title: Structural determinants of ligand recognition by type I insulin-like growth factor receptors: use of semisynthetic insulin analog probes.
abstract: We undertook a systematic analysis of the structural determinants necessary for ligand recognition by the type I insulin-like growth factor (IGF) receptor by investigating the binding of semisynthetic insulin analogs to IGF receptors from human placental cell membrane fragments. Analogs were prepared by synthetic and semisynthetic methods. Three groups of insulin analogs were synthesized: the first group contained insulin analogs modified at the amino-terminal position of the insulin A chain and included acetyl-insulin and human proinsulin; the second group included analogs in which B chain residues B26-B30 [despentapeptide insulin (DPI)], B25-B30 (deshexapeptide insulin), and B24-B30 (desheptapeptide insulin) were removed; the third group contained insulin analogs in which B chain residues B26-B30 were removed (DPI) and phenylalanine(B25) substituted with other amino acids, including alanine, serine, leucine, and tyrosine. Half-maximal inhibition of binding of radiolabeled IGF-I to placental cell membrane fragments was used as an index of relative binding affinity (K1/2). To determine further if semisynthetic insulin analogs bound to the type I IGF receptor, placental membrane fragments were affinity labeled with radiolabeled IGF-I in the presence and absence of submaximal concentrations of unlabeled hormone, insulin, or semisynthetic analogs, and the labeled proteins were analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Insulin had a 500 times lower affinity for the type I IGF receptor than IGF-I [K1/2 = 140 +/- 69 nM (mean +/- SD)] whereas proinsulin and acetyl insulin had a more than 100 times lower affinity than insulin for this receptor type. Removal of insulin B chain amino acid residues 26-30 (DPI) did not negatively affect the binding of the insulin-derived peptide and actually increased the apparent affinity of ligand-receptor association approximately 2-fold. However, further removal of phenylalanine(B25) (deshexapeptide insulin) and phenylalanine(B24) (desheptapeptide insulin) decreased the binding of ligand to the type I IGF receptor progressively by several orders of magnitude. Substitution of phenylalanine(B25) of DPI with tyrosine, a substitution that actually increased the homology of this analog to IGF-I, resulted in a 4- to 5-fold increase in the relative apparent affinity of the analog for the type I IGF receptor (K1/2 = 31 +/- 4 nM). On the other hand, substitution of phenylalanine(B25) with alanine, serine, and leucine decreased the relative apparent binding affinity approximately 2- to 8-fold.(ABSTRACT TRUNCATED AT 400 WORDS)
authors.affiliation: Department of Pediatrics, University of Chicago Pritzker School of Medicine, Illinois 60637.
authors.forename: J F
authors.initials: JF
authors.lastname: Cara
authors.forename: S H
authors.initials: SH
authors.lastname: Nakagawa
authors.forename: H S
authors.initials: HS
authors.lastname: Tager
mesh_terms: D000345:Affinity Labels; D002462:Cell Membrane / Q000378:metabolism; D004591:Electrophoresis, Polyacrylamide Gel; D005260:Female; D006801:Humans; D007328:Insulin / Q000031:analogs & derivatives* / Q000378:metabolism; D007334:Insulin-Like Growth Factor I / Q000378:metabolism; D008970:Molecular Weight; D010649:Phenylalanine; D010920:Placenta / Q000378:metabolism; D011247:Pregnancy; D011384:Proinsulin / Q000378:metabolism; D011972:Receptor, Insulin / Q000378:metabolism*; D017451:Receptors, Somatomedin; D013329:Structure-Activity Relationship
chemical_list: D000345:Affinity Labels; D007328:Insulin; D017451:Receptors, Somatomedin; C056000:acetylinsulin; D010649:Phenylalanine; D007334:Insulin-Like Growth Factor I; D011384:Proinsulin; D011972:Receptor, Insulin