Fluorescence resonance energy transfer analysis of merlin conformational changes.
Neurofibromatosis type 2 is an inherited autosomal disorder caused by biallelic inactivation of the NF2 tumor suppressor gene. The NF2 gene encodes a 70-kDa protein, merlin, which is a member of the ezrin-radixin-moesin (ERM) family. ERM proteins are believed to be regulated by a transition between a closed conformation, formed by binding of their N-terminal FERM domain and C-terminal tail domain (CTD), and an open conformation, in which the two domains do not interact. Previous work suggests that the tumor suppressor function of merlin is similarly regulated and that only the closed form is active. Therefore, understanding the mechanisms that control its conformation is crucial. We have developed a series of probes that measures merlin conformation by fluorescence resonance energy transfer, both as purified protein and in live cells. Using these tools, we find that merlin exists predominately as a monomer in a stable, closed conformation that is mediated by the central alpha-helical domain. The contribution from the FERM-CTD interaction to the closed conformation appears to be less important. Upon phosphorylation or interaction with an effector protein, merlin undergoes a subtle conformational change, suggesting a novel mechanism that modulates the interaction between the FERM domain and the CTD.
Hennigan
Robert F
RF
Department of Cancer & Cell Biology, University of Cincinnati, Colege of Medicine, Cincinnati, OH 45267, USA.
Foster
Lauren A
LA
Chaiken
Mary F
MF
Mani
Timmy
T
Gomes
Michelle M
MM
Herr
Andrew B
AB
Ip
Wallace
W
D000818:Animals; D002460:Cell Line; D031541:Fluorescence Resonance Energy Transfer; D051379:Mice; D008954:Models, Biological; D025581:Neurofibromin 2 / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D010750:Phosphoproteins / Q000737:chemistry; D010766:Phosphorylation; D017433:Protein Structure, Secondary; D017434:Protein Structure, Tertiary; D012583:Schwann Cells / Q000378:metabolism; D017923:Sodium-Hydrogen Exchangers / Q000737:chemistry
D025581:Neurofibromin 2; D010750:Phosphoproteins; D017923:Sodium-Hydrogen Exchangers; C093376:sodium-hydrogen exchanger regulatory factor