Subcellular localization and expression pattern of the neurofibromatosis type 2 protein merlin/schwannomin.
To elucidate the physiological function of the neurofibromatosis type 2 (NF2) tumor suppressor protein merlin/schwannomin, we studied the expression pattern and subcellular localization in human fibroblasts by Western blot analyses and immunofluorescence using a polyclonal antibody raised against the C-terminus of merlin. Three of the six merlin isoforms identified in this study (75 kDa, 58 kDa, 45 kDa) have been reported earlier and can be explained by alternative splicing. In addition, we detected higher molecular weight bands of about 110 kDa, 100 kDa and 84 kDa. Although the merlin bands of 100 kDa and 110 kDa may represent homo- or heterodimers, oligomerization due to formation of disulfide bonds was excluded. Furthermore, the isoforms of 84 kDa and 58 kDa were quantitatively extractable in Lubrol WX, indicating a localization in or close to the plasma membrane. The 45 kDa band, however, was not soluble in Lubrol WX compatible with a localization of this NF2 isoform in the endoplasmic reticulum. Applying confocal laser scanning microscopy, merlin was shown to be located in four subcellular compartments: (i) perinuclear in a compartment resembling endoplasmic reticulum, (ii) in ruffling membranes and at the leading edges, (iii) in filopodia, and (iv) at cell/substrate adhesion points. Codistribution of merlin and F-actin filaments was found in filopodia, ruffling membranes and at the insertion points of stress fibers at cell/substrate adhesion junctions as shown by phalloidin-rhodamine staining. Double immunofluorescence analyses of merlin and moesin revealed a colocalization in filopodia and ruffling membranes. The localization of merlin in the actin-rich cortical cytoskeleton corresponds to the ezrin-radixin-moesin family of proteins suggesting the NF2 protein to contribute to the regulation of cell growth by interaction with cytoskeleton-associated proteins.
Schmucker
B
B
Institute of Human Genetics, Friedrich Alexander University of Erlangen-Nürnberg, Germany.
Ballhausen
W G
WG
Kressel
M
M
D001483:Base Sequence; D015153:Blotting, Western; D002478:Cells, Cultured; D003902:Detergents / Q000494:pharmacology; D016515:Genes, Neurofibromatosis 2; D006801:Humans; D008565:Membrane Proteins / Q000235:genetics / Q000378:metabolism*; D008969:Molecular Sequence Data; D025581:Neurofibromin 2; D011092:Polyethylene Glycols / Q000494:pharmacology; D011993:Recombinant Fusion Proteins / Q000235:genetics; D012867:Skin / Q000166:cytology / Q000378:metabolism; D012995:Solubility; D013347:Subcellular Fractions / Q000378:metabolism
D003902:Detergents; D008565:Membrane Proteins; D025581:Neurofibromin 2; D011993:Recombinant Fusion Proteins; C017474:Lubrol WX; D011092:Polyethylene Glycols