Derivation of insertin

Insertin is an actin-binding protein that has been isolated from chicken gizzard smooth muscle that has been shown to be highly homologous to amino acids 962-1292 of tensin [Weigt et al., 1992]. Because of the high homology, we investigated the question whether the mRNAs of insertin and of tensin are derived from the same gene by alternative splicing, whether insertin and tensin are encoded by two different genes, or whether insertin is a proteolytic fragment of tensin. In a Northern blot analysis, mRNA from chicken gizzard was hybridized with oligonucleotides specific for tensin and for the insertin domain of tensin. The tensin-specific oligonucleotide hybridized only with the previously reported 8- and 10-kbp RNAs. However, the insertin domain-specific oligonucleotide hybridized with a 1.2 and a 1.6 kbp RNA in addition to the 8 and 10 kbp RNA. The 1.2- and 1.6-kbp RNA occurred in small amounts, as compared with the 8- and 10-kbp RNA. Southern blot analysis of DNA cleaved by the restriction endonucleases BamH1 and HindIII demonstrated that only one gene for the insertin and tensin exists. Insertin isolated from chicken gizzard smooth muscle was investigated by mass spectrometry. The N-termini of three isolated peptides were found to begin at adjacent amino acids and were likely to be formed from tensin by proteolysis. The results suggest that, for insertin, an mRNA exists that is derived from one gene common for insertin and tensin. However, the insertin-specific mRNA contributes relatively little to expression of insertin domains in cells. Insertin preparations from chicken gizzard contain mainly insertin domains formed from tensin by proteolysis.