Impairment of cell adhesion by expression of the mutant neurofibromatosis type 2 (NF2) genes which lack exons in the ERM-homology domain

Abstract

Neurofibromatosis 2 (NF2) is an inherited disorder characterized by a predisposition to multiple intracranial tumors. The protein encoded by the NF2 gene has striking similarities to ezrin, radixin and moesin (ERM) proteins which link membrane proteins to the cytoskeleton. Therefore, it can be speculated that the disruption of cytoskeletal organization by alterations in the NF2 gene is involved in the development of tumors. It has been reported that the majority of NF2 mutations were nonsense or frameshift mutations that result in premature termination of translation. To facilitate the detection of these mutations, we performed protein truncation test and found that 11 of 14 NF2 patients had truncational mutations (79%). Seven of the 11 patients (64%) had a splicing abnormality which lead to absence of exons in the ERM homology domain. To examine the biological significance of the exon-missing mutations in the ERM homology domain, we expressed the wild-type (wt-NF2) and the various mutant NF2s (mu-NF2s) in a fibroblast cell line by using both liposome-mediated transfection and nuclear microinjection of the expression plasmids. The wt-NF2 showed intense punctate staining in the perinuclear cytoplasm in addition to overall staining of the submembranous area, whereas the mu-NF2s lacking exons in the ERM homology domain showed granular staining at the perinuclear region without any accumulation at the submembrane region. Microinjection of wt-NF2 cDNA into the nucleus of VA13 cells revealed that wt-NF2 protein induced a progressive elongation of cell processes. Furthermore, cells that expressed mu-NF2 had decreased adhesion, which resulted in detachment from the substratum. These findings suggested that the exon-missing mutations in the ERM-homology domain may affect cell membrane-cytoskeleton signaling and consequently disrupt cell-to-cell or cell-to-matrix interaction.

Inhibition of the hyaluronan-CD44 interaction by merlin contributes to the tumor-suppressor activity of merlin

“…The first 50 amino acids of merlin are required for the binding of merlin to CD44 and for inhibition of the CD44-HA interaction Studies have shown that merlin mutants lacking different portions of the NH 2 -terminal ERM-homology domain lose their ability to interact with the plasma membrane (Deguen et al, 1998;Koga et al, 1998), a finding that is consistent with the possibility that merlin engages CD44 using the segment(s) located in its NH 2 -terminal half. To define the domain of merlin that is required for its inhibitory effect on the CD44-HA interaction, we established five mutants each of which bore a 50-amino acid deletion within the first 250 residues of the NH 2 -terminal portion of merlin.…”

“…These mutants are often associated with a more severe clinical outcome (Merel et al, 1995;Parry et al, 1996;Ruttledge et al, 1996;Deguen et al, 1998;Evans et al, 1998;Koga et al, 1998). It is widely held that tumor-suppressor mutants often act in a dominant-negative fashion to promote tumorigenesis (Nigro et al, 1989).…”

“…It is widely held that tumor-suppressor mutants often act in a dominant-negative fashion to promote tumorigenesis (Nigro et al, 1989). Various merlin deletion mutants have been shown to promote tumorigenesis (Koga et al, 1998;Lajeunesse et al, 1998;Giovannini et al, 2000), but whether their effects are the result of a dominantnegative or a gain-of-function prooncogenic activity has not been resolved. Tr6BC1 cells express merlin endogenously (Figure 1a), and the results of RT-PCR together with DNA sequencing indicated that both merlin isoforms I and II are expressed (data not shown).…”

“…The NH 2 -terminus of merlin shares high sequence homology with the NH 2 -terminal halves of ERM proteins that mediate the binding of these proteins to a variety of molecules in the plasma membrane (Tsukita et al, 1994;Hirao et al, 1996;Yonemura et al, 1998). Accordingly, merlin mutants that lack part of the NH 2 -terminal ERM-homology domains lose their ability to associate with the plasma membrane (Deguen et al, 1998;Koga et al, 1998). Genetic analysis of NF2 patients has shown that deletions in the NH 2 -terminal FERM domain occur frequently and are associated with early tumor onset and poor prognosis (Ruttledge et al, 1996).…”

Schwann cell hyperplasia and tumors in transgenic mice expressing a naturally occurring mutant NF2 protein

“…Koga et al (1998) have recently shown that nuclear microinjection of a mutant NF2 cDNA lacking exon 2 sequences induces loss of cell adhesion in VA13 cells. These results indicate that exon-skipping mutations in the amino terminus of schwannomin cause a dominant effect on cell attachment.…”