title: P-glycoprotein binds to ezrin at amino acid residues 149-242 in the FERM domain and plays a key role in the multidrug resistance of human osteosarcoma.
abstract: Overexpression of the mdr1 gene encoding P-glycoprotein (Pgp) exerts a major role in reducing the effectiveness of cytotoxic therapy in osteosarcoma. The interaction between actin and Pgp has been shown to be instrumental in the establishment of multidrug resistance (MDR) in human tumor cells. The cytoskeleton linker ezrin exerts a pivotal role in maintaining the functional connection between actin and Pgp. We investigated the role of ezrin in a human multidrug-resistant osteosarcoma cell line overexpressing Pgp and compared it to its counterpart that overexpresses an ezrin deletion mutant. The results showed that Pgp binds at amino acid residues 149-242 of the N-terminal domain of ezrin. The interaction between ezrin and Pgp occurs in the plasma membrane of MDR cells, where they also co-localize with the ganglioside G(M1)  located in lipid rafts. The overexpression of the ezrin deletion mutant entirely restored drug susceptibility of osteosarcoma cells, consistent with Pgp dislocation to cytoplasmic compartments and abrogation of G(M1) /Pgp co-localization at the plasma membrane. Our study provides evidence that ezrin exerts a key role in MDR of human osteosarcoma cells through a Pgp-ezrin-actin connection that is instrumental for the permanence of Pgp into plasma membrane lipid rafts. We also show for the first time that Pgp-binding site is localized to amino acid residues 149-242 of the ezrin Band 4.1, Ezrin/Radixin/Moesin (FERM) domain, thus proposing a specific target for future molecular therapy aimed at counteracting MDR in osteosarcoma patients.
authors.affiliation: Department of Therapeutic Research and Medicines Evaluation, Istituto Superiore di Sanità, Viale Regina Elena 299, Rome, Italy.
authors.forename: Daria
authors.initials: D
authors.lastname: Brambilla
authors.forename: Silvia
authors.initials: S
authors.lastname: Zamboni
authors.forename: Cristina
authors.initials: C
authors.lastname: Federici
authors.forename: Luana
authors.initials: L
authors.lastname: Lugini
authors.forename: Francesco
authors.initials: F
authors.lastname: Lozupone
authors.forename: Angelo
authors.initials: A
authors.lastname: De Milito
authors.forename: Serena
authors.initials: S
authors.lastname: Cecchetti
authors.forename: Maurizio
authors.initials: M
authors.lastname: Cianfriglia
authors.forename: Stefano
authors.initials: S
authors.lastname: Fais
mesh_terms: D020168:ATP Binding Cassette Transporter, Subfamily B, Member 1 / Q000096:biosynthesis / Q000378:metabolism*; D001859:Bone Neoplasms / Q000188:drug therapy / Q000378:metabolism; D045744:Cell Line, Tumor; D002462:Cell Membrane / Q000378:metabolism; D003598:Cytoskeletal Proteins / Q000235:genetics / Q000378:metabolism*; D019008:Drug Resistance, Neoplasm / Q000235:genetics; D005677:G(M1) Ganglioside / Q000378:metabolism; D006801:Humans; D021962:Membrane Microdomains; D012516:Osteosarcoma / Q000188:drug therapy* / Q000378:metabolism*; D000099149:Ezrin
chemical_list: D020168:ATP Binding Cassette Transporter, Subfamily B, Member 1; D003598:Cytoskeletal Proteins; D005677:G(M1) Ganglioside; D000099149:Ezrin; C478965:EPB41L4B protein, human