Abstract
We have used site-directed mutagenesis of a synthetic gene for insulin-like growth factor (IGF) I to prepare three analogs in which specific residues in the A region are replaced with the corresponding residues in the A chain of insulin. The analogs are [Ile41, Glu45, Gln46, Thr49, Ser50, Ile51, Ser53, Tyr55, Gln56]IGF I (A chain mutant), in which residue 41 is changed from threonine to isoleucine and residues 42 to 56 of the A region are replaced, [Thr49, Ser50, Ile51]IGF I, and [Tyr55, Gln56]IGF I. These analogs are all equipotent to IGF I at the type 1 IGF receptor in human placental membranes, and in stimulating the incorporation of [3H]thymidine into DNA in the rat vascular smooth muscle cell line A10. However, the A chain mutant and [Thr49, Ser50, Ile51]IGF I have greater than 20-fold lower relative affinity for the type 2 IGF receptor of rat liver membranes, respectively. In contrast, [Tyr55, Gln56]IGF I has 7-fold higher affinity than IGF I for the type 2 IGF receptor. Residues 49, 50, and 51 in IGF I are Phe-Arg-Ser and are strictly conserved in IGF II. Residues 55 and 56 of IGF I and the corresponding residues in IGF II are Arg-Arg and Ala-Leu, respectively. Thus, the presence of the charged residues at these positions in IGF I appears to be responsible, in part, for the lower affinity of IGF I for the type 2 IGF receptor. In addition to the alterations in affinity for the type 2 IGF receptor, the A chain mutant has a 7-fold increase in affinity for insulin receptors, and [Thr49, Ser50, Ile51]IGF I has a 4-fold lower affinity for acid-stable human serum binding protein. These data strongly suggest that specific determinants in the A region of IGF I are important for maintaining binding to the type 2 IGF receptor, and that these determinants are different from those required for maintaining high affinity for the type 1 IGF receptor.
Highlights
We have used site-directed mutagenesis of a syn- meric protein (3,5)
The A chain mutant has 7-fold higher relative ing residues in insulin leads to a drastic reduction in relative affinity for insulin receptors than does insulin-like growth factor (IGF) I, [Thr4', Ser", affinity for type 2 IGF receptors and a smaller loss in relative
As expected based on the potencies of these peptides at the type 1 IGF receptor, the dose-response curves show that the ED50 doses for the A chain mutant, [Thr4', Ser", IleS1]Insulin-like growth factor I (IGFI), and [Tyr5', Gln5'j]IGFI are the same as for IGF I
Summary
K G ATC TGC K T CTG TAC CAGCTC GAG ATG I A C TGC GCA membranes. Serum binding protein affinity was determined by meas-. Nonspecific binding to the receptors and binding proteins was determined by adding excess. Thr Gly I l e Val Asp Glu C y aC y s~ h h : ~ S e r ~ l l : ' C y s Aap Leu Arc AIS Leu G1U f k t IYr CYS Ala unlabeled ligand and was
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