Activity of human pregnancy insulin-like growth factor binding protein-3: determination by reconstituting recombinant complexes.

During pregnancy, IGF binding protein-3 (IGFBP-3) is completely proteolyzed to fragments with low affinities for IGFs but appears to transport IGFs normally in high-molecular-mass complexes. We previously reported that synthetic isolated amino- and carboxyl-terminal domains of IGFBP-3 cooperate to bind IGFs, and we investigated whether this is the mechanism whereby proteolyzed IGFBP-3 fragments bind IGFs normally in pregnancy serum. Two fragments of IGFBP-3 have been isolated from pregnancy serum, one with the same N-terminal sequence as intact IGFBP-3 (GASSG) and the other with an N-terminal sequence (160)KVDYE. Recombinant forms of these proteins, IGFBP-3(1-159) and IGFBP-3(160-264), have been synthesized and characterized, demonstrating that although the fragments individually have greatly reduced affinity for IGF complex formation, when combined they cooperate to form complexes with IGF with or without the acid-labile subunit, inhibit IGF transport across endothelial cell monolayers and inhibit IGF-I-induced IGF type I receptor phosphorylation. It is proposed that proteolysis of IGFBP-3 into two discrete complementary fragments does not significantly increase IGF bioavailability, consistent with previous findings that proteolyzed IGFBP-3 in pregnancy serum is functionally normal and circulates as part of the IGF ternary complexes.

The role of the acid-labile subunit in regulating insulin-like growth factor transport across human umbilical vein endothelial cell monolayers.

We have established a human umbilical vein endothelial cell (HUVEC) monolayer system to study the role of complex formation with IGF binding protein (IGFBP) and acid-labile subunit (ALS) on the transendothelial transport of IGF. Incubation with recombinant human IGFBP-3 alone did not retard (125)I-IGF-I or -II transport, but addition of ALS caused marked inhibition. Transport of (125)I-des(1-3)IGF-I was more rapid than (125)I-IGF-I, suggesting the presence of some endogenous IGFBPs, although these were undetectable by affinity labeling of cells or medium. In the presence of ALS, recombinant human IGFBP-5 also retarded IGF transport, although significantly less than IGFBP-3, despite their similar ternary complex formation. In contrast, IGFBP-3 mutated in its ALS-binding domain was not inhibitory. To study IGF transport by pregnancy-proteolyzed IGFBP-3, we prepared [Tyr(31)]monoiodoIGF-I, the only iodoIGF-I form that reacts normally with proteolyzed IGFBP-3. In the presence of ALS, IGFBP-3 isolated by immunoaffinity chromatography from second-trimester pregnancy serum significantly retarded IGF transport, but to a lower extent than IGFBP-3 isolated from normal serum, despite normal ALS binding. This study demonstrates the key role of ALS in regulating transendothelial IGF transport, but indicates that other factors are also involved. Our data suggest that pregnancy-proteolyzed IGFBP-3, despite forming normal ternary complexes, is less effective than intact IGFBP-3 in retarding IGF egress from the circulation.

Amino- and carboxyl-terminal fragments of insulin-like growth factor (IGF) binding protein-3 cooperate to bind IGFs with high affinity and inhibit IGF receptor interactions.

Both the amino-terminal and carboxyl-terminal domains of IGF binding protein (IGFBP)-3 are believed to contribute to high-affinity IGF binding. To investigate cooperativity in IGF binding by these domains, we expressed IGFBP-3 fragments 1-88 (NBP-3) and 185-264 (CBP-3) as FLAG and hexahistidine-tagged fusion proteins, respectively. IGF-I and IGF-II bound to NBP-3 poorly and to CBP-3 with moderate affinities, approximately 1 liter/nmol. Coincubating the fragments in equimolar concentrations caused a significant cooperative increase in IGF binding, demonstrated by immunoprecipitation with IGFBP-3, FLAG, or hexahistidine antibodies. Equimolar NBP-3 + CBP-3 bound IGF-II with an affinity (12.2 liter/nmol) only 4-fold lower than that of the IGFBP-3-IGF-II complex and IGF-I with an affinity (3.2 liter/nmol) 13-fold lower than IGFBP-3-IGF-I. Heterotrimeric complexes of NBP-3, CBP-3, and IGF, also demonstrated by affinity labeling, bound acid-labile subunit poorly. Coprecipitation assays with iodinated NBP-3 or CBP-3 indicated that the fragments cannot interact unless IGF is also present. Complexing with NBP-3 + CBP-3 inhibited IGF stimulation of type 1 IGF receptor activity and IGF-II binding to the type II receptor. This study demonstrates that isolated amino-terminal and carboxyl-terminal domains of IGFBP-3 cooperate in the presence of IGFs to form high-affinity complexes that retain the ability to block IGF activity.