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Abstract
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.
Highlights
Previous studies havesuggested that theCOOH-ter- and for the Torpedo cholinergic receptor (3)
(a)the structuresof molecules l and 2 of the hepatocytes, we show here that (a)the markedly de- insulin dimer within the 2-zinc insulin hexamer differ somecreasedaffinity of receptor for insulin analogs in what in distantregions involving both the mainchains of the which PheB2’is replaced by Ser is apparent foranalogs NHz-terminal A- and B-chain domains and thesidechains in in which up to 3 residues of the insulin B-chain have the COOH-terminal B-chain domain (9-11), (b)the structure been deleted, but is progressively reversed in the cor- of the NH2-terminal region of the B-chain in 4-zinc insulin responding des-tetrapeptide and des-pentapeptide an- differs considerably from the structureof the same region in alogs, and (b)unlike the case fdoerletion of TyrBaeand 2-zinc insulin (12, 13), and (c) the NH2-terminal domain of
Details of the semisynthetic methods used and of the cell preparation and incubation conditions are provided under "Materials and Methods." Relative receptor-binding potency is defined for these purposes as [(concentration of porcine insulin causing lecular locus that is involved in the effect of the COOH- half-maximal inhibition of binding of [('261)iodo~'']insulinto reterminal B-chain domain to decreasereceptor binding affinity ceptor)/(concentration of analog causing half-maximal inhibition of and the extent of conformational change that appears to be required for high-affinity insulinreceptor interactions
Summary
The truncated proinsulin analogs des-(30-65)-proinsulin (21) and des-(23-65)-proinsulin (22) (while they may be considerably strained) exhibit
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