a-amidation is a post-translational modification that occurs in some peptide hormones and involves the substitution of -NH2 (amide) for -OH (free-acid) residues at the carboxy-terminal amino acid. This chemical alteration is enzymatically mediated, and distinct amino acid sequence motifs code for the post-translation event to occur. Since a-amidation tracks with biological activity, we have used amidation signal motifs to identify cryptic peptides found within the precursor proteins of established human growth factors. Applying this investigative strategy to proinsulin-like growth factor-IB, a potential peptide amide (IBE1) has been found in the E domain of the precursor. A synthetic peptide homolog of IBE1 was shown to induce trophic effects, in a dose related manner, on both normal and malignant pulmonary cells. The peptide's proliferative action was shown to be dependent on the integrity of the amidated carboxy-terminal amino acid, the synthetic free-acid derivative being an impotent mitogen. The IBE1 peptide, though derived from IGF- IB precursor, does not mediate its action through the type 1 receptor of IGF-I. In a receptor binding assay with 125I-IBE1 neither insulin, IGF-I, IGF-II, nor GRP at 2mM concentration were able to block labeled ligand/receptor interaction. Scatchard analysis of IBE1 receptor expression on A549 cells (bronchioalveolar CA) gave a KD 2.8 x 10 to the -11 power and a receptor density of 1.5 x 10 to the 4th power per cell. Using polyclonal antisera to synthetic IBE1, we have demonstrated the existence of immunoreactive peptides with molecular weights of 25kDa, 12kDa, 8kDa, and 5kDa in small cell lung cancer cell lines. In addition, similar immunoreactive peptides have been identified in normal liver extracts from a variety of mammalian species, implicating evolutionary conservation. We are now following similar research strategies to identify alternative peptide amides which mediate tumor proliferation; these may represent rational biologic targets for the early diagnosis and prevention of malignant disease.
