Insulin-like growth factor binding protein (IGFBP) -5: A protein of two destinations.

Abstract

Insulin-like growth factor-binding protein (IGFBP)-5 is a secreted protein that binds to IGFs and modulates IGF actions. IGFBP-5 is also found in the nuclei of mammalian cells and has transactivation activity. The transactivation domain of IGFBP-5 was mapped to its N-terminal region. Sequence analysis revealed several amino acids in the IGFBP-5 N-domain that are not present in IGFBP-1. The activities of mutants in which these residues were changed to the corresponding IGFBP-1 sequence were determined. Mutations that changed acidic residues to neutral residues (<italic>e.g.</italic> E8A, D11S, E12A, E30S/P31A, E43L, and E52A) or a polar to a basic residue (<italic>e.g.</italic> Q56R) significantly reduced transactivation activity. These results suggest that IGFBP-5 may function as a transcriptional regulator in the nucleus. Previously, we used short double-stranded RNA (siRNA) to trigger RNA interference of IGFBP-5 in U2 osteosarcoma (U2OS) cells. Knock-down of IGFBP-5 resulted in significant increase in the number of TUNEL-positive cells. I tried to rescue this apoptotic phenotype by introducing nuclear localization mutations and ligand binding mutations into the siRNA-resistant IGFBP-5. The results showed that the nuclear localization mutant was able to rescue the apoptotic phenotype; however, the ligand binding mutant failed to rescue the apoptotic phenotype. These results indicate that IGFBP-5 promotes U2OS cell survival in an IGF-dependent but nuclear localization independent manner. We also investigated nuclear functions of IGFBP-5. IGFBP-5 overexpression reduced body length in the zebrafish embryos. This action of IGFBP-5 is due to its nuclear presence and its transcriptional activity and is independent of IGF binding. Using several cell biological and biochemical approaches, we showed that the nuclear IGFBP-5 is derived from the secretion pathway via endoplasmic reticulum-cytosol retro-translocation. We also found that PDI facilitates and ERp72 inhibits the IGFBP-5 retro-translocation. Injection of ERp72 mRNA suppressed the IGFBP-5 overexpression phenotype in zebrafish embryos. These results suggest that nuclear IGFBP-5 may exert cell autonomous functions in zebrafish embryonic development. Taken together, the evidences show that several acidic residues are important for IGFBP-5 transactivation activity, IGFBP-5 promotes U2OS cell survival in an IGF-dependent manner, and nuclear IGFBP-5 is derived from the secretion pathway and may exert IGF-independent functions.