Search

The neurofibromatosis 2 (NF2) tumor suppressor gene encodes an intracellular membrane-associated protein, called merlin (or schwannomin), that belongs to the band 4.1 family of cytoskeleton-associated proteins. Inactivating NF2 mutations occur in several sporadic tumor types and have been linked to the NF2 disease, whose hallmark is the development of bilateral Schwann cell tumors (schwannomas) of the eighth cranial nerve. Two major alternatively spliced NF2 variants are expressed in normal tissues: 'NF2-17' lacking exon 16 and 'NF2-16' that contains exon 16 and encodes a merlin protein truncated at the C-terminus. We report that overexpression of NF2-17 in rat schwannoma cells inhibits their growth in vitro and in vivo, while NF2-16 fails to influence schwannoma growth. Tumor growth inhibition by merlin depends on an interdomain association occurring either in cis or in trans between the N- and C-termini. This association does not occur in the truncated NF2-16 protein nor in a mutant NF2-17 protein lacking C-terminal sequences. These data indicate that merlin has a unique mechanism of tumor suppression, inhibiting cell proliferation via self-association.

The neurofibromatosis 2 (NF2) gene product, merlin, encodes a 595 amino acid protein with sequence similarity to a family of proteins linking cell membrane proteins to the cytoskeleton. Two isoforms of merlin have been described which differ by the presence (type 2 merlin) or absence (type 1 merlin) of exon 16 sequences inserted into the extreme carboxyl terminus of the protein. To determine the role of this important negative growth regulator during normal embryonic development, the expression of these two merlin isoforms was examined at representative stages of rat embryogenesis and in adult tissues. Partial sequence analysis of the rat merlin gene demonstrated striking amino acid identity to the published mouse and human merlin gene sequences. In situ hybridization and RT-PCR analyses demonstrated that rat merlin is widely expressed during embryogenesis and early postnatal life in most tissues but becomes restricted to the brainstem, cerebellum, dorsal root ganglia, spinal cord, adrenal gland and testis in adult animals. The elucidation of the pattern of merlin gene expression in adult and embryonic tissues provides the foundations for future studies aimed at determining the function(s) of this protein during cell differentiation and embryonic development.

Neurofibromatosis 2 (NF2) is an inherited cancer syndrome resulting from mutations in the NF2 tumor suppressor gene. Analysis of NF2 mutations has revealed some general genotype-phenotype correlations. Severe disease has been associated with mutations that produce a premature termination while more mild disease has been associated with missense mutations. Here, we provide experimental proof for these genotype-phenotype correlations by demonstrating that nonsense mutations fail to produce stable merlin protein while missense mutations result in the generation of merlin proteins defective in negative growth regulation. This inability to suppress cell growth may result from defects in the function of merlin at several levels, including failure to form an intramolecular complex. Based on these findings, we propose a model for merlin growth suppression that provides a framework for analyzing NF2 patient mutations and merlin function.

Tuberous sclerosis (TS) is an autosomal dominant disorder in which affected individuals manifest mental retardation, seizures, and a variety of benign and malignant tumors. The TSC2 tumor suppressor gene was recently identified by positional cloning and its protein product, tuberin, shown to represent one member of the rap GTPase activating protein (rapGAP) family. In order to determine the contribution of tuberin to the development of mental retardation and seizures in patients with TS, we examined the expression of tuberin in adult and developing nervous system tissues. Since tuberin is the second rapGAP found in the nervous system, the expression of tuberin was compared to the expression of rapGAP, rap1, and rap2. In this study, we demonstrate that tuberin is expressed at greatest levels in the spinal cord and cerebellum as opposed to rapGAP, which is not enriched in these tissues. Tuberin expression in the adult CNS is restricted to the olfactory bulb, several CNS neuronal populations, brainstem nuclei, cerebellar Purkinje cells, and motor neurons in the ventral spinal cord. In contrast, rapGAP is expressed in many different cell types in the adult CNS, but not in cerebellar Purkinje cells or motor neurons in the ventral spinal cord. However, there is significant expression of rapGAP in astrocytes. The restricted distribution of tuberin expression relative to rap1 and rapGAP suggests that tuberin may be the primary rap1 regulator in a subpopulation of CNS neurons.

The neurofibromatosis 1 (NF1) gene product, neurofibromin, is a tumor suppressor gene product capable of inhibiting the growth of cells in culture. If neurofibromin suppresses cell growth by arresting cells in G0 or G1, its expression might be regulated in a cell cycle-dependent fashion. In this study, we demonstrate that RAT-1A fibroblasts arrested in G0/G1 by serum starvation and then released to progress through the cell cycle do not demonstrate significant changes in NF1 expression. However, when arrested in G0/G1 by contact inhibition, NF1 expression in these cells is reversibly upregulated within 72 h, suggesting that NF1 expression is a late event associated with cell growth arrest which may contribute to the maintenance of the differentiated state.

The neurofibromatosis type 1 (NFl) gene encodes a tumor suppressor protein, termed neurofibromin, which is expressed pre- dominantly in neurons, Schwann cells, oligoden- drocytes, and leukocytes. There are at least three isoforms of neurofibromin produced by the alter- native use of exons 23a and 48a. Previously we described the identification of an NFl mRNA iso- form containing an additional 54 nucleotides from exon 48a (type 3 NFl) in human skeletal, cardiac and smooth muscle tissues by reverse-transcribed (RT)-PCR. To extend our initial observations, we have produced high titer chicken IgY antibodies which specifically recognize this muscle-specific neurofibromin isoform. An NF1 cDNA was gener- ated containing human exon 48a sequences and expressed as a fusion protein in bacteria. The muscle-specific neurofibromin antibodies de- tected this exon 48a fusion protein by Western im- munoblotting. Immunoprecipitation using these type 3 neurofibromin antibodies also specifically detected a 250 kDa protein in human and rat mus- cle tissues. Type 3 neurofibromin was found in rat heart and muscle, but not in liver, brain, kidney or spleen with levels of expression declining after postnatal day 7. Expression of total NFl RNA dur- ing rat embryonic development was detected at high levels in El5 heart, tongue, and limb bud. In addition, using type 2 neurofibromin-specific an- tibodies, the existence of a fourth isoform of neu- rofibromin (type 4 neurofibromin) containing both exon 23a and 48a sequences was demon- strated in rat heart muscle tissues. The identifica- tion of two muscle-specific isoforms of neurofibro- min expands our definition of this important tumor suppressor protein and suggests additional roles for neurofibromin in muscle development and differentiation. o 1995 WiIey-Liss, Inc.