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Expression level, subcellular distribution and rho-GDI binding affinity of merlin in comparison with
1Department of Cell Biology, Faculty of Medicine, Kyoto University, Sakyo-ku, Kyoto 606, Japan.
Abstract:
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.
Insights
Merlin, a neurofibromatosis type-2 tumor suppressor, interacts with Rho-GDI similarly to ERM proteins, but its distinct cellular localization suggests unique functions beyond simple redundancy or competition.
Area of Science:
- Cell Biology
- Molecular Biology
- Biochemistry
Background:
- Merlin is a neurofibromatosis type-2 tumor suppressor.
- Merlin shares sequence similarity with ERM (Ezrin/Radixin/Moesin) proteins, which link actin filaments to the plasma membrane and are Rho-dependent.
- The physiological functions of merlin, particularly in relation to ERM proteins, require elucidation.
Purpose of the Study:
- To compare the in vivo and in vitro properties of merlin and ERM proteins.
- To investigate the cellular localization and Rho-GDI binding of merlin.
- To determine if merlin functions redundantly or competitively with ERM proteins.
Main Methods:
- Quantitative immunoblotting to determine merlin/ERM molar ratios.
- Cellular fractionation to assess merlin and ERM solubility.
- Introduction of merlin and ERM into fibroblasts and epithelial cells to study localization.
- In vitro binding assays and immunoprecipitation to examine merlin and ERM binding to Rho GDP dissociation inhibitor (Rho-GDI).
Main Results:
- Merlin is present at lower molar ratios than ERM proteins in cells.
- Merlin is primarily insoluble, while ERM proteins are partially soluble.
- Merlin and ERM localize to microvilli in fibroblasts, but merlin localizes to lateral membranes with E-cadherin in epithelial cells, unlike ERM proteins in apical microvilli.
- Merlin isoforms bind Rho-GDI with similar affinity to ERM proteins.
Conclusions:
- Merlin's distinct cellular localization in epithelial cells suggests functions beyond ERM proteins.
- Merlin's interaction with Rho-GDI is comparable to ERM proteins.
- The findings do not support a simple redundant or competitive role for merlin in relation to ERM proteins.