A study on novel biological activities of pro-IGF-I E-peptides in human neuroblastoma cells

Insulin-like growth factors (IGFs) are mitogenic peptides that play important roles in development, growth and differentiation in vertebrates. Insulin-like growth factors (IGFs) are initially translated as pre-pro-peptides that require further processing, removing the signal peptide and the E-domain peptide, to result in the mature peptide hormones. Multiple forms of pro-IGF-I, displaying developmental regulation and tissue specificity, have been identified in various species from fish to mammals with differences only in the carboxyl-terminal E-domains. However, the biological significance of the diversity of E-domain and its differential expression has been unclear. ^ The primary goal of this thesis is to characterize the biological activities of pro IGF-I E-peptides using a human neuroblastom(SK-N-F1)a cell line (SK-N-F1) as a model system. In this study, we demonstrate that Ea-4-peptide of rainbow trout (rtEa-4) and hEb-peptide of human pro-IGF-I hEb-elicits unique biological activities in inducing morphological differentiation and inhibiting anchorage-independent growth in human neuroblastoma cells. Identical nature and range of biological activities displayed by E-peptides of rainbow trout and humans suggest existence of functional conservation of these E-peptides over a wide evolutionary scale. Not only have we demonstrated that E-peptides of pro-IGF-I are biologically active, but these peptides also exhibit distinct or contrast activities from those of the mature IGF-I. Thus, the notion that E-peptides, as part of pro-IGF peptides, are only involved in the biosynthesis of the mature IGFs and otherwise biologically inert has been seriously challenged. Furthermore, our binding studies demonstrated that hEb-peptide and rtEa-4-peptide bind to two distinct binding sites or two affinity states on SK-N-F1 cells. The second binding site (IC50 of 4.8 ± 2.6 × 10−6 M for hEb-peptide and 2.1 ± 0.6 × 10−6 M for rtEa-4-peptide, respectively) is in good agreement with the in vitro effective concentration of E-peptides. In addition, we demonstrated that hEb-peptide and rtEa-4-peptide share common binding sites, distinct from those of mature IGF-I and insulin. These results support the hypothesis that pro-IGF-I E-peptides interact with specific cell surface receptors that are conserved from fish to humans. This work sets a foundation for further elucidation of the molecular mechanism of E-peptide actions.