A novel naturally occurring splice variant of the human TGF-ß type II receptor (TßRII) encodes a truncated soluble molecule

BERTOLIO, MS; RODRIGUEZ, TM; CARREA, A; VELASCO ZAMORA, J; PERONE, MJ; DEWEY, RA

Congreso; LXI Reunión Anual de la Sociedad Argentina de Investigación Clínica; 2016

Here we describe for the first time the presence in human cells of a new splicing variant of TGF-ß type II receptor. Compared to the known TßRII-A variant mRNA sequence, the new molecule lacks the last 63 nucleotides of exon II and the first 86 nucleotides of exon III. This deletion of 149 nucleotides causes a frameshift with the appearance of an early stop codon rendering a truncated protein of 80 amino acids lacking the transmembrane domain. Here we show that this new splicing variant, named by us TßRII-Soluble endogenous (TßRII-Se), is transcribed by most cell types analyzed but absent/highly downregulated in solid tumor-derived cell lines (A549, HT1080, CaCo-2, SK-Mel). Moreover, overexpression of TßRII-Se by means of lentiviral vectors in A549 cells, increased the sensitive to TGF-ß growth inhibition, as determined by MTT proliferation assay. To allow increased expression in mammalian cells, we codon optimized the TßRII-Se cDNA, and fused it in frame with the human IgG1 Fc domain to ease protein purification, and to enhance in vivo half life. In addition, we generated a specific monoclonal antibody directed against TßRII-Se which allowed us the intra and extracellular detection of the novel secreted protein by means of Western blot and flow cytometry. Moreover, we identified the interactome of the TßRII-Se fusion protein by screening, in duplicates, the Huprot v3.0TM Human Proteome array (CDI), containing more than 15,000 genes covering around 75% of the proteome. This assay indicated that TßRII-Se fusion protein is able to bind to a panel of 150 proteins. Gene ontology analysis using the PANTHER gene list analysis, indicated that, at the molecular function, the novel protein interacts with binding proteins (56%). Of them, 59% are nucleic acid binding, and 33% protein binding proteins, being 54% of the latter, receptor binding proteins. Members of the TGF-ß pathway are known targets for diseases such as cancer, fibrosis, osteoarthritis, and Type II diabetes.