Protein 4.1R self-association: identification of the binding domain.
Erythroid protein 4.1 (4.1R) stabilizes the spectrin-actin network and anchors it to the plasma membrane. To contribute to the characterization of non-erythroid protein 4.1R, we used sedimentation, pull-down and co-immunoprecipitation assays to investigate the ability of protein 4.1R to establish inter-/intra-molecular associations. We demonstrated that the small 4.1R isoforms of 60 kDa (4.1R60), but not the larger isoforms of 80 and 135 kDa (4.1R80 and 4.1R135), were self-associated, and that a domain contained in all 4.1R isoforms, the core region, was responsible for 4.1R self-association. Results from denaturing-renaturing experiments, in which an initially non-self-associated 4.1R80 isoform became self-associated, suggested that an initially hidden core region was subsequently exposed. This hypothesis was supported by results from pull-down assays, which showed that the core region interacted with the N-terminal end of the FERM (4.1, ezrin, radixin, moesin) domain that is present in 4.1R80 and 4.1R135 isoforms but absent from 4.1R60 isoforms. Consistently, 4.1R80 isoforms bound neither to each other nor to 4.1R60 isoforms. We propose that 4.1R60 isoforms are constitutively self-associated, whereas 4.1R80 and 4.1R135 self-association is prevented by intramolecular interactions.
Pérez-Ferreiro
Carmen M
CM
Departamento de Biología Molecular, CBMSO (Centro de Biología Molecular Severo Ochoa), UAM/CSIC, Cantoblanco, E-28049 Madrid, Spain. cmperez@cbm.uam.es
Lospitao
Eva
E
Correas
Isabel
I
D000818:Animals; D001665:Binding Sites; D001798:Blood Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D019556:COS Cells; D002522:Chlorocebus aethiops; D003598:Cytoskeletal Proteins; D008565:Membrane Proteins; D008869:Microtubule-Associated Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D008958:Models, Molecular; D011485:Protein Binding; D011489:Protein Denaturation; D020033:Protein Isoforms; D017434:Protein Structure, Tertiary
D001798:Blood Proteins; D003598:Cytoskeletal Proteins; D008565:Membrane Proteins; D008869:Microtubule-Associated Proteins; D020033:Protein Isoforms; C028841:erythrocyte membrane band 4.1 protein