|Expression level, subcellular distribution and rho-GDI binding affinity of merlin in comparison with Ezrin/Radixin/Moesin proteins.|
Maeda, M, et al.
Oncogene, 18: 4788-97 (1999)
Merlin, a neurofibromatosis type-2 tumor suppressor, shows significant sequence similarity to ERM (Ezrin/Radixin/Moesin) proteins, general actin filament/plasma membrane cross-linkers, which are regulated in a Rho-dependent manner. To understand its physiological functions, we compared merlin with ERM proteins in vivo and in vitro. Quantitative immunoblotting revealed that the molar ratio of merlin/ERM in cultured epithelial or non-epithelial cells was approximately 0.14 or approximately 0.05, respectively. After centrifugation of cell homogenate, merlin was mostly recovered in the insoluble fraction, whereas almost half of ERM proteins were found in the soluble fraction. Merlin and ERM proteins were concentrated at microvilli when introduced into fibroblasts. In contrast, in epithelial cells, introduced merlin was co-distributed with E-cadherin in lateral membranes, whereas ERM proteins were concentrated in apical microvilli. Finally, we examined the binding affinity of merlin to Rho GDP dissociation inhibitor (Rho-GDI), to which N-terminal halves of ERM proteins but not the full-length molecules specifically bind. In vitro binding assays revealed that the N-terminal halves of merlin isoform-I and -II as well as full-length merlin isoform-II bound to Rho-GDI with similar binding affinity to ERM proteins. Immunoprecipitation confirmed these findings in vivo. These findings do not favor the notion that merlin functions simply in a redundant or competitive manner to ERM proteins.
|Moesin, like ezrin, colocalizes with actin in the cortical cytoskeleton in cultured cells, but its expression is more variable.|
Franck, Z, et al.
J. Cell. Sci., 105 ( Pt 1): 219-31 (1993)
The band 4.1 superfamily of proteins show approx. 30% sequence identity in their amino-terminal region to the membrane binding domain of erythrocyte band 4.1. Within this superfamily are three members, ezrin, radixin and moesin, that show approx. 75% overall sequence identity. A comparison of the domain structure and intracellular localization of ezrin and moesin in cultured cells is reported here. Limited proteolytic digestion of ezrin or moesin yields a relatively stable 32 kDa domain derived from the amino-terminal region that is homologous to the protease-resistant membrane binding domain of erythrocyte band 4.1. The remaining regions of the two proteins give rise to very different fragments, suggesting that the secondary/tertiary structures of the two proteins are different in these regions. We have generated polyclonal antibodies that discriminate between ezrin and moesin, and do not react with radixin. All cultured cell lines investigated contain ezrin, whereas moesin is variably expressed. Cells that contain both ezrin and moesin show a very similar pattern: both proteins are enriched and colocalize with actin in cell surface structures. Ezrin is also detected in the cytoplasm. In cells with few or no surface structures, both proteins show a patchy distribution in regions of the cell that contain fine networks of actin filaments. No staining of focal contacts or adherens junctions was observed. These results, together with those of others, lead to the conclusion that, of the members of this protein family, only radixin is an authentic component of adherens junctions and focal contacts. Ezrin and moesin are both found in cell surface structures after treatment of human A431 cells with epidermal growth factor, and ezrin, but not moesin, becomes phosphorylated on tyrosine. This study shows that ezrin and moesin have a similar subcellular distribution in cultured cells, yet are distinguishable in their expression, structure and ability to serve as a kinase substrate.