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.
Payet
Louis D
LD
Kolling Institute of Medical Research, University of Sydney, Royal North Shore Hospital, St. Leonards, New South Wales 2065, Australia.
Wang
Xiu-Hong
XH
Baxter
Robert C
RC
Firth
Sue M
SM
D002352:Carrier Proteins / Q000378:metabolism; D006023:Glycoproteins / Q000378:metabolism; D006801:Humans; D018972:Insulin-Like Growth Factor Binding Protein 3 / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D007334:Insulin-Like Growth Factor I / Q000378:metabolism*; D007335:Insulin-Like Growth Factor II / Q000378:metabolism; D007457:Iodine Radioisotopes; D010446:Peptide Fragments / Q000378:metabolism; D011233:Precipitin Tests; D017434:Protein Structure, Tertiary; D017526:Receptor, IGF Type 1 / Q000378:metabolism*; D017527:Receptor, IGF Type 2 / Q000378:metabolism
D002352:Carrier Proteins; D006023:Glycoproteins; D018972:Insulin-Like Growth Factor Binding Protein 3; D007457:Iodine Radioisotopes; D010446:Peptide Fragments; D017527:Receptor, IGF Type 2; C075944:insulin-like growth factor binding protein, acid labile subunit; D007334:Insulin-Like Growth Factor I; D007335:Insulin-Like Growth Factor II; D017526:Receptor, IGF Type 1