Structure of the Mr 140,000 growth hormone-dependent insulin-like growth factor binding protein complex: determination by reconstitution and affinity-labeling.

Abstract

To determine the structure of the high molecular weight, growth hormone-dependent complex between the insulin-like growth factors (IGF-I and IGF-II) and their binding proteins in human serum, we have reconstituted the complex from its purified component proteins and analyzed it by gel electrophoresis and autoradiography after covalent cross-linking. The proteins tested in reconstitution mixtures were an acid-labile Mr 84,000-86,000 glycoprotein doublet (.alpha. subunit), an acid-stable Mr 47,000-53,000 glycoprotein doublet with IGF-binding activity (BP-53 or .beta. subunit), and IGF-I or IGF-II (.gamma. subunit). In incubations containing any one of the three subunits 125I-labeled and the other two unlabeled, identical 125I-labeled .alpha.-.beta.-.gamma. complexes of Mr 140,000 were formed. Minor bands of Mr 120,000 and 90,000 were also seen, thought to represent a partially deglycosylated form of the .alpha.-.beta.-.gamma. complex, and an .alpha.-.gamma. complex arising as a cross-linking artifact. When serum samples from subjects of various growth hormone status were affinity-labeled with IGF-II tracer, a growth hormone-dependent Mr 140,000 and was seen, corresponding to the reconstituted .alpha.-.beta.-.gamma. complex. Other growth hormone-dependent labeled bands, of Mr 90,000 (corresponding to .alpha.-.gamma.), Mr 55,000-60,000 (corresponding to labeled .beta.-subunit doublet), and smaller bands of Mr 38,000, 28,000, and 23,000-25,000 (corresponding to labeled .beta.-subunit degradation products), were also seen in the affinity-labeled serum samples and in the complex reconstituded from pure proteins. All were immunoprecipitable with an anti-BP-53 antiserum. We conclude that the growth hormone-dependent Mr 140,000 IGF-binding protein complex in human serum has three components: the .alpha. (acid-labile) subunit, the .beta.(binding) subunit; and the .gamma. (growth factor) subunit.