Human recombinant insulin-like growth factor I. II. Binding characterization and radioreceptor assay development using Balb/c 3T3 mouse embryo fibroblasts.
The binding of human recombinant insulin-like growth factor I (IGF-I) to BALB/c 3T3 mouse embryo fibroblasts has been characterized, resulting in the development of a radioreceptor assay. Binding of radioiodinated IGF-I (125I-IGF-I) to washed monolayers of BALB/c 3T3 cells was specific, time dependent, and stable, being maximal after a 10-h incubation at 15 degrees C with no loss of bound ligand or cells through 25 h. Scatchard analysis identified a class of high affinity binding sites with Kd = 59.6 pM and an estimated 1.57 X 10(5) receptors/cell. Half-maximal displacement of bound 125I-IGF-I occurred with 15 to 20 ng/ml unlabeled IGF-I competitor. Insulin-like growth factor II and insulin were far less effective competitors, providing half-maximal displacement at concentrations of 130 to 170 ng/ml and 2 to 3 micrograms/ml, respectively. Epidermal growth factor, transforming growth factor type alpha, and acidic and basic fibroblast growth factors did not compete for 125I-IGF-I binding at 1 microgram/ml. Cells fixed with glutaraldehyde before ligand binding did remain attached to culture dishes more tightly; however such pretreatment destroyed approximately 70% of ligand binding. Crosslinking data indicated that 125I-IGF-I binds specifically to a 330-kDalton receptor as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis under nonreducing conditions. This receptor dissociated into 130-kDalton subunits when analyzed in the presence of dithiothreitol.
Karey
K P
KP
University of Texas Medical School, Department of Biochemistry and Molecular Biology, Houston 77225.
Riss
T L
TL
Burleigh
B D
BD
Parker
D
D
Sirbasku
D A
DA
D000818:Animals; D001667:Binding, Competitive; D003432:Cross-Linking Reagents; D007334:Insulin-Like Growth Factor I / Q000032:analysis / Q000378:metabolism*; D007700:Kinetics; D051379:Mice; D008807:Mice, Inbred BALB C / Q000196:embryology; D011869:Radioligand Assay; D011972:Receptor, Insulin / Q000378:metabolism*; D017451:Receptors, Somatomedin; D013002:Somatomedins / Q000378:metabolism*
D003432:Cross-Linking Reagents; D017451:Receptors, Somatomedin; D013002:Somatomedins; D007334:Insulin-Like Growth Factor I; D011972:Receptor, Insulin