Characterization of a novel Merlin and Sip1 interaction region. Namal Abeysundara, Albert Leung, Sarah C. Hughes. University of Alberta, Edmonton, Canada.

   Neurofibromatosis 2 (NF2) is a disorder characterized by the development of tumours of the central nervous system. The gene involved in NF2 encodes for the tumour suppressor protein, Merlin. Merlin is closely related to ezrin, radixin and moesin (ERM) proteins, which function together in maintaining cell integrity and coordinating cell proliferation. Even though these processes have been implicated in cancer, the specific mechanism behind Merlin function is not well understood. To investigate the mechanism(s) of Merlin activity, we analyze Merlin interacting proteins, including Sip1, the Drosophila orthologue of the Na+/H+ exchanger regulatory factor, NHERF1. Despite the biochemical evidence suggesting Merlin and NHERF1 interactions, the functional interaction between the two proteins still remains unclear. To further characterize the physical and functional interaction between Merlin and Sip1, a novel potential binding site in Merlin to the Sip1 protein was identified. A 100 amino acid region immediately downstream of the Four-point-one Ezrin-Radixin-Moesin (FERM) domain of Merlin, in addition to the FERM domain itself, was identified as being important for Sip1 binding. Within the 100 amino acid region, the substitution of two widely conserved arginine residues to the corresponding Moesin residues (R325A and R335L) resulted in reduced Sip1 binding, suggesting that these arginine residues are important for the Merlin and Sip1 interaction. To determine the functional importance of the interaction region, the over-expression of the R325A and R335L Merlin mutants were analyzed using the UAS-GAL4 system in wing imaginal discs. Immunofluorescence antibody staining of larval wing discs and adult wing size measurements provide insight into whether adhesion or proliferation is altered when Merlin and Sip1 binding is reduced. In addition, the effect of the Merlin mutants in Schneider 2 cells were analyzed using proliferation assays. Characterizing the physical and functional interaction between Merlin and Sip1 may provide insight into the mechanism behind Merlin function.