1.CD44-4.1R INTERACTION IN HELA CELLS
We have shown that erythroid protein 4.1 (4.1R) binds to the cytoplasmic domain of CD44 through its FERM (protein 4.1-ezrin-radixin-moesin) domain of the NH_2 terminal 300 amino acids with a dissociation constant (K_<(D)> of 275 nM. This binding prevents ankyrin binding to CD44, suggesting a potential functional role of 4.1R in modulating ankyrin-induced CD44 interaction with extracellular matrix proteins, such as hyaluronic acid (Nunomura et al. J. Biol. Chem. 1997). In the present study, using HeLa cell we wanted to demonstrate in vivo that 4.1R regulates ankyrin interaction with CD44 by overexpression of ezrin which may compete 4.1R on binding sites of CD44 through its FERM domain. Using the resonant mirror detection method (IAsys), we first measured the affinity of the FERM domain of ezrin to CD44 (K_<(D)>=78nM) which was higher than that for 4.1R. Importantly, in contrast to 4.1R, ezrin binding did not alter ankyrin interaction with CD44. In pa
rallel, we performed immunostaining of HeLa cells, showing colocalization of 4.1R and CD44 at the peripheral plasma membranes and both ankyrin and intrinsic ezrin in the cytoplasm. When the FERM domain of ezrin was overexpressed, this domain and ankyrin were observed at the membrane while 4.1R changed its distribution from the membrane to the cytoplasm. These results enable us to conclude that 4.1R regulate ankyrin interaction with CD44 in vivo. We also discuss the hyaluronic acid binding to CD44 which is induced by ankyrin interaction with CD44.
2.FUNCTIONAL ANALYSIS OF HEAD-PIECE REGION OF PROTEIN 4.1R AND 4.1G INTERACTIONS WITH MEMBRANE PROTEINS BY CHIMERA PROTEINS IN RED CELLS.
We have reported that the 297 amino acid (aa) NH_2-terminal region of 4.1R^<80> (30kDa domain) interacts with Band3 and glycophorin C (GPC), and that these interactions are regulated by Ca^<2+> and calmodulin (CaM) (JBC (2000) 275,6360 and 24540). Another isoform of 4.1R, 135kDa4.1R (4.1R^<135>) and 4.1G are expressed in erythroblasts. 4.1R^<135> and 4.1G have an additional 209 aa polypeptide, referred to as head-piece (HP), upstream of the NH_2-terminal end of the 30kDa domain. We have also reported that the HP region but not 4.1G regulated the binding of 4.1R^<135> to membrane proteins. To clarify the function of HP, we highlight the function of HP in by comparing the binding profiles of chimera proteins of 4.1RHP+4.1G30kDa and 4.1GHP+4.1R30kDa to membrane proteins. (1) The K_<(D)> values for chimera proteins binding to Band3 and GPC tails were 〜10^<-7> M as same as R30 and G30 ; (2) The K (D) values for RHP30 and GHP30 binding to CaM was 〜10^<-8>M in the presence of Ca^<2+> but those for the chimera proteins were was〜10^<-7>M in Ca^<2+>-independent manner; (3) Ca^<2+>/CaM reduced the binding affinity for RHG30 to Band3cyt and GPCcyt but not GHPR30. Our results support that HP in 4.1R^<135>and 4.1G may specifically regulate the 30kDa domain binding to membranes.
3.NOVEL INSIGHTS INTO BINDING PROPERTIES OF CYTOSKELETAL PROTEIN 4.1R AND 4.1G WITH CD44 IN HELA CELLS
We have reported that the 30kDa domain of 4.1R80 interacts with CD44, and that these interactions are regulated by Ca^<2+> and calmodulin (CaM). The 135kDa 4.1R isoform (4.1R^<135>) and 4.1G have an additional polypeptide, referred to as headpiece (HP), upstream of the 30kDa domain. Preliminary data suggest that the HP region of 4.1R regulates 4.1R^<135> interaction with membrane proteins but that it is not the case for 4.1G. In order to verify this hypothesis, we investigated the impact of HP on the binding affinity of two chimera proteins, 4.1RHP+4.1G30kDa (RHPG30) and 4.1GHP+4.1R30kDa (GHPR30), to CD44 and on the distribution of these chimera in HeLa cells.
4.IMMUNOCHEMICAL STUDIES ON THE ISOFORM OF PROTEIN 4.1R DURING ERYTHROPOIESIS.
Two major isoform of protein 4.1R, 135kDa type (4.1R^<135>) and 80kDa type (4.1R^<80>) are expressed in erythroblast and in erythrocyte, respectively. Only one difference between these molecules is 4.11V35 having additional 209 amino acids, named head-piece (HP) at the NH_2 termini of 4.1R^<80> Based on the in vitro binding assay, we suggested that the Ca^<2+> regulates the 4.1R^<135> biding to membrane proteins through the calmodulin binding to HP. In the present study, we studied on the immunocehmical localization of 4.1R isofrom during the erythropoiesis. A. Immunocytochemistry : The 4.1R^<135> broadly distributed in the cells and the GPC and GPA were stained at the peripheral membrane at D5. At D7, the 4.1R^<135> localized in the cytoplasm and at the peripheral membrane. The 4.1R^<135> was stained like a dot in the cytoplasm at D12. In the EGTA treated cells at D7,the 4.1R^<135> was strongly stained at the peripheral membrane. B. Immunoblot : The immunoblot detected 4.1R^<135> and 4.1R^<80> in the membrane fraction at D7. At D12, the 4.1R^<80> was more strongly detected in the membrane fraction but hardly see the band of 4.1R^<135> in the fraction.
5.PROTEIN 4.1R ISOFORM IN BLOOD CELLS OF ZEBRAFISH (Danio rerio): ANALYSIS OF MEMBRANE BINDING PROPERTIES.
We studied that the binding profiles of 30kDa domain of zebrafish protein 4.1R (ZF・R30) with human Band3, Glycophorin C (GPC) and p55. A complex of Ca^<2+> and calmodulin (CaM) regulation of the 4.1R interactions with those membrane proteins was investigated. (1) ZF・R30 co-precipitated with IOV ; (2) The K_<(D)> values of ZF・R30 binding to Band3cyt and GPCcyt were 〜10^<-6>M and 〜10^<-7>M, respectively ; (3) The K_<(D)> value for ZF・R30 binding to CaM was 〜10^<-7>M in the presence and the absence of Ca^<2+> ; (4) Ca^<2+>/CaM did not reduce the binding affinity for ZF・R30 to Band 3cyt and GPCcyt ; (5) PIP_2 was inhibited CaM binding to H・R30 but not ZF・R30 in the absence of Ca^<2+>. Our results suggest that ZF・R30 binding to membrane proteins is not affected by Ca^<2+>/CaM in erythrocytes regardless of Ca^<2+> level.
6.THERMODYNMIC ANAKYSIS OF CALMODULIN BINDING TO PEPTIDES DERIVED FROM PROTEIN 4.1R.
In NH_2-terminal 30kDa domain of the protein 4.1R (4.1R), an erythrocytes membrane skeletal protein, we have identified two calmodulin (CaM) binding sites, pep11 and pep9, which bind CaM in Ca^<2+>-independent and Ca^<2+>-dependent manner, respectively. To clarify the functional and physiochemical properties of these peptides binding to CaM, we measured the thermodynamic changes by microcaloriemeter. 1) The K(D) values of peps 9 and 11 to CaM were 10^<-6> and 10^<-7>M, respectively, in the presence of Ca^<2+> while that of pep9 was 10 times reduced in its absence. 2) The enthalpy change of pep11 binding to Ca^<2+>/CaM was -18.36KJ/mol, 3) Temperature of dependence of the partial molar heat capacity of Ca^<2+>/CaM, two peaks at 101.1 (p1) and 119.2 (p2) degree were appeared. The p1 was shifted to 100.7 and 106.7 deg. for Ca^<2+>/CaM complex with pep11 and pep9, respectively, but not p2. That of EGTA/CaM, 39.7 (p3) and 58.4 (p4) degree were appeared. The p3 was shifted to 58.9 and 49.1 deg. for EGAT/CaM complex with pep9 and pep11, respectively, but not p4. Changes of the pl and p3 reflect the change of C-lobe. The results indicate that these peptides bind to C-lobe of CaM. The pep11 may stabilize the structure of CaM.
7.CHARACTERIZATION OF MOUSE MONOCLONAL ANTIBODIES TO HUMAN PROTEIN 4.1R 30kDa DOMAIN.
We have reported that the 30kDa domain of 4.1R (R30) interacts with Band 3 and glycophorin C (GPC), and these interactions are regulated by Ca^<2+> and calmodulin (CaM). On the other hand, monoclonal antibody (mAb) is a powerful tool for not only immunochemical studies but also analysis of functional structures. In the present study, we established mAbs to R30 producing clones and characterized their binding profiles. In 13 clones producing IgG to R30, we selected clones #5, #7, #9 and #13 which recognized different epitope. #13 reacted with R30 and 4.1N while the other mAbs specifically reacted with 4.1R. #5 and #7 recognized different. epitope of exon 8 encoded C-half region. #9 and #13 recognized exon 4 and exon 11 encoded region, respectively. The K_<(D)> values of mAbs binding to R30 were 〜10^<-8> Immune complex of each mAb and R30 partially inhibited R30 binding to IOV. [Conclusion] These mAbs are useful tool for functional analysis of R30 binding to membrane proteins. Less