A mitogenic peptide amide encoded within the E peptide domain of the insulin-like growth factor IB prohormone

We have identified an amino acid sequence within the E peptide of the insulin-like growth factor IB (IGF-IB) precursor that is biologically active and designated this peptide insulin-like growth factor IB-(103-124) E₁ amide (IBE₁). Its existence was predicted by a flanking Gly-Lys-Lys-Lys, a signal sequence for sequential proteolytic cleavage and peptidyl C-terminal amidation. A synthetic analog of the predicted IBE₁ peptide, designated Y-23-R-NH₂, was generated with tyrosine added at position 0. This peptide at 2-20 nM had growth-promoting effects on both normal and malignant human bronchial epithelial cells. Y-23-R-NH₂ bound to specific high-affinity receptors (K_d = 2.8 ± 1.4 × 10^-11 M) present at 1-2 × 10⁴ binding sites per cell. Ligand binding was not inhibited by recombinant insulin or recombinant IGF-I. Furthermore, a monoclonal antibody antagonist to the IGF-I receptor (αIR3) did not suppress the proliferative response induced by Y-23-R-NH₂ . In addition, C-terminal amidation was shown to be important in receptor recognition since the free-acid analog of IBE₁ (Y-23-R-OH) did not effectively compete for binding and was not a potent agonist of proliferation. Immunoblot analysis of human lung tumor cell line extracts using an antibody raised against Y-23-R-NH₂ detected a low molecular mass band of ≈5 kDa, implying that a protein product is produced that has immunological similarity to IBE₁. Extracts of human, mammalian, and avian livers analyzed on an immunoblot with the anti-Y-23-R-NH₂ antibody contained proteins of ≈21 kDa that were specifically recognized by the antiserum and presumably represent an IGF-I precursor molecule. This implies that in species where an IGF-I mRNA with homology to the human IGF-IB E domain has not yet been described, an alternate mRNA must be produced that contains a sequence similar to that of the midportion of the human IGF-IB E domain. Our findings demonstrate that IBE₁ is a growth factor that mediates its effect through a specific high-affinity receptor and is most likely conserved in many species.