Neurofibromatosis (NF2) is an inherited disorder in which affected patients develop schwannomas, meningiomas, and gliomas. The NF2 tumor suppressor gene product, merlin, shares sequence similarity with a family of proteins that link integral membrane glycoproteins with the actin cytoskeleton (ERM protein family). This raises the possibility that merlin regulates cell growth by transducing an extracellular signal through cell surface-proteins and the actin cytoskeleton. This grant proposes to investigate how NF2 patient mutations lead to defects in merlin negative growth regulation. Specifically, we wish to test the hypothesis that defects in merlin function as a consequence of NF2 mutations result from the generation of (1) unstable merlin proteins, (2) merlin proteins with altered subcellular distributions, (3) merlin proteins with reduced abilities to form intra- or inter-molecular complexes, and/or (4) merlin proteins with reduced abilities to associate with merlin effector proteins. Some NF2 mutations are predicted to produce truncated and, therefore, unstable merlin proteins in vivo. In contrast, missense mutations may lead to the production of a stable merlin protein with reduced ability to suppress cell growth. We propose to determine whether NF2 patient mutations or alternatively spliced merlin isoforms result in the production of unstable merlin proteins. The normal rate of merlin turnover will be established in Schwann cells to provide the foundations for analyzing the effect of NF2 patient mutations and alternative splicing on merlin protein stability. NF2 patient mutations and merlin isoforms will be analyzed to determine the effect of alternative splicing and NF2 gene mutations on merlin's ability to function as a negative growth regulator both in vitro and in vivo. Merlin growth suppressor activity will be determined by growth rates, FACS analysis, anchorage-independent growth and ability to form tumors in athymic (nude) mice. Failure of merlin isoforms or mutant merlin proteins to suppress cell growth may result from impaired interactions with critical merlin effector proteins. Since merlin is a member of the ERM protein family, experiments are designed to determine which region of merlin are essential for interactions with cell membrane proteins and the actin cytoskeleton by analyzing the effect of NF2 patient mutations and alternative splicing on these interactions. Next, the molecular determinants required for merlin to form intra- and inter-molecular complexes necessary for merlin to function a growth suppressor will be analyzed. Finally, potential merlin effector proteins will be identified using a combination of approaches including biochemical and genetic interaction systems. The strategies outlined above to define how merlin functions to suppress growth through altered protein interactions are aimed at understanding the function of this novel tumor suppressor gene with an eye towards the design of more effective therapies for the tumors in which merlin expression is altered.
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