Insulin-like growth factors (IGFs) are crucial for many aspects of development, growth, and metabolism yet control of their activity by IGF-binding proteins (IGFBPs) remains controversial. The effect of IGFBP-1 depends on its phosphorylation status; phosphorylated IGFBP-1 inhibits IGF actions whereas the nonphosphorylated isoform is stimulatory. In order to understand this phenomenon, we purified phosphorylated IGFBP-1 from normal human plasma by immunoaffinity chromatography. Unexpectedly, the resulting preparation enhanced IGF-stimulated 3T3-L1 fibroblast proliferation, due to the presence of a co-purified protein of ≈700 kDa. Matrix-assisted laser desorption ionization-mass spectrometry and Western immunoblotting analysis identified this co-purified protein as α2-macroglobulin (α2M). Anti-α2M antibodies co-immunoprecipitated IGFBP-1 from human plasma and from125I-IGFBP-1·α2M complexes formed in vitro. The 125I-IGFBP-1/α2M association could be inhibited with excess unlabeled IGFBP-1. Surface plasmon resonance analysis indicated that α2M preferentially associates with the phosphorylated isoform of IGFBP-1 and that when complexed to α2M, IGFBP-1 can still bind IGF-I. These findings have functional significance since α2M protects IGFBP-1 from proteolysis and abrogates the inhibitory effect of phosphorylated IGFBP-1 on IGF-I stimulated 3T3-L1 cell proliferation. We conclude that α2M is a binding protein of IGFBP-1 which modifies IGF-I/IGFBP-1 actions resulting in enhanced IGF effects. In line with its role in regulating the clearance and activity of other growth factors, we predict that α2M has a novel and important role in controlling the transport and biological activity of IGFs.
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Published, JBC Papers in Press, August 23, 2001, DOI 1.1074/jbc.M102793200
This work was supported by The Royal Society and the Salford Royal Hospitals NHS Trust and Wellbeing.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
