Megalin and nonmuscle myosin heavy chain IIA interact with the adaptor protein Disabled-2 in proximal tubule cells
2009; Elsevier BV; Volume: 75; Issue: 12 Linguagem: Inglês
10.1038/ki.2009.85
ISSN1523-1755
AutoresKiyoko Hosaka, T. Takeda, Noriaki Iino, Michihiro Hosojima, Hiroyoshi Sato, Ryohei Kaseda, Keiko Yamamoto, Asako Kobayashi, Fumitake Gejyo, Akihiko Saito,
Tópico(s)Wnt/β-catenin signaling in development and cancer
ResumoMegalin plays a critical role in the endocytosis of albumin and other filtered low-molecular-weight proteins. Here we studied the interaction between megalin and Disabled-2 (Dab2), an adaptor protein that binds to the cytoplasmic domain of megalin and appears to control its trafficking. We co-immunoprecipitated megalin and Dab2 from cultured proximal tubule cells and identified the proteins by liquid chromatography and tandem mass spectrometry. We found two proteins associated with the megalin/Dab2 complex, nonmuscle myosin heavy chain IIA (NMHC-IIA) and β-actin. Subcellular fractionation followed by sucrose velocity gradient separation showed that megalin, Dab2, and NMHC-IIA existed as a complex in the same endosomal fractions. In vitro pull-down assays demonstrated that NMHC-IIA was bound to the carboxyl-terminal region of Dab2, but not to megalin's cytoplasmic domain. We then transfected COS-7 cells with plasmids that induced the expression of Dab2, NMHC-IIA, and the megalin minireceptor, a truncated form of megalin. Co-immunoprecipitation studies showed that the minireceptor and NMHC-IIA co-immunoprecipitated only with Dab2. Furthermore, the uptake of 125I-lactoferrin, an endocytic ligand of megalin, by rat yolk sac–derived megalin-expressing L2 cells was inhibited by blebbistatin, a specific inhibitor of nonmuscle myosin II. Our study shows that NMHC-IIA is functionally linked to megalin by interaction with Dab2 and is likely involved in megalin-mediated endocytosis in proximal tubule cells. Megalin plays a critical role in the endocytosis of albumin and other filtered low-molecular-weight proteins. Here we studied the interaction between megalin and Disabled-2 (Dab2), an adaptor protein that binds to the cytoplasmic domain of megalin and appears to control its trafficking. We co-immunoprecipitated megalin and Dab2 from cultured proximal tubule cells and identified the proteins by liquid chromatography and tandem mass spectrometry. We found two proteins associated with the megalin/Dab2 complex, nonmuscle myosin heavy chain IIA (NMHC-IIA) and β-actin. Subcellular fractionation followed by sucrose velocity gradient separation showed that megalin, Dab2, and NMHC-IIA existed as a complex in the same endosomal fractions. In vitro pull-down assays demonstrated that NMHC-IIA was bound to the carboxyl-terminal region of Dab2, but not to megalin's cytoplasmic domain. We then transfected COS-7 cells with plasmids that induced the expression of Dab2, NMHC-IIA, and the megalin minireceptor, a truncated form of megalin. Co-immunoprecipitation studies showed that the minireceptor and NMHC-IIA co-immunoprecipitated only with Dab2. Furthermore, the uptake of 125I-lactoferrin, an endocytic ligand of megalin, by rat yolk sac–derived megalin-expressing L2 cells was inhibited by blebbistatin, a specific inhibitor of nonmuscle myosin II. Our study shows that NMHC-IIA is functionally linked to megalin by interaction with Dab2 and is likely involved in megalin-mediated endocytosis in proximal tubule cells. Proximal tubule cells (PTC) are involved in the uptake and degradation of glomerular-filtered proteins by receptor-mediated endocytosis.1.Christensen E.I. Gburek J. Protein reabsorption in renal proximal tubule-function and dysfunction in kidney pathophysiology.Pediatr Nephrol. 2004; 19: 714-721Crossref PubMed Scopus (125) Google Scholar This process is important for proper metabolization of serum proteins, and also for the regulation of urinary protein excretion (such as albuminuria), which is a diagnostic and prognostic marker for chronic kidney disease and an indicator of cardiovascular risks.2.Remuzzi G. Bertani T. Pathophysiology of progressive nephropathies.N Engl J Med. 1998; 339: 1448-1456Crossref PubMed Scopus (1150) Google Scholar, 3.Go A.S. Chertow G.M. Fan D. et al.Chronic kidney disease and the risks of death, cardiovascular events, and hospitalization.N Engl J Med. 2004; 351: 1296-1305Crossref PubMed Scopus (8986) Google Scholar However, the molecular mechanisms of receptor-mediated endocytosis in PTC have not been fully understood. Megalin, a large (∼600 kDa) member of the low-density lipoprotein receptor family,4.Raychowdhury R. Niles J.L. McCluskey R.T. et al.Autoimmune target in Heymann nephritis is a glycoprotein with homology to the LDL receptor.Science. 1989; 244: 1163-1165Crossref PubMed Scopus (215) Google Scholar, 5.Saito A. Pietromonaco S. Loo A.K. et al.Complete cloning and sequencing of rat gp330/'megalin,' a distinctive member of the low density lipoprotein receptor gene family.Proc Natl Acad Sci USA. 1994; 91: 9725-9729Crossref PubMed Scopus (497) Google Scholar is found in the apical region of PTC. Megalin plays a critical role in the proximal tubular endocytosis of glomerular-filtered low-molecular weight proteins and albumin.6.Christensen E.I. Birn H.N. Megalin and cubilin: multifunctional endocytic receptors.Nat Rev Mol Cell Biol. 2002; 3: 256-266Crossref PubMed Scopus (651) Google Scholar Megalin knockout mice display symptoms of low-molecular weight proteinuria and albuminuria, as well as osteopathy.7.Nykjaer A. Dragun D. Walther D. et al.An endocytic pathway essential for renal uptake and activation of the steroid 25-(OH) vitamin D3.Cell. 1999; 96: 507-515Abstract Full Text Full Text PDF PubMed Scopus (836) Google Scholar, 8.Leheste J.R. Rolinski B. Vorum H. et al.Megalin knockout mice as an animal model of low molecular weight proteinuria.Am J Pathol. 1999; 155: 1361-1370Abstract Full Text Full Text PDF PubMed Scopus (351) Google Scholar, 9.Leheste J.R. Melsen F. Wellner M. et al.Hypocalcemia and osteopathy in mice with kidney-specific megalin gene defect.FASEB J. 2003; 17: 247-249Crossref PubMed Scopus (142) Google Scholar The C-terminal cytoplasmic tail domain of megalin encodes FXNPXY motifs for endocytosis signaling, as well as other protein interaction motifs (SH3 and PDZ domains) and phosphorylation sites.5.Saito A. Pietromonaco S. Loo A.K. et al.Complete cloning and sequencing of rat gp330/'megalin,' a distinctive member of the low density lipoprotein receptor gene family.Proc Natl Acad Sci USA. 1994; 91: 9725-9729Crossref PubMed Scopus (497) Google Scholar, 10.Hjalm G. Murray E. Crumley G. et al.Cloning and sequencing of human gp330, a Ca(2+)-binding receptor with potential intracellular signaling properties.Eur J Biochem. 1996; 239: 132-137Crossref PubMed Scopus (135) Google Scholar The cytoplasmic domain has been found to interact specifically with a variety of adaptor proteins including disabled-2 (Dab2),11.Oleinikov A.V. Zhao J. Makker S.P. Cytosolic adaptor protein Dab2 is an intracellular ligand of endocytic receptor gp600/megalin.Biochem J. 2000; 347: 613-621Crossref PubMed Google Scholar and others.12.Nagai M. Meerloo T. Takeda T. et al.The adaptor protein ARH escorts megalin to and through endosomes.Mol Biol Cell. 2003; 14: 4984-4996Crossref PubMed Scopus (89) Google Scholar, 13.Rader K. Orlando R.A. Lou X. et al.Characterization of ANKRA, a novel ankyrin repeat protein that interacts with the cytoplasmic domain of megalin.J Am Soc Nephrol. 2000; 11: 2167-2178PubMed Google Scholar, 14.Petersen H.H. Hilpert J. Militz D. et al.Functional interaction of megalin with the megalinbinding protein (MegBP), a novel tetratrico peptide repeat-containing adaptor molecule.J Cell Sci. 2003; 116: 453-461Crossref PubMed Scopus (42) Google Scholar, 15.Patrie K.M. Drescher A.J. Goyal M. et al.The membrane-associated guanylate kinase protein MAGI-1 binds megalin and is present in glomerular podocytes.J Am Soc Nephrol. 2001; 12: 667-677Crossref PubMed Google Scholar, 16.Lou X. McQuistan T. Orlando R.A. et al.GAIP, GIPC and Galphai3 are concentrated in endocytic compartments of proximal tubule cells: putative role in regulating megalin's function.J Am Soc Nephrol. 2002; 13: 918-927PubMed Google Scholar Although these adaptor proteins are hypothesized to function with other intracellular proteins during megalin-mediated endocytosis, their functional partners are largely unknown. Dab2 is an intracellular adaptor protein with an N-terminal phosphotyrosine interaction domain that binds to the second FXNPXY motif of megalin's cytoplasmic domain.11.Oleinikov A.V. Zhao J. Makker S.P. Cytosolic adaptor protein Dab2 is an intracellular ligand of endocytic receptor gp600/megalin.Biochem J. 2000; 347: 613-621Crossref PubMed Google Scholar, 17.Gallagher H. Oleinikov A.V. Fenske C. et al.The adaptor disabled-2 binds to the third psi xNPxY sequence on the cytoplasmic tail of megalin.Biochimie. 2004; 86: 179-182Crossref PubMed Scopus (24) Google Scholar Dab2 co-localizes with megalin in clathrin-coated pits and vesicles, but not in dense apical tubules or brush border.18.Nagai J. Christensen E.I. Morris S.M. et al.Mutually dependent localization of megalin and Dab2 in the renal proximal tubule.Am J Physiol Renal Physiol. 2005; 289: F569-F576Crossref PubMed Scopus (84) Google Scholar Dab2 conditional knockout mice show reduced numbers of clathrin-coated pits in PTC and also excrete megalin ligands in the urine.19.Morris S.M. Tallquist M.D. Rock C.O. et al.Dual roles for the Dab2 adaptor protein in embryonic development and kidney transport.EMBO J. 2002; 21: 1555-1564Crossref PubMed Scopus (174) Google Scholar Kidney-specific megalin knockout mice show a nearly complete loss of Dab2 expression in PTC.18.Nagai J. Christensen E.I. Morris S.M. et al.Mutually dependent localization of megalin and Dab2 in the renal proximal tubule.Am J Physiol Renal Physiol. 2005; 289: F569-F576Crossref PubMed Scopus (84) Google Scholar Reciprocally, knockout of Dab2 results in redistribution of megalin from endosomes to microvilli.18.Nagai J. Christensen E.I. Morris S.M. et al.Mutually dependent localization of megalin and Dab2 in the renal proximal tubule.Am J Physiol Renal Physiol. 2005; 289: F569-F576Crossref PubMed Scopus (84) Google Scholar These findings indicate that the subcellular localization of Dab2 requires megalin, and that Dab2 plays a critical role in megalin trafficking in PTC. Recently, Dab2 was found to interact with myosin VI,20.Inoue A. Sato O. Homma K. et al.DOC-2/DAB2 is the binding partner of myosin VI.Biochem Biophys Res Commun. 2002; 292: 300-307Crossref PubMed Scopus (59) Google Scholar, 21.Morris S.M. Arden S.D. Roberts R.C. et al.Myosin VI binds to and localises with Dab2, potentially linking receptor-mediated endocytosis and the actin cytoskeleton.Traffic. 2002; 3: 331-341Crossref PubMed Scopus (202) Google Scholar the 'unconventional myosin'—an actin-based molecular motor expressed only in higher eukaryotes.22.Buss F. Luzio J.P. Kendrick-Jones J. Myosin VI, an actin motor for membrane traffic and cell migration.Traffic. 2002; 3: 851-858Crossref PubMed Scopus (70) Google Scholar, 23.Hasson T. Myosin VI: two distinct roles in endocytosis.J Cell Sci. 2003; 116: 3453-3461Crossref PubMed Scopus (142) Google Scholar The interaction between Dab2 and myosin VI has been hypothesized to involve the formation of clathrin-coated vesicles.23.Hasson T. Myosin VI: two distinct roles in endocytosis.J Cell Sci. 2003; 116: 3453-3461Crossref PubMed Scopus (142) Google Scholar However, it is probable that Dab2 also interacts with other motor or adaptor proteins, because myosin VI is known to move only towards the minus end of actin filaments24.Wells A.L. Lin A.W. Chen L.Q. et al.Myosin VI is an actin-based motor that moves backwards.Nature. 1999; 401: 505-508Crossref PubMed Scopus (554) Google Scholar and additional machinery is likely to be required for megalin trafficking. Based on these previous findings and hypotheses, our study was designed to identify Dab2-interacting proteins and investigate their involvement in megalin trafficking in PTC. Here, we report that non-muscle myosin heavy chain IIA (NMHC-IIA) is a novel Dab2-binding protein that is functionally linked to megalin. Megalin and Dab2 are known to form a complex in immortalized rat proximal tubule cells (IRPTC) and rat yolk sac epithelium-derived L2 cells.11.Oleinikov A.V. Zhao J. Makker S.P. Cytosolic adaptor protein Dab2 is an intracellular ligand of endocytic receptor gp600/megalin.Biochem J. 2000; 347: 613-621Crossref PubMed Google Scholar To screen Dab2-interacting proteins that may be involved in megalin trafficking in PTC, IRPTC were used as a starting tool for the assay, and then L2 cells were used for its verification. Megalin- and Dab2-associated protein complexes were isolated from lysates of IRPTC by immunoprecipitation with a polyclonal antibody raised against immunopurified rat megalin (Supplementary Figure S1), and a polyclonal anti-Dab2 antibody, respectively. Immunoprecipitates were resolved by 4–15% gradient SDS-polyacrylamide gel electrophoresis (PAGE) and stained with SYPRO Ruby. Among the stained proteins shown in Figure 1, the identities of megalin and Dab2 were confirmed by immunoblot analyses (Supplementary Figure S2). To identify the two proteins that co-precipitated with either anti-megalin or anti-Dab2 antibodies, gel slices at ∼210 and ∼40 kDa in mass were excised, digested in a trypsin solution, and the resulting peptides were then analyzed by liquid chromatography coupled with tandem mass spectrometers (LC-MS/MS). Analysis of the ∼210 kDa band resulted in the identification of 31 peptides whose sequences corresponded completely to NMHC-IIA, with 20% total sequence coverage (Supplementary Figure S3). Similarly, the ∼40 kDa band was identified as β-actin, with 40% total sequence coverage (Supplementary Figure S4). Download .pdf (.37 MB) Help with pdf files Supplementary Figures 1 Download .pdf (.51 MB) Help with pdf files Supplementary Figures 2 Download .pdf (.05 MB) Help with pdf files Supplementary Figures 3 Download .pdf (.06 MB) Help with pdf files Supplementary Figures 4 To confirm the identity of the ∼210 kDa protein as NMHC-IIA, and to verify the association of this protein with megalin and Dab2, lysates of IRPTC and L2 cells were prepared. After incubation of the lysates with a monoclonal anti-megalin antibody, megalin-associated immunoprecipitates were analyzed by immunoblotting with anti-NMHC-IIA and anti-Dab2 antibodies. Both NMHC-IIA and Dab2 were detected in megalin-associated immunoprecipitates from the two different cell lines (Figure 2, lanes 2 and 5), but not in the control IgG immunoprecipitates (Figure 2, lanes 3 and 6). Also, we carried out subcellular fractionation of rat renal cortices on sucrose density gradients to investigate the fractions containing megalin, Dab2 and NMHC-IIA. As shown in Figure 3a and b, these three proteins were found in the same subcellular fractions where an endosome marker Rab 5A was contained. To validate that the three proteins form a complex in vivo, we next performed sucrose velocity gradient sedimentation experiments, followed by co-immunoprecipitation. As shown in Figure 3c and d, immunoprecipitation of the heavier fractions (no. 9 and no. 11) with the polyclonal anti-megalin revealed that the three proteins assembled together. Class II myosins are two-headed motor proteins composed of two heavy chains and two pairs of light chains. In humans, there are three non-muscle myosin II heavy chains (IIA, IIB, and IIC).25.Berg J.S. Powell B.C. Cheney R.E. A millennial myosin census.Mol Biol Cell. 2001; 12: 780-794Crossref PubMed Scopus (619) Google Scholar Because the N-termini of dimerized myosin II heavy chains are known to constitute an actin-binding domain, we hypothesized that this was the mechanism for co-immunoprecipitation of β-actin with NMHC-IIA. However, the molecular basis for NMHC-IIA associating with Dab2 and megalin was not known. We used in vitro Glutathione S-transferase (GST) pull-down assays to test whether NMHC-IIA binds directly to Dab2 or to the cytoplasmic domain of megalin. In vitro-translated NMHC-IIA bound to GST fused to full-length Dab2 (GST-Dab2) (Figure 4, lane 3), but not to GST fused to the cytoplasmic domain of megalin (Figure 4, lane 6), or GST alone (Figure 4, lane 2). Using the same procedure, the GST-megalin cytoplasmic domain protein did bind in vitro-translated autosomal recessive hypercholesterolemia (ARH), an adaptor protein of megalin12.Nagai M. Meerloo T. Takeda T. et al.The adaptor protein ARH escorts megalin to and through endosomes.Mol Biol Cell. 2003; 14: 4984-4996Crossref PubMed Scopus (89) Google Scholar (data not shown). In further experiments mapping the interaction of in vitro-translated NMHC-IIA to Dab2, NMHC-IIA bound to GST fused to the C-terminal region of Dab2 (GST-C-Dab2) (aa 447–770) (Figure 4, lane 4), but not to the N-terminal region (GST-N-Dab2) (aa 1-446) (Figure 4, lane 5). Thus, NMHC-IIA binds directly to the C-terminal region of Dab2, but not to megalin. As the C-terminal region of Dab2 binds to NMHC-IIA, although its N-terminal phosphotyrosine interaction domain binds to megalin's cytoplasmic domain,11.Oleinikov A.V. Zhao J. Makker S.P. Cytosolic adaptor protein Dab2 is an intracellular ligand of endocytic receptor gp600/megalin.Biochem J. 2000; 347: 613-621Crossref PubMed Google Scholar, 17.Gallagher H. Oleinikov A.V. Fenske C. et al.The adaptor disabled-2 binds to the third psi xNPxY sequence on the cytoplasmic tail of megalin.Biochimie. 2004; 86: 179-182Crossref PubMed Scopus (24) Google Scholar we hypothesize that one of the roles of Dab2 in vivo is to function as a physical link connecting these two proteins. To test this hypothesis, plasmids encoding green fluorescent protein (GFP)-tagged NMHC-IIA, V5-tagged Dab2 and a 'megalin minireceptor' were co-transfected into COS-7 cells, which express endogenously NMHC-IIB, but not NMHC-IIA.26.Kolega J. Cytoplasmic dynamics of myosin IIA and IIB: spatial 'sorting' of isoforms in locomoting cells.J Cell Sci. 1998; 111: 2085-2095PubMed Google Scholar The megalin minireceptor, which is truncated to maintain the fourth ligand-binding domain (LBD) through the cytoplasmic domain, has been shown to mimic the function and trafficking of native megalin.27.Takeda T. Yamazaki H. Farquhar M.G. Identification of an apical sorting determinant in the cytoplasmic tail of megalin.Am J Physiol Cell Physiol. 2003; 284: C1105-C1113Crossref PubMed Scopus (108) Google Scholar Lysates prepared from the co-transfected cells were used for immunoprecipitation analysis with the anti-megalin or anti-GFP antibody, followed by immunoblotting with the anti-GFP or anti-megalin antibody, respectively (Figure 5). In the lysates from the cells co-transfected with all the three plasmids, GFP-NMHC-IIA was detected clearly in the anti-megalin immunoprecipitates (Figure 5a, lane 5), and very little NMHC-IIA precipitated from the lysates lacking V5-tagged Dab2 (Figure 5a, lane 2; Figure 5b, lane 2). The low-level association of the megalin minireceptor and GFP-NMHC-IIA in the absence of transfected V5-tagged Dab2 is supposedly due to small amounts of endogenous Dab2 in the COS-7 cells (Figure 5a, lane 2). In a reciprocal experiment, the megalin minireceptor was clearly detected in GFP-immunoprecipitates when cells were co-transfected with the three plasmids (Figure 5b, lane 5). These results indicate that megalin associates with NMHC-IIA through interactions with Dab2, and that megalin, Dab2, and NMHC-IIA form a multimeric complex in co-transfected cells. Previous studies suggest that non-muscle myosin II plays a role in vesicle trafficking.28.Chan W. Calderon G. Swift A.L. et al.Myosin II regulatory light chain is required for trafficking of bile salt export protein to the apical membrane in Madin-Darby canine kidney cells.J Biol Chem. 2005; 280: 23741-23747Crossref PubMed Scopus (57) Google Scholar, 29.Steimle P.A. Fulcher F.K. Patel Y.M. A novel role for myosin II in insulin-stimulated glucose uptake in 3T3-L1 adipocytes.Biochem Biophys Res Commun. 2005; 331: 1560-1565Crossref PubMed Scopus (26) Google Scholar, 30.Rey M. Valenzuela-Fernandez A. Urzainqui A. et al.Myosin IIA is involved in the endocytosis of CXCR4 induced by SDF-1alpha.J Cell Sci. 2007; 120: 1126-1133Crossref PubMed Scopus (55) Google Scholar Non-muscle myosin II activity is specifically inhibited by blebbistatin,31.Straight A.F. Cheung A. Limouze J. et al.Dissecting temporal and spatial control of cytokinesis with a myosin II Inhibitor.Science. 2003; 299: 1743-1747Crossref PubMed Scopus (1113) Google Scholar which is cell permeable and does not alter the activities of other myosins.31.Straight A.F. Cheung A. Limouze J. et al.Dissecting temporal and spatial control of cytokinesis with a myosin II Inhibitor.Science. 2003; 299: 1743-1747Crossref PubMed Scopus (1113) Google Scholar, 32.Kovacs M. Toth J. Hetenyi C. et al.Mechanism of blebbistatin inhibition of myosin II.J Biol Chem. 2004; 279: 35557-35563Crossref PubMed Scopus (707) Google Scholar To examine whether blebbistatin affects the endocytic function of megalin, megalin-expressing L2 cells, with similar characteristics of PTC33.Orlando R.A. Farquhar M.G. Identification of a cell line that expresses a cell surface and a soluble form of the gp330/receptor-associated protein (RAP) Heymann nephritis antigenic complex.Proc Natl Acad Sci USA. 1993; 90: 4082-4086Crossref PubMed Scopus (73) Google Scholar and well-established for the analysis of megalin-mediated endocytosis34.Saito A. Kazama J.J. Iino N. et al.Bioengineered implantation of megalin-expressing cells: a potential intracorporeal therapeutic model for uremic toxin protein clearance in renal failure.J Am Soc Nephrol. 2003; 14: 2025-2032Crossref PubMed Scopus (31) Google Scholar, 35.Saito A. Nagai R. Tanuma A. et al.Role of megalin in endocytosis of advanced glycation end products: implications for a novel protein binding to both megalin and advanced glycation end products.J Am Soc Nephrol. 2003; 14: 1123-1131Crossref PubMed Scopus (38) Google Scholar, 36.Hama H. Saito A. Takeda T. et al.Evidence indicating that renal tubular metabolism of leptin is mediated by megalin but not by the leptin receptors.Endocrinology. 2004; 145: 3935-3940Crossref PubMed Scopus (87) Google Scholar, 37.Oyama Y. Takeda T. Hama H. et al.Evidence for megalin-mediated proximal tubular uptake of L-FABP, a carrier of potentially nephrotoxic molecules.Lab Invest. 2005; 85: 522-531Crossref PubMed Scopus (82) Google Scholar, 38.Kaseda R. Iino N. Hosojima M. et al.Megalin-mediated endocytosis of cystatin C in proximal tubule cells.Biochem Biophys Res Commun. 2007; 357: 1130-1134Crossref PubMed Scopus (71) Google Scholar were assayed in either the presence or absence of blebbistatin (100 μM) for the uptake of lactoferrin, an endocytic ligand of megalin.39.Willnow T.E. Goldstein J.L. Orth K. et al.Low density lipoprotein receptor-related protein and gp330 bind similar ligands, including plasminogen activator-inhibitor complexes and lactoferrin, an inhibitor of chylomicron remnant clearance.J Biol Chem. 1992; 267: 26172-26180Abstract Full Text PDF PubMed Google Scholar Blebbistatin treatment of the cells inhibited lactoferrin degradation by approximately 18 and 42% after two and four hours of incubation, respectively, compared with vehicle-only controls (Figure 6). Associations of the lactoferrin ligand to cells were also reduced by blebbistatin. Furthermore, we investigated the effects of blebbistatin on the endocytic trafficking of megalin by using monoclonal anti-megalin IgG (20B) that binds to megalin on L2 cell surface and tracking its endocytic pathway.12.Nagai M. Meerloo T. Takeda T. et al.The adaptor protein ARH escorts megalin to and through endosomes.Mol Biol Cell. 2003; 14: 4984-4996Crossref PubMed Scopus (89) Google Scholar We found that the internalization of 20B-bound megalin into L2 cells was much decreased by treating the cells with blebbistatin (Supplementary Figures S5 and S6). Blebbistatin was found not to affect fluid-phase endocytosis (Supplementary Figure S7) and did not induce apparent toxicity in L2 cells (Supplementary Figure S8). These data suggest that non-muscle myosin II activity is a necessary component of megalin's endocytic functions. Download .pdf (.93 MB) Help with pdf files Supplementary Figures 5 Download .pdf (.16 MB) Help with pdf files Supplementary Figures 6 Download .pdf (.08 MB) Help with pdf files Supplementary Figures 7 Download .pdf (.08 MB) Help with pdf files Supplementary Figures 8 In this study, we investigated protein complexes associated with Dab2-mediated trafficking of megalin, a multiligand endocytic receptor in PTC, by screening and characterizing proteins that co-immunoprecipitate with both Dab2 and megalin. We found that NMHC-IIA binds directly to the C-terminal region of Dab2 and is linked to megalin through the Dab2 interaction. Class II myosins (conventional type) are hexameric two-headed motor proteins composed of two heavy chains and two pairs of light chains. Dimerization of the heavy chains yields a polar structure; the N-termini form two globular heads with actin- and ATP-binding domains required for motor activity, whereas the α-helical C-termini form a single rod-like coiled-coil tail, which allows the molecules to polymerize into bipolar filaments. In humans, there are three non-muscle myosin II heavy chains (IIA, IIB, and IIC) encoded by separate genes (MYH9, MYH10, and MYH14, respectively).25.Berg J.S. Powell B.C. Cheney R.E. A millennial myosin census.Mol Biol Cell. 2001; 12: 780-794Crossref PubMed Scopus (619) Google Scholar Besides their well-characterized roles in contraction and force production in muscles, Class II myosins are also required for cytoskeleton organization and motility of non-muscle cells.40.Sellers J.R. Myosins: a diverse superfamily.Biochim Biophys Acta. 2000; 1496: 3-22Crossref PubMed Scopus (616) Google Scholar There are also reports suggesting the involvement of Class II myosins in vesicle trafficking,41.Neco P. Giner D. Viniegra S. et al.New roles of myosin II during vesicle transport and fusion in chromaffin cells.J Biol Chem. 2004; 279: 27450-27457Crossref PubMed Scopus (111) Google Scholar, 42.Togo T. Steinhardt R.A. Nonmuscle myosin IIA and IIB have distinct functions in the exocytosis-dependent process of cell membrane repair.Mol Biol Cell. 2004; 15: 688-695Crossref PubMed Scopus (85) Google Scholar but the mechanisms have been largely undetermined. Genetic alterations of NMHC-IIA are known to cause inherited human diseases, known as MYH9 disorders, that are characterized by giant platelets, thrombocytopenia and granulocyte inclusions.43.Kelley M.J. Jawien W. Ortel T.L. et al.Mutation of MYH9, encoding non-muscle myosin heavy chain A, in May-Hegglin anomaly.Nat Genet. 2000; 26: 106-108Crossref PubMed Scopus (204) Google Scholar, 44.Seri M. Cusano R. Gangarossa S. et al.Mutations in MYH9 result in the May-Hegglin anomaly, and Fechtner and Sebastian syndromes. The May-Heggllin/Fechtner Syndrome Consortium.Nat Genet. 2000; 26: 103-105Crossref PubMed Scopus (370) Google Scholar The spectrum of diseases due to mutations in MYH9 includes May-Hegglin anomaly, Sebastian syndrome, Fechtner syndrome and Epstein syndrome.43.Kelley M.J. Jawien W. Ortel T.L. et al.Mutation of MYH9, encoding non-muscle myosin heavy chain A, in May-Hegglin anomaly.Nat Genet. 2000; 26: 106-108Crossref PubMed Scopus (204) Google Scholar, 44.Seri M. Cusano R. Gangarossa S. et al.Mutations in MYH9 result in the May-Hegglin anomaly, and Fechtner and Sebastian syndromes. The May-Heggllin/Fechtner Syndrome Consortium.Nat Genet. 2000; 26: 103-105Crossref PubMed Scopus (370) Google Scholar, 45.Heath K.E. Campos-Barros A. Toren A. et al.Nonmuscle myosin heavy chain IIA mutations define a spectrum of autosomal dominant macrothrombocytopenias: May-Hegglin anomaly and Fechtner, Sebastian, Epstein, and Alport-like syndromes.Am J Hum Genet. 2001; 69: 1033-1045Abstract Full Text Full Text PDF PubMed Scopus (248) Google Scholar, 46.Arrondel C. Vodovar N. Knebelmann B. et al.Expression of the nonmuscle myosin heavy chain IIA in the human kidney and screening for MYH9 mutations in Epstein and Fechtner syndromes.J Am Soc Nephrol. 2002; 13: 65-74Crossref PubMed Google Scholar
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