, Found in the Same Protein Complex with Polycystin-2, Regulates Calcium Responses in Kidney Epithelia Mol. Cell. Biol., April 15, 2007; 27(8): 3241 - 3252. [Abstract] [Full Text] [PDF]

This Article Abstract FREE Full Text (PDF) Supplementary Data All Versions of this Article: 15/22/3280    most recent ddl404v1 Alert me when this article is cited Alert me if a correction is posted Services Email this article to a friend Similar articles in this journal Similar articles in ISI Web of Science Similar articles in PubMed Alert me to new issues of the journal Add to My Personal Archive Download to citation manager Search for citing articles in: ISI Web of Science (24) Request Permissions Google Scholar Articles by Wu, Y. Articles by Chen, X.-Z. Search for Related Content PubMed PubMed Citation Articles by Wu, Y. Articles by Chen, X.-Z. Social Bookmarking          What's this?

Online ISSN 1460-2083 - Print ISSN 0964-6906

Copyright © 2009 Oxford University Press

Oxford Journals Oxford University Press

• Site Map
• Privacy Policy
• Frequently Asked Questions

Other Oxford University Press sites: Oxford University Press Oxford Journals China Oxford Journals Japan American National Biography Booksellers' Information Service Children's Fiction and Poetry Children's Reference Corporate & Special Sales Dictionaries Dictionary of National Biography Digital Reference English Language Teaching Higher Education Textbooks Humanities International Education Unit Law Medicine Music Online Products Oxford English Dictionary Reference Rights and Permissions Science School Books Social Sciences Very Short Introductions World's Classics