Binding specificities and transducing function of the different molecular weight forms of insulin-like growth factor-II (IGF-II) on IGF-I receptors.
In a study that was reported from this laboratory, the mitogenic potency of an apparent mol wt (appMr) of 15,000 precursor form of human insulin-like growth factor-II (hIGF-II) was shown to be greater than that of completely processed hIGF-II for human fetal-derived fibroblasts, and both were more potent than rIGF-I. Since it is generally acknowledged that the stimulation of cell replication by the IGFs is mediated by IGF-I receptors, we undertook to determine whether differences between the receptors' affinity for the two Mr forms of hIGF-II and recombinant IGF-I (rIGF-I) or between its efficiency to couple specific growth factor occupancy to the activation of protein kinase could explain the greater replicating potential of appMr 15,000 hIGF-II. Equilibrium dissociation, i.e. Kd, and inhibition, i.e. Ki, constants were determined by measuring the ability of rIGF-I, hIGF-II, appMr 15,000 hIGF-II, insulin, and the antireceptor monoclonal antibody alpha IR-3 to compete with 125I-labeled rIGF-I and hIGF-II for binding to purified preparations of IGF-I receptors prepared from an enriched source of fetal membrane, i.e. human term placenta. The results of these experiments established that 1) hIGF-II and appMr 15,000 hIGF-II bind to the IGF-I receptor with the same affinity as rIGF-I, e.g. with Kd and Ki values between 0.03-0.07 nM; 2) the total binding capacity, i.e. Ro, for IGF-I binding was not statistically different from the Ro calculated for IGF-II binding; and 3) the statistical analysis of 12 data sets from the competitive binding experiments for goodness of fit indicated that a 1-site model for IGF-I and -II binding was a better fit of the data than a 2-site model. Measurements of the stimulation of IGF-I receptor autophosphorylation at low ligand concentrations established that appMr 15,000 hIGF-II and hIGF-II were more effective than rIGF-I in coupling receptor occupancy to the activation of its protein kinase. At saturating ligand concentrations, the 3 had similar potencies. The original preparation of appMr 15,000 hIGF-II contains a mixture of forms with acidic isoelectric points (pIs) and was more potent than Mr 7,500 IGF-II in stimulating receptor autophosphorylation. These results are consistent with the relative potencies of this preparation, hIGF-II, and rIGF-I in stimulating the replication of 12-week-old fetal dermal fibroblasts.(ABSTRACT TRUNCATED AT 400 WORDS)
Perdue
J F
JF
Jerome H. Holland Laboratory for the Biomedical Sciences, American Red Cross, Rockville, Maryland 20855.
LeBon
T R
TR
Kato
J
J
Hampton
B
B
Fujita-Yamaguchi
Y
Y
D000911:Antibodies, Monoclonal; D001667:Binding, Competitive; D004789:Enzyme Activation; D005260:Female; D006801:Humans; D007328:Insulin / Q000378:metabolism; D007334:Insulin-Like Growth Factor I / Q000378:metabolism / Q000494:pharmacology; D007335:Insulin-Like Growth Factor II / Q000378:metabolism* / Q000494:pharmacology; D008970:Molecular Weight; D010766:Phosphorylation; D010920:Placenta / Q000737:chemistry; D011494:Protein Kinases / Q000378:metabolism; D011956:Receptors, Cell Surface / Q000276:immunology / Q000378:metabolism*; D017451:Receptors, Somatomedin; D011994:Recombinant Proteins / Q000378:metabolism / Q000494:pharmacology; D015398:Signal Transduction
D000911:Antibodies, Monoclonal; D007328:Insulin; D011956:Receptors, Cell Surface; D017451:Receptors, Somatomedin; D011994:Recombinant Proteins; D007334:Insulin-Like Growth Factor I; D007335:Insulin-Like Growth Factor II; D011494:Protein Kinases