Marked difference in membrane-protein-binding properties of the two isoforms of protein 4.1R expressed at early and late stages of erythroid differentiation

Abstract

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.

Interaction between protein 4.1R and spectrin heterodimers

“…Deficiency of 4.1R in human RBCs leads to the pronounced disruption of cytoskeletal meshwork, which is closely associated with anemia and elliptocytosis (18)(19)(20)(21). In agreement with previous studies (22)(23)(24), the current study presents direct evidence demonstrating the interaction between 4.1R and spectrin heterodimers, suggesting the biological significance of this interaction in maintaining the stability of the plasma membrane skeleton. Additionally, we demonstrated for the first time the binding ratio of 4.1R to spectrin heterodimers, calculated to be 4.5-5.…”

Deep Intron Elements Mediate Nested Splicing Events at Consecutive AG Dinucleotides To Regulate Alternative 3′ Splice Site Choice in Vertebrate 4.1 Genes

“…The E1A-to-E2dis splice deletes AUG1 and produces an alternative mRNA isoform that encodes shorter proteins lacking the N-terminal headpiece of ϳ200 amino acids. Physiologically, the presence or absence of the headpiece likely has a substantial effect on protein 4.1B binding, since a similar phenomenon has already been reported with regard to binding of the paralogous 4.1R system to components of the erythroid membrane skeleton (23).…”

“…In contrast, upstream first exons execute iE R -or iE B -mediated intrasplicing to E2dis, excising AUG1 and resulting in production of smaller isoforms from the downstream AUG2 in exon 4. The presence or absence of the headpiece region modulates the affinity of protein 4.1 interactions with other cytoskeletal and membrane proteins (23). Thus, membrane functional properties can be regulated via a pathway that begins with differential transcriptional activation at alternative promoters, coupled to alternative splicing events that ultimately control alternative translation initiation sites responsible for synthesis of the different 4.1 protein isoforms.…”

Light Scattering: a Novel Approach to Analyze Protein 4.1R FERM Domain Interaction with Inside-out-vesicles in Solution

“…8,9 Briefly, 100 μg of IOVs were incubated with various concentrations of R30 in Buffer A for 10 min. The mixture was loaded onto a 2.5% sucrose solution in Buffer A and centrifuged at 60000 rpm (Optima TLX, TLA100.1 rotor, Beckman Coulter, USA) at 4 C for 10 min.…”

“…[7][8][9][10] Until now, the measurements of protein binding affinity have been relying on binding assays using radioisotope-labeled proteins, 7 or on the assessment of protein complex formation through a combination of sedimentation assays and immuno-blot analysis. [8][9][10] Although the binding analysis of radiolabeled proteins provides quantitative information, these methods are cumbersome, they involve the handling of hazardous reagents, and they cannot be used to measure binding affinity in real time. Immuno-blot analysis is also limited, this second type of experiments providing only semi-quantitative affinity measurements at equilibrium.…”