Molecular Cloning and Characterization of c-erb-A mRNA Species in Mouse Neuroblastoma Cells

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Abstract
The mouse neuroblastoma cell line is an excellent model in which to study thyroid hormone action and metabolism, particularly in neural tissue. We therefore undertook the molecular cloning and characterization in these cells of putative thyroid hormone receptors related to c-erb-A. Since rat brain tissue contains multiple cell types, and because of possible subtle differences between species (mouse and rat), we therefore screened a new cDNA library constructed from mouse neuroblastoma cell mRNA with synthetic oligonucleotide probes based on the published nucleotide sequence of the c-erb-A gene in whole rat brain. Despite the fact that this rat brain cDNA sequence is now recognized to represent a c-erb-A alpha 1 form, the cDNA clones that we isolated were all members of the newly-recognized c-erb-A alpha 2 form. This identification was made on the basis of nucleotide sequence divergence downstream of the nucleotide corresponding to amino-acid residue 370 in the predicted coding region. The two longest mouse neuroblastoma cDNA clones, clone 29 (1796 bp) and clone 32 (1410 bp), were 93% to 94% homologous with the c-erb-A alpha 2 and c-erb-A alpha 1 forms in their DNA binding and thyroid hormone binding domains (up to amino-acid residue 370 in the latter). Both clones 29 and 32 were incomplete in that they terminated at their 3'ends at an internal Eco R1 site. Fortunately this 120 bp (40 derived amino-acid) truncation was downstream of the reported thyroid hormone binding domain. The 5'untranslated end of clone 29 (446 bp) was of interest because a region of its nucleotide sequence (279 bp) revealed a high degree of homology (87%) with rat brain c-erb-A alpha 1. This highly conserved region in clone 29 appeared to be important in the regulation of translation because only clone 32, in which this region was truncated, efficiently translated protein in a cell-free system. The protein product of clone 32 did not bind thyroid hormone. Northern blot analysis of mouse neuroblastoma mRNA with site-specific synthetic oligonucleotides revealed that the c-erb-A alpha 2 species was dominant (major band of 2.4 kb), with a lesser amount of the c-erb-A alpha1 species (major band of 1.8 kb). 1) We report the molecular cloning of c-erb-A variants in a specific neural tissue cell type, namely mouse neuroblastoma cells; 2) The cells contained predominantly the c-erb-A alpha 2 subtype; 3) This c-erb-A alpha 2 form was unique up to bp 167 at its 5'untranslated end, and was then followed, up to the ATG initiation codon, by a highly conserved region common to all c-erb-A a species; 4) The 5'untranslated region appeared to play a role in the translational efficiency of this mRNA; 5) The protein product of the c-erb-A alpha 2 mRNA in these cells did not appear to bind thyroid hormone. In view of this finding, the physiological role of the c-erb-A alpha 2 protein remained speculative and may involve a represser function at the thyroid hormone responsive element.