EXPRESSION OF 2 NEW-PROTEIN ISOFORMS OF THE NEUROFIBROMATOSIS TYPE-1 GENE-PRODUCT, NEUROFIBROMIN, IN MUSCLE TISSUES

The neurofibromatosis type 1 (NF1) gene encodes a tumor suppressor pro tein, termed neurofibromin, which is expressed predominantly in neuron s, Schwann cells, oligodendrocytes, and leukocytes. There are at least three isoforms of neurofibromin produced by the alternative use of ex ons 23a and 48a. Previously we described the identification of an NF1 mRNA isoform containing an additional 54 nucleotides from exon 48a (ty pe 3 NF1) in human skeletal, cardiac and smooth muscle tissues by reve rse-transcribed (RT)-PCR. To extend our initial observations, we have produced high titer chicken IgY antibodies which specifically recogniz e this muscle-specific neurofibromin isoform. An NF1 cDNA was generate d containing human exon 48a sequences and expressed as a fusion protei n in bacteria. The muscle-specific neurofibromin antibodies detected t his exon 48a fusion protein by Western inmunoblotting. Immunoprecipita tion using these type 3 neurofibromin antibodies also specifically det ected a 250 kDa protein in human and rat muscle tissues. Type 3 neurof ibromin was found in rat heart and muscle, but not in liver, brain, ki dney or spleen with levels of expression declining after postnatal day 7. Expression of total NF1 RNA during rat embryonic development was d etected at high levels in E15 heart, tongue, and limb bud. In addition , using type 2 neurofibromin-specific antibodies, the existence of a f ourth isoform of neurofibromin (type 4 neurofibromin) containing both exon 23a and 48a sequences was demonstrated in rat heart muscle tissue s. The identification of two muscle-specific isoforms of neurofibromin expands our definition of this important tumor suppressor protein and suggests additional roles for neurofibromin in muscle development and differentiation. (C) 1995 Wiley-Liss, Inc.