@article {Kibbey833, author = {Megan M. Kibbey and Mark J. Jameson and Erin M. Eaton and Steven A. Rosenzweig}, title = {Insulin-Like Growth Factor Binding Protein-2: Contributions of the C-Terminal Domain to Insulin-Like Growth Factor-1 Binding}, volume = {69}, number = {3}, pages = {833--845}, year = {2006}, doi = {10.1124/mol.105.016998}, publisher = {American Society for Pharmacology and Experimental Therapeutics}, abstract = {Signaling by the insulin-like growth factor (IGF)-1 receptor (IGF-1R) has been implicated in the promotion and aggressiveness of breast, prostate, colorectal, and lung cancers. The IGF binding proteins (IGFBPs) represent a class of natural IGF antagonists that bind to and sequester IGF-1/2 from the IGF-1R, making them attractive candidates as therapeutics for cancer prevention and control. Recombinant human IGFBP-2 significantly attenuated IGF-1-stimulated MCF-7 cell proliferation with coaddition of 20 or 100 nM IGFBP-2 (50 or 80\% inhibition, respectively). We previously identified IGF-1 contact sites both upstream and downstream of the CWCV motif (residues 247-250) in human IGFBP-2 (J Biol Chem276:2880-2889, 2001OpenUrlAbstract/FREE Full Text). To further test their contributions to IGFBP-2 function, the single tryptophan in human IGFBP-2, Trp-248, was selectively cleaved with 2-(2'nitrophenylsulfenyl)-3-methyl-3 bromoindolenine (BNPS-skatole) and the BNPS-skatole products IGFBP-21-248 and IGFBP-2249-289 as well as IGFBP-21-190 were expressed as glutathione S-transferase-fusion proteins and purified. Based on competition binding analysis, deletion of residues 249 to 289 caused an \~{}20-fold decrease in IGF-1 binding affinity (IGFBP-2 EC50 = 0.35 nM and IGFBP-21-248 = 7 nM). Removal of the remainder of the C-terminal domain had no further effect on affinity (IGFBP-21-190 EC50 = 9.2 nM). In kinetic assays, IGFBP-21-248 and IGFBP-21-190 exhibited more rapid association and dissociation rates than full-length IGFBP-2. These results confirm that regions upstream and downstream of the CWCV motif participate in IGF-1 binding. They further support the development of full-length IGFBP-2 as a cancer therapeutic.}, issn = {0026-895X}, URL = {https://molpharm.aspetjournals.org/content/69/3/833}, eprint = {https://molpharm.aspetjournals.org/content/69/3/833.full.pdf}, journal = {Molecular Pharmacology} }