We have identified and characterized two proteins in rat brain that bind to the neuron-specific tropomyosin isoform, TMBr3. The two proteins were identified by blot overlay assay, in which the proteins immobilized on the membrane were probed by epitope-tagged TMBr3, followed by detection with anti-epitope antibody. We have purified these proteins using a TMBr3 affinity column. Peptide sequencing as well as immunoblotting showed that one of the two proteins is identical to tropomodulin, a tropomyosin-binding protein originally identified in erythrocytes. The cDNA for the other protein was cloned from an adult rat brain cDNA library using degenerate oligonucleotides that we designed based on the peptide sequences. Sequence analysis of the cDNA clone revealed this protein to be a novel isoform of tropomodulin which is the product of a distinct gene, and is herein referred to as N-tropomodulin. Recombinant N-tropomodulin bound to TMBr3 as well as to other low molecular mass tropomyosins (TM5a or TM5), but not to high molecular mass tropomyosins (TM2 or TMBr1). Northern blotting and RNase protection assays as well as immunoblotting showed that N-tropomodulin is expressed predominantly in brain. Furthermore, RNase protection assays revealed no alternatively spliced regions within the coding sequence. Developmentally, N-tropomodulin was detected in rat brain as early as embryonic day 14 and reaches the adult level before birth. Immunofluorescence of primary frontal cortex cell cultures showed that N-tropomodulin is specifically expressed in neurons. The neuron-specific expression of N-tropomodulin strongly suggests specialized roles of this TM-binding protein in neurons.
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