Insulin-like growth factor binding protein-2: contributions of the C-terminal domain to insulin-like growth factor-1 binding.
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 Chem 276:2880-2889, 2001). 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-2(1-248) and IGFBP-2(249-289) as well as IGFBP-2(1-190) were expressed as glutathione S-transferase-fusion proteins and purified. Based on competition binding analysis, deletion of residues 249 to 289 caused an approximately 20-fold decrease in IGF-1 binding affinity (IGFBP-2 EC50 = 0.35 nM and IGFBP-2(1-248) = 7 nM). Removal of the remainder of the C-terminal domain had no further effect on affinity (IGFBP-2(1-190) EC50 = 9.2 nM). In kinetic assays, IGFBP-2(1-248) and IGFBP-2(1-190) 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.
Kibbey
Megan M
MM
Department of Cell and Molecular Pharmacology and Experimental Therapeutics, Medical University of South Carolina, Charleston, SC 29425, USA.
Jameson
Mark J
MJ
Eaton
Erin M
EM
Rosenzweig
Steven A
SA
D020816:Amino Acid Motifs; D000595:Amino Acid Sequence; D000818:Animals; D002478:Cells, Cultured; D006801:Humans; D018971:Insulin-Like Growth Factor Binding Protein 2 / Q000096:biosynthesis / Q000737:chemistry* / Q000235:genetics; D007334:Insulin-Like Growth Factor I / Q000096:biosynthesis / Q000737:chemistry* / Q000235:genetics; D008024:Ligands; D008969:Molecular Sequence Data; D009154:Mutation; D017434:Protein Structure, Tertiary / Q000235:genetics; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000737:chemistry* / Q000235:genetics; D017384:Sequence Deletion; D012862:Skatole / Q000031:analogs & derivatives / Q000737:chemistry; D014364:Tryptophan / Q000737:chemistry
D018971:Insulin-Like Growth Factor Binding Protein 2; D008024:Ligands; D011994:Recombinant Proteins; C005904:BNPS-skatole; D007334:Insulin-Like Growth Factor I; D014364:Tryptophan; D012862:Skatole