Marked difference in membrane-protein-binding properties of the two isoforms of protein 4.1R expressed at early and late stages of erythroid differentiation.
Two major isoforms of protein 4.1R, a 135 kDa isoform (4.1R(135)) and an 80 kDa isoform (4.1R(80)), are expressed at distinct stages of terminal erythroid differentiation. The 4.1R(135) isoform is exclusively expressed in early erythroblasts and is not present in mature erythrocytes, whereas the 4.1R(80) isoform is expressed at late stages of erythroid differentiation and is the principal component of mature erythrocytes. These two isoforms differ in that the 4.1R(135) isoform includes an additional 209 amino acids designated as the HP (head-piece) at the N-terminus of 4.1R(80). In the present study, we performed detailed characterization of the interactions of the two 4.1R isoforms with various membrane-binding partners and identified several isoform-specific differences. Although both 4.1R(135) and 4.1R(80) bound to cytoplasmic domains of GPC (glycophorin C) and band 3, there is an order of magnitude difference in the binding affinities. Furthermore, although both isoforms bound CaM (calmodulin), the binding of 4.1R(80) was Ca2+-independent, whereas the binding of 4.1R(135) was strongly Ca2+-dependent. The HP of 4.1R(135) mediates this Ca2+-dependent binding. Ca2+-saturated CaM completely inhibited the binding of 4.1R(135) to GPC, whereas it strongly reduced the affinity of its binding to band 3. Interestingly, in spite of the absence of spectrin-binding activity, the 4.1R(135) isoform was able to assemble on to the membrane of early erythroblasts suggesting that its ability to bind to membrane proteins is sufficient for its membrane localization. These findings enable us to offer potential new insights into the differential contribution of 4.1R isoforms to membrane assembly during terminal erythroid differentiation.
Nunomura
Wataru
W
Department of Biochemistry, School of Medicine, Tokyo Women's Medical University, Tokyo 162-8666, Japan.
Parra
Marilyn
M
Hebiguchi
Miwa
M
Sawada
Ken-Ichi
K
Mohandas
Narla
N
Takakuwa
Yuichi
Y
D002118:Calcium / Q000378:metabolism; D002147:Calmodulin / Q000378:metabolism; D002454:Cell Differentiation; D002478:Cells, Cultured; D003598:Cytoskeletal Proteins / Q000378:metabolism*; D004591:Electrophoresis, Polyacrylamide Gel; D004900:Erythroblasts / Q000166:cytology / Q000378:metabolism; D004912:Erythrocytes / Q000166:cytology / Q000378:metabolism*; D005455:Fluorescent Antibody Technique; D006801:Humans; D007700:Kinetics; D008565:Membrane Proteins / Q000378:metabolism*; D011485:Protein Binding; D020033:Protein Isoforms / Q000378:metabolism
D002147:Calmodulin; D003598:Cytoskeletal Proteins; D008565:Membrane Proteins; D020033:Protein Isoforms; C028841:erythrocyte membrane band 4.1 protein; D002118:Calcium