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Annexin A5 Interacts with Polycystin-1 and Interferes with the Polycystin-1 Stimulated Recruitment of E-cadherin into Adherens Junctions
J. Mol. Biol. (2007) 369, 954–966
doi:10.1016/j.jmb.2007.03.070
Annexin A5 Interacts with Polycystin-1 and Interferes with the Polycystin-1 Stimulated Recruitment of E-cadherin into Adherens Junctions Arseni Markoff 1 ⁎, Nadia Bogdanova 2 , Markus Knop 1 , Claas Rüffer 1 Heidi Kenis 3 , Petra Lux 3 , Chris Reutelingsperger 3 , Vassil Todorov 4 Bernd Dworniczak 2 , Jürgen Horst 2 and Volker Gerke 1 1
Institute of Medical Biochemistry, WestfalianWilhelms University of Muenster, Von Esmarch Str. 56, 48149 Muenster, Germany 2
Institut für Humanegenetik, University Clinics Muenster, Vesaliusweg 12-14, 48149 Muenster, Germany
3
Department of Biochemistry, Cardiovascular Research Institute Maastricht, P.O. Box 616, 6200 MD Maastricht, Netherlands 4
Clinic of Nephrology and Haemodialysis, Medical University, Pleven, Bulgaria *Corresponding author
Polycystin-1 is the gene product of PKD1, the first gene identified to be causative for the condition of autosomal dominant polycystic kidney disease (ADPKD). Mutations in PKD1 are responsible for the majority of ADPKD cases worldwide. Polycystin-1 is a protein of the transient receptor potential channels superfamily, with 11 transmembrane spans and an extracellular N-terminal region of ∼3109 amino acid residues, harboring multiple putative ligand binding domains. We demonstrate here that annexin A5 (ANXA5), a Ca2+ and phospholipid binding protein, interacts with the N-terminal leucine-rich repeats of polycystin-1, in vitro and in a cell culture model. This interaction is direct and specific and involves a conserved sequence of the ANXA5 N-terminal domain. Using Madin-Darby canine kidney cells expressing polycystin-1 in an inducible manner we also show that polycystin-1 colocalizes with E-cadherin at cell–cell contacts and accelerates the recruitment of intracellular E-cadherin to reforming junctions. This polycystin-1 stimulated recruitment is significantly delayed by extracellular annexin A5. © 2007 Elsevier Ltd. All rights reserved.
Keywords: ANXA5; annexin A5; PKD1; polycystin-1; E-cadherin
Introduction Polycystins-1 and − 2 are the respective products of PKD1 and PKD2, genes responsible for the condition of autosomal dominant polycystic kidney disease (ADPKD). PKD1 mutations cause ADPKD in the majority of cases, leading to severe impairment of kidney function and accompanying systemic damage.1,2 Both polycystins interact via their cytosolic C-terminal regions3,4 and are integral parts of a common signal transduction pathway.1 Polycystin-1 (TRPP1) is the only 11 transmembrane domainscontaining protein that belongs to the superfamily of transient receptor potential channels (TRPs), normally characterized by six transmembrane spans. Abbreviations used: ADPKD, autosomal dominant polycystic kidney disease; LRR, leucine-rich repeat; GCT, glutathione-S-transferase. E-mail address of the corresponding author: [email protected]
Recent studies support possible mechanosensory function for TRPP1, which can be mediated by elastic properties of the extracellular region.5 The assembly of polycystins-1 and − 2 at the plasma membrane, when co-expressed in cultured CHO cells, has been reported to produce unique Ca2+-permeable nonselective currents.6 Moreover, recent studies demonstrate that the expression of polycystin-1 alone can result in such cation currents.7 Polycystin-1 consists of a large, extracellular Nterminal region of ∼ 3109 amino acid residues, 11 predicted transmembrane domains (∼ 993 amino acid residues), and a short, cytosolic C-terminal portion (∼ 200 amino acid residues).8,9 The N-terminal sequence of the protein harbors several domains predicted to represent ligand interaction sites: two leucine-rich repeats (LRRs), a C-type lectin domain, an LDL-A like domain, 15 copies of PKD domains, which are immunoglobulin (Ig)-like repeats, and a region homologous to the sea urchin sperm protein, REJ.9 The protein is cleaved specifically at position T3049, which releases an extracellular N-terminal
0022-2836/$ - see front matter © 2007 Elsevier Ltd. All rights reserved.
Interaction of Annexin A5 with Polycystin-1
fragment of ∼ 325 kDa.10 This fragment is tethered to the cell surface and the cleavage reaction is probably essential for the function of polycystin-1. There is accumulating evidence supporting regulatory function of polycystin-1 in organizing the cytoarchitecture of tubular structures in kidney and liver. Gene-targeting experiments indicate functions of polycystin-1 in the terminal differentiation of tubular epithelial cells11 and in the maintenance of blood vessels' structural integrity.12 Heterologous expression of polycystin-1 in a MDCK cell culture model results in slower growth, promotes resistance to apoptosis and induces spontaneous tubulogenesis.13 In further support for a role in cytoarchitecture, polycystin-1 has been identified in a complex with the cell adhesion molecules E-cadherin and α, β and γ-catenin14 and compromised cytoarchitecture has been demonstrated in ADPKD epithelia, characterized by subcellular sequestration of E-cadherin to an internal cellular compartment, among other protein mislocalizations.15 Furthermore a disruption of the polycystin-1 association with E-cadherin and the catenins is observed in primary cells from ADPKD patients as a result of increased polycystin-1 phosphorylation.16 Polycystin-1 also has the capacity of transducing signals from the extracellular environment to the inside of the cell, thereby regulating major aspects of cellular fate, such as terminal differentiation and cell polarization. The cytosolic C terminus of the protein has been shown to be a site of interaction with heterotrimeric G-proteins,17 which is underpinning a function for polycystin-1 as a G protein-coupled receptor.18 Moreover, polycystin-1 has been demonstrated to up-regulate p21, thereby inducing cellcycle arrest in a manner depending on polycystin-2 interaction.19 This p21 induction can be suppressed by the helix-loop-helix inhibitor Id2, which directly interacts with polycystin-2.20 Here we sought to identify interacting partners of polycystin-1 N-terminal region, concentrating on the LRRs at the very tip of the N-terminal sequence. Our studies show that annexin A5, a Ca2+ and phospholipids binding protein, is able to interact with the LRR domain of polycystin-1. In a cell culture model, inducible heterologous expression of polycystin-1 leads to more rapid recruitment of E-cadherin to cell junctions forming after a Ca2+ switch protocol. This enhanced mobilization of E-cadherin is inhibited by annexin A5, indicative of a function of the polycystin-1/annexin A5 interaction in regulating the formation of epithelial cell contacts.
Results Identification of a peptide ligand of the polycystin-1-LRR The polycystin-1-LRR domain with flanking regions (amino acid residues 27–203) was expressed
955 as a recombinant glutathione-S-transferase (GST) fusion protein in Escherichia coli, attached to glutathione-Sepharose, and used as a bait for selection of E.coli clones, displaying on their flagella random dodecapeptides encoded by the pFliTrx plasmid. Thirty insert-containing clones were selected through this procedure, their inserts were PCR amplified and sequenced. Among the selected clones, one clone emerged in duplicate. Its amino acid sequence QNMKALRGTVCV was queried for homology against the Protein Data Bank and SwissProt databases. This revealed significant identity with the ALRGTV sequence present at the very amino terminus of rat annexin A5 (pos. 1–6). In human annexin A5 this sequence is VLRGTV (pos. 4–9). Maximum likelihood alignment was obtained in six of the selected clones by random positioning of the ALRGTV segment (Table 1). The consensus, which can be deduced from the sequence alignment of clones selected is xLRG/xTV, where x is an aliphatic amino acid. Annexin A5 binds to the LRR domain of polycystin-1 in vitro In order to test a possible binding of annexin A5 to the LRR domain of polycystin-1, in vitro translated full-length annexin A5 was subjected to binding assays employing the (GST)-polycystin-1LRR. In vitro translation of annexin A5 resulted in a product, which separated in two bands on SDSPAGE (Figure 1(a), lane 3). The upper band is the result of an additional upstream translation initiation codon, generated by the amplification of the cDNA, whereas the lower band corresponds to authentic annexin A5, showing a relative molecular mass of 33 kDa. Analysis of the GST pull-down revealed binding of in vitro translated annexin A5 to the LRR domain of polycystin-1. Retained annexin A5 signal in lane 4 represents a 33.5% fraction of the input (right panel). Although exact quantification of protein stoichiometry in this type of assay is not possible, it appears that annexin A5 is present in a molar excess, since the amount of polycystin-1-LRR-bound annexin A5 retained under saturating binding conditions (lane 5) is reduced by only 4% when half of the in vitro translation product is used (lane 4 and right panel). Thus, in vitro translated annexin A5 shows Table 1. Sequences of six selected clones, using the biopanning procedure on the FliTrx random peptide display library with the polycystin-1 LRR as a bait QNMKALRGTVCVa QKNQVARGTVCG QFIALRGTVQSSL ATRGLRVTVTGN LQIVFSALRVTV SDSGGPLRVTAY Consensus amino acids are marked bold, aliphatic residues in consensus strings are underlined. a This clone was selected twice.
956
Interaction of Annexin A5 with Polycystin-1
Figure 1. Left panels: in vitro binding assays (GST pull-down) using (GST)PKD1-LRR as bait. Autoradiographs of [35S] methionine labeled in vitro translation products and their retained fractions after binding. Lanes, marked with (−) are in vitro translation controls and (+) lanes correspond to proteins of the in vitro translation reaction that bound to (GST)PKD1LRR. (a) Lane 1, in vitro translated ANX A1; lane 2, bound fraction; lane 3, in vitro translated ANX A5; lane 4, bound fraction, 0.5 of saturation input; lane 5, bound fraction, saturation input. (b) Lane 1, in vitro translated ANX A5, N-terminal fragment (see Materials and Methods); lane 2, bound fraction; lane 3, in vitro translated ANX A5, C-terminal fragment; lane 4, bound fraction; lane 5, bound fraction of in vitro translated ANX A5 ((a), lane 3) incubated with GST-Sepharose: no ANX A5 binding to the GST-beads alone. Right panels: signal intensities quantification of protein bands, using the NIH Image utility. Signal intensities were recorded as “mean average” of measured band areas, background values were subtracted and values were expressed as percent fractions, normalized to a “standard”, the most intensive signal occurring in a gel image. (a) Signal intensities were normalized to the lower band in lane 3 (100%), corresponding to the relative molecular mass of native annexin A5. Signal intensity of the band in lane 2 was not exceeding the background. (b) Signal intensities were normalized to the band in lane 1 (100%), corresponding to the N-terminal portion of in vitro translated annexin A5. Signal intensity of the band in lane 4 was 0.003% and 0.004% of the input signal (lane 3).
significant binding to immobilized polycystin-1LRR, in contrast to annexin A1 (a ∼ 37 kDa protein, lane 1), which has an unrelated N-terminal sequence and was used as a control in these experiments, and did not show any binding (lane 2 and right panel). To corroborate that this binding is due to the annexin A5 N-terminal domain, harboring the VLRGTV sequence, a 206 amino acid residue amino-terminal fragment and a 185 amino acid residue carboxyterminal fragment of the protein were subjected to in vitro binding assays (Figure 1(b)). Appreciable binding was observed when lysates containing the N-terminal 206 amino acid residues of annexin A5 (∼ 23 kDa, lane 1) were probed in the GST pull-down (lane 2). The signal intensity of retained annexin A5 N-terminal portion was 21.3% of the input (right panel). The carboxy-terminal portion of annexin A5 (∼ 21 kDa, lane 4) did not show any appreciable binding. Neither did GST alone bind to in vitro translated annexin A5 (lane 5 and right panel).
To address the relevance of the in vitro binding experiments with the LRR domain of polycystin-1, we carried out co-immunoprecipitation following addition of annexin A5 to PKD1-transfected HeLa cells and to PKD1-19 MDCK cells, which express a FLAG-tagged version of polycystin-1 in a tetracycline-inducible manner. Both, M2 anti-FLAG antibodies and anti-annexin A5 antibodies were employed in parallel experiments. Immunoprecipitations were performed after washing unbound material off the cell surface and, in one series of experiments, after applying moderate amounts of a mild cleavable cross-linker, DSP, to stabilize protein interactions. Expression of polycystin-1 was verified through immunoprecipitation and detection of the protein with anti-PKD1-LRR antibodies (Figure 2(a)). The ∼ 520 kDa band in the immunoprecipitates and cell lysates of PKD1–19 MDCK cells induced to express polycystin-1 corresponds to full-length polycystin-1 and the ∼ 325 kDa band represents a
Interaction of Annexin A5 with Polycystin-1
957
Figure 2. Immunoprecipitations of polycystin-1 and annexin A5 after chemical cross-link. Table indicates presence or lack of polycystin-1 expression and addition or absence of annexin A5, in the different experiments (a, b, c). a, b, c, Immunoprecipitation products from lysates of PKD1–19 MDCK cells in non-induced or induced state. (a) Polycystin-1 immunoprecipitations with M2 anti-FLAG antibodies. La, Lb, Lc in lanes 1, 2 and 3 denote lysate fractions of each experimental condition. Lanes 4, 5 and 6, immunoprecipitation products for experimental conditions a, b and c. (b) Coimmunoprecipitation of polycystin-1 with anti-ANX A5 antibodies. Lane 1, co-immunoprecipitation products for experimental condition a; lane 2, co-immunoprecipitation products for experimental condition b; lane 3, coimmunoprecipitation products for experimental condition c. (c) Co-immunoprecipitation of annexin A5 with anti-M2 anti-FLAG antibodies (polycystin-1). Lane 1, purified annexin A5 from human placenta, lanes 2, 3 and 4, immunoprecipitation products for experimental conditions a, b and c. (d) and (e) immunoprecipitation products from lysates of HeLa cells, not transfected or transfected with the PKD1-F plasmid. (d) Polycystin-1 co-immunoprecipitations with anti-ANX A5 antibodies. Lanes 1, 2 and 3, immunoprecipitation products for experimental conditions a, b and c. (e) Co-immunoprecipitation of annexin A5 with anti-M2 anti-FLAG antibodies (polycystin-1). Lane 1, purified annexin A5 from human placenta, lanes 2, 3 and 4, immunoprecipitation products for experimental conditions b, a and c, respectively.
cleaved N-terminal fragment (NTF).10 Non-induced cells seem to produce some low level of polycystin-1, which can be immunoprecipitated and detected with the anti-PKD1-LRR antibodies and most likely is a result of incomplete repression (lane 6). Figure 2(b) shows the result of an anti-annexin A5 immunoprecipitation probed in the Western blot
with anti-PKD1-LRR antibodies. Two bands of approximately 520 and 325 kDa, corresponding in size to full-length and NTF polycystin-1 were visible when annexin A5 was added to the PKD1-expressing cells and also when complexes were stabilized by DSP cross-link prior to cell lysis (lanes 1 and 2). Faint immunoprecipitation products with the same
958 relative molecular masses can also be detected in the case of non-induced cells (lane 3). It must be noted that the polycystin-1 NTF is more efficiently immunoprecipitated under these experimental conditions. The reciprocal experiment likewise reveals an annexin A5 co-immunoprecipitation with polycystin-1 however only following DSP cross-link (Figure 2(c), lane 2). Similarly, polycystin-1 coprecipitates with annexin A5 antibodies from lysates of PKD1-F plasmid6 transfected HeLa cells after a mild cross-link (Figure 2(d), lane 2) and reciprocally, ANXA5 can be detected in anti-M2-FLAG coimmunoprecipitates of polycystin-1 from the same lysates (Figure 2(e), lane 2). Bands at 25 kDa observed in Figure 2(c) and (e) and 50 kDa bands in Figure 2(e) across all lanes, accordingly, correspond to the light and heavy chains of the immunoprecipitating antibodies. Staining of blotted coimmunoprecipitation products from relevant after cross-link cell lysates with antibodies against transferrin receptor produced no signal. This suggests that the mild cross-linking applied did not result in unspecific binding to abundant transmembrane proteins. Surface binding of exogenous annexin A5 to polycystin-1 expressing PKD1–19 MDCK cells Expression of polycystin-1 in tetracycline-induced PKD1–19 MDCK cells was verified by Western blot of cell lysates and immunoprecipitated fractions (Figure 2(a)), as well as by immunofluorescence employing M2 anti-FLAG and anti-PKD1-LRR anti-
Interaction of Annexin A5 with Polycystin-1
bodies (Figure 3(a) and (b)). M2 anti-FLAG antibody staining of induced cells reveals some membrane localization in the region of cell–cell contacts as well as a vesicular intracellular signal (Figure 3(a), “PKD1 on”). In contrast, non-induced cells do not show any appreciable polycystin-1 staining over the background under these experimental conditions (Figure 3(a), “PKD1 off”). Similar results were obtained employing the anti-PKD1-LRR antibodies on non-permeabilized PKD1–19 cells. In addition to a general background signal seen on PKD1 on and PKD1 off cells (Figure 3(b)), this antibody produces a specific staining of cell contact regions in PKD1 on but not PKD1 off cells (arrow). To verify the binding of extracellular annexin A5 to cells expressing polycystin-1 in an inducible manner, PKD1–19 MDCK cells were induced for heterologous polycystin-1 expression, subjected to Ca2+ starvation and then incubated with fluorescently labeled annexin A5 together with normal Ca2+-containing medium for different periods of time. Since significant differences in the subcellular distribution of polycystin-1 were noted between 1 h and 2 h of Ca2+-replenishment (see below), we analyzed annexin A5 binding 2 h after the Ca2+ switch. Figure 4(a) shows confocal images of polycystin-1 expressing (PKD1 on) and non-expressing (PKD1 off) cells, stained with E-cadherin antibodies as a marker for adherens junctions (red channel) and fluorescent annexin A5 (Oregon Green 488 labeled, green channel). To ensure the intact physicochemical properties of stoichiometrically labeled annexin A5, phospholipid binding affinities were
Figure 3. Immunofluorescence staining of PKD1–19 MDCK cells, non-induced (PKD1 off) or induced (PKD1 on) to express polycystin-1 with (a) M2 anti-FLAG antibodies; (b) anti-PKD1-LRR antibodies. Arrow points at cell–cell contact staining. Scale bars represent 5 μm.
Interaction of Annexin A5 with Polycystin-1
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Figure 4. Annexin A5 binding to the surface of PKD1–19 MDCK cells, non-induced (PKD1 off) or induced (PKD1 on) to express polycystin-1. (a) Detection of fluorescent annexin A5, bound to the cell surface 2 h after Ca2+ switch. E-cadherin (red) marks cell contacts and the green fluorescent signal corresponds to Oregon Green 488-labeled annexin A5. Scale bars represent 5 μm. (b) Quantification of the annexin A5 fluorescent signal detected on the surface of non-permeabilized cells, integration per high-powered field for PKD1 off and PKD1 on cells. Light intensity per pixel is in arbitrary units. PKD1 on cells concentrate more green fluorescent label (n = 4 × 5 HPF, values are means ± SE, *P < 0.05 PKD1 off, t-test). (c) Annexin A1 binding to the surface of PKD1–19 MDCK cells, non-induced (PKD1 off) or induced (PKD1 on) to express polycystin-1. Annexin A1 was detected with fluorescently labeled anti-annexin A1 antibodies (green) 2 h after Ca2+ switch. Cell–cell contacts are marked by E-cadherin (red). Scale bar represents 5 μm.
compared for the labeled and non-labeled protein and identical results were obtained (not shown). Retention of annexin A5 on the cell surface of polycystin-1 expressing cells could be observed to a significant extent only in PKD1 on cells (Figure 4(a), right panel). The fluorescent signal of annexin A5 binding seems to be clustered at E-cadherin positive cell–cell contacts of the polycystin-1 expressing cells. To rule out the possibility that binding of annexin A5
to the surface of PKD1 on cells is due to phosphatidylserine exposure resulting from an enhanced apoptosis rate in these cells, we measured apoptosis by propidium iodide staining and flow cytometry of cell nuclei from PKD1 on and PKD1 off monolayers. No significant differences were observed (results not shown), strongly suggesting that the increased surface binding of annexin A5 is due to polycystin1 expressed at the cell surface. A quantitative
960 comparison of annexin A5 fluorescence signal intensities on the surface of PKD1 off and PKD1 on cells, integrated per pixel of high powered field, is presented in Figure 4(b). On average, 25–50% more annexin A5 binding was observed for polycystin-1 expressing cells as compared to similar cells in noninduced state. Annexin A1, a protein of the same annexin superfamily, has a homologous C-terminal “core” region and a distinct, unrelated N-terminal domain and was used as negative control for the in vitro binding experiments. Non-permeabilized PKD1 off and PKD1 on cells do not show any labeling signal after incubation with annexin A1 and staining with anti-annexin A1 antibodies (Figure 4(c)). In contrast, PKD1 on cells incubated with fluorescent annexin A5 under the same experimental conditions retain fluorescent signal on their surface, to some extent clustered at cell–cell contacts (Figure 4(a)). Polycystin-1 and E-cadherin recruitment to cell junctions of polycystin-1 expressing cells Since some enrichment of the annexin A5 fluorescence label was observed at or close to cell junctions, we performed E-cadherin and polycystin-1 costaining to visualize the subcellular distribution of both proteins upon Ca2+ replenishment. The subcellular localization was recorded 1, 2 and 4 h after Ca2+ replenishment. Figure 5 reveals that at all time points the two fluorescence signals largely overlap, with some differences in cytosolic staining pattern of Ca2+ -starved cells. Upon Ca 2+ starvation, both proteins are sequestered in cytosolic pools with perinuclear location and vesicles dispersed in the cytoplasm. Recruitment of E-cadherin and polycystin-1 to cell junctions, which was triggered by Ca2+ replenishment, was rather advanced at 1 h and almost complete at 2 h after the Ca2+ switch. Cells cultivated for 4 h following Ca2+ replenishment show the same distribution of E-cadherin and polycystin-1. Altogether we conclude that (a) polycystin-1 and E-cadherin show a high degree of colocalization in MDCK cells (as observed before16) and (b) polycystin-1 translocation to reforming cell– cell contacts follows the same overall kinetics as E-cadherin recruitment. Annexin A5 affects E-cadherin recruitment to cell junctions in polycystin-1 expressing cells To elucidate possible effects of the annexin A5/polycystin-1 interaction on the recruitment of E-cadherin to reforming cell adhesion contacts, we performed Ca2+ switch experiments in the presence and absence of annexin A5 added to PKD1–19 cells induced or not induced to express polycystin-1. As noted above, PKD1–19 MDCK cells do express some basal levels of polycystin-1 in non-induced state (Figure 2(a) and (b)), non-detectable by immunofluorescence staining. For E-cadherin recruitment we actually compared PKD1 off cells weakly expressing polycystin-1 with PKD1 on cells
Interaction of Annexin A5 with Polycystin-1
overexpressing the protein. Monolayers were stained for E-cadherin and its subcellular distribution was recorded 1 h and 2 h after Ca2+ replenishment. Typical images of annexin A5 treated and untreated PKD1 on and PKD1 off cells are shown in Figure 6(a). There was no notable difference in the subcellular distribution of E-cadherin between annexin A5 treated versus untreated PKD1 off cells. Interestingly in PKD1 on cells extracellular Ca2+ replenishment seemed to trigger a more rapid E-cadherin recruitment as compared to PKD1 off monolayers (see for examples the 1 h panels of Figure 6(a)). This rapid recruitment of E-cadherin was reduced to rates characteristic for PKD1 off monolayers when annexin A5 was added to PKD1 on cells. The effect of annexin A5 on E-cadherin recruitment to junctions of polycystin-1 expressing cells was also quantified by expressing the ratio of cell junctions, positively stained for E-cadherin, to the number of scored cells (Figure 6(b)). This “junction index”, I, can vary between 0 and 2 (see Materials and Methods) and represents an adequate measure of E-cadherin appearance in cell junctions. In PKD1 on cells I was generally higher than in PKD1 off cells. These differences for the 1 h and 2 h time points amounted to 60–70% and to 45–55%, respectively. When annexin A5 was added to PKD1 on cells, these differences were basically abolished (Figure 6(b)). Thus, the stimulating effect of polycystin-1 on the recruitment of E-cadherin to reforming cell contacts is abrogated by annexin A5, most likely due to the direct binding of annexin A5 to the N-terminal LRR domain of polycystin-1. Annexin A1 added together with normal Ca2+containing medium for different periods of time to PKD1–19 MDCK cells induced or not induced to express polycystin-1, subjected to calcium starvation, did not have any effect on the rates of Ecadherin recruitment as indicator of cell junction reestablishment (results not shown). Annexin A5 levels are elevated in cystic fluids of ADPKD patients To assess a possible influence of the annexin A5/ polycystin-1 interaction on extracellular annexin A5 concentrations in the relevant organ, we undertook measurements of annexin A5 levels in cystic fluids of patients with ADPKD and patients with renal solitary cysts, with similar progression of kidney function deterioration (Figure 7). The average annexin A5 levels we measured in fluids of solitary cysts amount to 0.86 ng/ml (n = 11). In contrast, mean annexin A5 levels in cystic fluids of ADPKD patients are 3.53 ng/ml (n = 3), or fourfold higher as compared to solitary cysts.
Discussion By employing a genetic screen we identified a novel protein ligand of the extracellular LRR domain of polycystin-1. The Ca2+ and phospholipid
Interaction of Annexin A5 with Polycystin-1
961
Figure 5. Ca2+ switch experiments using PKD1–19 MDCK cells induced to express polycystin-1. The red fluoreecent signal shows anti-FLAG M2 antibody staining for polycystin-1 and green fluorescence marks E-cadherin antibody staining. Medium containing normal Ca2+ concentrations was replenished for 1 h, 2 h and 4 h after Ca2+ starvation. Cells not subjected to Ca2+ starvation, i.e. kept in medium containing normal Ca2+, are shown in the bottom panels. Scale bar represents 5 μm. PKD1 off cells did not show any appreciable polycystin-1 anti-FLAG M2 antibody staining over the background.
binding protein annexin A5 showed a specific interaction with the LRRs of polycystin-1, mediated through the VLRGTV sequence located at the very amino-terminal end of the annexin protein, spanning residues 4–9. Annexin A5 is a member of the annexin protein superfamily, for which extracellular localizations have been documented in addition to its expression in various cells and tissues.21 The
sequence xxRGTV at the very N terminus of annexin A5 is conserved in vertebrates, from zebrafish through chicken up to various mammalian species. Crystallography studies of human and rat annexin A5 show that residues 1–15 are in an extended conformation. 22,23 Likewise, the two complete LRRs of polycystin-1, constrained by the cysteine-rich flanking regions, should fold as a
962
Interaction of Annexin A5 with Polycystin-1
Figure 6. Effect of annexin A5 on E-cadherin recruitment to cellular junctions of polycystin-1 expressing PKD1–19 MDCK cells. (a) Immunofluorescence staining revealing the subcellular distribution of E-cadherin, 1 h and 2 h after Ca2+ replenishment of Ca2+ starved PKD1–19 MDCK cells. Experiments were performed on cells either non-induced or induced for polycystin-1 expression, with and without the addition of annexin A5. Figure scale bar represents 5 μm. (b) Quantification of cell contact-staining with E-cadherin antibodies for the different experimental conditions, using junction index (see Materials and Methods) as measure for the cell contact recruitment of E-cadherin. PKD1 on cells have a higher junction index (*P < 0.05, PKD1 on versus PKD1 off cells, values are means ± SE, t-test, n = 4 × 5 HPF). PKD1 on cells treated with ANXA5 have lowered junction index, similar to PKD1 off cells (**P < 0.05, PKD1 on+ ANXA5 versus PKD1 on cells, values are means ± SE, t-test, n = 4 × 5 HPF).
separate unit and thus be accessible for ligand binding.8,9 The specificity of protein–protein interactions mediated by LRRs is likely to be provided by the hydrophilic, solvent accessible amino acids located between the repeated aliphatic residues.24 Such structure allows for a “sequential” rather than “conformational” mode of recognition, which in turn supports the notion of a specific “epitope”
involved in the interaction. Three lines of evidence indicate that the in vitro binding of annexin A5 to the LRR domain of polycystin-1 does not require the homologous annexin repeat sequences, which are conserved among annexins and constitute the C-terminal annexin core domain. First, the peptide sequence VLRGTV is capable of binding alone. Second, the carboxy-terminal portion of annexin A5
Interaction of Annexin A5 with Polycystin-1
Figure 7. Concentrations of annexin A5 (ng/ml), measured using standard ELISA, in solitary and ADPKD cystic fluids. Solitary cysts samples, n = 11; ADPKD cysts samples, n = 3.
used for the binding experiment shown in Figure 1(b) contains two annexin repeats but does not interact. Third, another annexin protein (annexin A1) though containing highly homologous annexin repeats does not bind to the polycystin-1-LRR (Figure 1(a)). The in vitro interaction of polycystin-1 with annexin A5 was confirmed by co-immunoprecipitation experiments, following addition of annexin A5 to polycystin-1 expressing cells. However detection of the respective partner of polycystin-1 in the immunoprecipitates required a mild in vivo crosslink. Having in mind that this interaction depends on a short peptide sequence at least in annexin A5, the cross-link most likely stabilizes the peptidemediated polycystin-1/annexin A5 interaction enabling co-immunopecipitation under the conditions chosen. On the other hand, no chemical crosslink is necessary to detect co-immunoprecipitated polycystin-1 in Figure 2(b). The cell surface binding experiments with fluorescently labeled annexin A5 also support the specificity of interaction, since they reveal a specific enrichment of annexin A5 on the surface of PKD1 on cells whereas annexin A1, serving as a negative conrol, shows no appreciable binding (Figure 4(c)). The relatively large variation (Figure 4(b)) can be explained with differing degrees of polycystin-1 expression among induced cells. Since externally bound annexin A5 was enriched at or close to cell junction regions of PKD1 on cells (Figure 4), we examined the subcellular distribution of polycystin-1 in Ca2+ switch experiments. Our results demonstrate a polycystin-1 recruitment to cell contacts upon Ca2+ replenishment (Figure 5) that follows the same kinetics as E-cadherin. Moreover, E-cadherin and polycystin-1 signals largely overlap also in intracellular structures. Association of polycystin-1 with E-cadherin has been reported previously14 and has been confirmed in more recent studies.16 Upon Ca2+ deprivation and subsequent replenishment, both proteins were targeted to cell junctions although a subfraction
963 could still be observed in cytosolic vesicles. Interestingly, the E-cadherin recruitment to cell junctions of PKD1 on cells proceeded with enhanced rate in comparison to PKD1 off monolayers (Figure 6). Cell surface levels of E-cadherin are generally regulated by membrane trafficking pathways and a “setting point” of E-cadherin turnover is the association with p120-catenin.25 Our data suggest that polycystin-1 can affect this trafficking, thereby accelerating the E-cadherin recruitment to reforming cell contacts. Altered E-cadherin recruitment dynamics to forming cell contacts in ADPKD epithelia after stress or injury, when disturbed due to lack of polycystin-1 function, could be at least partly responsible for the observed cysts phenotype. The effect of enhanced E-cadherin recruitment to reforming cell contacts is reversed by the addition of annexin A5 (Figure 6). This could be due either to disturbance of homophilic interactions between polycystin-1 extracellular regions of neighboring cells, as previously reported,26 or to an effect on the signal transduction properties of the polycystin-1 molecule. Such possible mechanisms would both involve the extracellular N-terminal region of polycystin-1 and thereby could be affected by annexin A5 binding to this domain. The physiological role of annexin A5 as a possible effector of polycystin-1-mediated junction reformation is in line with its abundant expression in kidney.27 Moreover, increased extracellular annexin A5 levels are observed in kidney disease patients and renal damage models.28,29 Our preliminary studies indicate significantly elevated annexin A5 levels in ADPKD cystic fluids, compared to fluids of solitary cysts upon progressing deterioration of kidney function (Figure 7). More studies with proper statistical backup are necessary, on renal cysts patients in various stages of kidney disease, to address the role of annexin A5 as a possible physiological factor. With this in mind, the identification of annexin A5 as a novel ligand of the polycystin-1 LRR domain affecting the polycystin-1 mediated recruitment of Ecadherin to reforming epithelial junctions could indicate a link between epithelial abnormalities and the dynamics of E-cadherin positive junctions.
Materials and Methods Recombinant expression of polycystin-1-LRR The LRR domain of polycystin-1 and its flanking regions (bp 290–813 of the cDNA sequence, acc. no. L33243) was SalI/NotI subcloned into the pGEX4T-2 expression vector (Amersham-Pharmacia Biotech, Freiburg, Germany). The sequence of the resulting construct was verified across the site of gene fusion before use. The LRR polycystin-1 polypeptide was expressed as a GST fusion protein (∼48 kDa) and coupled to glutathione-Sepharose 4B resin according to manufacturers instructions (Amersham-Pharmacia Biotech, Freiburg, Germany).
964 Selection of random peptides (peptide panning) A FliTrx random peptide display library (Invitrogen, Groningen, Netherlands) was grown and induced for expression according to the guidelines of the manufacturer. Glutathione-Sepharose bound polycystin-1-LRR was loaded into a polyalomere column (10 ml) and the induced peptide display library was passed through in five successive rounds of selection. Washes and elution of bound E.coli were performed under sterile conditions, according to the manufacturer's instructions. After the fifth round of selection the remaining clones were plated and individual clones were subjected to PCR. Plasmid inserts were directly amplified using the M13 sequencetailed oligonucleotides M13.FliTrxF 5′-CGACGTTGTAAAACGACGGCCAGTATTCACCT GACTGACGAC-3′ and M13.FliTrxR 5′-CAGGAAACAGCTATGACCCCTGATATTCGTCA GCG-3′. The underlined M13 sequences were added to facilitate sequencing with universal primers. Amplicons were purified and directly sequenced using M13 fluorescently labeled forward and reverse primers on an ALF Express automated sequencer (Amersham-Pharmacia Biotech, Freiburg, Germany). Computer analysis (homology searches) Peptide sequences obtained more than once were compared against the PDB and Swiss-Prot databases, using the Smith-Waterman homology search algorithm. Sequences of additional clones were aligned with the redundants using the maximum likelihood rule. In vitro transcription and translation Annexin A1 and annexin A5 templates for in vitro transcription and translation were obtained from cloned cDNAs. Transcription/translation reactions were performed on 250–500 ng of template using the T7 TNT™ Coupled Reticulocyte Lysate System (Promega, Mannheim, Germany) in the presence of [35S]methionine according to the manufacturer's instructions. In vitro binding assay (GST pull-down) About 0.2 μg protein immobilized on glutathioneSepharose was incubated overnight at 4 °C with programmed lysates (10 or 20 μl) in NTEN buffer (20 mM TrisCl (pH 8), 100 mM NaCl, 1 mM EDTA, 0.5% Nonidet P-40 (NP-40)) containing protease inhibitors (1 mM phenylmethylsulfonyl fluoride, 1 μg/ml leupeptin, 1 μg/ml pepstatin and 10 μg/ml aprotinin), 0.5 mM Na3VO4, 80 μM β-glycerophosphate and 10 mM NaF. Beads were washed four times in NTEN buffer at room temperature and then mixed with 0.2 volumes of 6× SDS loading buffer, boiled for 5 min and released proteins were analyzed by SDS-PAGE. Immunoprecipitation and Western blot analysis PKD1–19 MDCK cells were induced with tetracycline (1 μg/ml) to express polycystin-1 for 48 h. Induced and non-induced cells were washed thoroughly with 4 °C cold PBS, placed on ice, and 100 nM annexin A5, purified from human placenta (Sigma, Deisenhofen, Germany), and dissolved in PBS, was added for 1 h to cell monolayers. Cells were washed three times with cold PBS and a cross-
Interaction of Annexin A5 with Polycystin-1 linker, di-thio-bis(succinimidylpropionate) (DSP), (PiercePerbio Science, Bonn, Germany), was added at concentration of 100 μg/ml for an additional 20 min on ice. After thoroughly washing the monolayers, 0.2 M glycine/PBS was added for 5 min to quench traces of reactive crosslinker and cells were washed again 3× with PBS and harvested for immunoprecipitation and Western blotting. Cells were lysed in 10 mM sodium phosphate buffer with 0.5% Triton X-100, as described.6 Cleared lysates were incubated with 2 μg of sheep anti-annexin A5 antibodies (kind gift from John Dedman, Univeristy of Cincinnati, Ohio) for 1 h at 4 °C or with 50 μl of Red EasyView™ M2anti-FLAG agarose (Sigma, St. Louis, MO, USA) for 2 h at 4 °C. Fifty microliter of Protein G Sepharose beads (Amersham Biosciences, Freiburg, Germany) were added to the samples containing anti-annexin A5 antibodies and they were incubated for another hour at 4 °C. Immunoprecipitates were washed five times with 1 ml of lysis buffer and eluted in 60 μl of 1× Laemmli loading buffer. Eluted products (10–15 μl) were subjected to polyacrylamide gel electrophoresis (4% (w/v) acrylamide for anti-annexin A5 co-precipitates and 12% acrylamide for M2 anti-FLAG co-precipitates) and analyzed using standard Western blot protocols (ECL, Applichem, Darmstadt, Germany). The same protocol was previously applied to HeLa cells transiently transfected with the PKD1-F plasmid.6 Annexin A5 fluorescent labeling Recombinantly expressed annexin A5 was purified to homogeneity as assessed by silver-stained PAGE and Western blotting. The protein was labeled with Oregon Green 488 and Alexa 568-succinimidylester according to the manufacturers protocol (Molecular Probes, Leiden, Netherlands). The 1:1 stoichiometric complex of fluorescently labeled annexin A5 was purified from the mixtures by MonoQ chromatography with the Äcta Explorer system (Amersham Biosciences, Roosendaal, Netherlands). Cell-based assays Stably transfected (MDCK)-cell line for heterologous inducible expression of human polycystin-1 (clone PKD1– 19) was obtained from Gregory G. Germino (Johns Hopkins University, Baltimore, MD). Expression of endogenous polycystin-1 in these cell lines was very low and detectable only at the mRNA level. The inducible cell line PKD1–19 was employed in Ca2+ switch experiments. For these experiments, PKD1–19 cells, induced or non-induced to express polycystin-1, were grown to confluence on LabTek slide chambers (Nalge Nunc, Wiesbaden, Germany). Cells were then depleted of Ca2+ by washing with Ca2+and magnesium-free PBS and incubating for 16 to 18 h in a Ca2+/glutamine-free DMEM medium (Invitrogen, Karlsruhe, Germany), supplemented with 10% (v/v) dialyzed foetal bovine serum (FBS) and 3 μM Ca2+ chloride. Ca2+containing normal DMEM with 10% FBS was then replenished, with or without 100 nM fluorescently labeled annexin A5. Cells were removed at different time points and processed for immunostaining. Immunofluorescence PKD1–19 MDCK cells, induced or non-induced to express polycystin-1 were washed 3 x in PBS, then fixed
Interaction of Annexin A5 with Polycystin-1 with 4% paraformaldehyde in PBS (pH 7.5) for 10 min, followed by incubation in 50 mM NH4Cl/PBS for 5 min and 1.5% goat serum (Sigma, Deisenhofen, Germany) in PBS for 20 min. Non-permeabilized cells were incubated with anti-PKD1-LRR rabbit polyclonal antibodies10 for 30 min, washed six times with PBS and and stained with Texas Red™- conjugated goat anti-rabbit immunoglobulin (Ig)G antibodies (Jackson ImmunoResearch Laboratories, West Grove, PA, USA) for 20 min. Specimens were washed six times with PBS and then mounted for imaging. Cells to be stained employing M2 anti-FLAG antibodies were permeabilized for 20 min with 0.1% Triton X-100 and 1.5% goat serum in PBS after fixation. Specimens were incubated with M2 anti-FLAG mouse monoclonal antibodies (Sigma, Deisenhofen, Germany), washed six times with 0.1% Triton X-100 in PBS and stained with Texas Red™- conjugated goat anti-mouse IgG Fab' fragments (Jackson ImmunoResearch Laboratories, West Grove, PA, USA) for 20 min. Specimens were washed six times with 0.1% Triton X-100 in PBS and then mounted for imaging. PKD1–19 cells, induced or non-induced to express polycystin-1 were washed 3 x in PBS and incubated with PBS solutions of 100 nM fluorescent annexin A5 or with 100 nM recombinantly expressed and purified annexin A1 for 1 h, then fixed with 4% paraformaldehyde and quenched as indicated above. Annexin A1-treated cells were then incubated with mouse anti-annexin A1 monoclonal antibodies (BD Transduction Laboratories, Lexington, KY, USA) for 30 min, PBS washed and stained with Cy2™-conjugated goat anti-mouse IgG antibodies (Jackson ImmunoResearch Laboratories, West Grove, PA, USA) for 20 min. Cells were permeabilized and specimens were then incubated with rat monoclonal DECMA-1 (anti-Ecadherin, Sigma, Deisenhofen, Germany) antibodies for 30 min, washed (see above) and stained with Texas Red™conjugated goat anti-rat IgG antibodies (Jackson ImmunoResearch Laboratories, West Grove, PA, USA) for 20 min. Specimens were washed six times with 0.1% Triton X-100 in PBS and then mounted for imaging. For E-cadherin/polycystin-1 staining permeabilized cells were incubated first with rat monoclonal DECMA-1 (anti-E-cadherin, Sigma, Deisenhofen, Germany) antibodies, stained with Cy2™-conjugated goat anti-rat IgGs (Jackson ImmunoResearch Laboratories, West Grove, PA, USA), then incubated with M2 anti-FLAG monoclonal antibodies (Sigma, Deisenhofen, Germany) and stained with Texas Red™-conjugated goat anti-mouse IgG Fab' fragments (same supplier). Specimens were washed six times with 0.1% Triton X-100 in PBS and then mounted for imaging. Confocal laser scanning microscopy was performed on a Zeiss LSM 510 Meta microscope (Carl Zeiss, Göttingen, Germany). The PKD1 off cells, as well as a control cell line clone, MDCKpcDNA.Fcγ, which was stained in tetracyclineinduced or non-induced state, did not show any staining over the background with the M2 anti-FLAG antibodies or with fluorescently labeled annexin A5 under these experimental conditions. Quantification of annexin A5 surface binding and E-cadherin recruiment For each time point, five high-powered fields (HPF) amounting to 200 to 250 cells, from four different experiments were scored for annexin A5 binding or E-cadherin recruitment to cell junctions. Fluorescent images of cells, replenished with Ca2+-containing normal
965 medium for 1 and 2 h, were taken with the same settings and magnification, and image files were transferred for evaluation with MetaMorph imaging software (Universal Imaging Corporation, Marlow, UK). Light intensities of surface bound Oregon Green 488-labeled annexin A5 were measured in the green channel for polycystin-1-induced and non-induced cells and integrated per HPF (1024 × 1024 pixel). E-cadherin recruitment to cell junctions was scored as junction index, I, where I = J/N. J is the number of positively stained cell junctions (scored manually as membrane outlines), divided by the number of cells in the field, N (usually 30–50). It can be easily shown that for a regular polygonal array of cells, when counting the sideto-side and corner junctions, but not the external outlines, Imax = 2n/n+1, where n is the polygon sides number. Thus, I ∈ (0.2). Measurement of annexin A5 concentrations in cystic kidney fluids Material for analysis was collected from renal disease patients referred for treatment to the Clinic of Nephrology and Haemodialysis at the Medical University Pleven. Informed consent was obtained from all subjects examined and the study complied with the ethical guidelines of all institutions involved. Members of the studied group had comparable progression of kidney disease and similar decline of renal function as assessed by relevant clinical tests. Cystic fluids were drawn upon subcutaneous puncture with a syringe from patients with solitary kidney cysts and such with ADPKD cysts as a first step in a standard therapeutic procedure of subsequent alcoholic cyst cauterization. Obtained fluids (typically 3 to 5 ml volume) were centrifuged at 100,000 g for 15 min at 4 °C to remove cell remnants. Concentrations of soluble annexin A5 were measured in samples of the supernatants following standard ELISA protocol.30
Acknowledgements This work was supported by grants from the Deutsche Forschungsgemeinschaft (DFG) to B. D., J. H. and to V. G. and from Interdisziplinäres Zentrum für Klinische Forschung (IZKF), Universitätsklinikum Münster, to B. D., J. H. and to V. G. We are grateful to Gregory G. Germino and Feng Qian for the continuous support with crucial reagents and the critical reading of the manuscript. We thank Jonathan Tait for providing the annexin A5 cDNA plasmid, John Dedman for the anti-annexin A5 antibodies, Klaus Ebnet for the Ca2+ switch protocol and valuable discussions, Carsten Lange for assistance with the flow cytometer, and Ursula Rescher for the help with MetaMorph.
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S. A. & Wandinger-Ness, A. (2004). A polycystin-1 multiprotein complex is disrupted in polycystic kidney disease cells. Mol. Biol. Cell, 15, 1334–1346. Parnell, S. C., Magenheimer, B. S., Maser, R. L., Rankin, C. A., Smine, A., Okamoto, T. & Calvet, J. P. (1998). The polycystic kidney disease-1 protein, polycystin-1, binds and activates heterotrimeric G-proteins in vitro. Biochem. Biophys. Res. Commun. 251, 625–631. Delmas, P., Nomura, H., Li, X., Lakkis, M., Luo, Y., Segal, Y. et al. (2002). Constitutive activation of G-proteins by polycystin-1 is antagonized by polycystin-2. J. Biol. Chem. 277, 11276–11283. Bhunia, A. K., Piontek, K., Boletta, A., Liu, L., Qian, F., Xu, P. N. et al. (2002). PKD1 induces p21(waf1) and regulation of the cell cycle via direct activation of the JAK-STAT signaling pathway in a process requiring PKD2. Cell, 109, 157–168. Li, X., Luo, Y., Starremans, P. G., McNamara, C. A., Pei, Y. & Zhou, J. (2005). Polycystin-1 and polycystin-2 regulate the cell cycle through the helix-loop-helix inhibitor Id2. Nature Cell Biol. 7, 1202–1212. Gerke, V. & Moss, S. (2002). Annexins: from structure to function. Phys. Rev. 82, 331–371. Huber, R., Berendes, R., Burger, A., Schneider, M., Karshikov, A., Luecke, H. et al. (1992). Crystal and molecular structure of human annexin V after refinement. Implications for structure, membrane binding and ion channel formation of the annexin family of proteins. J. Mol. Biol. 223, 683–704. Concha, N. O., Head, J. F., Kaetzel, M. A., Dedman, J. R. & Seaton, B. A. (1993). Rat annexin V crystal structure: Ca2+ -induced conformational changes. Science, 261, 1321–1324. Kobe, B. & Deisenhofer, J. (1995). A structural basis of the interactions between leucine-rich repeats and protein ligands. Nature, 374, 183–186. Kowalczyk, A. P. & Reynolds, A. B. (2004). Protecting your tail: regulation of cadherin degradation by p120catenin. Curr. Opin. Cell. Biol. 16, 522–527. Streets, A. J., Newby, L. J., O'hare, M., Bukanov, N. O., Ibraghimov-Beskrovnaya, O. & Ong, A. C. (2003). Functional analysis of PKD1 transgenic lines reveals a direct role for polycystin-1 in mediating cell–cell adhesion. J. Am. Soc. Nephrol. 14, 1804–1815. Markoff, A. & Gerke, V. (2005). Expression and functions of annexins in the kidney. Am. J. Physiol. Renal Physiol. 289, F949–F956. Matsuda, R., Kaneko, N., Horikawa, Y., Chiwaki, F., Shinozaki, M., Abe, S. et al. (2000). Measurement of urinary annexin V by ELISA and its significance as a new urinary-marker of kidney disease. Clin. Chim. Acta, 298, 29–43. Matsuda, R., Kaneko, N., Horikawa, Y., Chiwaki, F., Shinozaki, M., Ieri, T. et al. (2001). Localization of annexin V in rat normal kidney and experimental glomerulonephritis. Res. Exp. Med. (Berl), 200, 77–92. Van Heerde, W. L., Reutelingsperger, C. P., Maassen, C., Lux, P., Derksen, R. H. & De Groot, P. G. (2003). The presence of antiphospholipid antibodies is not related to increased levels of annexin A5 in plasma. J. Thromb. Haemost. 1, 532–536.
Edited by J. Karn (Received 13 February 2007; received in revised form 21 March 2007; accepted 27 March 2007) Available online 31 March 2007
doi:10.1016/j.jmb.2007.03.070
Annexin A5 Interacts with Polycystin-1 and Interferes with the Polycystin-1 Stimulated Recruitment of E-cadherin into Adherens Junctions Arseni Markoff 1 ⁎, Nadia Bogdanova 2 , Markus Knop 1 , Claas Rüffer 1 Heidi Kenis 3 , Petra Lux 3 , Chris Reutelingsperger 3 , Vassil Todorov 4 Bernd Dworniczak 2 , Jürgen Horst 2 and Volker Gerke 1 1
Institute of Medical Biochemistry, WestfalianWilhelms University of Muenster, Von Esmarch Str. 56, 48149 Muenster, Germany 2
Institut für Humanegenetik, University Clinics Muenster, Vesaliusweg 12-14, 48149 Muenster, Germany
3
Department of Biochemistry, Cardiovascular Research Institute Maastricht, P.O. Box 616, 6200 MD Maastricht, Netherlands 4
Clinic of Nephrology and Haemodialysis, Medical University, Pleven, Bulgaria *Corresponding author
Polycystin-1 is the gene product of PKD1, the first gene identified to be causative for the condition of autosomal dominant polycystic kidney disease (ADPKD). Mutations in PKD1 are responsible for the majority of ADPKD cases worldwide. Polycystin-1 is a protein of the transient receptor potential channels superfamily, with 11 transmembrane spans and an extracellular N-terminal region of ∼3109 amino acid residues, harboring multiple putative ligand binding domains. We demonstrate here that annexin A5 (ANXA5), a Ca2+ and phospholipid binding protein, interacts with the N-terminal leucine-rich repeats of polycystin-1, in vitro and in a cell culture model. This interaction is direct and specific and involves a conserved sequence of the ANXA5 N-terminal domain. Using Madin-Darby canine kidney cells expressing polycystin-1 in an inducible manner we also show that polycystin-1 colocalizes with E-cadherin at cell–cell contacts and accelerates the recruitment of intracellular E-cadherin to reforming junctions. This polycystin-1 stimulated recruitment is significantly delayed by extracellular annexin A5. © 2007 Elsevier Ltd. All rights reserved.
Keywords: ANXA5; annexin A5; PKD1; polycystin-1; E-cadherin
Introduction Polycystins-1 and − 2 are the respective products of PKD1 and PKD2, genes responsible for the condition of autosomal dominant polycystic kidney disease (ADPKD). PKD1 mutations cause ADPKD in the majority of cases, leading to severe impairment of kidney function and accompanying systemic damage.1,2 Both polycystins interact via their cytosolic C-terminal regions3,4 and are integral parts of a common signal transduction pathway.1 Polycystin-1 (TRPP1) is the only 11 transmembrane domainscontaining protein that belongs to the superfamily of transient receptor potential channels (TRPs), normally characterized by six transmembrane spans. Abbreviations used: ADPKD, autosomal dominant polycystic kidney disease; LRR, leucine-rich repeat; GCT, glutathione-S-transferase. E-mail address of the corresponding author: [email protected]
Recent studies support possible mechanosensory function for TRPP1, which can be mediated by elastic properties of the extracellular region.5 The assembly of polycystins-1 and − 2 at the plasma membrane, when co-expressed in cultured CHO cells, has been reported to produce unique Ca2+-permeable nonselective currents.6 Moreover, recent studies demonstrate that the expression of polycystin-1 alone can result in such cation currents.7 Polycystin-1 consists of a large, extracellular Nterminal region of ∼ 3109 amino acid residues, 11 predicted transmembrane domains (∼ 993 amino acid residues), and a short, cytosolic C-terminal portion (∼ 200 amino acid residues).8,9 The N-terminal sequence of the protein harbors several domains predicted to represent ligand interaction sites: two leucine-rich repeats (LRRs), a C-type lectin domain, an LDL-A like domain, 15 copies of PKD domains, which are immunoglobulin (Ig)-like repeats, and a region homologous to the sea urchin sperm protein, REJ.9 The protein is cleaved specifically at position T3049, which releases an extracellular N-terminal
0022-2836/$ - see front matter © 2007 Elsevier Ltd. All rights reserved.
Interaction of Annexin A5 with Polycystin-1
fragment of ∼ 325 kDa.10 This fragment is tethered to the cell surface and the cleavage reaction is probably essential for the function of polycystin-1. There is accumulating evidence supporting regulatory function of polycystin-1 in organizing the cytoarchitecture of tubular structures in kidney and liver. Gene-targeting experiments indicate functions of polycystin-1 in the terminal differentiation of tubular epithelial cells11 and in the maintenance of blood vessels' structural integrity.12 Heterologous expression of polycystin-1 in a MDCK cell culture model results in slower growth, promotes resistance to apoptosis and induces spontaneous tubulogenesis.13 In further support for a role in cytoarchitecture, polycystin-1 has been identified in a complex with the cell adhesion molecules E-cadherin and α, β and γ-catenin14 and compromised cytoarchitecture has been demonstrated in ADPKD epithelia, characterized by subcellular sequestration of E-cadherin to an internal cellular compartment, among other protein mislocalizations.15 Furthermore a disruption of the polycystin-1 association with E-cadherin and the catenins is observed in primary cells from ADPKD patients as a result of increased polycystin-1 phosphorylation.16 Polycystin-1 also has the capacity of transducing signals from the extracellular environment to the inside of the cell, thereby regulating major aspects of cellular fate, such as terminal differentiation and cell polarization. The cytosolic C terminus of the protein has been shown to be a site of interaction with heterotrimeric G-proteins,17 which is underpinning a function for polycystin-1 as a G protein-coupled receptor.18 Moreover, polycystin-1 has been demonstrated to up-regulate p21, thereby inducing cellcycle arrest in a manner depending on polycystin-2 interaction.19 This p21 induction can be suppressed by the helix-loop-helix inhibitor Id2, which directly interacts with polycystin-2.20 Here we sought to identify interacting partners of polycystin-1 N-terminal region, concentrating on the LRRs at the very tip of the N-terminal sequence. Our studies show that annexin A5, a Ca2+ and phospholipids binding protein, is able to interact with the LRR domain of polycystin-1. In a cell culture model, inducible heterologous expression of polycystin-1 leads to more rapid recruitment of E-cadherin to cell junctions forming after a Ca2+ switch protocol. This enhanced mobilization of E-cadherin is inhibited by annexin A5, indicative of a function of the polycystin-1/annexin A5 interaction in regulating the formation of epithelial cell contacts.
Results Identification of a peptide ligand of the polycystin-1-LRR The polycystin-1-LRR domain with flanking regions (amino acid residues 27–203) was expressed
955 as a recombinant glutathione-S-transferase (GST) fusion protein in Escherichia coli, attached to glutathione-Sepharose, and used as a bait for selection of E.coli clones, displaying on their flagella random dodecapeptides encoded by the pFliTrx plasmid. Thirty insert-containing clones were selected through this procedure, their inserts were PCR amplified and sequenced. Among the selected clones, one clone emerged in duplicate. Its amino acid sequence QNMKALRGTVCV was queried for homology against the Protein Data Bank and SwissProt databases. This revealed significant identity with the ALRGTV sequence present at the very amino terminus of rat annexin A5 (pos. 1–6). In human annexin A5 this sequence is VLRGTV (pos. 4–9). Maximum likelihood alignment was obtained in six of the selected clones by random positioning of the ALRGTV segment (Table 1). The consensus, which can be deduced from the sequence alignment of clones selected is xLRG/xTV, where x is an aliphatic amino acid. Annexin A5 binds to the LRR domain of polycystin-1 in vitro In order to test a possible binding of annexin A5 to the LRR domain of polycystin-1, in vitro translated full-length annexin A5 was subjected to binding assays employing the (GST)-polycystin-1LRR. In vitro translation of annexin A5 resulted in a product, which separated in two bands on SDSPAGE (Figure 1(a), lane 3). The upper band is the result of an additional upstream translation initiation codon, generated by the amplification of the cDNA, whereas the lower band corresponds to authentic annexin A5, showing a relative molecular mass of 33 kDa. Analysis of the GST pull-down revealed binding of in vitro translated annexin A5 to the LRR domain of polycystin-1. Retained annexin A5 signal in lane 4 represents a 33.5% fraction of the input (right panel). Although exact quantification of protein stoichiometry in this type of assay is not possible, it appears that annexin A5 is present in a molar excess, since the amount of polycystin-1-LRR-bound annexin A5 retained under saturating binding conditions (lane 5) is reduced by only 4% when half of the in vitro translation product is used (lane 4 and right panel). Thus, in vitro translated annexin A5 shows Table 1. Sequences of six selected clones, using the biopanning procedure on the FliTrx random peptide display library with the polycystin-1 LRR as a bait QNMKALRGTVCVa QKNQVARGTVCG QFIALRGTVQSSL ATRGLRVTVTGN LQIVFSALRVTV SDSGGPLRVTAY Consensus amino acids are marked bold, aliphatic residues in consensus strings are underlined. a This clone was selected twice.
956
Interaction of Annexin A5 with Polycystin-1
Figure 1. Left panels: in vitro binding assays (GST pull-down) using (GST)PKD1-LRR as bait. Autoradiographs of [35S] methionine labeled in vitro translation products and their retained fractions after binding. Lanes, marked with (−) are in vitro translation controls and (+) lanes correspond to proteins of the in vitro translation reaction that bound to (GST)PKD1LRR. (a) Lane 1, in vitro translated ANX A1; lane 2, bound fraction; lane 3, in vitro translated ANX A5; lane 4, bound fraction, 0.5 of saturation input; lane 5, bound fraction, saturation input. (b) Lane 1, in vitro translated ANX A5, N-terminal fragment (see Materials and Methods); lane 2, bound fraction; lane 3, in vitro translated ANX A5, C-terminal fragment; lane 4, bound fraction; lane 5, bound fraction of in vitro translated ANX A5 ((a), lane 3) incubated with GST-Sepharose: no ANX A5 binding to the GST-beads alone. Right panels: signal intensities quantification of protein bands, using the NIH Image utility. Signal intensities were recorded as “mean average” of measured band areas, background values were subtracted and values were expressed as percent fractions, normalized to a “standard”, the most intensive signal occurring in a gel image. (a) Signal intensities were normalized to the lower band in lane 3 (100%), corresponding to the relative molecular mass of native annexin A5. Signal intensity of the band in lane 2 was not exceeding the background. (b) Signal intensities were normalized to the band in lane 1 (100%), corresponding to the N-terminal portion of in vitro translated annexin A5. Signal intensity of the band in lane 4 was 0.003% and 0.004% of the input signal (lane 3).
significant binding to immobilized polycystin-1LRR, in contrast to annexin A1 (a ∼ 37 kDa protein, lane 1), which has an unrelated N-terminal sequence and was used as a control in these experiments, and did not show any binding (lane 2 and right panel). To corroborate that this binding is due to the annexin A5 N-terminal domain, harboring the VLRGTV sequence, a 206 amino acid residue amino-terminal fragment and a 185 amino acid residue carboxyterminal fragment of the protein were subjected to in vitro binding assays (Figure 1(b)). Appreciable binding was observed when lysates containing the N-terminal 206 amino acid residues of annexin A5 (∼ 23 kDa, lane 1) were probed in the GST pull-down (lane 2). The signal intensity of retained annexin A5 N-terminal portion was 21.3% of the input (right panel). The carboxy-terminal portion of annexin A5 (∼ 21 kDa, lane 4) did not show any appreciable binding. Neither did GST alone bind to in vitro translated annexin A5 (lane 5 and right panel).
To address the relevance of the in vitro binding experiments with the LRR domain of polycystin-1, we carried out co-immunoprecipitation following addition of annexin A5 to PKD1-transfected HeLa cells and to PKD1-19 MDCK cells, which express a FLAG-tagged version of polycystin-1 in a tetracycline-inducible manner. Both, M2 anti-FLAG antibodies and anti-annexin A5 antibodies were employed in parallel experiments. Immunoprecipitations were performed after washing unbound material off the cell surface and, in one series of experiments, after applying moderate amounts of a mild cleavable cross-linker, DSP, to stabilize protein interactions. Expression of polycystin-1 was verified through immunoprecipitation and detection of the protein with anti-PKD1-LRR antibodies (Figure 2(a)). The ∼ 520 kDa band in the immunoprecipitates and cell lysates of PKD1–19 MDCK cells induced to express polycystin-1 corresponds to full-length polycystin-1 and the ∼ 325 kDa band represents a
Interaction of Annexin A5 with Polycystin-1
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Figure 2. Immunoprecipitations of polycystin-1 and annexin A5 after chemical cross-link. Table indicates presence or lack of polycystin-1 expression and addition or absence of annexin A5, in the different experiments (a, b, c). a, b, c, Immunoprecipitation products from lysates of PKD1–19 MDCK cells in non-induced or induced state. (a) Polycystin-1 immunoprecipitations with M2 anti-FLAG antibodies. La, Lb, Lc in lanes 1, 2 and 3 denote lysate fractions of each experimental condition. Lanes 4, 5 and 6, immunoprecipitation products for experimental conditions a, b and c. (b) Coimmunoprecipitation of polycystin-1 with anti-ANX A5 antibodies. Lane 1, co-immunoprecipitation products for experimental condition a; lane 2, co-immunoprecipitation products for experimental condition b; lane 3, coimmunoprecipitation products for experimental condition c. (c) Co-immunoprecipitation of annexin A5 with anti-M2 anti-FLAG antibodies (polycystin-1). Lane 1, purified annexin A5 from human placenta, lanes 2, 3 and 4, immunoprecipitation products for experimental conditions a, b and c. (d) and (e) immunoprecipitation products from lysates of HeLa cells, not transfected or transfected with the PKD1-F plasmid. (d) Polycystin-1 co-immunoprecipitations with anti-ANX A5 antibodies. Lanes 1, 2 and 3, immunoprecipitation products for experimental conditions a, b and c. (e) Co-immunoprecipitation of annexin A5 with anti-M2 anti-FLAG antibodies (polycystin-1). Lane 1, purified annexin A5 from human placenta, lanes 2, 3 and 4, immunoprecipitation products for experimental conditions b, a and c, respectively.
cleaved N-terminal fragment (NTF).10 Non-induced cells seem to produce some low level of polycystin-1, which can be immunoprecipitated and detected with the anti-PKD1-LRR antibodies and most likely is a result of incomplete repression (lane 6). Figure 2(b) shows the result of an anti-annexin A5 immunoprecipitation probed in the Western blot
with anti-PKD1-LRR antibodies. Two bands of approximately 520 and 325 kDa, corresponding in size to full-length and NTF polycystin-1 were visible when annexin A5 was added to the PKD1-expressing cells and also when complexes were stabilized by DSP cross-link prior to cell lysis (lanes 1 and 2). Faint immunoprecipitation products with the same
958 relative molecular masses can also be detected in the case of non-induced cells (lane 3). It must be noted that the polycystin-1 NTF is more efficiently immunoprecipitated under these experimental conditions. The reciprocal experiment likewise reveals an annexin A5 co-immunoprecipitation with polycystin-1 however only following DSP cross-link (Figure 2(c), lane 2). Similarly, polycystin-1 coprecipitates with annexin A5 antibodies from lysates of PKD1-F plasmid6 transfected HeLa cells after a mild cross-link (Figure 2(d), lane 2) and reciprocally, ANXA5 can be detected in anti-M2-FLAG coimmunoprecipitates of polycystin-1 from the same lysates (Figure 2(e), lane 2). Bands at 25 kDa observed in Figure 2(c) and (e) and 50 kDa bands in Figure 2(e) across all lanes, accordingly, correspond to the light and heavy chains of the immunoprecipitating antibodies. Staining of blotted coimmunoprecipitation products from relevant after cross-link cell lysates with antibodies against transferrin receptor produced no signal. This suggests that the mild cross-linking applied did not result in unspecific binding to abundant transmembrane proteins. Surface binding of exogenous annexin A5 to polycystin-1 expressing PKD1–19 MDCK cells Expression of polycystin-1 in tetracycline-induced PKD1–19 MDCK cells was verified by Western blot of cell lysates and immunoprecipitated fractions (Figure 2(a)), as well as by immunofluorescence employing M2 anti-FLAG and anti-PKD1-LRR anti-
Interaction of Annexin A5 with Polycystin-1
bodies (Figure 3(a) and (b)). M2 anti-FLAG antibody staining of induced cells reveals some membrane localization in the region of cell–cell contacts as well as a vesicular intracellular signal (Figure 3(a), “PKD1 on”). In contrast, non-induced cells do not show any appreciable polycystin-1 staining over the background under these experimental conditions (Figure 3(a), “PKD1 off”). Similar results were obtained employing the anti-PKD1-LRR antibodies on non-permeabilized PKD1–19 cells. In addition to a general background signal seen on PKD1 on and PKD1 off cells (Figure 3(b)), this antibody produces a specific staining of cell contact regions in PKD1 on but not PKD1 off cells (arrow). To verify the binding of extracellular annexin A5 to cells expressing polycystin-1 in an inducible manner, PKD1–19 MDCK cells were induced for heterologous polycystin-1 expression, subjected to Ca2+ starvation and then incubated with fluorescently labeled annexin A5 together with normal Ca2+-containing medium for different periods of time. Since significant differences in the subcellular distribution of polycystin-1 were noted between 1 h and 2 h of Ca2+-replenishment (see below), we analyzed annexin A5 binding 2 h after the Ca2+ switch. Figure 4(a) shows confocal images of polycystin-1 expressing (PKD1 on) and non-expressing (PKD1 off) cells, stained with E-cadherin antibodies as a marker for adherens junctions (red channel) and fluorescent annexin A5 (Oregon Green 488 labeled, green channel). To ensure the intact physicochemical properties of stoichiometrically labeled annexin A5, phospholipid binding affinities were
Figure 3. Immunofluorescence staining of PKD1–19 MDCK cells, non-induced (PKD1 off) or induced (PKD1 on) to express polycystin-1 with (a) M2 anti-FLAG antibodies; (b) anti-PKD1-LRR antibodies. Arrow points at cell–cell contact staining. Scale bars represent 5 μm.
Interaction of Annexin A5 with Polycystin-1
959
Figure 4. Annexin A5 binding to the surface of PKD1–19 MDCK cells, non-induced (PKD1 off) or induced (PKD1 on) to express polycystin-1. (a) Detection of fluorescent annexin A5, bound to the cell surface 2 h after Ca2+ switch. E-cadherin (red) marks cell contacts and the green fluorescent signal corresponds to Oregon Green 488-labeled annexin A5. Scale bars represent 5 μm. (b) Quantification of the annexin A5 fluorescent signal detected on the surface of non-permeabilized cells, integration per high-powered field for PKD1 off and PKD1 on cells. Light intensity per pixel is in arbitrary units. PKD1 on cells concentrate more green fluorescent label (n = 4 × 5 HPF, values are means ± SE, *P < 0.05 PKD1 off, t-test). (c) Annexin A1 binding to the surface of PKD1–19 MDCK cells, non-induced (PKD1 off) or induced (PKD1 on) to express polycystin-1. Annexin A1 was detected with fluorescently labeled anti-annexin A1 antibodies (green) 2 h after Ca2+ switch. Cell–cell contacts are marked by E-cadherin (red). Scale bar represents 5 μm.
compared for the labeled and non-labeled protein and identical results were obtained (not shown). Retention of annexin A5 on the cell surface of polycystin-1 expressing cells could be observed to a significant extent only in PKD1 on cells (Figure 4(a), right panel). The fluorescent signal of annexin A5 binding seems to be clustered at E-cadherin positive cell–cell contacts of the polycystin-1 expressing cells. To rule out the possibility that binding of annexin A5
to the surface of PKD1 on cells is due to phosphatidylserine exposure resulting from an enhanced apoptosis rate in these cells, we measured apoptosis by propidium iodide staining and flow cytometry of cell nuclei from PKD1 on and PKD1 off monolayers. No significant differences were observed (results not shown), strongly suggesting that the increased surface binding of annexin A5 is due to polycystin1 expressed at the cell surface. A quantitative
960 comparison of annexin A5 fluorescence signal intensities on the surface of PKD1 off and PKD1 on cells, integrated per pixel of high powered field, is presented in Figure 4(b). On average, 25–50% more annexin A5 binding was observed for polycystin-1 expressing cells as compared to similar cells in noninduced state. Annexin A1, a protein of the same annexin superfamily, has a homologous C-terminal “core” region and a distinct, unrelated N-terminal domain and was used as negative control for the in vitro binding experiments. Non-permeabilized PKD1 off and PKD1 on cells do not show any labeling signal after incubation with annexin A1 and staining with anti-annexin A1 antibodies (Figure 4(c)). In contrast, PKD1 on cells incubated with fluorescent annexin A5 under the same experimental conditions retain fluorescent signal on their surface, to some extent clustered at cell–cell contacts (Figure 4(a)). Polycystin-1 and E-cadherin recruitment to cell junctions of polycystin-1 expressing cells Since some enrichment of the annexin A5 fluorescence label was observed at or close to cell junctions, we performed E-cadherin and polycystin-1 costaining to visualize the subcellular distribution of both proteins upon Ca2+ replenishment. The subcellular localization was recorded 1, 2 and 4 h after Ca2+ replenishment. Figure 5 reveals that at all time points the two fluorescence signals largely overlap, with some differences in cytosolic staining pattern of Ca2+ -starved cells. Upon Ca 2+ starvation, both proteins are sequestered in cytosolic pools with perinuclear location and vesicles dispersed in the cytoplasm. Recruitment of E-cadherin and polycystin-1 to cell junctions, which was triggered by Ca2+ replenishment, was rather advanced at 1 h and almost complete at 2 h after the Ca2+ switch. Cells cultivated for 4 h following Ca2+ replenishment show the same distribution of E-cadherin and polycystin-1. Altogether we conclude that (a) polycystin-1 and E-cadherin show a high degree of colocalization in MDCK cells (as observed before16) and (b) polycystin-1 translocation to reforming cell– cell contacts follows the same overall kinetics as E-cadherin recruitment. Annexin A5 affects E-cadherin recruitment to cell junctions in polycystin-1 expressing cells To elucidate possible effects of the annexin A5/polycystin-1 interaction on the recruitment of E-cadherin to reforming cell adhesion contacts, we performed Ca2+ switch experiments in the presence and absence of annexin A5 added to PKD1–19 cells induced or not induced to express polycystin-1. As noted above, PKD1–19 MDCK cells do express some basal levels of polycystin-1 in non-induced state (Figure 2(a) and (b)), non-detectable by immunofluorescence staining. For E-cadherin recruitment we actually compared PKD1 off cells weakly expressing polycystin-1 with PKD1 on cells
Interaction of Annexin A5 with Polycystin-1
overexpressing the protein. Monolayers were stained for E-cadherin and its subcellular distribution was recorded 1 h and 2 h after Ca2+ replenishment. Typical images of annexin A5 treated and untreated PKD1 on and PKD1 off cells are shown in Figure 6(a). There was no notable difference in the subcellular distribution of E-cadherin between annexin A5 treated versus untreated PKD1 off cells. Interestingly in PKD1 on cells extracellular Ca2+ replenishment seemed to trigger a more rapid E-cadherin recruitment as compared to PKD1 off monolayers (see for examples the 1 h panels of Figure 6(a)). This rapid recruitment of E-cadherin was reduced to rates characteristic for PKD1 off monolayers when annexin A5 was added to PKD1 on cells. The effect of annexin A5 on E-cadherin recruitment to junctions of polycystin-1 expressing cells was also quantified by expressing the ratio of cell junctions, positively stained for E-cadherin, to the number of scored cells (Figure 6(b)). This “junction index”, I, can vary between 0 and 2 (see Materials and Methods) and represents an adequate measure of E-cadherin appearance in cell junctions. In PKD1 on cells I was generally higher than in PKD1 off cells. These differences for the 1 h and 2 h time points amounted to 60–70% and to 45–55%, respectively. When annexin A5 was added to PKD1 on cells, these differences were basically abolished (Figure 6(b)). Thus, the stimulating effect of polycystin-1 on the recruitment of E-cadherin to reforming cell contacts is abrogated by annexin A5, most likely due to the direct binding of annexin A5 to the N-terminal LRR domain of polycystin-1. Annexin A1 added together with normal Ca2+containing medium for different periods of time to PKD1–19 MDCK cells induced or not induced to express polycystin-1, subjected to calcium starvation, did not have any effect on the rates of Ecadherin recruitment as indicator of cell junction reestablishment (results not shown). Annexin A5 levels are elevated in cystic fluids of ADPKD patients To assess a possible influence of the annexin A5/ polycystin-1 interaction on extracellular annexin A5 concentrations in the relevant organ, we undertook measurements of annexin A5 levels in cystic fluids of patients with ADPKD and patients with renal solitary cysts, with similar progression of kidney function deterioration (Figure 7). The average annexin A5 levels we measured in fluids of solitary cysts amount to 0.86 ng/ml (n = 11). In contrast, mean annexin A5 levels in cystic fluids of ADPKD patients are 3.53 ng/ml (n = 3), or fourfold higher as compared to solitary cysts.
Discussion By employing a genetic screen we identified a novel protein ligand of the extracellular LRR domain of polycystin-1. The Ca2+ and phospholipid
Interaction of Annexin A5 with Polycystin-1
961
Figure 5. Ca2+ switch experiments using PKD1–19 MDCK cells induced to express polycystin-1. The red fluoreecent signal shows anti-FLAG M2 antibody staining for polycystin-1 and green fluorescence marks E-cadherin antibody staining. Medium containing normal Ca2+ concentrations was replenished for 1 h, 2 h and 4 h after Ca2+ starvation. Cells not subjected to Ca2+ starvation, i.e. kept in medium containing normal Ca2+, are shown in the bottom panels. Scale bar represents 5 μm. PKD1 off cells did not show any appreciable polycystin-1 anti-FLAG M2 antibody staining over the background.
binding protein annexin A5 showed a specific interaction with the LRRs of polycystin-1, mediated through the VLRGTV sequence located at the very amino-terminal end of the annexin protein, spanning residues 4–9. Annexin A5 is a member of the annexin protein superfamily, for which extracellular localizations have been documented in addition to its expression in various cells and tissues.21 The
sequence xxRGTV at the very N terminus of annexin A5 is conserved in vertebrates, from zebrafish through chicken up to various mammalian species. Crystallography studies of human and rat annexin A5 show that residues 1–15 are in an extended conformation. 22,23 Likewise, the two complete LRRs of polycystin-1, constrained by the cysteine-rich flanking regions, should fold as a
962
Interaction of Annexin A5 with Polycystin-1
Figure 6. Effect of annexin A5 on E-cadherin recruitment to cellular junctions of polycystin-1 expressing PKD1–19 MDCK cells. (a) Immunofluorescence staining revealing the subcellular distribution of E-cadherin, 1 h and 2 h after Ca2+ replenishment of Ca2+ starved PKD1–19 MDCK cells. Experiments were performed on cells either non-induced or induced for polycystin-1 expression, with and without the addition of annexin A5. Figure scale bar represents 5 μm. (b) Quantification of cell contact-staining with E-cadherin antibodies for the different experimental conditions, using junction index (see Materials and Methods) as measure for the cell contact recruitment of E-cadherin. PKD1 on cells have a higher junction index (*P < 0.05, PKD1 on versus PKD1 off cells, values are means ± SE, t-test, n = 4 × 5 HPF). PKD1 on cells treated with ANXA5 have lowered junction index, similar to PKD1 off cells (**P < 0.05, PKD1 on+ ANXA5 versus PKD1 on cells, values are means ± SE, t-test, n = 4 × 5 HPF).
separate unit and thus be accessible for ligand binding.8,9 The specificity of protein–protein interactions mediated by LRRs is likely to be provided by the hydrophilic, solvent accessible amino acids located between the repeated aliphatic residues.24 Such structure allows for a “sequential” rather than “conformational” mode of recognition, which in turn supports the notion of a specific “epitope”
involved in the interaction. Three lines of evidence indicate that the in vitro binding of annexin A5 to the LRR domain of polycystin-1 does not require the homologous annexin repeat sequences, which are conserved among annexins and constitute the C-terminal annexin core domain. First, the peptide sequence VLRGTV is capable of binding alone. Second, the carboxy-terminal portion of annexin A5
Interaction of Annexin A5 with Polycystin-1
Figure 7. Concentrations of annexin A5 (ng/ml), measured using standard ELISA, in solitary and ADPKD cystic fluids. Solitary cysts samples, n = 11; ADPKD cysts samples, n = 3.
used for the binding experiment shown in Figure 1(b) contains two annexin repeats but does not interact. Third, another annexin protein (annexin A1) though containing highly homologous annexin repeats does not bind to the polycystin-1-LRR (Figure 1(a)). The in vitro interaction of polycystin-1 with annexin A5 was confirmed by co-immunoprecipitation experiments, following addition of annexin A5 to polycystin-1 expressing cells. However detection of the respective partner of polycystin-1 in the immunoprecipitates required a mild in vivo crosslink. Having in mind that this interaction depends on a short peptide sequence at least in annexin A5, the cross-link most likely stabilizes the peptidemediated polycystin-1/annexin A5 interaction enabling co-immunopecipitation under the conditions chosen. On the other hand, no chemical crosslink is necessary to detect co-immunoprecipitated polycystin-1 in Figure 2(b). The cell surface binding experiments with fluorescently labeled annexin A5 also support the specificity of interaction, since they reveal a specific enrichment of annexin A5 on the surface of PKD1 on cells whereas annexin A1, serving as a negative conrol, shows no appreciable binding (Figure 4(c)). The relatively large variation (Figure 4(b)) can be explained with differing degrees of polycystin-1 expression among induced cells. Since externally bound annexin A5 was enriched at or close to cell junction regions of PKD1 on cells (Figure 4), we examined the subcellular distribution of polycystin-1 in Ca2+ switch experiments. Our results demonstrate a polycystin-1 recruitment to cell contacts upon Ca2+ replenishment (Figure 5) that follows the same kinetics as E-cadherin. Moreover, E-cadherin and polycystin-1 signals largely overlap also in intracellular structures. Association of polycystin-1 with E-cadherin has been reported previously14 and has been confirmed in more recent studies.16 Upon Ca2+ deprivation and subsequent replenishment, both proteins were targeted to cell junctions although a subfraction
963 could still be observed in cytosolic vesicles. Interestingly, the E-cadherin recruitment to cell junctions of PKD1 on cells proceeded with enhanced rate in comparison to PKD1 off monolayers (Figure 6). Cell surface levels of E-cadherin are generally regulated by membrane trafficking pathways and a “setting point” of E-cadherin turnover is the association with p120-catenin.25 Our data suggest that polycystin-1 can affect this trafficking, thereby accelerating the E-cadherin recruitment to reforming cell contacts. Altered E-cadherin recruitment dynamics to forming cell contacts in ADPKD epithelia after stress or injury, when disturbed due to lack of polycystin-1 function, could be at least partly responsible for the observed cysts phenotype. The effect of enhanced E-cadherin recruitment to reforming cell contacts is reversed by the addition of annexin A5 (Figure 6). This could be due either to disturbance of homophilic interactions between polycystin-1 extracellular regions of neighboring cells, as previously reported,26 or to an effect on the signal transduction properties of the polycystin-1 molecule. Such possible mechanisms would both involve the extracellular N-terminal region of polycystin-1 and thereby could be affected by annexin A5 binding to this domain. The physiological role of annexin A5 as a possible effector of polycystin-1-mediated junction reformation is in line with its abundant expression in kidney.27 Moreover, increased extracellular annexin A5 levels are observed in kidney disease patients and renal damage models.28,29 Our preliminary studies indicate significantly elevated annexin A5 levels in ADPKD cystic fluids, compared to fluids of solitary cysts upon progressing deterioration of kidney function (Figure 7). More studies with proper statistical backup are necessary, on renal cysts patients in various stages of kidney disease, to address the role of annexin A5 as a possible physiological factor. With this in mind, the identification of annexin A5 as a novel ligand of the polycystin-1 LRR domain affecting the polycystin-1 mediated recruitment of Ecadherin to reforming epithelial junctions could indicate a link between epithelial abnormalities and the dynamics of E-cadherin positive junctions.
Materials and Methods Recombinant expression of polycystin-1-LRR The LRR domain of polycystin-1 and its flanking regions (bp 290–813 of the cDNA sequence, acc. no. L33243) was SalI/NotI subcloned into the pGEX4T-2 expression vector (Amersham-Pharmacia Biotech, Freiburg, Germany). The sequence of the resulting construct was verified across the site of gene fusion before use. The LRR polycystin-1 polypeptide was expressed as a GST fusion protein (∼48 kDa) and coupled to glutathione-Sepharose 4B resin according to manufacturers instructions (Amersham-Pharmacia Biotech, Freiburg, Germany).
964 Selection of random peptides (peptide panning) A FliTrx random peptide display library (Invitrogen, Groningen, Netherlands) was grown and induced for expression according to the guidelines of the manufacturer. Glutathione-Sepharose bound polycystin-1-LRR was loaded into a polyalomere column (10 ml) and the induced peptide display library was passed through in five successive rounds of selection. Washes and elution of bound E.coli were performed under sterile conditions, according to the manufacturer's instructions. After the fifth round of selection the remaining clones were plated and individual clones were subjected to PCR. Plasmid inserts were directly amplified using the M13 sequencetailed oligonucleotides M13.FliTrxF 5′-CGACGTTGTAAAACGACGGCCAGTATTCACCT GACTGACGAC-3′ and M13.FliTrxR 5′-CAGGAAACAGCTATGACCCCTGATATTCGTCA GCG-3′. The underlined M13 sequences were added to facilitate sequencing with universal primers. Amplicons were purified and directly sequenced using M13 fluorescently labeled forward and reverse primers on an ALF Express automated sequencer (Amersham-Pharmacia Biotech, Freiburg, Germany). Computer analysis (homology searches) Peptide sequences obtained more than once were compared against the PDB and Swiss-Prot databases, using the Smith-Waterman homology search algorithm. Sequences of additional clones were aligned with the redundants using the maximum likelihood rule. In vitro transcription and translation Annexin A1 and annexin A5 templates for in vitro transcription and translation were obtained from cloned cDNAs. Transcription/translation reactions were performed on 250–500 ng of template using the T7 TNT™ Coupled Reticulocyte Lysate System (Promega, Mannheim, Germany) in the presence of [35S]methionine according to the manufacturer's instructions. In vitro binding assay (GST pull-down) About 0.2 μg protein immobilized on glutathioneSepharose was incubated overnight at 4 °C with programmed lysates (10 or 20 μl) in NTEN buffer (20 mM TrisCl (pH 8), 100 mM NaCl, 1 mM EDTA, 0.5% Nonidet P-40 (NP-40)) containing protease inhibitors (1 mM phenylmethylsulfonyl fluoride, 1 μg/ml leupeptin, 1 μg/ml pepstatin and 10 μg/ml aprotinin), 0.5 mM Na3VO4, 80 μM β-glycerophosphate and 10 mM NaF. Beads were washed four times in NTEN buffer at room temperature and then mixed with 0.2 volumes of 6× SDS loading buffer, boiled for 5 min and released proteins were analyzed by SDS-PAGE. Immunoprecipitation and Western blot analysis PKD1–19 MDCK cells were induced with tetracycline (1 μg/ml) to express polycystin-1 for 48 h. Induced and non-induced cells were washed thoroughly with 4 °C cold PBS, placed on ice, and 100 nM annexin A5, purified from human placenta (Sigma, Deisenhofen, Germany), and dissolved in PBS, was added for 1 h to cell monolayers. Cells were washed three times with cold PBS and a cross-
Interaction of Annexin A5 with Polycystin-1 linker, di-thio-bis(succinimidylpropionate) (DSP), (PiercePerbio Science, Bonn, Germany), was added at concentration of 100 μg/ml for an additional 20 min on ice. After thoroughly washing the monolayers, 0.2 M glycine/PBS was added for 5 min to quench traces of reactive crosslinker and cells were washed again 3× with PBS and harvested for immunoprecipitation and Western blotting. Cells were lysed in 10 mM sodium phosphate buffer with 0.5% Triton X-100, as described.6 Cleared lysates were incubated with 2 μg of sheep anti-annexin A5 antibodies (kind gift from John Dedman, Univeristy of Cincinnati, Ohio) for 1 h at 4 °C or with 50 μl of Red EasyView™ M2anti-FLAG agarose (Sigma, St. Louis, MO, USA) for 2 h at 4 °C. Fifty microliter of Protein G Sepharose beads (Amersham Biosciences, Freiburg, Germany) were added to the samples containing anti-annexin A5 antibodies and they were incubated for another hour at 4 °C. Immunoprecipitates were washed five times with 1 ml of lysis buffer and eluted in 60 μl of 1× Laemmli loading buffer. Eluted products (10–15 μl) were subjected to polyacrylamide gel electrophoresis (4% (w/v) acrylamide for anti-annexin A5 co-precipitates and 12% acrylamide for M2 anti-FLAG co-precipitates) and analyzed using standard Western blot protocols (ECL, Applichem, Darmstadt, Germany). The same protocol was previously applied to HeLa cells transiently transfected with the PKD1-F plasmid.6 Annexin A5 fluorescent labeling Recombinantly expressed annexin A5 was purified to homogeneity as assessed by silver-stained PAGE and Western blotting. The protein was labeled with Oregon Green 488 and Alexa 568-succinimidylester according to the manufacturers protocol (Molecular Probes, Leiden, Netherlands). The 1:1 stoichiometric complex of fluorescently labeled annexin A5 was purified from the mixtures by MonoQ chromatography with the Äcta Explorer system (Amersham Biosciences, Roosendaal, Netherlands). Cell-based assays Stably transfected (MDCK)-cell line for heterologous inducible expression of human polycystin-1 (clone PKD1– 19) was obtained from Gregory G. Germino (Johns Hopkins University, Baltimore, MD). Expression of endogenous polycystin-1 in these cell lines was very low and detectable only at the mRNA level. The inducible cell line PKD1–19 was employed in Ca2+ switch experiments. For these experiments, PKD1–19 cells, induced or non-induced to express polycystin-1, were grown to confluence on LabTek slide chambers (Nalge Nunc, Wiesbaden, Germany). Cells were then depleted of Ca2+ by washing with Ca2+and magnesium-free PBS and incubating for 16 to 18 h in a Ca2+/glutamine-free DMEM medium (Invitrogen, Karlsruhe, Germany), supplemented with 10% (v/v) dialyzed foetal bovine serum (FBS) and 3 μM Ca2+ chloride. Ca2+containing normal DMEM with 10% FBS was then replenished, with or without 100 nM fluorescently labeled annexin A5. Cells were removed at different time points and processed for immunostaining. Immunofluorescence PKD1–19 MDCK cells, induced or non-induced to express polycystin-1 were washed 3 x in PBS, then fixed
Interaction of Annexin A5 with Polycystin-1 with 4% paraformaldehyde in PBS (pH 7.5) for 10 min, followed by incubation in 50 mM NH4Cl/PBS for 5 min and 1.5% goat serum (Sigma, Deisenhofen, Germany) in PBS for 20 min. Non-permeabilized cells were incubated with anti-PKD1-LRR rabbit polyclonal antibodies10 for 30 min, washed six times with PBS and and stained with Texas Red™- conjugated goat anti-rabbit immunoglobulin (Ig)G antibodies (Jackson ImmunoResearch Laboratories, West Grove, PA, USA) for 20 min. Specimens were washed six times with PBS and then mounted for imaging. Cells to be stained employing M2 anti-FLAG antibodies were permeabilized for 20 min with 0.1% Triton X-100 and 1.5% goat serum in PBS after fixation. Specimens were incubated with M2 anti-FLAG mouse monoclonal antibodies (Sigma, Deisenhofen, Germany), washed six times with 0.1% Triton X-100 in PBS and stained with Texas Red™- conjugated goat anti-mouse IgG Fab' fragments (Jackson ImmunoResearch Laboratories, West Grove, PA, USA) for 20 min. Specimens were washed six times with 0.1% Triton X-100 in PBS and then mounted for imaging. PKD1–19 cells, induced or non-induced to express polycystin-1 were washed 3 x in PBS and incubated with PBS solutions of 100 nM fluorescent annexin A5 or with 100 nM recombinantly expressed and purified annexin A1 for 1 h, then fixed with 4% paraformaldehyde and quenched as indicated above. Annexin A1-treated cells were then incubated with mouse anti-annexin A1 monoclonal antibodies (BD Transduction Laboratories, Lexington, KY, USA) for 30 min, PBS washed and stained with Cy2™-conjugated goat anti-mouse IgG antibodies (Jackson ImmunoResearch Laboratories, West Grove, PA, USA) for 20 min. Cells were permeabilized and specimens were then incubated with rat monoclonal DECMA-1 (anti-Ecadherin, Sigma, Deisenhofen, Germany) antibodies for 30 min, washed (see above) and stained with Texas Red™conjugated goat anti-rat IgG antibodies (Jackson ImmunoResearch Laboratories, West Grove, PA, USA) for 20 min. Specimens were washed six times with 0.1% Triton X-100 in PBS and then mounted for imaging. For E-cadherin/polycystin-1 staining permeabilized cells were incubated first with rat monoclonal DECMA-1 (anti-E-cadherin, Sigma, Deisenhofen, Germany) antibodies, stained with Cy2™-conjugated goat anti-rat IgGs (Jackson ImmunoResearch Laboratories, West Grove, PA, USA), then incubated with M2 anti-FLAG monoclonal antibodies (Sigma, Deisenhofen, Germany) and stained with Texas Red™-conjugated goat anti-mouse IgG Fab' fragments (same supplier). Specimens were washed six times with 0.1% Triton X-100 in PBS and then mounted for imaging. Confocal laser scanning microscopy was performed on a Zeiss LSM 510 Meta microscope (Carl Zeiss, Göttingen, Germany). The PKD1 off cells, as well as a control cell line clone, MDCKpcDNA.Fcγ, which was stained in tetracyclineinduced or non-induced state, did not show any staining over the background with the M2 anti-FLAG antibodies or with fluorescently labeled annexin A5 under these experimental conditions. Quantification of annexin A5 surface binding and E-cadherin recruiment For each time point, five high-powered fields (HPF) amounting to 200 to 250 cells, from four different experiments were scored for annexin A5 binding or E-cadherin recruitment to cell junctions. Fluorescent images of cells, replenished with Ca2+-containing normal
965 medium for 1 and 2 h, were taken with the same settings and magnification, and image files were transferred for evaluation with MetaMorph imaging software (Universal Imaging Corporation, Marlow, UK). Light intensities of surface bound Oregon Green 488-labeled annexin A5 were measured in the green channel for polycystin-1-induced and non-induced cells and integrated per HPF (1024 × 1024 pixel). E-cadherin recruitment to cell junctions was scored as junction index, I, where I = J/N. J is the number of positively stained cell junctions (scored manually as membrane outlines), divided by the number of cells in the field, N (usually 30–50). It can be easily shown that for a regular polygonal array of cells, when counting the sideto-side and corner junctions, but not the external outlines, Imax = 2n/n+1, where n is the polygon sides number. Thus, I ∈ (0.2). Measurement of annexin A5 concentrations in cystic kidney fluids Material for analysis was collected from renal disease patients referred for treatment to the Clinic of Nephrology and Haemodialysis at the Medical University Pleven. Informed consent was obtained from all subjects examined and the study complied with the ethical guidelines of all institutions involved. Members of the studied group had comparable progression of kidney disease and similar decline of renal function as assessed by relevant clinical tests. Cystic fluids were drawn upon subcutaneous puncture with a syringe from patients with solitary kidney cysts and such with ADPKD cysts as a first step in a standard therapeutic procedure of subsequent alcoholic cyst cauterization. Obtained fluids (typically 3 to 5 ml volume) were centrifuged at 100,000 g for 15 min at 4 °C to remove cell remnants. Concentrations of soluble annexin A5 were measured in samples of the supernatants following standard ELISA protocol.30
Acknowledgements This work was supported by grants from the Deutsche Forschungsgemeinschaft (DFG) to B. D., J. H. and to V. G. and from Interdisziplinäres Zentrum für Klinische Forschung (IZKF), Universitätsklinikum Münster, to B. D., J. H. and to V. G. We are grateful to Gregory G. Germino and Feng Qian for the continuous support with crucial reagents and the critical reading of the manuscript. We thank Jonathan Tait for providing the annexin A5 cDNA plasmid, John Dedman for the anti-annexin A5 antibodies, Klaus Ebnet for the Ca2+ switch protocol and valuable discussions, Carsten Lange for assistance with the flow cytometer, and Ursula Rescher for the help with MetaMorph.
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Edited by J. Karn (Received 13 February 2007; received in revised form 21 March 2007; accepted 27 March 2007) Available online 31 March 2007