The HEI-193 cell collection, immortalized from your schwannoma cells of an NF2 patient tumor, does not suffer from these disadvantages, although until now, its characterization was incomplete. merlin, isoform 3 is as resistant to proteasomal degradation as isoforms 1 and 2 and can interact with each of these isoforms transcript can be alternatively spliced to form many variants [4,5], the most abundant of which are isoforms 1 and 2, which comprise approximately 90% of the mature transcript within cells ([6], observe Fig. 1). Only isoform 1 has been shown to suppress cell growth in cell model systems [7]. Open in a separate window Physique 1 Schematic of isoformsisoforms 1, 2 and 3 are diagrammatically represented and aligned with the gene to show the contribution of the different exons to the resultant mRNA structure and protein sequence. isoform 2 differs from isoform 1 by the addition of exon 16, resulting in the substitution of the last 16 amino acids of isoform 1 with 11 different ones. Isoform 3 lacks both exon 15 and 16, resulting in a protein C-terminus different from both merlin 1 and 2. The arrows represent the relative positions of primers utilized for PCR and RT-PCR analysis. The open rectangles represent the non-translated region whereas the black rectangles represent the translated region of the mRNA. The asterisks denote positions of quit codons. The mechanism by which merlin regulates cell proliferation is not fully comprehended. Merlin can block Rac-mediated signaling [8], perhaps directly through inhibition of Pak activity [9]. Consistent with this notion, tumor-derived gene at position ?1 of the intron 14 / exon 15 border. This mutation is Rabbit Polyclonal to AIBP usually predicted to eliminate the donor sequence of exon 15 and result in exon skipping [16]. The presence of a shorter transcript in HEI-193 cells was confirmed by RT-PCR [15]. However, the molecular alterations in the transcript and the encoded merlin protein were not fully described. In this paper we statement that this merlin protein expressed in HEI-193 cells has amino acid sequence identical to that of a splice variant previously designated as isoform 3 [17]. This isoform was first described in a family with a history of a moderate form of NF2 and was shown to arise because of an AT mutation within the gene at the +3 position of the donor site of intron 15 [17]. Interestingly, isoforms 1, 2 and 3 are simultaneously and equivalently expressed both at the RNA and protein levels in fibroblasts derived from this family, but in schwannoma cells only isoform 3 is usually expressed [17]. Based on the moderate nature of the NF2 disease phenotype seen Stattic in this family, the authors of this study concluded that merlin isoform 3 retained moderate tumor suppressive activity. Here we present evidence that Stattic HEI-193 cells express merlin isoform Stattic 3 with no detectable isoform 1 or 2 2. The level of merlin isoform 3 in HEI-193 cells is comparable to levels of merlin found in normal human Schwann cells and several immortalized cell lines, and merlin isoform 3 appears to be as stable as isoforms 1 and 2. Although the presence of merlin isoform 3 has no apparent negative effect on the growth of HEI-193 cells, when exogenously expressed in NF2?/? mouse embryonic fibroblasts, isoform 3 suppresses growth, but much less effectively than similarly expressed merlin isoform 1. Merlin isoform 3 also interacts readily with both isoforms I and 2 and transcription/translation was performed using the TnT? system; both systems were purchased from Promega (Madison, WI). Cell Culture HEI-193, NIH3T3, MEF3flox2, MEF32, U251 and Cos-7 cells were managed in DMEM supplemented with 10% fetal calf serum (U.S. Bio-Technologies Inc., Parkerford, PA) and 100.