IMPAIRED INTERACTION OF NATURALLY-OCCURRING MUTANT NF2 PROTEIN WITH ACTIN-BASED CYTOSKELETON AND MEMBRANE

Although schwannomin, the product of the neurofibromatosis type 2 gene , shaves homology with three cytoskeleton-to-membrane protein linkers defining the ERM family, the mechanism by which it exerts a tumor supp ressive activity remains elusive, Based on the knowledge of naturally occurring mutations, a functional study of schwannomin was initiated, Constructs encoding the two wild-type isoforms and nine mutant forms w ere transfected into HeLa cells. Transiently expressed wild-type isofo rms were both observed underneath the plasma membrane, At this locatio n they were detergent insoluble and redistributed by a cytochalasin D treatment, suggesting interaction with actin-based cytoskeletal struct ures, Proteins with single amino acid substitutions at positions 219 a nd 220 demonstrated identical properties, Three different truncated sc hwannomins, that are prototypic for most naturally occurring NF2 mutat ions, were affected neither in their location nor in their cytochalasi n D sensitivity, However, they were revealed to be detergent soluble, indicating a relaxed interaction with the actin-based structures. An i ncreased solubility was also observed for a mutant with a single amino acid substitution at position 360 in the C-terminal half of the prote in, Mutant proteins with either a single amino acid deletion at positi on 118 or an 83 amino acid deletion within the N-terminal domain had l ost the submembraneous localization and tended to accumulate in perinu clear patches that were unaffected by cytochalasin D treatment, A simi lar behavior was observed when the N-terminal domain was entirely dele ted, Taken together these observations suggest that the N-terminal dom ain is the main determinant that localizes the protein at the membrane where it interacts weakly with actin-based cytoskeletal structures, T he C-terminal domain potentiates this interaction, With rare exception s, most naturally occurring mutant schwannomins that have lost their t umor suppressive activity are impaired in an interaction involving act in-based structures and are no longer firmly maintained at the membran e.