title: Megalin and nonmuscle myosin heavy chain IIA interact with the adaptor protein Disabled-2 in proximal tubule cells.
abstract: 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 beta-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 (125)I-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.
authors.affiliation: Division of Clinical Nephrology and Rheumatology, Niigata University Graduate School of Medical and Dental Sciences, Niigata, Japan.
authors.forename: Kiyoko
authors.initials: K
authors.lastname: Hosaka
authors.affiliation: Division of Clinical Nephrology and Rheumatology, Niigata University Graduate School of Medical and Dental Sciences, Niigata, Japan. Electronic address: tetakeda@med.niigata-u.ac.jp.
authors.forename: Tetsuro
authors.initials: T
authors.lastname: Takeda
authors.affiliation: Department of Applied Molecular Medicine, Niigata University Graduate School of Medical and Dental Sciences, Niigata, Japan.
authors.forename: Noriaki
authors.initials: N
authors.lastname: Iino
authors.affiliation: Division of Clinical Nephrology and Rheumatology, Niigata University Graduate School of Medical and Dental Sciences, Niigata, Japan.
authors.forename: Michihiro
authors.initials: M
authors.lastname: Hosojima
authors.affiliation: Department of Applied Molecular Medicine, Niigata University Graduate School of Medical and Dental Sciences, Niigata, Japan; Center for Transdisciplinary Research, Niigata University, Niigata, Japan.
authors.forename: Hiroyoshi
authors.initials: H
authors.lastname: Sato
authors.affiliation: Division of Clinical Nephrology and Rheumatology, Niigata University Graduate School of Medical and Dental Sciences, Niigata, Japan.
authors.forename: Ryohei
authors.initials: R
authors.lastname: Kaseda
authors.affiliation: Division of Clinical Nephrology and Rheumatology, Niigata University Graduate School of Medical and Dental Sciences, Niigata, Japan.
authors.forename: Keiko
authors.initials: K
authors.lastname: Yamamoto
authors.affiliation: Department of Applied Molecular Medicine, Niigata University Graduate School of Medical and Dental Sciences, Niigata, Japan.
authors.forename: Asako
authors.initials: A
authors.lastname: Kobayashi
authors.affiliation: Division of Clinical Nephrology and Rheumatology, Niigata University Graduate School of Medical and Dental Sciences, Niigata, Japan.
authors.forename: Fumitake
authors.initials: F
authors.lastname: Gejyo
authors.affiliation: Department of Applied Molecular Medicine, Niigata University Graduate School of Medical and Dental Sciences, Niigata, Japan; Center for Transdisciplinary Research, Niigata University, Niigata, Japan.
authors.forename: Akihiko
authors.initials: A
authors.lastname: Saito