Heterogeneity of insulin‐like growth factor binding proteins and relationships between structure and affinity

Circulating insulin-like growth factors (IGFs) are bound to specific, high-affinity binding proteins (BPs), and form complexes with relative molecular masses of about 150000 (‘large’ complex) and 40000 (‘small’ complex). The large complex appears to be under growth-hormone control and its proportions vary with those of the IGFs. Molecular heterogeneity among the binding proteins was revealed by polydcrylamide gel electrophoresis (SDS-PAGE) of serum in which they were cross-linked to 125I-labelled IGF I or II. Out of the six specific bands observed, of 150000, 120000, 49000, 46000, 40000 and 37000 Mr, the last three appeared in both complexes, whereas the first three were visible only in the large complex. Some or all of the 49000–37000-Mr species may constitute the subunits of 150000-Mr and/or 120000-Mr IGF-BP complexes. With electrophoresis followed by transfer onto nitrocellulose and incubation with either 125I-labelled IGF I or II (western blot), the different binding proteins were identified per se. There were five molecular forms with Mr of 41 500, 38 500, 34000, 30000 and 24000. In normal serum the 41 500 and 38 500-Mr forms were the major binding proteins. They appeared in both complexes, but were predominant in the large complex where they constitute the elementary binding units. These two proteins therefore bind to IGFs to form both ‘monomeric’ IGF-BP and ‘oligomeric’ (IGF-BP)n complexes. The 34000, 30000 and 24000-Mr forms, by contrast, were visible only in the small complex. Different mechanisms appear to regulate the different binding proteins: in acromegalic serum the 41 500 and 38 500-Mr forms were augmented and the 34000-Mr form diminished, whereas in hypopituitary serum the reverse was true and, in addition, the 30000-Mr form was augmented. With chromatofocusing, the 34000, 30000 and 24000-Mr forms eluted in three peaks between pH 6.0 and 4.0, whereas the 41 500 and 38 500-Mr forms eluted throughout the gradient, principally at pH 7.5 and 7.0. Competitive binding studies, done on binding proteins separated either by chromatofocusing or by SDS-PAGE and transfer onto nitrocellulose, revealed different affinities for the IGFs among the different molecular forms. The 41 500 and 24 000-Mr binding proteins preferentially bound IGF I and the 38 500, 34 000 and 30 000-Mr proteins preferentially bound IGF II. Our findings demonstrate the molecular heterogeneity of the binding proteins and the existence of a relationship between their structure and their affinities for the IGFs. They also suggest that, apart from their function as IGF carriers, the binding proteins also play a modulating role in the interaction between IGFs and their target cells.