Role of the COOH-terminal B-chain domain in insulin-receptor interactions. Identification of perturbations involving the insulin mainchain.
Previous studies have suggested that the COOH-terminal pentapeptide of the insulin B-chain can play a negative role in ligand-receptor interactions involving insulin analogs having amino acid replacements at position B25 (Nakagawa, S. H., and Tager, H. S. (1986) J. Biol. Chem. 261, 7332-7341). We undertook by the current investigations to identify the molecular site in insulin that induces this negative effect and to explore further the importance of conformational changes that might occur during insulin-receptor interactions. By use of semisynthetic insulin analogs containing amino acid replacements or deletions and of isolated canine hepatocytes, we show here that (a) the markedly decreased affinity of receptor for insulin analogs in which PheB25 is replaced by Ser is apparent for analogs in which up to 3 residues of the insulin B-chain have been deleted, but is progressively reversed in the corresponding des-tetrapeptide and des-pentapeptide analogs, and (b) unlike the case for deletion of TyrB26 and ThrB27, replacement of residue TyrB26 or ThrB27 has no effect to reverse the decreased affinity of full length analogs containing Ser for Phe substitutions at position B25. Additional experiments demonstrated that introduction of a cross-link between Lys epsilon B29 and Gly alpha A1 of insulin decreases the affinity of ligand-receptor interactions whether or not PheB25 is replaced by Ser. We conclude that the negative effect of the COOH-terminal B-chain domain on insulin-receptor interactions arises in greatest part from the insulin mainchain near the site of the TyrB26-ThrB27 peptide bond and that multiple conformational perturbations may be necessary to induce a high-affinity state of receptor-bound insulin.
Nakagawa
S H
SH
Tager
H S
HS
D000818:Animals; D001665:Binding Sites; D004285:Dogs; D007328:Insulin / Q000031:analogs & derivatives / Q000378:metabolism*; D008099:Liver / Q000378:metabolism; D010446:Peptide Fragments / Q000378:metabolism*; D011972:Receptor, Insulin / Q000032:analysis* / Q000378:metabolism; D013329:Structure-Activity Relationship
D007328:Insulin; D010446:Peptide Fragments; C029491:insulin, 3-iodo-Tyr(A14)-; D011972:Receptor, Insulin