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The adaptor disabled-2 binds to the third ΨxNPxY sequence on the cytoplasmic tail of megalin
Biochimie 86 (2004) 179–182 www.elsevier.com/locate/biochi
The adaptor disabled-2 binds to the third WxNPxY sequence on the cytoplasmic tail of megalin Hugh Gallagher a,*, Andrew V. Oleinikov b, Christiane Fenske c, David J. Newman a a
SW Thames Institute for Renal Research, St. Helier Hospital, Carshalton, Surrey SM5 1AA, UK b CombiMatrix Corporation, Mukilteo, WA 98275, USA c Department of Medical Genetics, St. George’s Hospital Medical School, London SW17 0RE, UK Received 20 November 2003; accepted 9 March 2004 Available online 26 March 2004
Abstract The cytoplasmic tail (CT) of megalin possesses several functional motifs likely to participate in protein–protein interactions within the proximal tubular epithelial cell (PTEC) of the kidney. One such interaction is with the phosphotyrosine interaction domain (PID) of the adaptor protein disabled-2 (Dab2), a mitogen-responsive phosphoprotein, which interacts via its PID with WxNPxY (where W represents a hydrophobic residue) motifs on its binding partners. Megalin CT has three such motifs; it has been established that there is no interaction of Dab2 with the first (from N to C) (Biochem. J. 3 (2000) 613). Here, we analyse in real-time the binding of recombinant megalin CT, and of synthetic peptide sequences encompassing the second and third WxNPxY motifs, to Dab2PID in real-time using surface plasmon resonance (SPR). We report a binding affinity of DabPID for megalin CT of KD = 2.6 × 10–7 ± 5.3 × 10–8. Direct binding and competition studies indicate that this interaction is with the third WxNPxY motif. The dissociation of Dab2 from the third WxNPxY peptide was significantly slower than that from the second (koff (mean ± S.E.M.) (per s) = 0.002 ± 0.002 vs. 0.007 ± 0.002, P < 0.05). Synthetic peptide sequences encompassing the third WxNPxY but not the second inhibited Dab2PID binding both to intact megalin CT and to the third WxNPxY motif. Tyrosine phosphorylation of either motif did not exert a major effect upon competition efficacy. We further demonstrate for the first time the presence of Dab2 expression in primary human PTEC. © 2004 Elsevier SAS. All rights reserved. Keywords: Megalin; Disabled-2; Surface plasmon resonance; Endocytosis
1. Introduction The 600 kDa glycoprotein megalin, a member of the low density lipoprotein (LDL) receptor family, is the most important receptor mediating the endocytic uptake of filtered proteins from the renal proximal tubular lumen. Recent data suggest that albumin may interact with the proximal tubular epithelial cell (PTEC) in the manner of a signalling molecule to, for example, modulate proliferative pathways within the cell [2].
Abbreviations: CT, cytoplasmic tail; Dab2, disabled-2; GST, glutathione S-transferase; LDL, low density lipoprotein; PID, phosphotyrosine interaction domain; PTEC, proximal tubular epithelial cell; SPR, surface plasmon resonance. * Corresponding author. Tel.: +44-78-7981-2696; fax: +44-20-8549-5747. E-mail address: [email protected] (H. Gallagher). © 2004 Elsevier SAS. All rights reserved. doi:10.1016/j.biochi.2004.03.001
The mechanisms underlying these effects are unknown. However, the participation of megalin in signalling events is suggested by the presence of potentially functional amino acid motifs on its cytoplasmic tail (CT) [3], and recent studies have documented several interactions of possible significance [1,4]. One such intracellular ligand is the mitogen-responsive phosphoprotein disabled-2 (Dab2) which interacts via its phosphotyrosine interaction domain (PID) with WxNPxY/WxNPxY-like (where W represents a hydrophobic residue) motifs on megalin CT. Megalin possesses three such motifs, which are highly conserved within the CT of all LDL receptor family members. Current data indicate a strong interaction with the third (from N to C) WxNPxY sequence, a weaker interaction with the second, and no binding to the first [1]. Here, we analyse the interaction of Dab2 with megalin CT in real-time using surface plasmon resonance (SPR) and
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H. Gallagher et al. / Biochimie 86 (2004) 179–182
demonstrate for the first time the presence of Dab2 expression in primary human PTEC.
third WxNPxY motif (biotin-QVTVSENVDNKNYGSPI). The first WxNPxY was not studied.
2. Materials and methods
2.4. Inhibition of Dab2-megalin CT binding with synthetic peptide sequences
SPR measurements were performed on a BIACORE®X optical biosensor and kinetic data evaluated using BIA evaluation software (Biacore AB, Uppsala, Sweden). Peptide sequences were synthesised by Sigma-Genosys (Cambridge, UK). Experiments were conducted at 25 °C with a flow rate of 20 µl/min in HBS-P buffer (10 mM HEPES, pH 7.4, 150 mM NaCl, 0.005% surfactant P20). Association and dissociation were modelled assuming 1:1 (Langmuir) binding. Reported affinity parameters represent the mean of at least three independent experiments. 2.1. Expression and purification of recombinant proteins Glutathione S-transferase (GST) constructs encoding wild-type megalin CT and the PID of Dab2 (containing amino acid residues 1–176) were generated in pGEX4T-1 and pGEX4T-2 vectors, respectively, as previously reported [1]. The two proteins were expressed as GST fusions in the BL21 Escherichia coli strain and affinity purified using standard techniques. The identities of the fusion products were confirmed by Western blotting using 1:20 000 rabbit anti-rat megalin CT antibody (a gift from David Sundin, University of Indianapolis) and 1:100 goat anti-human N-terminal Dab2 antibody (Santa Cruz, USA). 2.2. Measurement of Dab2-megalin CT binding affınity Twenty micrograms per millilitre wild-type megalin CTGST in 10 mM sodium acetate, pH 4.8 was coupled to one surface of a CM5 sensor chip using NHS/EDC chemistry. Seven micrograms per millilitre recombinant GST (Santa Cruz, USA) in 10 mM sodium formate, pH 4.0 was coupled to the second surface. Remaining active sites on both surfaces were blocked with 1 M ethanolamine. The specific binding of Dab2PID-GST to megalin CT, measured as the net difference in signal between the two surfaces, was studied over the range 20 nM–4 µM. There was no binding of purified GST to either surface. 2.3. Measurement of Dab2 affınity for synthetic peptide sequences A biotinylated 17-mer peptide encompassing the second WxNPxY motif (biotin-KSKQTTNFENPIYAQME) was immobilised upon one surface of a streptavidin-coated cuvette, and the binding of Dab2PID-GST followed over the range 200 nM–7 µM. Biotin alone coupled to the second surface served as a negative control. There was no binding of purified GST to either surface. The experiments were repeated with an immobilised 17-mer synthetic peptide encompassing the
Synthetic 14-mer peptides encompassing the second (VSENVDNKNYGSPI) and third (QTTNFENPIYAQME) WxNPxY motifs were synthesised in tyrosine phosphorylated and non-phosphorylated forms. 1:1 mixtures of Dab2PID-GST (4 µM) and a large molar excess of each peptide (2 mM) that had been incubated overnight at 4 °C served as the substrates for the inhibited reactions. The specific binding of 2 µM Dab2PID to megalin CT and to the third WxNPxY motif was measured, both uninhibited and inhibited by the four peptides. Peptides were studied in a random order and each inhibited interaction was preceded by a round of uninhibited binding. Uninhibited and inhibited results were expressed as initial binding rates (resonance units/s) over the first 10 s. Reported data represent the average of six independent experiments. 2.5. Expression of Dab2 in human brush border preparations and primary PTEC Human primary PTEC were isolated from nephrectomy specimens, cultured on collagen I-coated flasks and characterised as previously described [5]. Cell lysates were prepared by solubilisation of cells at passage 4 in boiling lysis buffer (10 mM Tris-pH 7.4, 1% SDS, 1 mM sodium orthovanadate) to which a mixture of protease inhibitors had been added. Insoluble material was pelleted by centrifugation and the supernatant boiled for 5 min in an equal volume of 2× SDS sample buffer. Renal brush border preparations were isolated from human whole cortical tissue using 10 mM CaCl2 as a precipitating agent [6]. PTEC lysate and brush border proteins were separated by SDS-PAGE and subjected to Western blot analyses with 0.1 µg/ml mouse anti-murine Dab2 (Transduction Laboratories, Lexington, USA)/1:10 000 peroxidase-labelled universal secondary antibody (Dako, Ely, UK). 2.6. Statistical analyses Statistics were analysed using Statview for Windows version 4.53 (Abacus Concepts). Direct measurements for equilibrium (KD) and off-rate (koff) constants for the interaction of Dab2 with megalin CT and synthetic WxNPxY sequences were compared using the paired t-test. Differences in initial rates of binding in the competition experiments were also analysed by paired t-testing. 3. Results 3.1. Direct affınity measurements Western blot analyses of purified recombinant material revealed specific bands at 50 and 55 kDa for Dab2PID-GST
H. Gallagher et al. / Biochimie 86 (2004) 179–182
181
Fig. 1. SPR analysis of the specific binding of Dab2PID (20 nM–4 µM) to megalin CT. Representative association and dissociation curves. Estimated KD = 2.6 × 10–7 ± 5.3 × 10–8 M (n = 3).
and megalin CT-GST, respectively. The interactions observed were not dependent upon the presence of GST on the recombinant proteins or biotin on the synthetic peptides. We found that the Dab2PID bound strongly to intact megalin CT (KD = 2.6 × 10–7 ± 5.3 × 10–8 M) (Fig. 1) and with comparable affinity to peptides encompassing both the second (KD = 3.2 × 10–7 ± 1.1 × 10–7 M) and third (KD = 6.1 × 10–8 ± 2.7 × 10–8 M) WxNPxY motifs. There was a trend (P = 0.1) towards higher affinity binding, as measured by the equilibrium rate constant, to the third as compared with the second WxNPxY motif. However, the dissociation of Dab2 from the third WxNPxY peptide was significantly slower than that from the second (koff (mean ± S.E.M.) (per s) = 0.002 ± 0.002 vs. 0.007 ± 0.002, P < 0.05), consistent with a higher affinity of binding to the former sequence.
Fig. 2. Inhibition of Dab2PID binding to immobilised megalin CT (above) and to immobilised third WxNPxY peptide (below) by synthetic tyrosine phosphorylated (pY) and non-phosphorylated second WxNPxY and third WxNPxY sequences. The third but not the second WxNPxY exerted a major inhibitory effect upon Dab2PID binding. Tyrosine phosphorylation had little impact upon competition efficacy. P < 0.05 compared with uninhibited.
3.2. Competition experiments An uninhibited Dab2PID concentration of 2 µM was selected on the basis that it offered a large and reproducible signal to inhibit. The experimental design was controlled for any systematic effects over time. The inhibition of Dab2PID binding both to intact megalin CT and to the third WxNPxY (Fig. 2) were studied on the basis of the results observed in the direct binding studies. The third WxNPxY was a more effective competitor of Dab2PID binding than the second in both series of experiments, consistent with a higher affinity of the PID for this motif. Tyrosine phosphorylation of either motif did not significantly influence competition efficacy on the peptide surface. On the intact CT surface the tyrosine phosphorylated but not the non-tyrosine phosphorylated second WxNPxY had a small, although statistically significant, effect on Dab2PID binding. 3.3. Dab2 expression in human primary PTEC and brush border preparations Immunoblotting with anti-Dab2 monoclonal antibody of microvillar membranes and lysates derived from primary PTEC confirm the potential for relevance of this protein in a human setting (Fig. 3). Control incubations in the absence of primary antibody were negative.
Fig. 3. Dab2 is expressed by both human brush border preparations and by cultured human primary PTEC. Lane 1, Dab2 purified from human foreskin fibroblasts; lane 2, molecular weight standards; lane 3, brush border preparations from fresh human kidney cortex; lanes 4 and 5, lysates from two human primary PTEC lines.
4. Discussion We have confirmed the interaction of Dab2 with megalin CT using SPR and measured an affinity which is in accordance with that documented for other PID–WxNPxY interactions and is sufficiently high as to afford probable biological relevance. Two lines of evidence support the contention
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that the interaction is with the third (from N to C) of megalin CT’s three WxNPxY motifs. The third WxNPxY displays slower dissociation from Dab2 than the second; and inhibition studies indicate that this motif is the more efficient competitor of Dab2 binding, both to intact CT and to itself. The presence of a b-turn within the PID-binding targets is a structural prerequisite for binding [7]. Residues critical for binding have been identified in many cases. However, other non-critical residues may also play a role [8]. Further studies are required to delineate the precise sequence determinants for binding to the Dab2PID. Despite the terminology, the presence of phosphotyrosine is not always required for the interaction of PID with their targets [9]. Preliminary evidence that tyrosine phosphorylation of megalin CT occurs on agonist stimulation has been presented [10], providing a rationale for study. However, on the basis of these experiments, agonist-induced phosphorylation of megalin CT does not appear to provide a link between the binding of extracellular ligand and any subsequent putative Dab2-mediated intracellular events. The role of Dab2PID-megalin CT binding remains uncertain. By analogy to disabled-1, which relays neuronal positional information in the central nervous system through LDL receptor family members [11], Dab2-megalin binding may play a role in the positioning of epithelial cells relative to basement membrane structures [12]. Alternatively, the interaction of Dab2 with megalin within the kidney may impact upon proliferative pathways via the Dab2-mediated uncoupling of receptor tyrosine kinase stimulation from Ras activation [13]. An involvement in endocytosis is a third possibility, given that the NPxY motif is recognised as an internalisation signal for clathrin-mediated endocytosis among LDL receptor family members. Dab2–/– mice exhibit proteinuria consistent with reduced megalin-mediated endocytosis [14], perhaps via the action of Dab2 as a novel endocytic adaptor [15]. In conclusion, this work has provided new evidence that Dab2 binds to the third WxNPxY on the CT of megalin. Although functional studies are required, it is likely the interaction plays a role within the PTEC in the regulation of cell positioning, proliferative or endocytic activity. References [1]
A.V. Oleinikov, J. Zhao, S.P. Makker, Cytosolic adaptor protein Dab2 is an intracellular ligand of endocytic receptor gp600/megalin, Biochemical Journal 3 (2000) 613–621.
[2]
R. Dixon, N.J. Brunskill, Albumin stimulates p44/p42 extracellularsignal-regulated mitogen-activated protein kinase in opossum kidney proximal tubular cells, Clinical Science 98 (2000) 295–301.
[3]
A. Saito, S. Pietromonaco, A.K. Loo, M.G. Farquhar, Complete cloning and sequencing of rat gp330/“megalin,” a distinctive member of the low density lipoprotein receptor gene family, Proceedings of the National Academy of Sciences of the United States of America 91 (1994) 9725–9729.
[4]
M. Gotthardt, M. Trommsdorff, M.F. Nevitt, J. Shelton, J.A. Richardson, W. Stockinger, et al., Interactions of the low density lipoprotein receptor gene family with cytosolic adaptor and scaffold proteins suggest diverse biological functions in cellular communication and signal transduction, Journal of Biological Chemistry 275 (2000) 25616–25624.
[5]
D.J. Newman, H. Thakkar, H. Gallagher, Progressive renal disease: does the quality of the proteinuria matter or only the quantity? Clinical Chemistry Acta 297 (2000) 43–54.
[6]
P. Malathi, H. Preiser, P. Fairclough, P. Mallett, R.K. Crane, A rapid method for the isolation of kidney brush border membranes, Biochimica and Biophysica Acta 554 (1979) 259–263.
[7]
B. Margolis, J.P. Borg, S. Straight, D. Meyer, The function of PTB domain proteins, Kidney International 56 (1999) 1230–1237.
[8]
Z. Zhang, C.H. Lee, V. Mandiyan, J.P. Borg, B. Margolis, J. Schlessinger, et al., Sequence-specific recognition of the internalization motif of the Alzheimer’s amyloid precursor protein by the X11 PTB domain, EMBO Journal 16 (1997) 6141–6150.
[9]
B.W. Howell, L.M. Lanier, R. Frank, F.B. Gertler, J.A. Cooper, The disabled 1 phosphotyrosine-binding domain binds to the internalization signals of transmembrane glycoproteins and to phospholipids, Molecular and Cellular Biology 19 (1999) 5179–5188.
[10] R.J. Baines, J. Brown, A.B. Tobin, H. Gallagher, D.J. Newman, N.J. Brunskill, Phosphorylation of megalin in human proximal tubule cells indicates a novel role in cell signalling and regulation of tubular function (abstract), UK Renal Association Autumn meeting, 2003. [11] M. Trommsdorff, M. Gotthardt, T. Hiesberger, J. Shelton, W. Stockinger, J. Nimpf, et al., Reeler/disabled-like disruption of neuronal migration in knockout mice lacking the VLDL receptor and ApoE receptor 2, Cell 97 (1999) 689–701. [12] Z. Sheng, W. Sun, E. Smith, C. Cohen, X.X. Xu, Restoration of positioning control following disabled-2 expression in ovarian and breast tumor cells, Oncogene 19 (2000) 4847–4854. [13] X.X. Xu, T. Yi, B. Tang, J.D. Lambeth, Disabled-2 (Dab2) is an SH3 domain-binding partner of Grb2, Oncogene 16 (1998) 1561–1569. [14] S.M. Morris, M.D. Tallquist, C.O. Rock, J.A. Cooper, Dual roles for the Dab2 adaptor protein in embryonic development and kidney transport, EMBO Journal 21 (2002) 1555–1564. [15] S.K. Mishra, P.A. Keyel, M.J. Hawryluk, N.R. Agostinelli, S.C. Watkins, L.M. Traub, Disabled-2 exhibits the properties of a cargoselective endocytic clathrin adaptor, EMBO Journal 21 (2002) 915– 916.
The adaptor disabled-2 binds to the third WxNPxY sequence on the cytoplasmic tail of megalin Hugh Gallagher a,*, Andrew V. Oleinikov b, Christiane Fenske c, David J. Newman a a
SW Thames Institute for Renal Research, St. Helier Hospital, Carshalton, Surrey SM5 1AA, UK b CombiMatrix Corporation, Mukilteo, WA 98275, USA c Department of Medical Genetics, St. George’s Hospital Medical School, London SW17 0RE, UK Received 20 November 2003; accepted 9 March 2004 Available online 26 March 2004
Abstract The cytoplasmic tail (CT) of megalin possesses several functional motifs likely to participate in protein–protein interactions within the proximal tubular epithelial cell (PTEC) of the kidney. One such interaction is with the phosphotyrosine interaction domain (PID) of the adaptor protein disabled-2 (Dab2), a mitogen-responsive phosphoprotein, which interacts via its PID with WxNPxY (where W represents a hydrophobic residue) motifs on its binding partners. Megalin CT has three such motifs; it has been established that there is no interaction of Dab2 with the first (from N to C) (Biochem. J. 3 (2000) 613). Here, we analyse in real-time the binding of recombinant megalin CT, and of synthetic peptide sequences encompassing the second and third WxNPxY motifs, to Dab2PID in real-time using surface plasmon resonance (SPR). We report a binding affinity of DabPID for megalin CT of KD = 2.6 × 10–7 ± 5.3 × 10–8. Direct binding and competition studies indicate that this interaction is with the third WxNPxY motif. The dissociation of Dab2 from the third WxNPxY peptide was significantly slower than that from the second (koff (mean ± S.E.M.) (per s) = 0.002 ± 0.002 vs. 0.007 ± 0.002, P < 0.05). Synthetic peptide sequences encompassing the third WxNPxY but not the second inhibited Dab2PID binding both to intact megalin CT and to the third WxNPxY motif. Tyrosine phosphorylation of either motif did not exert a major effect upon competition efficacy. We further demonstrate for the first time the presence of Dab2 expression in primary human PTEC. © 2004 Elsevier SAS. All rights reserved. Keywords: Megalin; Disabled-2; Surface plasmon resonance; Endocytosis
1. Introduction The 600 kDa glycoprotein megalin, a member of the low density lipoprotein (LDL) receptor family, is the most important receptor mediating the endocytic uptake of filtered proteins from the renal proximal tubular lumen. Recent data suggest that albumin may interact with the proximal tubular epithelial cell (PTEC) in the manner of a signalling molecule to, for example, modulate proliferative pathways within the cell [2].
Abbreviations: CT, cytoplasmic tail; Dab2, disabled-2; GST, glutathione S-transferase; LDL, low density lipoprotein; PID, phosphotyrosine interaction domain; PTEC, proximal tubular epithelial cell; SPR, surface plasmon resonance. * Corresponding author. Tel.: +44-78-7981-2696; fax: +44-20-8549-5747. E-mail address: [email protected] (H. Gallagher). © 2004 Elsevier SAS. All rights reserved. doi:10.1016/j.biochi.2004.03.001
The mechanisms underlying these effects are unknown. However, the participation of megalin in signalling events is suggested by the presence of potentially functional amino acid motifs on its cytoplasmic tail (CT) [3], and recent studies have documented several interactions of possible significance [1,4]. One such intracellular ligand is the mitogen-responsive phosphoprotein disabled-2 (Dab2) which interacts via its phosphotyrosine interaction domain (PID) with WxNPxY/WxNPxY-like (where W represents a hydrophobic residue) motifs on megalin CT. Megalin possesses three such motifs, which are highly conserved within the CT of all LDL receptor family members. Current data indicate a strong interaction with the third (from N to C) WxNPxY sequence, a weaker interaction with the second, and no binding to the first [1]. Here, we analyse the interaction of Dab2 with megalin CT in real-time using surface plasmon resonance (SPR) and
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H. Gallagher et al. / Biochimie 86 (2004) 179–182
demonstrate for the first time the presence of Dab2 expression in primary human PTEC.
third WxNPxY motif (biotin-QVTVSENVDNKNYGSPI). The first WxNPxY was not studied.
2. Materials and methods
2.4. Inhibition of Dab2-megalin CT binding with synthetic peptide sequences
SPR measurements were performed on a BIACORE®X optical biosensor and kinetic data evaluated using BIA evaluation software (Biacore AB, Uppsala, Sweden). Peptide sequences were synthesised by Sigma-Genosys (Cambridge, UK). Experiments were conducted at 25 °C with a flow rate of 20 µl/min in HBS-P buffer (10 mM HEPES, pH 7.4, 150 mM NaCl, 0.005% surfactant P20). Association and dissociation were modelled assuming 1:1 (Langmuir) binding. Reported affinity parameters represent the mean of at least three independent experiments. 2.1. Expression and purification of recombinant proteins Glutathione S-transferase (GST) constructs encoding wild-type megalin CT and the PID of Dab2 (containing amino acid residues 1–176) were generated in pGEX4T-1 and pGEX4T-2 vectors, respectively, as previously reported [1]. The two proteins were expressed as GST fusions in the BL21 Escherichia coli strain and affinity purified using standard techniques. The identities of the fusion products were confirmed by Western blotting using 1:20 000 rabbit anti-rat megalin CT antibody (a gift from David Sundin, University of Indianapolis) and 1:100 goat anti-human N-terminal Dab2 antibody (Santa Cruz, USA). 2.2. Measurement of Dab2-megalin CT binding affınity Twenty micrograms per millilitre wild-type megalin CTGST in 10 mM sodium acetate, pH 4.8 was coupled to one surface of a CM5 sensor chip using NHS/EDC chemistry. Seven micrograms per millilitre recombinant GST (Santa Cruz, USA) in 10 mM sodium formate, pH 4.0 was coupled to the second surface. Remaining active sites on both surfaces were blocked with 1 M ethanolamine. The specific binding of Dab2PID-GST to megalin CT, measured as the net difference in signal between the two surfaces, was studied over the range 20 nM–4 µM. There was no binding of purified GST to either surface. 2.3. Measurement of Dab2 affınity for synthetic peptide sequences A biotinylated 17-mer peptide encompassing the second WxNPxY motif (biotin-KSKQTTNFENPIYAQME) was immobilised upon one surface of a streptavidin-coated cuvette, and the binding of Dab2PID-GST followed over the range 200 nM–7 µM. Biotin alone coupled to the second surface served as a negative control. There was no binding of purified GST to either surface. The experiments were repeated with an immobilised 17-mer synthetic peptide encompassing the
Synthetic 14-mer peptides encompassing the second (VSENVDNKNYGSPI) and third (QTTNFENPIYAQME) WxNPxY motifs were synthesised in tyrosine phosphorylated and non-phosphorylated forms. 1:1 mixtures of Dab2PID-GST (4 µM) and a large molar excess of each peptide (2 mM) that had been incubated overnight at 4 °C served as the substrates for the inhibited reactions. The specific binding of 2 µM Dab2PID to megalin CT and to the third WxNPxY motif was measured, both uninhibited and inhibited by the four peptides. Peptides were studied in a random order and each inhibited interaction was preceded by a round of uninhibited binding. Uninhibited and inhibited results were expressed as initial binding rates (resonance units/s) over the first 10 s. Reported data represent the average of six independent experiments. 2.5. Expression of Dab2 in human brush border preparations and primary PTEC Human primary PTEC were isolated from nephrectomy specimens, cultured on collagen I-coated flasks and characterised as previously described [5]. Cell lysates were prepared by solubilisation of cells at passage 4 in boiling lysis buffer (10 mM Tris-pH 7.4, 1% SDS, 1 mM sodium orthovanadate) to which a mixture of protease inhibitors had been added. Insoluble material was pelleted by centrifugation and the supernatant boiled for 5 min in an equal volume of 2× SDS sample buffer. Renal brush border preparations were isolated from human whole cortical tissue using 10 mM CaCl2 as a precipitating agent [6]. PTEC lysate and brush border proteins were separated by SDS-PAGE and subjected to Western blot analyses with 0.1 µg/ml mouse anti-murine Dab2 (Transduction Laboratories, Lexington, USA)/1:10 000 peroxidase-labelled universal secondary antibody (Dako, Ely, UK). 2.6. Statistical analyses Statistics were analysed using Statview for Windows version 4.53 (Abacus Concepts). Direct measurements for equilibrium (KD) and off-rate (koff) constants for the interaction of Dab2 with megalin CT and synthetic WxNPxY sequences were compared using the paired t-test. Differences in initial rates of binding in the competition experiments were also analysed by paired t-testing. 3. Results 3.1. Direct affınity measurements Western blot analyses of purified recombinant material revealed specific bands at 50 and 55 kDa for Dab2PID-GST
H. Gallagher et al. / Biochimie 86 (2004) 179–182
181
Fig. 1. SPR analysis of the specific binding of Dab2PID (20 nM–4 µM) to megalin CT. Representative association and dissociation curves. Estimated KD = 2.6 × 10–7 ± 5.3 × 10–8 M (n = 3).
and megalin CT-GST, respectively. The interactions observed were not dependent upon the presence of GST on the recombinant proteins or biotin on the synthetic peptides. We found that the Dab2PID bound strongly to intact megalin CT (KD = 2.6 × 10–7 ± 5.3 × 10–8 M) (Fig. 1) and with comparable affinity to peptides encompassing both the second (KD = 3.2 × 10–7 ± 1.1 × 10–7 M) and third (KD = 6.1 × 10–8 ± 2.7 × 10–8 M) WxNPxY motifs. There was a trend (P = 0.1) towards higher affinity binding, as measured by the equilibrium rate constant, to the third as compared with the second WxNPxY motif. However, the dissociation of Dab2 from the third WxNPxY peptide was significantly slower than that from the second (koff (mean ± S.E.M.) (per s) = 0.002 ± 0.002 vs. 0.007 ± 0.002, P < 0.05), consistent with a higher affinity of binding to the former sequence.
Fig. 2. Inhibition of Dab2PID binding to immobilised megalin CT (above) and to immobilised third WxNPxY peptide (below) by synthetic tyrosine phosphorylated (pY) and non-phosphorylated second WxNPxY and third WxNPxY sequences. The third but not the second WxNPxY exerted a major inhibitory effect upon Dab2PID binding. Tyrosine phosphorylation had little impact upon competition efficacy. P < 0.05 compared with uninhibited.
3.2. Competition experiments An uninhibited Dab2PID concentration of 2 µM was selected on the basis that it offered a large and reproducible signal to inhibit. The experimental design was controlled for any systematic effects over time. The inhibition of Dab2PID binding both to intact megalin CT and to the third WxNPxY (Fig. 2) were studied on the basis of the results observed in the direct binding studies. The third WxNPxY was a more effective competitor of Dab2PID binding than the second in both series of experiments, consistent with a higher affinity of the PID for this motif. Tyrosine phosphorylation of either motif did not significantly influence competition efficacy on the peptide surface. On the intact CT surface the tyrosine phosphorylated but not the non-tyrosine phosphorylated second WxNPxY had a small, although statistically significant, effect on Dab2PID binding. 3.3. Dab2 expression in human primary PTEC and brush border preparations Immunoblotting with anti-Dab2 monoclonal antibody of microvillar membranes and lysates derived from primary PTEC confirm the potential for relevance of this protein in a human setting (Fig. 3). Control incubations in the absence of primary antibody were negative.
Fig. 3. Dab2 is expressed by both human brush border preparations and by cultured human primary PTEC. Lane 1, Dab2 purified from human foreskin fibroblasts; lane 2, molecular weight standards; lane 3, brush border preparations from fresh human kidney cortex; lanes 4 and 5, lysates from two human primary PTEC lines.
4. Discussion We have confirmed the interaction of Dab2 with megalin CT using SPR and measured an affinity which is in accordance with that documented for other PID–WxNPxY interactions and is sufficiently high as to afford probable biological relevance. Two lines of evidence support the contention
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that the interaction is with the third (from N to C) of megalin CT’s three WxNPxY motifs. The third WxNPxY displays slower dissociation from Dab2 than the second; and inhibition studies indicate that this motif is the more efficient competitor of Dab2 binding, both to intact CT and to itself. The presence of a b-turn within the PID-binding targets is a structural prerequisite for binding [7]. Residues critical for binding have been identified in many cases. However, other non-critical residues may also play a role [8]. Further studies are required to delineate the precise sequence determinants for binding to the Dab2PID. Despite the terminology, the presence of phosphotyrosine is not always required for the interaction of PID with their targets [9]. Preliminary evidence that tyrosine phosphorylation of megalin CT occurs on agonist stimulation has been presented [10], providing a rationale for study. However, on the basis of these experiments, agonist-induced phosphorylation of megalin CT does not appear to provide a link between the binding of extracellular ligand and any subsequent putative Dab2-mediated intracellular events. The role of Dab2PID-megalin CT binding remains uncertain. By analogy to disabled-1, which relays neuronal positional information in the central nervous system through LDL receptor family members [11], Dab2-megalin binding may play a role in the positioning of epithelial cells relative to basement membrane structures [12]. Alternatively, the interaction of Dab2 with megalin within the kidney may impact upon proliferative pathways via the Dab2-mediated uncoupling of receptor tyrosine kinase stimulation from Ras activation [13]. An involvement in endocytosis is a third possibility, given that the NPxY motif is recognised as an internalisation signal for clathrin-mediated endocytosis among LDL receptor family members. Dab2–/– mice exhibit proteinuria consistent with reduced megalin-mediated endocytosis [14], perhaps via the action of Dab2 as a novel endocytic adaptor [15]. In conclusion, this work has provided new evidence that Dab2 binds to the third WxNPxY on the CT of megalin. Although functional studies are required, it is likely the interaction plays a role within the PTEC in the regulation of cell positioning, proliferative or endocytic activity. References [1]
A.V. Oleinikov, J. Zhao, S.P. Makker, Cytosolic adaptor protein Dab2 is an intracellular ligand of endocytic receptor gp600/megalin, Biochemical Journal 3 (2000) 613–621.
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