RESUMEN
The landscape paradigm of protein folding can enable preferred pathways on a funnel-like energy surface. Hierarchical preferences may be manifest as a nonrandom pathway of disulfide pairing. Stepwise stabilization of structural subdomains among on-pathway intermediates is proposed to underlie the disulfide pathway of proinsulin and related molecules. Here, effects of pairwise serine substitution of insulin's exposed interchain disulfide bridge (Cys(A7)-Cys(B7)) are characterized as a model of a late intermediate. Untethering cystine A7-B7 in an engineered monomer causes significantly more marked decreases in the thermodynamic stability and extent of folding than occur on pairwise substitution of internal cystine A6-A11 [Weiss, M. A., Hua, Q. X., Jia, W., Chu, Y. C., Wang, R. Y., and Katsoyannis, P. G. (2000) Biochemistry 39, 15429-15440]. Although substantially disordered and without significant biological activity, the untethered analogue contains a molten subdomain comprising cystine A20-B19 and a native-like cluster of hydrophobic side chains. Remarkably, A and B chains make unequal contributions to this folded moiety; the B chain retains native-like supersecondary structure, whereas the A chain is largely disordered. These observations suggest that the B subdomain provides a template to guide folding of the A chain. Stepwise organization of insulin-like molecules supports a hierarchic view of protein folding.
Asunto(s)
Insulina/análogos & derivados , Insulina/química , Dicroismo Circular , Disulfuros/química , Estabilidad de Medicamentos , Humanos , Técnicas In Vitro , Insulina/síntesis química , Espectroscopía de Resonancia Magnética , Modelos Moleculares , Oxidación-Reducción , Proinsulina/síntesis química , Proinsulina/química , Conformación Proteica , Desnaturalización Proteica , Pliegue de Proteína , Estructura Terciaria de Proteína , Subunidades de Proteína , TermodinámicaRESUMEN
Previous studies have demonstrated that the potency and thermodynamic stability of human insulin are enhanced in concert by substitution of Thr(A8) by arginine or histidine. These surface substitutions stabilize the N-terminal alpha-helix of the A chain, a key element of hormone-receptor recognition. Does enhanced stability necessarily imply enhanced activity? Here, we test by structure-based mutagenesis the relationship between the stability and activity of the hormone. To circumvent confounding effects of insulin self-association, A chain analogs were combined with a variant B chain (Asp(B10), Lys(B28), and Pro(B29) (DKP)) to create a monomeric template. Five analogs were obtained by chain combination; disulfide pairing proceeded in each case with native yield. CD and (1)H NMR spectra of the DKP analogs are essentially identical to those of DKP-insulin, indicating a correspondence of structures. Receptor binding affinities were determined by competitive displacement of (125)I-insulin from human placental membranes. Thermodynamic stabilities were measured by CD titration; unfolding was monitored as a function of guanidine concentration. In this broader collection of analogs receptor binding affinities are uncorrelated with stability. We suggest that receptor binding affinities of A8 analogs reflect local features of the hormone-receptor interface rather than the stability of the free hormone or the intrinsic C-capping propensity of the A8 side chain.
Asunto(s)
Insulina/análogos & derivados , Secuencia de Aminoácidos , Estabilidad de Medicamentos , Guanidina , Calor , Insulina/química , Modelos Moleculares , Datos de Secuencia Molecular , Unión Proteica , Conformación Proteica , Desnaturalización Proteica , Receptor de Insulina/metabolismo , Homología de Secuencia de Aminoácido , Relación Estructura-Actividad , TermodinámicaRESUMEN
An invariant residue, valine B12, is part of the insulin B-chain central alpha-helix (B9-B19), and its aliphatic side chain lies at the surface of the hydrophobic core of the insulin monomer in close contact with the neighboring aromatic side chains of phenylalanines (B24 and B25) and tyrosines (B26 and B16). This surface contributes to the dimerization of insulin, maintains the active conformation of the insulin monomer, and has been suspected to be directly involved in receptor recognition. To investigate in detail the role of the B12 residue in insulin-receptor interactions, we have synthesized nine analogues bearing natural or unnatural amino acid replacements for valine B12 by chemical synthesis of modified insulin B-chains and the subsequent combination of each synthetic B-chain with natural insulin A-chain. The receptor binding potencies of the synthetic B12 analogues relative to porcine insulin were determined by use of isolated canine hepatocytes, and the following results were obtained: isoleucine, 13%; allo-isoleucine, 77%; tert-leucine, 107%; cyclopropylglycine, 43%; threonine, 5.4%; D-valine, 3.4%; alpha-amino-n-butyric acid, 14%; alanine, 1.0%; and glycine, 0.32%. Selected analogues were also analyzed by far-UV circular dichroic spectroscopy and by absorption spectroscopy of their complexes with Co(2+). Our results indicate that beta-branched aliphatic amino acids are generally tolerated at the B12 position with specific steric preferences and that the receptor binding potencies of these analogues correlate with their abilities to form dimers. Furthermore, the structure-activity relationships of valine B12 are quite similar to those of valine A3, suggesting that valine residues at both A3 and B12 contribute to the insulin-receptor interactions in a similar manner.
Asunto(s)
Sustitución de Aminoácidos/genética , Insulina/análogos & derivados , Insulina/genética , Insulina/metabolismo , Receptor de Insulina/metabolismo , Valina/genética , Animales , Sitios de Unión/genética , Dicroismo Circular , Análisis Mutacional de ADN , Perros , Espectrofotometría , PorcinosRESUMEN
Maturity-onset diabetes of the young, a monogenic form of Type II diabetes mellitus, is most commonly caused by mutations in hepatic nuclear factor 1alpha (HNF-1alpha). Here, the dimerization motif of HNF-1alpha is shown to form an intermolecular four-helix bundle. One face contains an antiparallel coiled coil whereas the other contains splayed alpha-helices. The "mini-zipper" is complementary in structure and symmetry to the top surface of a transcriptional coactivator (dimerization cofactor of homeodomains). The bundle is destabilized by a subset of mutations associated with maturity-onset diabetes of the young. Impaired dimerization of a beta-cell transcription factor thus provides a molecular mechanism of metabolic deregulation in diabetes mellitus.
Asunto(s)
Proteínas de Unión al ADN , Diabetes Mellitus Tipo 2/genética , Islotes Pancreáticos/metabolismo , Mutación , Proteínas Nucleares , Estructura Secundaria de Proteína , Factores de Transcripción/química , Secuencia de Aminoácidos , Dimerización , Factor Nuclear 1 del Hepatocito , Factor Nuclear 1-alfa del Hepatocito , Factor Nuclear 1-beta del Hepatocito , Humanos , Concentración de Iones de Hidrógeno , Modelos Moleculares , Datos de Secuencia Molecular , Factores de Transcripción/genética , Factores de Transcripción/metabolismoRESUMEN
[GlyB24]insulin is a novel insulin analog which maintains nearly full biological activity [Mirmira, R. G., & Tager, H. S. (1989) J. Biol. Chem. 264, 6349-6354] even though its structure, as determined by 2D NMR, shows complete loss of the characteristic B-chain beta-turn [Hua, Q. X., Shoelson, S. E., Kochoyan, M., & Weiss, M. A. (1991) Nature 354, 238-241], which in native insulin allows the extended B-chain C-terminal region to fold against the central B-chain helix. In these studies, steady-state anisotropy measurements and fluorescence quenching analysis of the tryptophan-substituted analogs [TrpB25]insulin and [GlyB24,TrpB25]insulin have been used to study the structure of the C-terminal region of the B-chain and have demonstrated that [GlyB24]insulin mutants maintain the normal B-chain conformation to a degree comparable to that of native (PheB24) insulin at neutral pH. The tryptophan-substituted, B-chain C-terminally truncated analogs [TrpB25-alpha-carboxamide]despentapeptide(B26-B30)-insulin (DPI) and [GlyB24,TrpB25-alpha-carboxamide]DPI also significantly retain the characteristic insulin B-chain fold in solution with [GlyB24,TrpB25-alpha-carboxamide]DPI being more tightly folded than its corresponding PheB24-analog ([TrpB25-alpha-carboxamide]DPI), as assessed by these methods. The results of anisotropy measurements are consistent with the existence of a correlation between the high-affinity receptor binding of [GlyB24]insulin and the partial maintenance of the B-chain beta-turn under physiologic conditions. Thus we conclude that only analogs which possess, or can readily assume, this oriented structure can form high-affinity binding complexes with insulin receptor.
Asunto(s)
Insulina/química , Modelos Químicos , Acrilamidas , Polarización de Fluorescencia , Glicina , Humanos , Insulina/genética , Mutagénesis , Estructura Secundaria de Proteína , Receptor de Insulina/metabolismo , TriptófanoRESUMEN
To evaluate more thoroughly the importance of main-chain structure and flexibility in ligand interactions with the insulin receptor, we undertook to synthesize analogues with reduced peptide bonds in the COOH-terminal B chain domain of the hormone (a stable, but adjustable beta-strand region). By use of solid-phase, solution-phase and semisynthetic methods, analogues were prepared in which ArgB22 of des-octapeptide(B23-B30)-insulin was extended by the sequences Gly-Phe-psi (CH2-NH)-Phe-NH2, Gly-Gly-psi(CH2-NH)-Phe-Phe-NH2, Gly-Phe-psi (CH2-NH)-Phe-Phe-Thr-Pro-Ala-Thr-OH, and Gly-Phe-Phe-psi (CH2-NH)-Phe-Thr-Pro-Ala-Thr-OH, and were studied with respect to their abilities both to interact with the hepatocyte insulin receptor and to form soluble anion-stabilized hexamers in the presence of Co2+ and phenol. Additional analogues of des-pentapeptide(B26-B30)-insulin were also examined. Overall, our results show that, whereas all analogues retain considerable ability to form organized metal ion-coordinated complexes in solution, the reduction of peptide bonds both proximal and distal to the critical side chain of PheB25 results in analogues with severely diminished receptor binding potency. We conclude that the peptide carbonyls from both PheB24 and PheB25 are important for insulin-receptor interactions and that the structural organization of the region when insulin is bound to its receptor differs from that occurring during simple monomer-monomer and higher-order interactions of the hormone.
Asunto(s)
Insulina/análogos & derivados , Receptor de Insulina/metabolismo , Secuencia de Aminoácidos , Animales , Relación Dosis-Respuesta a Droga , Hígado/metabolismo , Modelos Químicos , Datos de Secuencia Molecular , Conformación Proteica , Relación Estructura-ActividadRESUMEN
We have investigated the effects of altering the disposition between the COOH-terminal B chain domain of insulin and the core of the insulin molecule on ligand interactions with the hepatocyte insulin receptor. Analogues include those in which ArgB22 of des-octapeptide(B23-B30)-insulin is extended by one to three residues of glycine prior to termination in Phe-NH2, by one to five residues of glycine prior to termination in Phe-Phe-NH2, or by an additional residue of glycine prior to termination in more extended sequences derived from insulin or [GlyB24]insulin. Analogues were also examined with respect to their abilities to form hexamers in solution in the presence of Co2+, phenol, and NaSCN. Overall, our studies of ligand-receptor interactions identify that (a) the energetic penalty for the introduction of a single residue of glycine is uniform in all classes of analogues for up to three residues of glycine but diminishes somewhat for analogues with longer insertions and (b) the COOH-terminal residues of the B chain retain their importance for all classes of analogues, no matter the number of glycine residues introduced. Analogues with glycine insertions, but not those with glycine substitutions, readily form thiocyanate-stabilized complexes with Co2+ in the presence of phenol.(ABSTRACT TRUNCATED AT 250 WORDS)
Asunto(s)
Insulina/química , Insulina/metabolismo , Pliegue de Proteína , Receptor de Insulina/metabolismo , Secuencia de Aminoácidos , Aminoácidos/química , Animales , Células Cultivadas , Perros , Hígado/citología , Hígado/metabolismo , Datos de Secuencia Molecular , PorcinosRESUMEN
We have examined, by use of a hybrid insulin/insulin-like growth factor-I analog and chimeric insulin/type I insulin-like growth factor receptors, the interplay between ligand and receptor structure in determining the affinity and specificity of hormone-receptor interactions in the insulin and insulin-like growth factor-I systems. Our findings, obtained through the study of radiolabeled peptide binding to detergent-solubilized full-length receptors and to soluble truncated receptors, show that (a) the two-chain hybrid analog exhibits significant cross-reactivity with both receptor systems, (b) the exchange of appropriate domains in chimeric receptors enhances the receptor binding affinity of the analog by 3.5-21-fold, and (c) the affinity of the hybrid analog for the chimeric receptors actually exceeds that of either natural insulin or natural insulin-like growth factor-I. We conclude that the specificity-conferring domains of the insulin and type I insulin-like growth factor receptors reside in different regions of a common binding site, and that the exchange of domains between pairs of related hormones and between pairs of related receptors can yield new ligand-receptor systems with significantly altered affinities and selectivities.
Asunto(s)
Factor I del Crecimiento Similar a la Insulina/metabolismo , Insulina/metabolismo , Receptor IGF Tipo 1/metabolismo , Proteínas Recombinantes de Fusión/metabolismo , Sitios de Unión , Humanos , Insulina/genética , Factor I del Crecimiento Similar a la Insulina/genética , Cinética , Ligandos , Modelos Estructurales , Multimerización de Proteína , Receptor IGF Tipo 1/genética , Eliminación de SecuenciaRESUMEN
In order to evaluate the cause of the greatly decreased receptor-binding potency of the naturally occurring mutant human insulin Insulin Wakayama ([LeuA3]insulin, 0.2% relative potency), we examined (by the semisynthesis of insulin analogues based on N alpha-PheB1,N epsilon-LysB29-bisacetyl-insulin) the importance of aliphatic side chain structure at positions A2 and A3 (Ile and Val, respectively) in directing the interaction of insulin with its receptor. Analogues bearing glycine, alanine, alpha-amino-n-butyric acid, norvaline, norleucine, valine, isoleucine, allo-isoleucine, threonine, tert-leucine, or leucine at positions A2 or A3 were assayed for their potencies in competing for the binding of 125I-labeled insulin to isolated canine hepatocytes, as were analogues bearing deletions from the A-chain amino terminus or the B-chain carboxyl terminus. Selected analogues were also analyzed by far-UV CD and absorption spectroscopy of Co2+ complexes. Our results identify that (a) Ile and Val serve well at position A2, whereas residues with other side chains (including those with straight chains, alternatively configured beta-branches, or a gamma-branch) exhibit relative receptor-binding potencies in the range 1-5%; (b) greater flexibility is allowed side-chain structure at position A3, with Ile, allo-Ile, alpha-amino-n-butyric acid, and tert-Leu exhibiting relative receptor-binding potencies in the range 11-36%; and (c) simultaneous replacements at positions A2 and A3, and deletions of the COOH-terminal domain of the insulin B chain in related analogues, yield cumulative effects. These findings are discussed with respect to a model for insulin-receptor interactions that involves a structure-orienting role for residue A2, the direct interaction of residue A3 with receptor, and multiple separately defined elements of structure and of conformational adjustment.
Asunto(s)
Insulina/química , Receptor de Insulina/química , Secuencia de Aminoácidos , Animales , Dicroismo Circular , Perros , Insulina/análogos & derivados , Datos de Secuencia Molecular , Conformación Proteica , Espectrofotometría , Relación Estructura-Actividad , PorcinosRESUMEN
By the chemical synthesis of modified insulin B chains and the combination of the synthetic B chains with natural insulin A chains, we have prepared insulin analogs with natural and unnatural amino acid replacements of invariant residue LeuB6. Analogs have been investigated by reference to their potencies for interaction with the insulin receptor (as assessed by competition for 125I-labeled binding to isolated canine hepatocytes) and to their abilities to undergo the structural transitions that are characteristic of insulin self-aggregation (as assessed by the spectroscopic analysis of analog complexes with cobalt). Our results identify that (a) replacement of LeuB6 by glycine has nearly the equivalent effect as deletion of residues B1-B6 in decreasing receptor binding potency of the analog to only about 0.05% of that of insulin; (b) relative to the GlyB6 derivative, replacements that increase the relative hydrophobicity of the residue B6 side chain also increase the relative receptor binding potencies of the resulting analogs; (c) negative steric effects resulting from substitutions by valine, phenylalanine, and gamma-ethylnorleucine limit the potential for enhancing potency as the result of increased hydrophobicity; and (d) two analogs with disparate potency for receptor interaction (those with alanine and gamma-ethylnorleucine at position B6, analogs exhibiting about 1 and 48% of the potency of insulin, respectively) undergo the T6----R6 structural transition in the presence of Co2+ and phenol which is typical of insulin but result in hexameric complexes with greatly reduced stability. We conclude that leucine provides a closely determined best fit at insulin position B6, and we discuss our findings in terms of insulin conformations that may apply to the receptor-bound state of the hormone.
Asunto(s)
Insulina/metabolismo , Leucina/metabolismo , Receptor de Insulina/metabolismo , Animales , Cromatografía Líquida de Alta Presión , Dicroismo Circular , Insulina/análogos & derivados , Espectrofotometría Ultravioleta , PorcinosRESUMEN
By use of isolated canine hepatocytes and insulin analogs prepared by trypsin-catalyzed semisynthesis, we have investigated the importance of the aromatic triplet PheB24-PheB25-TyrB26 of the COOH-terminal B-chain domain of insulin in directing the affinity of insulin-receptor interactions. Analysis of the receptor binding potencies of analogs bearing transpositions or replacements (by Tyr, D-Tyr or their corresponding 3,5-diiodo derivatives) in this region demonstrates a wide divergence in the acceptance both of configurational change (with [D-TyrB24,PheB26]insulin and [D-TyrB25,PheB26]insulin exhibiting 160 and 0.1% of the receptor binding potency of insulin, respectively) and of detailed side chain structure (with [TyrB24,PheB26]insulin and [TyrB25,PheB26]insulin exhibiting 2 and 80% of the receptor binding potency of insulin, respectively). Additional experiments addressed the solvent accessibilities of the 4 tyrosine residues of insulin and the insulin analogs at selected peptide concentrations by use of analytical radioiodination. Whereas two analogs ([TyrB25,PheB26]insulin and [D-TyrB24,PheB26]insulin) were found to undergo self aggregation, no strict correlation was found between the ability of an analog to aggregate and its potency for interaction with the insulin receptor. Related findings are discussed in terms of the interplay between side chain and main chain structure in the COOH-terminal domain of the insulin B-chain and the structural attributes of insulin that determine the affinity of insulin-receptor interactions.
Asunto(s)
Insulina/metabolismo , Receptor de Insulina/metabolismo , Secuencia de Aminoácidos , Animales , Sitios de Unión , Perros , Técnicas In Vitro , Insulina/química , Radioisótopos de Yodo/química , Hígado/metabolismo , Datos de Secuencia Molecular , Mapeo Peptídico , Unión Proteica , Relación Estructura-Actividad , Tirosina/químicaRESUMEN
The conformational stability and flexibility of insulin containing a cross-link between the alpha-amino group of the A-chain to the epsilon-amino group of Lys29 of the B-chain was examined. The cross-link varied in length from 2 to 12 carbon atoms. The conformational stability was determined by guanidine hydrochloride-induced equilibrium denaturation and flexibility was assessed by H2O/D2O amide exchange. The cross-link has substantial effects on both conformational stability and flexibility which depend on its length. In general, the addition of a cross-link enhances conformational stability and decreases flexibility. The optimal length for enhanced stability and decreased flexibility was the 6-carbon link. For the 6-carbon link the Gibbs free energy of unfolding was 8.0 kcal/mol compared to 4.5 kcal/mol for insulin, and the amide exchange rate decreased by at least 3-fold. A very short cross-link (i.e. the 2-carbon link) caused conformational strain that was detectable by a lack of stabilization in the Gibbs free energy of unfolding and enhancement in the amide exchange rate compared to insulin. The effect of the cross-link length on insulin hydrodynamic properties is discussed relative to previously obtained receptor binding results.
Asunto(s)
Insulina , Animales , Dicroismo Circular , Reactivos de Enlaces Cruzados , Deuterio/química , Guanidina , Guanidinas/farmacología , Técnicas In Vitro , Insulina/química , Movimiento (Física) , Conformación Proteica , Desnaturalización Proteica/efectos de los fármacos , PorcinosRESUMEN
We have prepared by semisynthetic methods a two-chain insulin/insulin-like growth factor I hybrid that contains a synthetic peptide related to residues 22-41 of insulin-like growth factor I linked via peptide bond to ArgB22 of des-octapeptide-(B23-B30)-insulin and have applied the analog to the analysis of ligand interactions with the type I insulin-like growth factor and insulin receptors of placental plasma membranes. Relative potencies for the inhibition of 125I-labeled insulin-like growth factor I binding to type I insulin-like growth factor receptors were 1.0:0.20:0.003 for insulin-like growth factor I, the hybrid analog, and insulin, respectively. Corresponding relative potencies for the inhibition of 125I-labeled insulin binding to insulin receptors were 0.007:0.28:1 for the three respective peptides. Additional studies identified that the hybrid analog interacts with only one of two populations of insulin-like growth factor I binding sites on placental plasma membranes and permitted the analysis of insulin-like growth factor I interactions with the separate populations of binding sites. We conclude that (a) des-octapeptide-(B23-B30)-insulin can serve well as a scaffold to support structural elements of insulin-like growth factor I and insulin necessary for high affinity binding to their receptors, (b) major aspects of structure relevant to the conferral of receptor binding affinity lie in the COOH-terminal region of the insulin B chain and in the COOH-terminal region of the insulin-like growth factor I B domain and in its C domain, and (c) the evolution of ligand-receptor specificity in these systems has relied as much on restricting interactions (through the selective introduction of negative structural elements) as it has on enhancing interactions (through the introduction of affinity conferring elements of structure).
Asunto(s)
Factor I del Crecimiento Similar a la Insulina/metabolismo , Insulina/metabolismo , Placenta/metabolismo , Receptor de Insulina/metabolismo , Receptores de Superficie Celular/metabolismo , Secuencia de Aminoácidos , Membrana Celular/metabolismo , Femenino , Humanos , Insulina/genética , Factor I del Crecimiento Similar a la Insulina/genética , Cinética , Modelos Moleculares , Datos de Secuencia Molecular , Embarazo , Conformación Proteica , Multimerización de Proteína , Receptores de SomatomedinaRESUMEN
We have evaluated, by use of isolated canine hepatocytes, the importance of intramolecular hormone cross-linking (and of concomitant changes in molecular flexibility) to the interaction of insulin with its plasma membrane receptor. Cross-linked hormone analogs were prepared by reacting porcine insulin, N alpha A1-t-butyloxycarbonyl insulin or N alpha A1-t-butyloxycarbonyl [D-LysA1]insulin with various dicarboxylic acid active esters to obtain alpha-GlyA1/epsilon-LysB29-, alpha-PheB1/epsilon-LysB29-, and epsilon-D-LysA1/epsilon-LysB29-cross-linked insulins, respectively. In the aggregate, insulin analogs cross-linked by groups containing 2-12 atoms retained 1.4-35% of the receptor binding potency of native insulin. Analysis of our results suggests that: (a) loss of chemical functionality, steric interference, and restriction of potential intramolecular movement can all play roles in determining the receptor binding potencies of cross-linked insulin analogs; (b) restriction of intramolecular movement between residues A1 and B29 affects negatively the binding of insulin to its receptor (but accounts for only a fraction of the conformational change which insulin must undergo to achieve a high affinity state of ligand-receptor interaction); and (c) introduction of a cross-link between residues B1 and B29 (residues that are in fact in proximity in one crystalline form of the hormone) decreases markedly the receptor binding potencies of the corresponding analogs. The importance of these findings is discussed in relation to the potential structure of insulin when it is bound to its plasma membrane receptor.
Asunto(s)
Reactivos de Enlaces Cruzados/farmacología , Insulina/análogos & derivados , Insulina/farmacología , Hígado/metabolismo , Receptor de Insulina/metabolismo , Animales , Unión Competitiva , Membrana Celular/metabolismo , Perros , Técnicas In Vitro , Insulina/metabolismo , Cinética , Sustancias Macromoleculares , Modelos Moleculares , Conformación ProteicaRESUMEN
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)
Asunto(s)
Insulina/análogos & derivados , Receptor de Insulina/metabolismo , Marcadores de Afinidad , Membrana Celular/metabolismo , Electroforesis en Gel de Poliacrilamida , Femenino , Humanos , Insulina/metabolismo , Factor I del Crecimiento Similar a la Insulina/metabolismo , Peso Molecular , Fenilalanina , Placenta/metabolismo , Embarazo , Proinsulina/metabolismo , Receptores de Somatomedina , Relación Estructura-ActividadRESUMEN
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.
Asunto(s)
Insulina/metabolismo , Fragmentos de Péptidos/metabolismo , Receptor de Insulina/análisis , Animales , Sitios de Unión , Perros , Insulina/análogos & derivados , Hígado/metabolismo , Receptor de Insulina/metabolismo , Relación Estructura-ActividadRESUMEN
To gain an understanding of the causes of decreased biological activity in insulins bearing amino acid substitutions at position B25 and the importance of the PheB25 side chain in directing hormone-receptor interactions, we have prepared a variety of insulin analogs and have studied both their interactions with isolated canine hepatocytes and their abilities to stimulate glucose oxidation by isolated rat adipocytes. The semisynthetic analogs fall into three structural classes: (a) analogs in which the COOH-terminal 5, 6, or 7 residues of the insulin B-chain have been deleted, but in which the COOH-terminal residue of the B-chain has been derivatized by alpha-carboxamidation; (b) analogs in which PheB25 has been replaced by unnatural aromatic or natural L-amino acids; and (c) analogs in which the COOH-terminal 5 residues of the insulin B-chain have been deleted and in which residue B25 has been replaced by selected alpha-carboxamidated amino acids. Our results showed that (a) insulin residues B26-B30 can be deleted without decrease in biological potency, whereas deletion of residues B25-B30 and B24-B30 causes a marked and cumulative decrease in potency; (b) replacement of PheB25 in insulin by Leu or Ser results in analogs with biological potency even less than that observed when residues B25-B30 are deleted; (c) the side chain bulk of naphthyl(1)-alanine or naphthyl(2)-alanine at position B25 is well tolerated during insulin interactions with receptor, whereas that of homophenylalanine is not; and (d) the decreased biological potency attending substitution of insulin PheB25 by Ala, Ser, Leu, or homophenylalanine is reversed, in part or in total, by deletion of COOH-terminal residues B26-B30. Additional experiments showed that the rate of dissociation of receptor-bound 125I-labeled insulin from isolated hepatocytes is enhanced by incubating cells with insulin or [naphthyl(2)-alanineB25]insulin, but not with analogs in which PheB25 is replaced by serine, leucine, or homophenylalanine; deletion of residues B26-B30, however, results in analogs that enhance the rate of dissociation of receptor-bound insulin in all cases studied. We conclude that (a) steric hindrance involving the COOH-terminal domain of the B chain plays a major role in directing the interaction of insulin with its receptor; (b) the initial negative effect of this domain is reversed upon the filling of a site reflecting interaction of the receptor and the beta-aromatic ring of the PheB25 side chain.(ABSTRACT TRUNCATED AT 400 WORDS)
Asunto(s)
Insulina/metabolismo , Fenilalanina/fisiología , Receptor de Insulina/metabolismo , Animales , Perros , Insulina/farmacología , Radioisótopos de Yodo , Ligandos , Conformación Proteica , Relación Estructura-ActividadRESUMEN
As part of our studies on the design of agonists of the luteinizing hormone-releasing hormone (LH-RH), we have synthesized the [des-Gly-NH2(10)]-LH-RH N-methylhydrazide (1), the corresponding thiosemicarbazide (2), and the N-formyl- (3) N-acetyl- (4) and N-(trifluoroacetyl)hydrazide (5). Analogue 1 may be regarded as isosteric with [des-Gly-NH2(10)]-LH-RH N-alkylamides which are, in general, potent agonists. Analogues 2-5 may be regarded as isosteric with [aza-Gly-NH2(10)]-OH-RH, which is equipotent with the hormone. The required protected intermediates were prepared by solid-phase synthesis, and the free peptides were prepared from them by deprotection with HF, followed by purification on Sephadex G-25. Bioassay of these analogues with rat hemipituitaries in vitro showed the following values as percentages of the hormonal values for the release of LH and FSH respectively: N-methylhydrazide (1), 17 and 11%; semithiocarbazide (2), 6.5 and 4.6%; N-formylhydrazide (3), 15.3 and 10%; N-acetylhydrazide (4), 1.2 and 0.6%; N-(trifluoroacetyl)hydrazide (5), 1.0 and 0.9%. Thus, these types of isosteric substitutions are inimical to the preservation of the high biological activity of LH-RH.
Asunto(s)
Hormona Liberadora de Gonadotropina/análogos & derivados , Animales , Fenómenos Químicos , Química , Hormona Liberadora de Gonadotropina/síntesis química , Hormona Liberadora de Gonadotropina/farmacología , Técnicas In Vitro , Masculino , Hipófisis/efectos de los fármacos , RatasAsunto(s)
Hormona Liberadora de Gonadotropina , Animales , Bioensayo , Hormona Folículo Estimulante/metabolismo , Hormona Liberadora de Gonadotropina/farmacología , Radioisótopos de Yodo , Marcaje Isotópico/métodos , Hormona Luteinizante/metabolismo , Masculino , Rotación Óptica , Hipófisis/efectos de los fármacos , Hipófisis/metabolismo , RatasRESUMEN
[3-Iodo-Tyr2]oxytocin (MIOT), [3,5-diiodo-Tyr2]oxytocin (DIOT), [3-iodo-Tyr2,Lys8]vasopressin (MILVP), [3,5-diiodo-Tyr2,Lys8]vasopressin (DILVP), [3-iodo-Tyr2,Arg8]vasopressin (MIAVP), and [3,5-diiodo-Tyr2,Arg8]vasopressin (DIAVP) were synthesized by iodination of the respective hormones, pruified, and characterized. All the monoiodo hormones had to be freshly prepared prior to bioassays, since on storage they gave rise to hormonal-like biological activity. The biological activities of these iodo analogues were measured in an adenylate cyclase assay employing neurohypophyseal hormone (NHH) sensitive bovine renal medullary membranes, and/or the rat oxytocic assay. In the cyclase assay, DIOT, DILVP, and DIAVP were inactive as agonists or antagonists. MIOT shows no agonistic activity in the renal cyclase system and uterus, but is a weak reversible inhibitor of oxytocin (OT) in both systems. When MIOT (10(-4) M) was preincubated with renal membranes for 10 min at 37 degrees C before addition of OT, it behaved as a noncompetitive inhibitor of NHH-stimulated adenylate cyclase. MILVP and MIAVP appear to be partial agonists with Km (half maximal response) 3 X 10(-6) and 3 X 10(-7) M, respectively, as determined in the cyclase assay. Upon preincubation with renal medullary membranes, MILVP (10(-6) M) behaves as a more potent noncompetitive inhibitor of OT than MIOT. Accordingly, iodo derivatives of NHH do not exhibit sufficient affinity to serve an specific ligands to measure OT, LVP, or AVP receptors in the uterus and kidney. Study of the specificity of inhibition produced by MIOT revealed that this analogue does not act selectively upon NHH receptors. Thus, MIOT modified adenylate cyclase systems which do not have NHH receptors, e.g., the PTH-sensitive adenylate cyclase in bovine renal cortex and the glucagon-sensitive adenylate cyclase in rat liver. DIOT, DILVP, and DIAVP were subjected to catalytic tritiation (employing carrier free tritium) and were converted to [3H]OT (25, 31, and 25 Ci/mmol), [3H]LVP (26 and 23 Ci/mmol), and [3H]AVP (17 Ci/mmol), respectively. These tritiated ligands have been successfully used to measure NHH receptor sites both in kidney and uterine membranes as described in other studies.