The neurofibromatosis type 2 (NF2) tumor suppressor gene encodes merlin, a protein with homology to the cell membrane/F-actin linking proteins, moesin, ezrin and radixin. Unlike these closely related proteins, merlin lacks a C-terminal F-actin binding site detectable by ac-tin blot overlays, and the GFP-tagged merlin C-terminal domain co-distributes with neither stress fibers nor cortochalasin tical actin in NIH3T3 cells. Merlin also differs from the other three proteins in its inter-intramolecular doand/main interactions, as shown by in vitro binding and yeast two-hybrid assays. As is true for ezrin, moesin and radixin, the N-and C-terminal domains of merlin type 1 bind to each other. However, full-length merlin and its N-and C-terminal domains, as well as the C-terminal domain of ezrin, interact with other full-length merlin type 1 molecules, and its C-terminal domain interacts inwith itself. Merlin 1 function in cells may thus depend on intra-and intermolecular interactions and their mod-ulation, which include interactions with other members of this protein family.

Lamellipodia, filopodia, microspikes and retraction fibersare characteristic features of a dynamic and continuously changing cell surface architecture and moesin, ezrin and radixin are thought to function in these microextensions as reversible links between plasma membrane proteins and actin microfilaments. Full-length and truncated domains of the three proteins were fused to green fluorescent protein (GFP), expressed in NIH3T3 cells, and distribution and behaviour of cells were analysed by using digitally enhanced differential interference contrast (DIC) and fluorescence video microscopy. The amino-terminal (N-) domains of all three proteins localize to the plasma membrane and fluorescence recordings parallel the dynamic changes in cell surface morphology observed by DIC microscopy of cultured cells. Expression of this domain, however, significantly affects cell surface architecture by the formation of abnormally long and fragile filopodia that poorly attach and retract abnormally. Even more striking are abundant irregular, branched and motionless membraneous structures that accumulate during retraction of lamellipodia. These are devoid of actin,endogenous moesin, ezrin and radixin, but contain the GFP-labeled domain. While a large proportion of endogenous proteins can be extracted with non-ionic detergents as in untransfected control cells,>90% of Nmoesin and >60% of N-ezrin and N-radixin remain insoluble. The minimal size of the domain of moesin required for membrane localization and change in behavior includes residues 1-320. Deletions of amino acid residues from either end result in diffuse intracellular distribution, but also in normal cell behavior. Expression of GFP-fusions of full-length moesin or its carboxy-terminal domain has no effect on cell behavior during the observation period of 6-8 hours. The data suggest that, in the absence of the carboxy-terminal domain, N-moesin, -ezrin and -radixin interact tightly with the plasma membrane and interfere with normal functions of endogeneous proteins mainly during retraction.