RESUMEN
The relative contributions of type I and type II insulinlike growth factor (IGF) receptors and IGF carrier proteins to the binding of IGF-I tracer to cultured human fibroblasts were determined in competitive binding experiments that used unlabeled insulin and synthetic insulin-IGF-I hybrid molecules containing the A chain of insulin and the B domain of IGF-I. Whereas insulin binds only to type I IGF receptors, the B-IGF-I hybrids bind to type I receptors and IGF carrier proteins but not to type II receptors. In suspended human fibroblasts, IGF-I tracer binds predominantly to type I IGF receptors (inhibition by IGF-I much greater than insulin greater than B-IGF-I hybrid molecules). By contrast, in fibroblast monolayers, IGF-I binding was minimally inhibited by insulin or hybrid molecules, suggesting predominant binding to the type II IGF receptor. The type I receptor appears to be masked on fibroblast monolayers, and to require suspension or detergent solubilization of the cells to be demonstrated. In the course of the monolayers binding experiments, we noted that low concentrations of unlabeled IGF-I (5-10 ng/ml) or B-IGF-I hybrids (100 ng/ml) paradoxically increased IGF-I tracer binding up to twofold. We postulated that during the binding incubation (5 h, 15 degrees C), IGF-I tracer partitioned between binding sites on the cell surface and IGF carrier proteins released to the incubation media. Preferential occupancy of binding sites in the media by unlabeled ligand increased the tracer available to bind to the cells. In support of this hypothesis, carrier proteins were demonstrated in the media at the end of the binding incubation with fibroblast monolayers, and the concentration of unsaturated binding sites in the media correlated inversely with tracer binding to the cells. Thus carrier proteins released to the media during the binding incubation modulate the binding of IGF-I tracer to cell receptors, suggesting that the carrier proteins may play an important role in regulating cellular responsiveness to the IGFs.
Asunto(s)
Proteínas Portadoras/metabolismo , Fibroblastos/metabolismo , Factor I del Crecimiento Similar a la Insulina/metabolismo , Receptores de Superficie Celular/metabolismo , Somatomedinas/metabolismo , Adolescente , Adulto , Sitios de Unión , Unión Competitiva , Sangre , Células Cultivadas , Medios de Cultivo , Femenino , Humanos , Insulina/metabolismo , Proteínas de Unión a Factor de Crecimiento Similar a la Insulina , Factor II del Crecimiento Similar a la Insulina/metabolismo , Masculino , Multimerización de Proteína , Receptores de SomatomedinaRESUMEN
A study of the sites of insulin binding in subcellular fractions of rat liver is reported. A method for the isolation of liver plasma membranes, which permits one to follow quantitatively the distribution of all the parameters of interest, was modified and applied to the study of the cellular topography of insulin binding. The insulin binding capacity did not follow closely the enzyme marker (5'-nucleotidase) for plasma membranes when differential centrifugation schemes were used, and the divergence from this marker was more prominent when separations were performed on discontinous sucrose gradients. A significant amount of insulin binding capacity was always present in fractions with higher density than those containing the majority of 5'-nycleotidase. Results of studies on linear sucrose gradients have disclosed in some of the purified membrane fractions small but consistent differences in density of the insulin binding, and plasma membrane particles. It is suggested that there may be several types of intracellular membranes to which insulin can bind besides the plasma membranes.
Asunto(s)
Insulina/metabolismo , Hígado/metabolismo , Receptores de Superficie Celular , Animales , Sitios de Unión , Fraccionamiento Celular , Membrana Celular/metabolismo , Centrifugación por Gradiente de Densidad , Glucosa-6-Fosfatasa/análisis , Hexosaminidasas/análisis , Nucleotidasas/análisis , Unión Proteica , Ratas , Fracciones Subcelulares/metabolismoRESUMEN
Insulin and the insulin-like growth factors IGF-I and IGF-II are thought to exert their mitogenic effects in cultured chick embryo fibroblasts and human skin fibroblasts via IGF receptors rather than via insulin receptors. These effects appear to be mediated by the type I subtype of IGF receptor, which is structurally similar to the insulin receptor and exhibits significant cross-reactivity with insulin. As a first step in our long-range goal of defining those features of the IGF-I and IGF-II molecules that confer enhanced mitogenic activity and reactivity with these mitogenic type I IGF receptors, we have prepared two hybrid insulin-IGF molecules and examined their mitogenic and binding activities: (1) A27-insulin, containing an elongated 27-residue A-chain (in which the 6-residue D-domain of IGF-II was added to the carboxy-terminus of the 21-residue A-chain of insulin) combined with the B-chain of insulin; and (2) A insulin-B IGF-1, containing the A-chain of insulin and the synthetic 30-residue B-domain of IGF-I. Both hybrid molecules stimulated DNA synthesis and inhibited 125I-IGF-I binding to type I IGF receptors in both chick embryo and human fibroblast cultures. A27-insulin had considerably greater mitogenic potency and binding potency than A insulin-B IGF-I. Neither hybrid molecule was more potent in these assays than insulin, indicating that the presence of D IGF-II or B IGF-I by itself was not sufficient to increase the mitogenic potency of insulin in fibroblasts. By contrast, A insulin-B IGF-I showed enhanced reactivity with an antiserum to IGF-I. A27-insulin retained significant insulin-like metabolic activity despite the presence of the D-domain of IGF-II.
Asunto(s)
Factor II del Crecimiento Similar a la Insulina/farmacología , Factor I del Crecimiento Similar a la Insulina/farmacología , Insulina/farmacología , Mitógenos/farmacología , Receptores de Superficie Celular/efectos de los fármacos , Somatomedinas/farmacología , Animales , Células Cultivadas , Embrión de Pollo , Fibroblastos/efectos de los fármacos , Humanos , Radioinmunoensayo , Receptores de Superficie Celular/metabolismo , Receptores de Somatomedina , Piel/citologíaRESUMEN
Functional surfaces of a protein are often mapped by combination of X-ray crystallography and mutagenesis. Such studies of insulin have yielded paradoxical results, suggesting that the native state is inactive and reorganizes on receptor binding. Of particular interest is the N-terminal alpha-helix of the A-chain. Does this segment function as an alpha-helix or reorganize as recently proposed in a prohormone-convertase complex? To correlate structure and function, we describe a mapping strategy based on protein design. The solution structure of an engineered monomer ([AspB10, LysB28, ProB29]-human insulin) is determined at neutral pH as a template for synthesis of a novel A-chain analogue. Designed by analogy to a protein-folding intermediate, the analogue lacks the A6-A11 disulphide bridge; the cysteine residues are replaced by serine. Its solution structure is remarkable for segmental unfolding of the N-terminal A-chain alpha-helix (A1 to A8) in an otherwise native subdomain. The structure demonstrates that the overall orientation of the A and B chains is consistent with reorganization of the A-chain's N-terminal segment. Nevertheless, the analogue's low biological activity suggests that this segment, a site of clinical mutation causing diabetes mellitus, functions as a preformed recognition alpha-helix.
Asunto(s)
Insulina/química , Secuencia de Aminoácidos , Humanos , Insulina/análogos & derivados , Espectroscopía de Resonancia Magnética , Datos de Secuencia Molecular , Estructura Molecular , Pliegue de Proteína , Soluciones , Relación Estructura-ActividadRESUMEN
Protein minimization highlights essential determinants of structure and function. Minimal models of proinsulin and insulin-like growth factor I contain homologous A and B domains as single-chain analogues. Such models (designated mini-proinsulin and mini-IGF-I) have attracted wide interest due to their native foldability but complete absence of biological activity. The crystal structure of mini-proinsulin, determined as a T3R3 hexamer, is similar to that of the native insulin hexamer. Here, we describe the solution structure of a monomeric mini-proinsulin under physiologic conditions and compare this structure to that of the corresponding two-chain analogue. The two proteins each contain substitutions in the B-chain (HisB10-->Asp and ProB28-->Asp) designed to destabilize self-association by electrostatic repulsion; the proteins differ by the presence or absence of a peptide bond between LysB29 and GlyA1. The structures are essentially identical, resembling in each case the T-state crystallographic protomer. Differences are observed near the site of cross-linking: the adjoining A1-A8 alpha-helix (variable among crystal structures) is less well-ordered in mini-proinsulin than in the two-chain variant. The single-chain analogue is not completely inactive: its affinity for the insulin receptor is 1500-fold lower than that of the two-chain analogue. Moreover, at saturating concentrations mini-proinsulin retains the ability to stimulate lipogenesis in adipocytes (native biological potency). These results suggest that a change in the conformation of insulin, as tethered by the B29-A1 peptide bond, optimizes affinity but is not integral to the mechanism of transmembrane signaling. Surprisingly, the tertiary structure of mini-proinsulin differs from that of mini-IGF-I (main-chain rms deviation 4.5 A) despite strict conservation of non-polar residues in their respective hydrophobic cores (side-chain rms deviation 4.9 A). Three-dimensional profile scores suggest that the two structures each provide acceptable templates for threading of insulin-like sequences. Mini-proinsulin and mini-IGF-I thus provide examples of homologous protein sequences encoding non-homologous structures.
Asunto(s)
Factor I del Crecimiento Similar a la Insulina/química , Proinsulina/química , Secuencia de Aminoácidos , Animales , Humanos , Insulina/metabolismo , Factor I del Crecimiento Similar a la Insulina/metabolismo , Espectroscopía de Resonancia Magnética , Datos de Secuencia Molecular , Proinsulina/metabolismo , Unión Proteica , Conformación Proteica , Pliegue de Proteína , Ratas , Soluciones , Electricidad EstáticaAsunto(s)
Insulina , Secuencia de Aminoácidos , Animales , Fenómenos Químicos , Química , Humanos , Técnicas In Vitro , OvinosRESUMEN
The synthesis and isolation in purified form of [Leu9-B]insulin, a biologically active analogue of human insulin, is described. This analogue differs from the parent molecule in that the polar residue, serine, occupying position 9 in the B chain and located on the outside of the insulin molecule, has been replaced with the hydrophobic leucine residue. For the synthesis of this analogue the [Leu9]B chain of human insulin was chemically synthesized by the fragment condensation approach and isolated in the S-sulfonated form. Combination of this compound with the sulfhydryl form of human A chain afforded [Leu9-B]insulin. Separation of this analogue from the combination mixture and isolation as the hydrochloride in purified form were accomplished by chromatography on a carboxymethylcellulose column with an acetate buffer (pH 3.3) and an exponential sodium chloride gradient. [Leu9-B]Insulin possesses a potency of 13-14 IU/mg when assayed by the mouse convulsion method and 11-12 IU/mg by the radioimmunoassay method as compared to 23-25 IU/mg possessed by the natural hormone.
Asunto(s)
Insulina/análogos & derivados , Aminoácidos/análisis , Animales , Bioensayo , Humanos , Insulina/síntesis química , Leucina , Ratones , Péptidos CíclicosRESUMEN
The sythesis and isolation in purified form of an analog of insulin with the interchain disulfide bridges elongated by a methylene group is described. This analog differs from the parent molecule in that the cystein residues occupying positions A-7 and A-20 and involved in the formation of the two interchain disulfide bridges of insulin have been replaced by homocysteine residues. For the synthesis of this compound the Hcy-7, 20-A chain of sheep insulin was chemically synthesized and isolated in the S-sulfonated form. Conversion of the latter product to the sulfhydryl derivative and combination with the S-sulfonated form of the B chain of sheep insulin yielded the [Hcy-7, 20-A] insulin. Isolation of the analog from the combination mixture was effected by chromatography on a carboxymethylcellulose column with acetate buffer (pH 3.3) and an exponential sodium chloride gradient. This analog, by the mouse convulsion assay methods and in doses at least 40-fold higher than those normally used for insulin assay, was inactive. By the radioimmunoassay method this synthetic analog was found to possess a potency of 2 i.u./mg. It is concluded that the biological activity of insulin depends critically on a particular geometry conferred on the molecule by the proper placement of the A and B chains.
Asunto(s)
Insulina/análogos & derivados , Aminoácidos/análisis , Animales , Bioensayo , Bovinos , Cromatografía en Gel , Disulfuros , Electroforesis , Insulina/síntesis química , Ratones , Radioinmunoensayo , Convulsiones , OvinosRESUMEN
Two analogues of human insulin, des(tetrapeptide B(1-4))-and des (pentapeptide B(1-5))-insulin, which differ from the parent molecule in that the N-terminal tetrapeptide and pentapeptide sequences, respectively, have been eliminated, have been synthesized. The des(tetrapeptide B(1-4))-insulin shows potencies of 13 IU/mg by the mouse convulsion assay method and 7.6 IU/mg by the radioimmunoassay method. The des(pentapeptide B(1-5))-insulin possesses a potency of 1.2 IU/mg when assayed by the glucose-oxidation method in isolated fat cells and 3.7 IU/mg by the radioimmunoassay technique. The natural hormone has a potency of 23--25 IU/mg by both assay methods.
Asunto(s)
Insulina/análogos & derivados , Tejido Adiposo/efectos de los fármacos , Tejido Adiposo/metabolismo , Secuencia de Aminoácidos , Aminoácidos/análogos & derivados , Aminoácidos/análisis , Animales , Bioensayo , Glucosa/metabolismo , Humanos , Insulina/farmacología , Ratones , Oligopéptidos , Radioinmunoensayo , Especificidad de la EspecieRESUMEN
The synthesis and isolation in purified form of two analogues of insulin is described. [21-Isoasparagine-A] ([Iasn21-A]) insulin differs from the parent molecule in that the amino acid residue, asparagine, found at the C terminus of the A chain (A21) has been replaced by isoasparagine. [Sar1, Iasn21-A] insulin differs from insulin in that both the A1 and A21 amino acid residues, glycine and asparagine, have been substituted by sarcosine and isoasparagine, respectively. The synthesis of these analogues followed the pattern employed in this laboratory for the synthesis of insulin and its analogues. The S-sulfonated derivatives of the A chain analogues were chemically synthesized, converted to their sulfhydryl forms, and then combined with the S-sulfonated B chain to produce the respective insulin analogues. Isolation of the insulin analogues from the combination mixtures was effected by chromatography on a carboxymethylcellulose column with an exponential sodium chloride gradient. By the mouse convulsion assay method [Iasn21-A]insulin possessed a potency of 21 IU/mg and [Sar1, Iasn21-A] insulin 15 IU/mg. The radioimmunoassay method gave values of 16 IU/mg for the former and 7IU/mg for the altter analogue. The natural hormone has a potency of 23-25 IU/mg by both assay methods. These data indicate that the alpha- and beta-carboxyl groups of the A21 amino acid residue are nearly equivalent in terms of their contribution to the expression of the biological activity of insulin. Furthermore, these data strengthen the speculation (Cosmatos, A., Johnson, S., Breier, B., and Katsoyannis, P. G. (1975), J. Chem. Soc. Perkin Trans. 1, 2157) that the change in the relative positive charge at the N-terminal amino acid residue of the A chain is responsible for the considerable decrease in the immunoreactivity observed in such modified insulins.
Asunto(s)
Insulina/análogos & derivados , Secuencia de Aminoácidos , Bioensayo , Fenómenos Químicos , Química , Humanos , Insulina/síntesis química , Insulina/inmunología , Métodos , Rotación ÓpticaRESUMEN
Two analogs of bovine insulin, [des(tetrapeptide B27--30), Tyr(NH2)26-B] and [des(pentapeptide B26--30), Phe(NH2)25-B] insulin, which differ from the parent molecule in that the C-terminal tetrapeptide and pentapeptide sequences, respectively, from the B chain have been eliminated and the newly exposed residues are amidated, have been synthesized. The [des(tetrapeptide B27--30), Tyr(NH2)26-B] insulin shows potencies of 16.8 IU/mg by the mouse convulsion assay method and 10.8 IU/mg by the radioimmunoassay method. The [des(pentapeptide B26--30), Phe(NH2)25-B] insulin possesses a potency of 10.5 IU/mg when assayed by the mouse convulsion method and 14 IU/mg by the radioimmunoassay technique. The potencies of these analogs are higher than the potencies of the respective non-amidated derivatives (Katsoyannis et al., 1973, 1974). It is speculated that the gradual decline of biological activity observed as amino acid residues are eliminated from the C-terminal region of the B chain of insulin is due to the proximity of a hydrophilic carboxyl group to the hydrophobic core of the protein molecule.
Asunto(s)
Insulina/análogos & derivados , Péptidos/síntesis química , Secuencia de Aminoácidos , Animales , Bioensayo , Bovinos , Cromatografía por Intercambio Iónico , Electroforesis , Insulina/análisis , Insulina/síntesis química , Focalización Isoeléctrica , Ratones , Músculos/análisis , Péptidos/análisis , Relación Estructura-ActividadRESUMEN
We report the synthesis and biological evaluation of a two-chain, disulfide-linked, insulin-like compound consisting of the B-chain of bovine insulin and an A-chain corresponding to the A- and D- domains of human insulin-like growth factor-I (IGF-I) in which the A-domain amino-acid residues -Phe49-Arg50-Ser51-found in IGF-I have been replaced by -Ala-Gly-Val-, the homologous region of sheep insulin. The compound is indistinguishable from a previously reported compound whose A-chain corresponds to the A- and D-domains of IGF-I without the substitution, in assays for insulin-like activity as well as in assays for growth-promoting activity. We conclude that these A-domain residues do not contribute significantly to the interaction of IGF-I with either insulin or IGF-I receptors.
Asunto(s)
Insulina/síntesis química , Secuencia de Aminoácidos , Animales , Bovinos , Humanos , Factor I del Crecimiento Similar a la Insulina , Datos de Secuencia Molecular , Fragmentos de Péptidos/síntesis química , Ratas , OvinosRESUMEN
As part of our aim to study the conformation of insulin in solution by time-resolved fluorescence spectroscopy, we have synthesized the analogue [19-Tryptophan-A]insulin. In this compound, the tyrosine residue at position 19 of the A-chain of insulin, one of the most strongly conserved residues in insulins from various species, is substituted with the strongly fluorescent tryptophan residue. [19-Tryptophan-A]insulin displays 4.1 +/- 1.9% of the potency of natural insulin in binding to the insulin receptor from rat liver plasma membranes, 5.0 +/- 2.3% in stimulating lipogenesis in rat adipocytes, and 75.7 +/- 4% of the potency of insulin in radioimmunoassay. In connection with our previous work, these data indicate that an aromatic side chain at position A19 of insulin seems necessary but not sufficient for high biological activity. We further conclude that in regard to the immunogenic determinants of insulin, tryptophan in position A19 is an essentially neutral substitution for tyrosine in that position, in sharp contrast to the situation with regard to biological activity.
Asunto(s)
Insulina/análogos & derivados , Tejido Adiposo/metabolismo , Aminoácidos/análisis , Animales , Fluorescencia , Cobayas , Insulina/análisis , Insulina/síntesis química , Insulina/metabolismo , Lípidos/biosíntesis , Hígado/ultraestructura , Proteínas de la Membrana , Radioinmunoensayo , Ratas , Receptor de Insulina/metabolismo , OvinosRESUMEN
An insulin-like hybrid molecule consisting of the A chain of insulin and a B chain corresponding to the B domain of human insulin-like growth factor I (growth factor I sequence 1-30) has been synthesized essentially by the procedures developed in this laboratory for the synthesis of insulin and analogues. The hybrid competed with 125I-insulin for insulin receptors in rat liver plasma membranes and was a full agonist in stimulating incorporation of [3(-3)H]glucose into lipids in rat adipocytes. In both assays, the compound displayed ca. 2% of the potency of insulin. The compound was recognized by anti-insulin antibodies but was only ca. 0.25% as potent as insulin in this activity. The hybrid exhibited growth-promoting activity in fibroblasts, displaying 3-8% of the activity of insulin. In contrast, the compound was recognized by insulin-like growth factor carrier proteins, a property not associated with insulin. Two points of nonhomology between the B chain of insulin and the B domain of insulin-like growth factor I are considered in connection with these observations.
Asunto(s)
Factor I del Crecimiento Similar a la Insulina/síntesis química , Insulina/síntesis química , Multimerización de Proteína , Somatomedinas/síntesis química , Tejido Adiposo/efectos de los fármacos , Tejido Adiposo/metabolismo , Secuencia de Aminoácidos , Aminoácidos/análisis , Animales , Bovinos , Membrana Celular/metabolismo , Humanos , Insulina/farmacología , Lípidos/biosíntesis , Hígado/metabolismo , Sustancias Macromoleculares , Fragmentos de Péptidos , Ratas , Receptor de Insulina/metabolismoRESUMEN
We report the synthesis and biological evaluation of five insulin analogues in which one or both of the B-chain tyrosine residues have been substituted. [B16 Phe]insulin and [B16 Trp]insulin display a very modest reduction in potency (c. 65%) relative to porcine insulin; [B26 Phe]insulin is less active (30-50%), and the doubly substituted [B16 Phe, B26 Phe]insulin displays still lower potency (c. 35%). The further substitution of Asp for B10 His in [B16 Phe, B26 Phe]insulin raises its activity to approximately twofold greater than natural insulin, an increase of approximately fivefold over the parent compound. We conclude that the bulk and/or aromaticity of the amino acid residue at position B16, but not its hydrogen-bonding capacity, contributes to the biological activity of the hormone. We further conclude that hydrogen-bonding capacity or special side-chain packing characteristics are required at the B26 position for insulin to display high biological activity.
Asunto(s)
Insulina/análogos & derivados , Insulina/química , Tirosina , Secuencia de Aminoácidos , Bioensayo , Humanos , Insulina/síntesis química , Insulina/farmacología , Conformación ProteicaRESUMEN
We have synthesized an insulin-like compound, consisting of the B-chain of bovine insulin and an A-chain corresponding to the A-domain of human insulin-like growth factor-I (IGF-I), in which the isoleucine residue normally present in position 2 of the A-domain of IGF-I has been replaced with glycine. Biological evaluation of the compound indicated that its insulin-like activity (insulin receptor-binding and stimulation of lipogenesis) was 0.2%, and its growth-factor activity (stimulation of thymidine incorporation) was less than 1%, both relative to natural insulin. We conclude that interactions between IleA2 and TyrA19, which are crucial to high biological activity in insulin, are also present in IGF-I, and are equally critical for its biological activity.