Synthesis and characterization of insulin-like growth factor (IGF)-1 photoprobes selective for the IGF-binding proteins (IGFBPS). photoaffinity labeling of the IGF-binding domain on IGFBP-2.
Elevated insulin-like growth factor (IGF)-1 levels are prognostic for the development of prostate and breast cancers and exacerbate the complications of diabetes. In each case, perturbation of the balance between IGF-1/2, the IGF-1 receptor, and the IGF-binding proteins (IGFBPs) leads to elevated IGF-1 sensitivity. Blockade of IGF action in these diseases would be clinically significant. Unfortunately, effective IGF antagonists are currently unavailable. The IGFBPs exhibit high affinity and specificity for the IGFs and serve as natural IGF antagonists, limiting their mitogenic/anti-apoptotic effects. As an initial step in designing IGFBP-based agents that antagonize IGF action, we have begun to analyze the structure of the IGF-binding site on IGFBP-2. To this end, two IGF-1 photoprobes, N(alphaGly1)-(4-azidobenzoyl)-IGF-1 (abG(1)IGF-1) and N(alphaGly1)-([2-6-(biotinamido)-2(p-azidobenzamido)hexanoamido]ethyl-1,3'-dithiopropionoyl)-IGF-1 (bedG(1)IGF-1), selective for the IGFBPs were synthesized by derivatization of the alpha-amino group of Gly(1), known to be part of the IGFBP-binding domain. Mass spectrometric analysis of the reduced, alkylated, and trypsin-digested abG(1)IGF-1.recombinant human IGFBP-2 (rhIGFBP-2) complex indicated photoincorporation near the carboxyl terminus of rhIGFBP-2, between residues 266 and 287. Mass spectrometric analysis of avidin-purified tryptic peptides of the bedG(1)IGF-1.rhIGFBP-2 complex revealed photoincorporation within residues 212-227. Taken together, these data indicate that the IGFBP-binding domain on IGF-1 contacts the distal third of IGFBP-2, providing evidence that the IGF-1-binding domain is located within the C terminus of IGFBP-2.
Horney
M J
MJ
Department of Cell and Molecular Pharmacology and Experimental Therapeutics, Medical University of South Carolina, Charleston 29425, USA.
Evangelista
C A
CA
Rosenzweig
S A
SA
D000595:Amino Acid Sequence; D001386:Azides / Q000138:chemical synthesis / Q000378:metabolism*; D001665:Binding Sites; D001667:Binding, Competitive; D018971:Insulin-Like Growth Factor Binding Protein 2 / Q000378:metabolism*; D007334:Insulin-Like Growth Factor I / Q000031:analogs & derivatives / Q000037:antagonists & inhibitors* / Q000138:chemical synthesis / Q000378:metabolism*; D013058:Mass Spectrometry; D008958:Models, Molecular; D008969:Molecular Sequence Data; D010446:Peptide Fragments / Q000378:metabolism; D019920:Photoaffinity Labels; D011485:Protein Binding; D017526:Receptor, IGF Type 1 / Q000037:antagonists & inhibitors
D001386:Azides; D018971:Insulin-Like Growth Factor Binding Protein 2; C428452:Nalpha(Gly1)-((2-6-(biotinamido)-2-(4-azidobenzamido)hexanoamido)ethyl-1.'-dithiopropionyl)-insulin-like growth factor-1; C428451:Nalpha(Gly1)-(4-azidobenzoyl)-insulin-like growth factor-1; D010446:Peptide Fragments; D019920:Photoaffinity Labels; D007334:Insulin-Like Growth Factor I; D017526:Receptor, IGF Type 1