In addition to being important for normal fetal and postnatal growth, there is increasing evidence that insulin-like growth factors I and II (IGF-I, IGF-II) also support the growth of certain cancers. Biologic responses to IGF-I and IGF-II are signaled by the IGF-I receptor. Therefore, we are focusing our research effort on understanding signaling by the IGF-I receptor. We have used the yeast two-hybrid system to identify new binding partners for the IGF-I receptor. We have inserted the cytoplasmic domain of the IGF-I receptor into the LexA DNA binding vector. Poly A mRNA was prepared from the human osteosarcoma cell line, MG-63, and given to a commercial laboratory for preparation of a cDNA library in the yeast two-hybrid activation domain vector. The library screen identified IGF-I receptor binding partners that we and others had previously identified (p85 regulatory subunit of PI3-K, p66 Shc, Grb10, 14-3-3 beta and zeta, SOCS-1, SH2-B/PSM, RACK-1) as well as new interactors (STAT5a, GIPC, Ril, and 4 clones that are not fully characterized). We are focusing on GIPC which represented 25% of the most strongly interacting clones. GIPC did not interact with the insulin receptor in the yeast two hybrid assay. GIPC is a 333 amino acid protein with a central SH2 domain. Mutational analysis in the yeast two-hybrid system showed that GIPC PDZ domain binds to the carboxy tail of the IGF-I receptor. GIPC dimerizes and the dimerization domain has been shown to include the amino terminal domain as well as a portion of the PDZ domain. IGF-I receptor constructs that have been mutated to prevent GIPC binding have been transfected into NIH 3T3 cells in order to explore the function of GIPC in IGF-I receptor signaling. Cells transfected with receptors mutated for GIPC binding did not form colonies in soft agar, suggesting that GIPC binding to the receptor may be required for a transformation pathway. We have developed a mouse monoclonal antibody (4G11) directed against the human IGF-I receptor. This monoclonal antibody blocks the binding of radiolabeled IGF-I to MCF-7 breast cancer cells and MG-63 osteosarcoma cells and downregulates the receptor. Receptor downregulation results in inhibition of IGF-I stimulated activation of Akt and MAPK in MCF-7 cells. The Fab fragment of the monoclonal antibody inhibits binding of IGF-I to the receptor but does not cause receptor downregulation, suggesting that receptor aggregation is required for downregulation. Full recovery of receptor expression following downregulation requires 48 hrs. This result suggests that intermittent treatment with the monoclonal antibody in vivo may be sufficient to block IGF-I receptor signaling in cancers.
| Jackson-Booth, P-G; Terry, C; Lackey, B et al. (2003) Inhibition of the biologic response to insulin-like growth factor I in MCF-7 breast cancer cells by a new monoclonal antibody to the insulin-like growth factor-I receptor. The importance of receptor down-regulation. Horm Metab Res 35:850-6 |
| Spence, Susan L; Dey, Bhakta R; Terry, Cheryl et al. (2003) Interaction of 14-3-3 proteins with the insulin-like growth factor I receptor (IGFIR): evidence for a role of 14-3-3 proteins in IGFIR signaling. Biochem Biophys Res Commun 312:1060-6 |
