Characterization of the increased biological potency in BALB/C 3T3 cells of two analogs of human insulinlike growth factor I which have reduced affinity for the 28 K cell-derived binding protein.
We have characterized the biological activity of two analogs of insulinlike growth factor I (IGF I) which have significantly reduced affinity for the soluble 28 K binding proteins which are secreted by various cell types. The analogs, which were made by site-directed mutagenesis of a synthetic gene encoding for IGF I, are [Gln 3, Ala 4, Tyr 15, Leu 16] IGF I and an analog in which the first 16 amino acids of IGF I were replaced with the first 17 amino acids of insulin (B-chain mutant). These two peptides have 100-fold and greater than 1,000-fold lower affinity, respectively, than IGF I for the 28 K binding protein present in the conditioned medium of two cell types, the clonal rat vascular smooth muscle line A10, and BALB/C 3T3 cells. The 28 K protein secreted by BALB/C 3T3 cells has fivefold-lower apparent affinity for both IGF I and [Gln 3, Ala 4, Tyr 15, Leu 16] IGF I than does the 28 K protein secreted by A 10 cells. Conditioned medium from these two cell types has similar amounts of unoccupied 28 K protein as evidenced by the ability of 125I-IGF I to specifically bind to and be covalently bound to the protein after treatment with the bifunctional cross-linking reagent disuccinimidyl suberate. In the presence of 0.1% calf serum, IGF I and [Gln 3, Ala 4, Tyr 15, Leu 16] IGF I stimulate DNA synthesis in A10 cells with ED50 = 0.4 nM, and in BALB/C 3T3 cells with ED50 = 10 nM and 1.3 nM, respectively. Thus, these peptides are equipotent in A10 cells, but the mutant peptide is ten times more active than IGF I in BALB/C 3T3 cells. A10 cells can be made ten times less sensitive to IGF I by performing the incubation in the presence of conditioned media from BALB/C 3T3 cells but not from A10 cells. The activity of [Gln 3, Ala 4, Tyr 15, Leu 16] IGF I is not altered under these conditions. Thus, the conditioned media, which contain 28 K proteins secreted by A10 cells and BALB/C 3T3 cells, have different effects on the biological action of IGF I. These data suggest that the 28 K binding proteins can have important effects on the sensitivity of tissues to IGF I and that the B-chain mutant and [Gln 3, Ala 4, Tyr 15, Leu 16] IGF I will be useful in assessing the biological role of these proteins.
Cascieri
M A
MA
Department of Biochemical Endocrinology, Merck Sharp and Dohme Research Laboratories, Rahway, New Jersey 07065.
Hayes
N S
NS
Bayne
M L
ML
D000818:Animals; D001667:Binding, Competitive; D002352:Carrier Proteins / Q000378:metabolism*; D003470:Culture Media; D004247:DNA / Q000096:biosynthesis; D005347:Fibroblasts / Q000187:drug effects* / Q000378:metabolism; D007334:Insulin-Like Growth Factor I / Q000031:analogs & derivatives* / Q000235:genetics; D051379:Mice; D008807:Mice, Inbred BALB C; D008970:Molecular Weight; D009131:Muscle, Smooth, Vascular / Q000166:cytology / Q000378:metabolism; D009154:Mutation; D013002:Somatomedins / Q000031:analogs & derivatives*
D002352:Carrier Proteins; D003470:Culture Media; D013002:Somatomedins; D007334:Insulin-Like Growth Factor I; D004247:DNA