title: Biochemical analysis of potential sites for protein 4.1-mediated anchoring of the spectrin-actin skeleton to the erythrocyte membrane.
abstract: Erythrocyte protein 4.1 has been hypothesized to link the spectrin-actin junctional complex directly to the cytoplasmic domain of glycophorin C, but this bridging function has never been directly demonstrated. Because an alternative protein-mediated bridge between the junctional complex and the cytoplasmic domain of band 3 is also plausible, we have undertaken to characterize the membrane sites to which protein 4.1 can anchor the spectrin and actin skeleton. We demonstrate that proteolytic removal of the cytoplasmic domain of band 3 has minimal effect on the ability of protein 4.1 to promote 125I-labeled spectrin and actin binding to KI-stripped erythrocyte membrane vesicles. We also show that quantitative blockade of all band 3 sites with either monoclonal or polyclonal antibodies to band 3 is equally ineffective in preventing protein 4.1-mediated association of spectrin and actin with the membrane. In contrast, obstruction of protein 4.1 binding to its docking site on the cytoplasmic pole of glycophorin C is demonstrated to reduce the same protein 4.1 bridging function by approximately 85%. We conclude from these data that (i) glycophorin C contributes the primary anchoring site of the protein 4.1-mediated bridge to the spectrin-actin skeleton; (ii) band 3 is incapable of serving the same function; and (iii) additional minor protein 4.1 bridging sites may exist on the human erythrocyte membrane.
authors.affiliation: Department of Chemistry, Purdue University, West Lafayette, Indiana 47907-1393, USA.
authors.forename: R F
authors.initials: RF
authors.lastname: Workman
authors.forename: P S
authors.initials: PS
authors.lastname: Low
mesh_terms: D000199:Actins / Q000378:metabolism*; D000595:Amino Acid Sequence; D001665:Binding Sites; D003598:Cytoskeletal Proteins; D004910:Erythrocyte Membrane / Q000378:metabolism* / Q000648:ultrastructure; D006021:Glycophorins / Q000378:metabolism*; D008565:Membrane Proteins / Q000378:metabolism*; D008969:Molecular Sequence Data; D009479:Neuropeptides; D011485:Protein Binding; D013049:Spectrin / Q000378:metabolism*
chemical_list: D000199:Actins; D003598:Cytoskeletal Proteins; D006021:Glycophorins; D008565:Membrane Proteins; D009479:Neuropeptides; C028841:erythrocyte membrane band 4.1 protein; C120557:erythrocyte membrane protein band 4.1-like 1; D013049:Spectrin