Structural Analysis of Drosophila Merlin Reveals Functional Domains Important for Growth Control and Subcellular Localization

Recent studies have identified a rapidly growing number of tumor suppressor genes whose normal function in cells directly or indirectly regulates cellular proliferation. Not surprisingly, many of these genes, such as Rb and p53, have been shown to encode proteins that regulate aspects of the cell cycle (for review see Brown, 1997). However, other tumor suppressor genes appear to function in processes less clearly related to control of the cell cycle. One of the most intriguing genes of this latter class is the Neurofibromatosis type 2 (NF2)1 gene, which encodes a member of the protein 4.1 superfamily, Merlin (Rouleau et al., 1993; Trofatter et al., 1993). Members of the protein 4.1 superfamily, a large group of membrane-associated cytoplasmic proteins, include protein 4.1; talin; the ezrin, radixin, moesin (ERM) proteins; Merlin; Drosophila Expanded; several protein phosphatases; and at least two nonmuscle myosins (for review see McCartney and Fehon, 1997). The defining feature of this superfamily is a conserved region of 200–300 amino acids usually located in the NH2 terminus of the protein. This region is particularly well conserved between Merlin and the ERM proteins. The ERM proteins appear to function as molecular linkers by binding to transmembrane proteins through the NH2-terminal domain and linking them to the cortical actin cytoskeleton through a COOH-terminal actin-binding domain. Consistent with this role, ERM proteins localize to actin-rich structures such as the adherens junction and microvilli (Franck et al., 1993; Takeuchi et al., 1994). Although the structural similarities between Merlin and the ERM proteins suggest that they may have similar functions, the exact nature of Merlin's cellular functions has not been defined. Recent studies in cultured cells indicate that expressed Merlin protein accumulates in some actin-rich membrane domains, such as membrane ruffles at the leading edge of migrating cells (Gonzalez-Agosti et al., 1996; Sainio et al., 1997), consistent with the localization of ERM proteins. However, several lines of evidence indicate that Merlin has functions that are clearly distinct from those of ERM proteins. First, although the NH2-terminal domains of Merlin and the ERM proteins are similar (the protein 4.1 superfamily domain), Merlin lacks the well- defined COOH-terminal actin-binding domain found in ERM proteins (Turunen et al., 1994; Gary and Bretscher, 1995). In addition, while the ERM proteins are functionally redundant (Takeuchi et al., 1994), there is no evidence for redundancy between the ERM proteins and Merlin. The NF2/Merlin gene can be mutated to lethality in both Drosophila and mouse (Fehon et al., 1997; McClatchey et al., 1997). Finally, in vivo studies of subcellular localization of Merlin and Moesin reveal that they can be distinct, again supporting the idea that these proteins have different functions (McCartney and Fehon, 1996). To define the cellular functions of Merlin and the ERM proteins, we have isolated and characterized two Drosophila genes, Moesin, the sole ERM gene in Drosophila, and Merlin, a well conserved NF2 homologue (McCartney and Fehon, 1996). In the present study, we show that Merlin is essential for viability in Drosophila and is required for the proper regulation of cell proliferation. Furthermore, our analysis indicates that essential Merlin functions occur at the cytoplasmic face of the cell membrane and that all of these functions can be mediated by the conserved NH2-terminal region.