MODIFICATIONS IN THE B10-REGION AND B26-30-REGION OF THE B-CHAIN OF HUMAN INSULIN ALTER AFFINITY FOR THE HUMAN IGF-I-RECEPTOR MORE THAN FORTHE INSULIN-RECEPTOR

Inversion of the natural sequence of the B chain of human insulin (HI) from pro(B28)Lys(B29) to Lys(B28)pro(B29) generates an insulin analog ue with reduced tendency to self-associate. Since this substitution in creases the homology of insulin to insulin-like growth factor-I (IGF-I ), we have examined the affinity of a series of insulin analogues with the general modified structure Xaa(B28)Pro(B29) HI for binding to bot h human placental insulin and IGF-I receptors. The Xaa(B28)pro(B29) HI series is approximately equipotent to HI in binding to the insulin re ceptor with the exception of when Xaa = Phe, Trp, Leu, Ile, and Gly (4 0-60 % relative to HI). Substitution with basic residues in the B28 po sition increased the relative affinity to the IGF-I receptor approxima tely 1.5-2-fold (Arg(B28)pro(B29) > Orn(B28)Pro(B29) = Lys(B28)pro(B29 )). Substitution with acidic residues reduced relative affinity for th e IGF-I receptor approximately 2-fold Cya(B28)pro(B29) = GlU(B28)pro(B 29) > Asp(B28)Pro(B29)). Combination of Asp(B10) substitution in conju nction with a modification in the B28-29 position (e.g. Asp(B10)Lys(B2 8)pro(B29) HI) showed an additional 2-fold selective increase in affin ity for the IGF-I receptor, suggesting that these two effects are addi tive. Addition of Arg residues at B31-32, on the backbone of either HI or Asp(B10) HI, increased affinity for the IGF-I receptor 10 and 28 f old, respectively, compared to HI, confirming the significance of enha nced positive charge at the C-terminal end of the insulin B-chain in i ncreasing selectivity for the IGF-I receptor. This relative increase i n IGF-I receptor affinity correlated largely, but not completely, with enhanced growth promoting activity in human mammary epithelial cells. In the case of Lys(B28)Pro(B29) III, growth activity correlated with dissociation kinetics from the insulin receptor which were shown to be identical with those of human insulin.