Fibrocystin interacts with CAML, a protein involved in Ca2+ signaling

BBRC Biochemical and Biophysical Research Communications 338 (2005) 880–889 www.elsevier.com/locate/ybbrc

Fibrocystin interacts with CAML, a protein involved in Ca2+ signaling Junko Nagano a,b, Kenichiro Kitamura b,*, Kristine M. Hujer a, Christopher J. Ward c, Richard J. Bram d, Ulrich Hopfer e, Kimio Tomita b, Chunfa Huang a, R. Tyler Miller a,e,f a

Division of Nephrology, Department of Medicine, Case Western Reserve University, Lois Stokes Veterans Affairs Medical Center, Cleveland, OH, USA b Department of Nephrology, Graduate School of Medical Sciences, Kumamoto University, Kumamoto, Japan c Division of Nephrology, Mayo Clinic, Rochester, MN, USA d Pediatrics and Immunology, Mayo Clinic, Rochester, MN, USA e Physiology and Biophysics, Case Western Reserve University, Cleveland, OH, USA f Rammelkamp Center for Research and Education, MetroHelath Medical Center, Cleveland, OH, USA Received 29 September 2005 Available online 14 October 2005

Abstract The predicted structure of the autosomal recessive polycystic kidney disease protein, fibrocystin, suggests that it may function as a receptor, but its function remains unknown. To understand its function, we searched for proteins that interact with the intracellular C-terminus of fibrocystin using the yeast two-hybrid system. From the screening, we found calcium modulating cyclophilin ligand (CAML), a protein involved in Ca2+ signaling. Immunofluorescent analysis showed that both proteins are co-localized in the apical membrane, primary cilia, and the basal body of cells derived from the distal nephron Epitope-tagged expression constructs of both proteins were co-immunoprecipitated from COS7 cells. The intracellular C-terminus of fibrocystin interacts with CAML, a protein with an intracellular distribution that is similar to that of PKD2. Fibrocystin may participate in regulation of intracellular Ca2+ in the distal nephron in a manner similar to PKD1 and PKD2 that are involved in autosomal dominant polycystic kidney disease.
2005 Elsevier Inc. All rights reserved. Keywords: ARPKD; Fibrocystin; CAML; Ca2+signaling; Cilia; PKD1; PKD2

Autosomal recessive polycystic kidney disease (ARPKD) is an important cause of renal and liver-related morbidity and mortality in childhood with an incidence of 1 in 20,000 live births. The disease leads to fusiform dilitation of renal collecting ducts and biliary dysgenesis [1,2]. Recently, mutations in the human PKHD1 gene on chromosome 6p21.1-p12 were identified as the cause of ARPKD [3,4]. The longest continuous open reading frame (ORF) of the predicted primary sequence encodes a 4074amino acid receptor-like protein with a predicted MW of 447 kDa, a long extracellular amino terminus, a single

*

Corresponding author. Fax: +81 96 366 8458. E-mail address: [email protected] (K. Kitamura).

0006-291X/$ - see front matter
2005 Elsevier Inc. All rights reserved. doi:10.1016/j.bbrc.2005.10.022

transmembrane (TM) domain, and a short intracellular C-terminus. The mouse homologue has an open reading frame of 4059 AA and is highly similar [5]. Although both the human and mouse proteins have large open reading frames, the expressed proteins appear to represent a number of splice variants with tissue-specific patterns of expression [5]. Fibrocystin is expressed in kidney, liver (bile ducts), and testis, and is found in the plasma membrane, and intracellular distribution is believed to be the basal body, and primary cilia [6–9]. Expression of fibrocystin appears to be regulated by HNF-1b [10,11]. Although fibrocystin contains regions with structural motifs found in other proteins, it appears to be a member of a new family of proteins, whose function remains unknown [3,4,12]. Additionally, proteins that interact with fibrocystin and

J. Nagano et al. / Biochemical and Biophysical Research Communications 338 (2005) 880–889

that could provide clues to its function have not been identified. A number of recent studies show that disruption of genes that code for proteins that are expressed in the central cilium or that are required for its structure results in various forms of human polycystic kidney disease or a polycystic phenotype in animals [13–15]. The central cilium appears to function as a mechano- or chemosensor in the lumen of the tubule. Polycystin 1 (PKD1), mutations in which cause autosomal dominant polycystic kidney disease type 1, is found in the central cilium as well as adherens junctions along the nephron. PKD1 has a long extracellular N-terminus that may interact with proteins on neighboring cells and an intracellular C-terminus that interacts with PKD2. PKD2, a Ca2+ channel related to the TRP family of Ca2+ channels, is also expressed throughout the nephron, is primarily found in the endoplasmic reticulum, but is also present in the central cilium [13,16,17]. The PKD1–PKD2 complex contributes to Ca2+ signaling in response to movement of the central cilium, presumably in response to fluid flow [13]. Fibrocystin is expressed in the distal nephron, on the apical surface and the central cilium of epithelial cells, and in an intracellular distribution consistent with expression in the basal body [6–9]. The products of the four genes responsible for hereditary nephronophthesis, nephrocystin 1, inversin, nephrocystin 3, and nephrocystin 4, are all found in the central cilium and appear to form a complex [14]. In mouse and rat animal models, mutations in, or inactivation of genes including pkd1, pkd2, pkhd1, invs (inversin), and kif3a (kinesin II 3a subunit that is involved in vesicle transport in the cilium), result in a polycystic kidney phenotype [18]. Based on analysis of its sequence, fibrocystin appears to be a signaling protein, possibly a receptor. Although it has many splice variants and could function as a secreted protein, one mutation that leads to ARPKD is in the intracellular C-terminus (exon 65, W7831X) indicating that the intracellular C-terminus is important for the function of the protein [1]. However, the intracellular region of the C-terminus does not contain known structural motifs that could suggest a function for it, and interacting proteins have not been described. In an effort to identify proteins that interact with the intracellular C-terminus of fibrocystin, we used that portion of the protein as bait to screen a human adult kidney cDNA library using the yeast twohybrid approach. We found that fibrocystin interacts with a protein that is involved in Ca2+ signaling, calcium modulating cyclophilin ligand (CAML). These results support the possibility that fibrocystin may act as a membrane receptor and suggest that it may participate in Ca2+ signaling like other proteins that lead to polycystic kidney disease. Materials and methods Materials. Chemicals were purchased from Sigma Chemical (St. Louis, MO, USA) or Fisher Scientific (Pittsburgh, PA, USA) unless specified otherwise. Tissue culture medium and serum were obtained from Invit-

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rogen (Carlsbad, CA, USA), plasticware was from BD Biosciences (Palo Alto, CA, USA), the COS7 and MDCK cells were from the American Tissue Culture Collection (Manassas, VA, USA), restriction and DNA modifying enzymes were purchased from Promega (Madison, WI, USA), and plasmid preparation kits were from Qiagen (Valencia, CA, USA). Millicell-CM 12 mm diameter 0.4 lm tissue culture plate inserts were obtained from Millipore (Billerica, MA, USA). The yeast strain (AH109 and Y187), the cDNA library (Pretransformed Human Kidney MATCHMAKER cDNA libraries), and the GAL4 Two-Hybrid system (MATCHMAKER Two-Hybrid System 3) were from BD Biosciences. The full-length CAML cDNAs and poly- and monoclonal anti-CAML antibodies were described previously [19]. The fibrocystin mouse monoclonal antibody 5A that recognizes in the intracellular C-terminus of fibrocystin was characterized and described recently [8]. cDNA constructs. The mouse PKHD1 C-terminus (exons 65–67, the C-terminal 214 AA of mouse fibrocystin, AA 3845–4059, including the predicted transmembrane domain) was obtained by PCR with a mouse kidney mRNA (Clontech) as a template and ligated into the TOPO-TA cloning vector (Invitrogen) [5]. The cDNAs corresponding to mouse fibrocystin AA 3875–3939 (derived from exons 65 to 66 and excluding the predicted transmembrane domain) and AA 3940–4059 (derived form exon 67) were amplified by PCR using the mouse fibrocystin full-length C-terminus constructs as a template and ligated in pGBKT7. The PCR products containing the predicted transmembrane domain (AA 3845– 4059) were ligated into pCRUZ-Myc (Santa Cruz Biotechnology). A cDNA, representing the C-terminal 222 AA of human fibrocystin (AA 3855–4074; exons 65–67) including the transmembrane domain, was amplified by PCR using a human kidney cDNA library as a template and ligated into the TA-TOPO cloning vector (Invitrogen) [3]. The human fibrocystin cDNAs corresponding to AA 3882–3929 (derived from exons 65–66), and AA 3930–4074 (exon 67) were amplified by PCR using the human fibrocystin full-length C-terminus construct as a template and ligated into pGBKT7. The 222 AA human fibrocyctien Cterminus (AA 3852–4074) was ligated into pCRUZ-Myc. Four CAML deletion mutants were made from the full-length CAML cDNA. To make deletion mutants of CAML corresponding to AA 1–130, AA 131–296, and AA 54–296, full-length CAML in the pACT2 vector was digested at the restriction sites XmaI and SacI, the fragments were removed using gel electrophoresis, and the plasmids were religated. The cDNA corresponding to AA 1–251 of CAML was amplified by PCR and ligated into pACT2. The sequences of all cDNAs generated by PCR were verified by direct sequencing. Yeast twohybrid interactions. The bait plasmids containing the mouse and human fibrocystin C-termini (excluding the predicted transmembrane domain) were transformed into Saccharomyces cerevisiae strain AH 109 by electroporation and the yeast mated with the pretransformed library in S. cerevisiae strain Y187. The yeast were plated on SD medium deficient in leucine, tryptophan, and histidine. The colonies that grew under these conditions were replicated on medium lacking leucine, tryptophan, histidine and adenine, and that contained X-a-Gal. The mouse and human C-terminus constructs were transformed into Y187 and used for small scale matings to verify their interaction with the full-length CAML in AH109. All constructs above were tested for auto-activation before use. Each CAML construct in pACT2 and human or mouse fibrocystin C-terminus construct in pGBKT7 was tested for interaction in the yeast two-hybrid system assaying for survival with quadruple selection and in situ X-a-galactosidase activity. The pACT2 vector was transformed into AH109, and pGBKT7 was transformed into Y187, and this mating was used as a negative control. All constructs above were tested for auto-activation before use. Immunoblotting. MDCK and COS7 cells were harvested in buffer containing 20 mM Hepes, pH 7.5, 2 mM MgCl2, 1 mM EDTA, and protease inhibitors, and homogenized with 30 strokes of a Dounce homogenizer. Homogenates were centrifuged at 1500 rpm for 10 min at 4 C to yield a post-nuclear pellet. The resultant supernatants were centrifuged at 15,000 rpm for 1 h at 4 C to yield crude membrane and cytosol fractions. Protein concentrations were determined using the BCA assay

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with BSA as the standard and then adjusted to the same concentration with buffer. The samples were subjected to 11 or 6% SDS–PAGE, processed for immunoblotting with the antibodies indicated, and visualized with enhanced chemiluminescence kit (Amersham Biosciences, Uppsala, Sweden). Immunofluorescence. MDCK cells were grown on collagen-coated Millicell-CM 12 mm diameter 0.4 lm Culture Plate Inserts and fixed for 20 min in 4% paraformaldehyde. The cells were rinsed using PBS and permeabilized with buffer containing 0.1% Triton X-100, 0.05% Saponin, made up in blocking buffer (5% goat serum, 5% donkey Serum, 1% bovine serum albumin, and 1% fetal bovine serum, in PBS) for 5 min. The cells were rinsed in PBS and incubated with primary antibodies (mouse monoclonal anti-fibrocystin 1:500 dilution and rabbit polyclonal antiCAML 1:500) for 1 h. The cells were washed with PBS and then incubated with secondary antibodies (goat-anti-mouse Alexa Fluor 597 antibody for ARPKD [red] and a goat-anti-rabbit Alexa Fluor 488 antibody for CAML [green], Molecular Probes, Eugene, OR, USA) both at 1:500 dilution in blocking buffer (above) for 45 min. The filters were removed from the plastic inserts and placed on slides between dabs of nail polish as spacers. The cells were mounted using Vectashield mounting medium with DAPI with a size 1.5 coverslip. Images were obtained with a Zeiss Axiovert 200M microscope (Carl Zeiss, Thornwood, NY, USA) using Metamorph Imaging System software (Universal Imaging, Downingtown, PA, USA). Deconvolution was done using AutoDeblur and AutoVisualize software (AutoQuant Imaging, Watervliet, NY, USA). Two-month-old BALB/C mice were anesthetized with phenobarbitone. The mouse kidneys were perfused first with 10 ml saline and then with 2% paraformaldehyde. The kidneys were then removed, placed in 2% paraformaldehyde, and embedded in paraffin. Five micrometer serial sections were then taken, mounted on glass slides, and baked at 60  C overnight. The slides were deparaffinized in serial xylene and ethanol, and then washed in PBS. The slides were placed in boiling 10 mM Na tricitrate for 10 min, washed for 10 min in PBS with 0.1 M glycine, and then twice in PBS for 5 min. The sections were delineated with a hydrophobic barrier using a DAKO pen and blocked with 10% goat serum in 1% BSA for 2 h. The sections were then incubated with primary antibodies, a 1:200 dilution of anti-fibrocystin antibody (Christopher J. Ward, Mayo clinic, Rochester, MN) or a 1:500 dilution of acetylated-a-tubulin antibody (Sigma, T6793) and a 1:50 dilution of anti-CAML antibody (Richard Bramm, Mayo Clinic), at 4 C overnight [8,19,20]. Serial sections were also incubated with anti-aquaporin-2 antibody (BD Bioscience, 550649) to identify collecting duct cells. After washing three times in PBS, the sections were incubated with secondary antibodies, a goat anti-mouse IgG Alexa Fluor 488 (Molecular Probes, A-11029) for anti-fibrocystin and tubulin, and a goat anti-rabbit IgG Alexa Fluor 568 (Molecular Probes, A-11036) for anti-CAML and aquaporin-2. The sections were then washed three times in PBS, mounted, covered with thin coverslips, and sealed with clear nail polish. Images were analyzed using a confocal laser scanning microscope (Olympus, Fluoview 300). Expression of cDNAs in cultured cells. The mouse and human fibrocystin C-terminus constructs expressed in mammalian cells contained the transmembrane domain fused to a Myc tag at the N-terminus. The Myctagged 219 AA human fibrocystin or the Myc-tagged 198 AA mouse fibrocystin C-terminus in pcDNA3 and the FLAG-tagged full-length CAML in pBJ5 [20] were transiently transfected into COS7 cells using FuGENE6 transfection reagent (Roche, Basel, Switzerland). After 48 h, cells were harvested. Co-immunoprecipitation. COS7 cells that were co-transfected with the Myc-tagged human or mouse fibrocystin C-terminus and FLAG-tagged full-length CAML were lysed on ice in immunoprecipitation buffer containing 125 mM NaCl, 62.5 mM NaH2PO4, pH 7.2, 0.625% C12E10 (Lubrol), and protease inhibitors. The extract was mixed with Dynabeads– protein A coated with polyclonal anti-Myc antibody by slow rotation at 4 C overnight [21]. The antibody–protein complexes were isolated magnetically. The isolated protein samples were size-fractionated by SDS– PAGE and immunoblotted with either the monoclonal anti-Myc antibody (9E10) or the monoclonal anti-FLAG antibody [21].

Results Identification of proteins that interact with the C-terminus of fibrocystin in yeast In order to identify proteins that interact with the intracellular C-terminus of fibrocystin, and that might provide clues to its function, we used a construct derived from the mouse fibrocystin C-terminus, the C-terminal 184 AA of mouse fibrocystin (AA 3875–4059, the intracellular C-terminus, excluding the predicted transmembrane domain), to screen a pretransformed human adult kidney cDNA library. A total of 13 positive clones were isolated from the library screening using the mouse bait construct and sequenced, two of which coded for CAML. The CAML cDNA clones were 849 nucleotides in length and lacked the first 39 nucleotides of the coding region (nt 75–1128) [22]. Two other groups have identified CAML as an interacting protein by isolating full length cDNAs using the yeast two-hybrid system [22,23]. The interaction of fibrocystin with CAML was verified using cDNAs representing the C-terminal 192 AA of human fibrocystin (AA 3855–4074; excluding the predicted transmembrane domain) and the mouse C-terminus in small scale matings with the full-length CAML in the prey plasmid pACT2. Both the human and mouse fibrocystin C-termini interacted with CAML based on growth on selective media and in situ X-a-galactosidase activity. To identify the regions of fibrocystin and CAML that are required for their interaction, we used deletion mutants of fibrocystin (human and mouse) and CAML assayed in the yeast two-hybrid system (Fig. 1). The full-length CAML cDNA and its deletion mutants, corresponding to AA 1–251 (truncated before the last predicted transmembrane domain), AA 1–130 (truncated before the first of three predicted transmembrane domains), AA 131–296 (lacking most of the predicted N-terminal domain), and AA 54–296 (lacking the N-terminal 53 AA), were cloned into pACT2 and then transformed into AH109. The human and mouse C-termini differ in length by 8 AA (human 184 AA and mouse 192 AA). Three equivalent constructs were made from each. The first construct corresponds to the full length intracellular C-terminus, but excludes the transmembrane domain (mouse AA 3845–4059 and human AA 3882–4074). The second construct corresponds to the region closest to the transmembrane domain and contains part of exon 65 and 66 (AA 3882–3939 human and 3861– 3922 mouse), and the third construct corresponds to the distal C-terminus or exon 67 (mouse AA 3940–4059 and human AA 3930–4074). Combinations of the CAML and Human or Mouse fibrocystin C-terminus constructs were tested for interaction in the yeast two-hybrid system. Fig. 1 shows that the N-terminal region of CAML (AA 1–53 CAML) and the region of fibrocystin nearest the transmembrane domain (exons 65–66 of fibrocystin from both human and mouse) are the regions needed for interaction.

J. Nagano et al. / Biochemical and Biophysical Research Communications 338 (2005) 880–889

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Fig. 1. Yeast two-hybrid analysis of the interaction of human and mouse fibrocystin C-termini and CAML. Figure shows the results of matings of yeast strains containing the fibrocystin and CAML constructs indicated. A schematic diagram of each construct is shown along with the amino acids it includes. Fine lines represent exon boundaries (fibrocystin constructs). The left-hand side of the figure shows the interaction of the full-length human and mouse C-termini with the full-length CAML C-terminus and the four truncation mutants. The right-hand side of the figure shows the interaction of the full length CAML construct with the three human or mouse C-terminus constructs. The fibrocystin C-terminus constructs in pGBKT7 and yeast strain Y187 were mated with the CAML constructs in pACT2 and yeast strain AH109, and plated on selective medium with X-a-Gal. The + indicates that the yeast grew in the presence of quadruple selection (-Leu, -His, -Trp, and -Ad) and produced X-a-galactosidase activity in situ. The indicates that the yeast did not survive selection.

Western analysis of MDCK cells The interaction of fibrocystin with CAML identified in the yeast two hybrid system provides no information on the cell type(s) that express the proteins or the regions of the kidney in which they are expressed. In order to demonstrate that the two proteins are both expressed in the same cells and in renal epithelial cells from the distal nephron, we performed Western blots on membrane and cytosol fractions from MDCK cells that are of distal nephron origin (Fig. 2). The left panel shows fibrocystin expression and the right panel shows CAML expression. Both proteins are expressed strongly in the membrane fraction, although fibrocystin is also present in the cytosolic fraction, possibly in small vesicles.

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MDCK cells express a number of fibrocystin isoforms based on the number of bands seen. A band is seen at approximately 440 kDa in the membrane fraction, but bands of approximately 180 kDa appear to be the predominant isoforms. The bands present in the cytosolic fraction appear to be of slightly lower molecular weight than bands in the membrane fraction, suggesting that they may not be fully glycosylated. The small amount of the 440 kDa band in the cytosolic fraction may represent imperfect separation of the fractions. CAML is present as a single band at approximately 35 kDa in the membrane fraction of MDCK cells. The cytosolic fraction appears not to contain CAML, suggesting that compared to fibrocystin, the levels of expressed protein in the membrane fraction are higher.

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48 212 34 28 135 Fibrocystin

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Fig. 2. Endogenous expression of fibrocystin and CAML in cultured renal cells. MDCK cells were grown to confluence and separated into membrane and cytosolic fractions with centrifugation. Duplicate samples were normalized for protein, size-fractionated on a 6% gel for fibrocystin (left), or an 11% gel for CAML (right), and blotted with the antibodies indicated.

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Localization of fibrocystin and CAML in MDCK cells Although fibrocystin and CAML are both expressed in the membrane fraction of renal epithelial cells, and fibrocystin is present in the apical membrane, central cilium, and basal body, the localization of CAML in epithelial cells is not defined. Using MDCK cells grown on filters to maximize differentiation and polarization, we analyzed the subcellular distribution of fibrocystin and CAML with confocal microscopy and deconvolution analysis. Fig. 3 shows a group of MDCK cells in which fibrocystin is localized near the apical surface in a ring pattern compatible with the basal body, consistent with reports by others (Fig. 3A) [6–8]. CAML is expressed in a diffuse cytosolic distribution with increased density in the region of the basal body and the central cilium (panel 3B). Panel 3C shows an overlay of 3A and 3B where yellow indicates that fibrocystin and CAML are in close proximity in the basal body. Panel 3D shows a more apical image and overlap of fibrocystin and CAML in the central cilium. Panels 3E and 3F are negative controls for the fibrocystin antibody (hybridoma medium) and CAML antibody (normal rat serum) and are shown in gray scale. This pattern of co-localization is reasonable for two proteins that participate in Ca2+ signaling. Immunohistochemistry to co-localize fibrocystin and CAML in mouse kidneys If fibrocystin and CAML are both expressed in, and interact in mammalian kidney cells, they should co-localize

in mammalian kidney tissues. We used normal adult mouse kidney sections to study their co-localization in intact tissue (Figs. 4 and 5). Serial sections were used to stain with antibodies against fibrocystin, CAML, acetylated-a-tubulin, and aquaporin-2 to confirm localization in the distal nephron and central cilium. In Fig. 4, the top row (4A, 4B, and 4C) shows expression of fibrocystin and CAML at low magnification (40·). The bottom row shows negative controls for the fibrocystin and CAML antibodies (4D and 4E, respectively, omission of the primary antibody) and staining for aquaporin-2 (4F) at 40· magnification. The sections were stained with the anti-fibrocystin antibody (green secondary antibody, 4A) and the antiCAML antibody (red secondary antibody, panels 4B, and the images were merged in panels 4C). Areas of overlap of the two proteins are seen as yellow or orange, indicating their co-localization. CAML is found in the cytosol and in a membrane pattern, predominantly in the apical membrane, as well as the central cilium, consistent with results previously published with this antibody [8]. CAML is present in a diffuse cytosolic pattern with concentration in the region of the plasma membrane, as well as the cilium. Co-localization of fibrocystin and CAML was found predominantly in cilia and the region of the apical plasma membrane. CAML is not a plasma membrane protein, so the apparent co-localization probably reflects presence of CAML in a membrane compartment that is close to the plasma membrane and/or CAML and fibrocystin together in a membrane compartment that is close to the plasma membrane [19]. Staining for aquaporin-2 (panel 4F) demonstrates that the tubules are collecting ducts.

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10 um Fig. 3. Co-localization of fibrocystin and CAML in MDCK cells. MDCK cells were grown on filters for 4–5 days. The cells were fixed in paraformaldehyde, permeabilized with saponin, incubated with the antibodies indicated, and analyzed using a confocal microscope with deconvolution analysis. The images are shown at 63· magnification, and a scale is shown at the bottom left. (A, green) cells stained with the antibody to fibrocystin, (B, red) the cells stained with the antibody to CAML, and (C, yellow or orange) the overlay of the two images. (D) A more apical plane and a central cilium staining for fibrocystin and CAML (lower right) and a cell where the focal plane is below the apical plasma membrane. (E) Cells stained with hybridoma medium and the secondary antibody for fibrocystin (red), and (F) shows the cells stained with normal rat serum and the secondary antibody used for CAML (green).

J. Nagano et al. / Biochemical and Biophysical Research Communications 338 (2005) 880–889

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Fig. 4. Co-localization of fibrocystin and CAML in the distal nephron of mouse kidneys. Paraformaldehyde-fixed, parafin-embedded sections of adult mouse kidney were sectioned serially and processed for immunofluorescence using antibodies for fibrocystin, CAML, and aquaporin-2. The top row (A, fibrocystin; B, CAML; and C, overlay) shows serial mouse kidney sections at low magnification (40·). The bottom row shows negative controls for the fibrocystin antibody (D), CAML antibody (E), and staining for aquaporin-2 (F) (all at 40· magnification). Areas of overlap of fibrocystin and CAML are seen as yellow or orange, indicating co-localization (C).

Although the pattern of expression of fibrocystin is well defined by others, the pattern of expression of CAML has not been addressed in renal tissue. In order to be certain of its localization to cilia, we stained serial sections with the anti-CAML antibody and anti-acetylated a-tubulin antibody, a cilium marker. Fig. 5, top row (A, B, and C) shows

the expression of acetylated-a-tubulin, CAML, and the overlap of acetylated tubulin and CAML at low magnification (40·). The second row shows acetylated tubulin, CAML, and their overlap at higher magnifications (5D– 5F, 100·). The third row shows negative controls for acetylated tubulin (5G) and CAML (5H). Staining for aquapo-

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Fig. 5. Co-localization of acetylated tubulin, a marker for cilia, and CAML. Paraformaldehyde-fixed, parafin-embedded sections of adult mouse kidney were sectioned serially and processed for immunofluorescence using antibodies for acetylated tubulin, CAML, and aquaporin-2. The top row (A, B, and C) shows a low magnification of serial sections of mouse kidney (40·) stained for acetylated tubulin (A), CAML (B), and overlay of the two (C). Regions marked with a box are magnified below. The second row (D, E, and F) shows higher magnification of selected regions of the sections in the first row (100·). The third row shows negative controls for the acetylated tubulin antibody (G), CAML antibody (H), and positive staining for aquaporin (I) (all at 40· magnification).

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rin-2 (panel 5I) demonstrates that the tubules are collecting ducts. The cells were stained with the anti-acetylated tubulin antibody (green secondary antibody, far left; 5A, 5D) and the anti-CAML antibody (red secondary antibody, middle; 5B, 5E) and the images were merged in panels 5C and 5F. This result confirms localization of CAML in cilia of collecting duct cells in the distal nephron. Interaction of fibrocystin and CAML expression constructs in mammalian cells We used expressed CAML and fibrocystin constructs to study the interaction of these two proteins in mammalian cells for a number of reasons. Fibrocystin appears to be a low-abundance protein, while CAML is expressed at relatively high levels. Their distributions overlap, but are not identical (Figs. 2 and 3), meaning that fibrocystin may not always be associated with CAML. Consequently, the amount of fibrocystin and CAML that interact at a particular time may be small and difficult to detect. Fibrocystin is expressed as a number of splice variants that could result in reduced sensitivity in detecting it by Western blot. To document that our expressed forms of fibrocystin and CAML are found in the membrane fraction like the endogenous proteins, we transiently transfected FLAG-tagged CAML and mouse or human fibrocystin constructs (containing the transmembrane domain and a Myc tag at the N-terminus) into COS7 cells. Cells that were not transfected were used as a negative control. The cells were harvested and separated into membrane and cytosolic fractions by centrifugation. The Myc-tagged fibrocystin constructs and the FLAG-tagged CAML construct were predominantly found in the membrane fraction, demonstrating that the fribrocystin and CAML expression constructs localize correctly in cultured cells. For immunoprecipitation, we co-expressed the FLAGtagged CAML construct with either the human or mouse Myc-tagged fibrocystin C-terminus constructs in COS7 cells and immunoprecipitated the proteins using a polyclonal anti-Myc antibody (Fig. 6). Duplicate samples of cell extracts were incubated with the polyclonal anti-Myc antibody and blots were probed with a monoclonal antiMyc antibody and a monoclonal anti-FLAG antibody. In both panels, the first pair of lanes contains extracts from cells that were not transfected, the second pair of lanes contains extracts from cells that were co-transfected with the human fibrocystin C-terminus and CAML constructs, the third pair of lanes contains extracts from cells that were co-transfected with the mouse fibrocystin C-terminus and CAML constructs, and the last pair of lanes contains extracts from cells co-transfected with either the mouse or human fibrocystin C-terminus and CAML constructs, but in which the immunoprecipitating antibody was omitted. In the first and last pair of lanes in both panels, bands that correspond to either the fibrocystin C-terminus or CAML are not present. However, in the top panel, bands are present at the appropriate positions for the human and mouse

IP- AB 1 WB-AB Anti-Myc (9E10)

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8 hFC mFC CAML

Anti-FLAG Fig. 6. Co-immunoprecipitation of fibrocystin and CAML in cultured cells. COS7 cells were transiently co-transfected with human or mouse Myc-tagged fibrocystin and the full-length FLAG-tagged CAML construct. After the transfection, cells were lysed and immunoprecipitation was performed with an anti-Myc antibody. The immunoprecipitate was divided, size-fractionated, and blotted with a monoclonal anti-Myc antibody (top panel) or an anti-FLAG antibody (bottom panel). Lanes 1 and 2 are untransfected cells, 3 and 4 are cells co-transfected with the Myc-tagged human fibrocystin C-terminus and Flag-tagged CAML, lanes 5 and 6 are cells co-transfected with Myc-tagged mouse fibrocystin Cterminus and FLAG-tagged CAML, lane 7 is cells co-transfected with the Myc-tagged human fibrocystin C-terminus and Flag-tagged CAML, and lane 8 is cells co-transfected with the Myc-tagged mouse fibrocystin Cterminus and Flag-tagged CAML. In lane 7 and 8, the immunoprecipitating antibodies were omitted.

C-terminus constructs, and in the right panel, bands are seen in the second and third pairs of lanes that correspond to FLAG-tagged CAML. These results demonstrate that CAML and fibrocystin expression constructs co-immunoprecipitate from COS7 cells and indicate that they can interact in mammalian cells. Discussion We report that fibrocystin, the product of the PKHD1 gene, interacts with calcium modulating cyclophilin ligand (CAML), a protein that participates in the regulation of cytosolic calcium pools [9,19,20,22,24,25]. These studies support the possibility that fibrocystin acts as a receptor and add evidence that fibrocystin may act at least in part via Ca2+ signaling like PKD1 and PKD2. We demonstrate that these two proteins interact in the yeast two-hybrid system indicating a direct interaction, and that the interaction involves the N-terminus of CAML and the juxtamembrane region of the intracellular C-terminus of fibrocystin. The studies in yeast also demonstrate that the same region of the mouse and human fibrocystin C-terminus interacts with CAML. We further find that these two proteins co-localize in cilia, the basal body, and the plasma membrane in cells from the distal nephron, and finally that expression constructs of the two proteins co-immunoprecipitate from mammalian cells. In adult animals, fibrocystin is expressed in the kidney, biliary tree, and pancreatic ducts. Within the kidney, fibrocystin is found in the distal nephron, specifically in the thick ascending limb of Henle, and the collecting duct [6– 8,26]. Light and electron microscopic studies demonstrate that fibrocystin is present in the apical membrane, the cen-

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tral cilium, and that it is also present in a cytoplasmic distribution that is less well defined but that involves the basal body [6–8,26]. We find significant cytoplasmic expression and predominant apical labeling with some basolateral labeling. Our sections of kidney did not permit us to address subcellular localization more precisely, but in MDCK cells, we found expression in the cilium and basal body as do others [7,8,26]. Some investigators report that fibrocystin is not detected in renal or liver tissue from a limited number of patients with ARPKD or the pck rat [7,8,26]. Fibrocystin has several structural motifs, TIG domains, TIG-like domains, and regions with homology to TMEM2 and DKFZ regions. Other than the suggestion that some of these domains appear in plexins, they provide no firm evidence for the function of fibrocystin, and interacting proteins that could also provide clues to the function of fibrocystin have not been identified. Another member of the fibrocystin family has been identified, PKHDL-1, a protein of similar size and putative structure that is expressed in lymphocytes, also with unknown function [27]. CAML, the protein that interacts with fibrocystin, was originally found in a yeast two-hybrid screen as a protein that interacts with cyclophilin-B in lymphocytes [22]. CAML is a 296 AA ubiquitously expressed protein that in lymphocytes is located in the endoplasmic reticulum, and that is in the same membrane fractions as the markers of calcium storage organelles, calreticulin and SERCA2 [19]. We found that CAML is expressed in all cells we evaluated (HEK-293 cells, COS7 cells, and MDCK cells) and in all cells of the mouse kidney in the membrane fraction, but in a cytoplasmic pattern when evaluated with microscopy. Confocal imaging of MDCK cells also demonstrates that CAML is present in a pattern that is consistent with its presence in the region of the basal body where it could either participate in Ca2+ signaling or where it could be part of a protein complex being transported to the cilium or apical membrane. CAML is present in the central cilium although the central cilium does not contain endoplasmic reticulum. Consequently, its expression is not limited to the endoplasmic reticulum, and its role in this organelle is not clear. Homozygous deletion of CAML in mice results in early embryonic lethality, but cells grown from embryonic stem cells are viable, indicating that CAML has an essential function in development, in some cases perhaps via its interaction with fibrocystin, but perhaps not for individual cells [28]. The CAML protein has three predicted membranespanning domains and hydrophilic N- and C-terminal domains. The N-terminal domain is directed toward the cytoplasm and interacts with other proteins including TACI (transmembrane activator and CAML interactor), a T cell-specific protein, the EGF receptor, the KaposiÕs sarcoma virus K7 protein, and fibrocystin. The C-terminal domain is in the lumen of the ER and interacts with effector proteins which are not defined, but that may include Ca2+ transport proteins [9,19,20,24,28]. TACI is a member of the TNF family of receptors and activates NF-AT [25]. Expression of a dominant negative form

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of CAML inhibits TACI-mediated NF-AT activation and overexpression of CAML results in depletion of thapsigargin-sensitive intracellular Ca2+ pools and a rise in intracellular Ca2+ [20,25]. CAML interacts with ATRAP (AT1 receptor-associated protein), a protein that interacts directly with the C-terminus of the AT1 receptor where it contributes to regulation of NF-AT by the AT1 receptor [23]. The N-terminal domain of CAML interacts with the kinase domain of the EGF receptor in a ligand-dependent manner and contributes to the recycling of internalized receptors to the plasma membrane [28]. This interaction appears to take place in the ER. CAML also interacts with p56Lck and regulates its intracellular location in T cells [29]. Finally, the KaposiÕs sarcoma virus mitochondrial K7 protein, an antiapoptotic protein produced by the KaposiÕs sarcoma virus, interacts with the N-terminal domain of CAML to raise intracellular Ca2+ and block apoptosis [24]. We found that fibrocystin is a membrane protein and that it is expressed in cilia and the basal body of renal epithelial cells from the distal nephron consistent with reports of others [6–8]. The precise patterns of staining may depend on the stage of differentiation of the cells [6,7]. Although the multiple splice variants of fibrocystin suggest that a cleaved form may function as a secreted protein, the studies with antibodies directed at the intracellular C-terminus indicate that a significant amount of the protein is resident in the plasma membrane where it is in a position to interact with intracellular proteins or other membrane proteins. We used an antibody directed at the intracellular C-terminus, so we could not have seen forms of fibrocystin that lack the C-terminus. Like fibrocystin, CAML is a membrane protein and is found in cilia on the apical surface of renal epithelial cells. However, CAML is localized to intracellular compartments, the ER or other intracellular Ca2+ stores where it presumably interacts with plasma membrane proteins to regulate the state of filling of intracellular Ca2+ stores. Consistent with previous studies of CAML, we found that fibrocystin interacts with the N-terminus of CAML which is exposed to the cytosol and available to interact with membrane proteins [19,20]. The N-terminus of CAML interacts with fibrocystin in a span of 83 AA that begins at the end of the transmembrane domain and extends to the end of exon 66 (AA 3875–3938 mouse and AA 3882–3939 human). This region does not contain known consensus sequences for interaction with other proteins. The part of the cell where fibrocystin and CAML interact is not fully defined in these studies. The two proteins predominantly co-localize in the cilium and basal body, but may also interact elsewhere in the cell such as the ER where their density may not be sufficient to allow visualization with microscopy. At this point, we do not know the significance of the interaction between fibrocystin and CAML. The fact that these two proteins interact suggests that either fibrocystin participates in regulation of intracellular Ca2+ stores like

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TACI, ATRAP, or the KaposiÕs sarcoma virus K7 protein, or that the interaction of the two proteins relates to protein trafficking as is the case with the EGF receptor and p56Lck [20,23,24,28,29]. Co-localization of fibrocystin and CAML in the cilium is intriguing because of the fact that PKD1 and PKD2 are also localized in the cilium and presumably function to regulate intracellular Ca2+ [13,16]. The intracellular pattern of distribution of CAML is similar to that of PKD2 in that both are present in the endoplasmic reticulum and the central cilium where the endoplasmic reticulum is not present [30]. Bending of the cilium in response to fluid flow increases intracellular Ca2+ from the extracellular space by a mechanism that requires PKD1 and PKD2 [16]. The behavior of intracellular Ca2+ stores was not studied in these experiments, but presumably is important in Ca2+ signaling. In the distal nephron, bile ducts, and pancreatic ducts, fibrocystin could interact with CAML to modulate intracellular Ca2+ by affecting the filling of the intracellular stores. Based on analogy to TACI and expression studies of CAML and its mutants in Jurkat cells, homozygous loss of function mutations in fibrocystin could result in loss of function of CAML which would lead to more filling of stores and a lower intracellular Ca2+ [9,19,24]. This scenario is compatible with the phenotypes of loss of function mutations in PKD-1 and PKD-2 that also result in reduced Ca2+ entry in response to fluid flow. In the distal nephron, loss of function mutations in fibrocystin that result in lower levels of intracellular Ca2+ could lead to cAMP-stimulated epithelial cell growth [31]. Future studies will be required to define the physiologic significance of this interaction. Acknowledgments We thank Dr. Junichi Sato for help with cDNA cloning and Dr. Yukimasa Koda for technical support with immunohistochemistry and confocal microscopy. This work was supported by grants from the American Heart Association (C.H. and R.T.M.), the National Institutes of Health (DK59985, PO1-HL41618), the Rainbow, Babies and ChildrenÕsÕ Polycystic Kidney Disease Center, and a VA Merit Review (R.T.M.), the Veterans Administration, the Leonard Rosenberg Research Foundation and by the Grants-in-Aid for Scientific Research from the Ministry of Education, Culture, Sports, Science and Technology in Japan (15790432 to K.K.), the Salt Science Research Foundation (0529 to K.K.), Uehara Memorial Foundation (to K.K.), and the Japan Heart Foundation Research Grant (to K.K.). References [1] C. Bergmann, J. Senderek, B. Sedlacek, I. Pegiazoglou, P. Puglia, T. Eggermann, S. Rudnick-Schoneborn, L. Furu, L.F. Onuchic, M. De Baca, G.G. Germino, L. Guay-Woodford, S. Somlo, M. Moser, R. Buttner, K. Zerres, Spectrum of mutations in the gene for autosomal recessive polycystic kidney disease (ARPKD/PKHD1), J. Am. Soc. Nephrol. 13 (2003) 76–89.

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