RESUMEN
Insulin regulates glucose homeostasis via binding and activation of the insulin receptor dimer at two distinct pairs of binding sites 1 and 2. Here, we present cryo-EM studies of full-length human insulin receptor (hIR) in an active state obtained at non-saturating, physiologically relevant insulin conditions. Insulin binds asymmetrically to the receptor under these conditions, occupying up to three of the four possible binding sites. Deletion analysis of the receptor together with site specific peptides and insulin analogs used in binding studies show that both sites 1 and 2 are required for high insulin affinity. We identify a homotypic interaction of the fibronectin type III domain (FnIII-3) of IR resulting in tight interaction of membrane proximal domains of the active, asymmetric receptor dimer. Our results show how insulin binding at two distinct types of sites disrupts the autoinhibited apo-IR dimer and stabilizes the active dimer. We propose an insulin binding and activation mechanism, which is sequential, exhibits negative cooperativity, and is based on asymmetry at physiological insulin concentrations with one to three insulin molecules activating IR.
Asunto(s)
Antígenos CD , Insulina , Receptor de Insulina , Antígenos CD/química , Antígenos CD/metabolismo , Sitios de Unión , Microscopía por Crioelectrón , Humanos , Insulina/metabolismo , Unión Proteica , Dominios Proteicos , Multimerización de Proteína , Receptor de Insulina/química , Receptor de Insulina/metabolismo , Transducción de SeñalRESUMEN
The potentially fatal cardiovascular effects of hypoglycaemia are not well understood and large animal models of the counter-regulatory responses and cardiovascular consequences of insulin-induced hypoglycaemia are needed to understand the mechanisms in humans. The aim of this study was to develop a human-like minipig model of hypoglycaemia including healthy and diabetic pigs to investigate endocrine, electrocardiographic and platelet effects. Hypoglycaemia was induced using a hyperinsulinaemic, hypoglycaemic clamp and an insulin bolus protocol. Plasma glucose, glucagon, C-peptide, insulin, epinephrine and platelet aggregation responses were measured before, during and after hypoglycaemia. Continuous electrocardiographic recordings were obtained. Hypoglycaemia at a plasma glucose concentration of 0.8-1.0 mM in the clamp induced 25-fold increase in epinephrine and sixfold and threefold increase in glucagon for healthy and diabetic pigs, respectively. The hypoglycaemic clamp induced QTc-interval prolongation and increase in cardiac arrhythmias. In the bolus approach, the non-diabetic group reached plasma glucose target of 1.5 mM and QTc-interval was prolonged after insulin injection, but before glucose nadir. The diabetic group did not reach hypoglycaemic target, but still demonstrated QTc-interval prolongation. These results demonstrate effects of hyperinsulinaemic hypoglycaemia closely resembling human physiology, indicating the minipig as a translational animal model of counter-regulatory endocrine and myocardial effects of hypoglycaemia.
Asunto(s)
Hiperinsulinismo Congénito/complicaciones , Hiperinsulinismo Congénito/veterinaria , Cardiopatías/diagnóstico , Cardiopatías/etiología , Enfermedades de los Porcinos/sangre , Animales , Biomarcadores/sangre , Glucemia , Plaquetas/metabolismo , Plaquetas/ultraestructura , Manejo de la Enfermedad , Modelos Animales de Enfermedad , Susceptibilidad a Enfermedades , Electrocardiografía , Sistema Endocrino/metabolismo , Agregación Plaquetaria , Pruebas de Función Plaquetaria , Porcinos , Enfermedades de los Porcinos/diagnóstico , Porcinos Enanos , Evaluación de SíntomasRESUMEN
Insulin analogue X10 has a higher mitogenic potency than native human insulin in vitro and supra-pharmacological doses of insulin X10 increased the incidence of mammary tumours in rats. Compared to native human insulin, insulin X10 has increased binding affinity to the insulin receptor and the IGF-1 receptor, but it is not known whether either or both characteristics are important for stimulation of cell proliferation in vivo. The aim of this study was to explore how increased binding affinity to the insulin receptor or the IGF-1 receptor contributes to stimulation of cell proliferation in vivo. A mouse xenograft model was established with rat L6 myoblast cells transfected with the human insulin receptor (L6hIR cells) and effects of supra-pharmacological doses of native human insulin, insulin X10 or novel insulin analogues with increased binding affinity to either the insulin receptor or the IGF-1 receptor were examined. Treatment with insulin X10 and insulin analogues with increased binding affinity to either the insulin receptor or the IGF-1 receptor increased growth of L6hIR cell xenografts significantly compared to native human insulin. Thus, increased binding affinity to the insulin receptor and the IGF-1 receptor are each independently linked to increased growth of L6hIR cell xenografts in vivo.
Asunto(s)
Receptor IGF Tipo 1/metabolismo , Receptor de Insulina/metabolismo , Animales , Xenoinjertos , Humanos , Insulina/metabolismo , Ratones , Unión Proteica , RatasRESUMEN
Insulin and insulin-like growth factor-1 stimulate specific responses in arteries, which may be disrupted by diet-induced obesity. We examined (1) temporal effects of high-fat diet compared to low-fat diet in mice on insulin receptor, insulin-like growth factor-1 receptor, insulin receptor/insulin-like growth factor-1 receptor hybrid receptor expression and insulin/insulin-like growth factor-1-mediated Akt phosphorylation in aorta; and (2) effects of high-fat diet on insulin and insulin-like growth factor-1-mediated Akt phosphorylation and vascular tone in resistance arteries. Medium-term high-fat diet (5 weeks) decreased insulin-like growth factor-1 receptor expression and increased hybrid expression (~30%) only. After long-term (16 weeks) high-fat diet, insulin receptor expression was reduced by ~30%, insulin-like growth factor-1 receptor expression decreased a further ~40% and hybrid expression increased a further ~60%. Independent correlates of hybrid receptor expression were high-fat diet, duration of high-fat diet and plasma insulin-like growth factor-1 (all p < 0.05). In aorta, insulin was a more potent activator of Akt than insulin-like growth factor-1, whereas in resistance arteries, insulin-like growth factor-1 was more potent than insulin. High-fat diet blunted insulin-mediated vasorelaxation ( p < 0.01) but had no effect on insulin-like growth factor-1-mediated vasorelaxation in resistance arteries. Our findings support the possibility that hybrid receptor level is influenced by nutritional and metabolic cues. Moreover, vessel-dependent effects of insulin and insulin-like growth factor-1 on vascular tone and Akt activation may have implications in treating obesity-related vascular disease.
Asunto(s)
Aorta/efectos de los fármacos , Insulina/farmacología , Arterias Mesentéricas/efectos de los fármacos , Obesidad/enzimología , Proteínas Proto-Oncogénicas c-akt/metabolismo , Receptor IGF Tipo 1/metabolismo , Resistencia Vascular/efectos de los fármacos , Animales , Antígenos CD/metabolismo , Aorta/enzimología , Células Cultivadas , Dieta con Restricción de Grasas , Dieta Alta en Grasa , Modelos Animales de Enfermedad , Activación Enzimática , Células Endoteliales de la Vena Umbilical Humana/efectos de los fármacos , Células Endoteliales de la Vena Umbilical Humana/enzimología , Humanos , Factor I del Crecimiento Similar a la Insulina/farmacología , Masculino , Arterias Mesentéricas/enzimología , Arterias Mesentéricas/fisiopatología , Ratones Endogámicos C57BL , Obesidad/sangre , Obesidad/fisiopatología , Fosforilación , Receptor IGF Tipo 1/genética , Receptor de Insulina/metabolismo , Receptores de Somatomedina/metabolismo , Transducción de Señal/efectos de los fármacos , Vasodilatación/efectos de los fármacosRESUMEN
Translation across species of immunoassay results is often challenging due to the lack of cross-species reactivity of antibodies. In order to investigate the biology of insulin and IGF1 receptors, we generated new versatile monoclonal assay antibodies using the extracellular domain of the insulin/IGF1 hybrid receptor as the bait protein in the Adimab yeast antibody discovery platform and as the antigen in a rabbit monoclonal antibody platform. The resulting antibody clones were screened for receptor specificity as well as cross-species reactivity to both tissue and cell line derived samples. Using these strategies, we were able to identify highly specific insulin receptor monoclonal antibodies that lack cross-reactivity to the IGF1 receptor using the Adimab platform and a highly specific IGF1 receptor monoclonal antibody that lacks cross-reactivity to the insulin receptor using the rabbit antibody platform. Unlike earlier monoclonal antibodies reported in the literature, these antibodies show cross-species reactivity to the extracellular domains of mouse, rat, pig, and human receptors, indicating that they bind conserved epitopes. Furthermore, the antibodies work well in several different assay formats, including ELISA, flow cytometry, and immunoprecipitation, and therefore provide new tools to study insulin and IGF1 receptor biology with translation across several species and experimental model systems.
Asunto(s)
Anticuerpos Monoclonales/inmunología , Receptor IGF Tipo 1/inmunología , Receptor de Insulina/inmunología , Animales , Anticuerpos Monoclonales/química , Reacciones Cruzadas , Células HCT116 , Humanos , Ratones , Conejos , Ratas , Especificidad de la Especie , PorcinosRESUMEN
AIMS/HYPOTHESIS: Recent studies with normal rats and mouse allograft models have reported that insulin and insulin analogues do not activate the IGF-1 receptor in vivo, and that this characteristic therefore cannot be responsible for the increased incidence of mammary tumours observed for the insulin analogue X10 in chronic toxicity studies with Sprague Dawley rats. This is in clear contrast to reports of insulin and insulin analogues in vitro. Clarification of this is important for understanding the mechanisms behind possible growth-promoting effects of insulin analogues, and will have implications for the development of novel insulin analogues. METHODS: We established a xenograft model in BALB/c nude mice with the human colon cancer cell line COLO-205, which expresses human insulin and IGF-1 receptors, and explored the acute and chronic effects of treatment with supra-pharmacological doses of human insulin, insulin analogue X10 and human IGF-1. With a novel antibody, acute IGF-1 receptor activation was also examined in various tissues from normal rats treated with human insulin, insulin analogue X10 or human IGF-1. Finally, the effects of pharmacologically relevant doses of human insulin and insulin analogue X10 on receptor activation and growth of COLO-205 xenograft were explored in BALB/c nude mice with alloxan-induced hyperglycaemia. RESULTS: In normal rats and in BALB/c nude mice bearing a COLO-205 cell xenograft, treatment with supra-pharmacological doses of human insulin, insulin analogue X10 or human IGF-1 resulted in activation of insulin receptors as well as IGF-1 receptors. Treatment of diabetic nude mice with pharmacologically relevant doses of human insulin or insulin analogue X10, which decreased blood glucose from hyperglycaemic levels to the normoglycaemic range, did not increase IGF-1 receptor activation. Furthermore, repeated treatment with supra-pharmacological as well as pharmacological doses of human insulin or insulin analogue X10 did not influence the growth of COLO-205 xenografts. CONCLUSIONS/INTERPRETATION: This study demonstrates that activation of IGF-1 receptors in cancer cells by insulin and insulin analogues cannot be considered as a purely in vitro phenomenon. It does occur in vivo in animal models, although only after treatment with supra-pharmacological doses. Furthermore, treatment with insulin or insulin analogue X10 did not influence the growth of COLO-205 xenografts under normo- or hypoglycaemic conditions. Further studies are needed before a conclusion can be reached on whether IGF-1 receptor activation by insulin analogues correlates with increased growth in vivo.
Asunto(s)
Hipoglucemia/tratamiento farmacológico , Hipoglucemia/metabolismo , Receptor IGF Tipo 1/metabolismo , Receptor de Insulina/metabolismo , Aloxano/toxicidad , Animales , Línea Celular Tumoral , Neoplasias del Colon/metabolismo , Humanos , Hipoglucemia/inducido químicamente , Hipoglucemiantes/uso terapéutico , Insulina/uso terapéutico , Ratones , Ratones Endogámicos BALB C , Ratones Desnudos , Trasplante HeterólogoRESUMEN
METHODS: Alanine scan of insulin receptor (IR)-B exon 11 and site-directed mutagenesis of amino acid 718 in human IR-A and IR-B were performed. Ligand affinities to wild type and mutated receptors were studied by displacement of radioactive insulin in binding assay on secreted soluble midi receptors or solubilized semi-purified full length receptors stably expressed in Baby Hamster Kidney cells. Phosphorylation of IR in response to insulin, IGF1 and IGF2 was measured using ELISA. RESULTS: Insulin, insulin detemir and insulin glargine maximally showed two fold differences in affinity for human IR-A and IR-B, but IGF1 and IGF2 had up to 10 fold preference for IR-A. Alanine scan of exon 11 revealed that position 718 is important for low IGF1 affinity to IR-B. Mutational analysis of amino acid residue 718 in IR-A and IR-B demonstrated that charge is important for IGF1 and IGF2 affinity but not important for insulin affinity. The affinity of IGF1 and IGF2 for the mutant IR-A P718K was comparable to the wild type IR-B whereas the affinity of IGF1 and IGF2 for the mutant IR-B K718P was comparable to the wild type IR-A. Changes in affinity were also reflected in the IR activation pattern. CONCLUSION: Mutating position 718 in human IR-B to the proline found at position 718 in human IR-A increased IGF1 and IGF2 affinity to a level comparable to IR-A and mutating position 718 in IR-A to the lysine found at position 718 in IR-B decreased IGF1 and IGF2 affinity to a level comparable to IR-B, whereas a negatively charged glutamate did not. These changes in the affinities were also reflected in the IR phosphorylation pattern, meaning that position 718 is important for both affinity and activation of the receptor. It should be emphasized that none of the mutations affected insulin affinity, indicating that the mutations did not alter the overall receptor structure and that the effect is ligand specific.
Asunto(s)
Aminoácidos/metabolismo , Factor II del Crecimiento Similar a la Insulina/metabolismo , Factor I del Crecimiento Similar a la Insulina/metabolismo , Isoformas de Proteínas/metabolismo , Receptor de Insulina/metabolismo , Aminoácidos/química , Animales , Línea Celular , Cricetinae , Humanos , Receptor de Insulina/químicaRESUMEN
This novel method enables specific measurement of the activation of hybrid receptors formed between the Insulin Receptor (IR) and the Insulin-like Growth Factor 1 Receptor (IGF1R). These hybrid receptors are present in tissues and cell lines expressing both IR and IGF1R. It is therefore challenging to separate the homodimer and hybrid receptor activation properties. This ELISA method enabled fast and quantitative measurements of activated hybrid receptors. The hybrid receptor specificity is obtained from a combination of two specific antibodies for IGF1R and for an IR tyrosine phosphorylation site. The specificity was shown by immunoprecipitations and Western blot analysis. IR exists as two splice variants; consequently, two splice variants of hybrid receptors can be expressed. It is reported here that both splice variants of insulin/IGF1 receptor hybrids are activated by IGF1 with >20-fold higher potency than insulin.
Asunto(s)
Factor I del Crecimiento Similar a la Insulina , Receptor de Insulina , Receptores de Somatomedina , Animales , Cricetinae , Ensayo de Inmunoadsorción Enzimática/métodos , Células Hep G2 , Humanos , Factor I del Crecimiento Similar a la Insulina/genética , Factor I del Crecimiento Similar a la Insulina/metabolismo , Receptor IGF Tipo 1 , Receptor de Insulina/genética , Receptor de Insulina/metabolismo , Receptores de Somatomedina/genética , Receptores de Somatomedina/metabolismo , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/metabolismoRESUMEN
AIMS/HYPOTHESIS: There is controversy with respect to molecular characteristics of insulin analogues. We report a series of experiments forming a comprehensive characterisation of the long acting insulin analogues, glargine and detemir, in comparison with human insulin, IGF-1, and the super-mitogenic insulin, X10. METHODS: We measured binding of ligands to membrane-bound and solubilised receptors, receptor activation and mitogenicity in a number of cell types. RESULTS: Detemir and glargine each displayed a balanced affinity for insulin receptor (IR) isoforms A and B. This was also true for X10, whereas IGF-1 had a higher affinity for IR-A than IR-B. X10 and glargine both exhibited a higher relative IGF-1R than IR binding affinity, whereas detemir displayed an IGF-1R:IR binding ratio of ≤ 1. Ligands with high relative IGF-1R affinity also had high affinity for IR/IGF-1R hybrid receptors. In general, the relative binding affinities of the analogues were reflected in their ability to phosphorylate the IR and IGF-1R. Detailed analysis revealed that X10, in contrast to the other ligands, seemed to evoke a preferential phosphorylation of juxtamembrane and kinase domain phosphorylation sites of the IR. Sustained phosphorylation was only observed from the IR after stimulation with X10, and after stimulation with IGF-1 from the IGF-1R. Both X10 and glargine showed an increased mitogenic potency compared to human insulin in cells expressing many IGF-1Rs, whereas only X10 showed increased mitogenicity in cells expressing many IRs. CONCLUSIONS: Detailed analysis of receptor binding, activation and in vitro mitogenicity indicated no molecular safety concern with detemir.
Asunto(s)
Hipoglucemiantes/farmacocinética , Insulina de Acción Prolongada/farmacocinética , Insulina Regular Humana/farmacocinética , Factor I del Crecimiento Similar a la Insulina/análogos & derivados , Receptor IGF Tipo 1/metabolismo , Células Cultivadas , Femenino , Humanos , Hipoglucemiantes/farmacología , Insulina Detemir , Insulina Glargina , Insulina de Acción Prolongada/farmacología , Insulina Regular Humana/farmacología , Factor I del Crecimiento Similar a la Insulina/farmacocinética , Factor I del Crecimiento Similar a la Insulina/farmacología , Mitosis/efectos de los fármacos , Fosforilación/efectos de los fármacos , Unión Proteica , Receptor IGF Tipo 1/genéticaRESUMEN
The insulin receptor (IR) belongs to the receptor tyrosine kinase super family and plays an important role in glucose homeostasis. The receptor interacts with several large docking proteins that mediate signaling from the receptor, including the insulin receptor substrate (IRS) family and Src homology-2-containing proteins (Src). Here, we applied the bioluminescence resonance energy transfer 2 (BRET2) technique to study the IR signaling pathways. The interaction between the IR and the substrates IRS1, IRS4 and Shc was examined in response to ligands with different signaling properties. The association between IR and the interacting partners could successfully be monitored when co-expressing green fluorescent protein 2 (GFP2) tagged substrates with Renilla reniformis luciferase 8 (Rluc8) tagged IR. Through additional optimization steps, we developed a stable and flexible BRET2 assay for monitoring the interactions between the IR and its substrates. Furthermore, the insulin analogue X10 was characterized in the BRET2 assay and was found to be 10 times more potent with respect to IRS1, IRS4 and Shc recruitment compared to human insulin. This study demonstrates that the BRET2 technique can be applied to study IR signaling pathways, and that this assay can be used as a platform for screening and characterization of IR ligands.
Asunto(s)
Proteínas Fluorescentes Verdes/análisis , Proteínas Sustrato del Receptor de Insulina/metabolismo , Insulina/farmacología , Mediciones Luminiscentes , Receptor de Insulina/metabolismo , Proteínas Adaptadoras de la Señalización Shc/metabolismo , Células Cultivadas , Humanos , Insulina/análogos & derivados , Riñón/citología , Riñón/efectos de los fármacos , Riñón/metabolismo , Unión Proteica , Dominios y Motivos de Interacción de Proteínas/efectos de los fármacos , Proteínas Recombinantes de Fusión/metabolismo , Proteína Transformadora 1 que Contiene Dominios de Homología 2 de SrcRESUMEN
In this publication we describe a peptide insulin receptor antagonist, S661, which is a single chain peptide of 43 amino acids. The affinity of S661 for the insulin receptor is comparable to that of insulin and the selectivity for the insulin receptor versus the IGF-1 receptor is higher than that of insulin itself. S661 is also an antagonist of the insulin receptor of other species such as pig and rat, and it also has considerable affinity for hybrid insulin/IGF-1 receptors. S661 completely inhibits insulin action, both in cellular assays and in vivo in rats. A biosynthetic version called S961 which is identical to S661 except for being a C-terminal acid seems to have properties indistinguishable from those of S661. These antagonists provide a useful research tool for unraveling biochemical mechanisms involving the insulin receptor and could form the basis for treatment of hypoglycemic conditions.
Asunto(s)
Antagonistas de Insulina/farmacología , Péptidos/farmacología , Receptor de Insulina/antagonistas & inhibidores , Secuencia de Aminoácidos , Animales , Humanos , Insulina/metabolismo , Insulina/farmacología , Antagonistas de Insulina/química , Antagonistas de Insulina/metabolismo , Datos de Secuencia Molecular , Péptidos/química , Péptidos/metabolismo , Ratas , Ratas Zucker , Receptor de Insulina/metabolismoRESUMEN
OBJECTIVE: To determine whether different albumins have an effect on IGF-I binding assays. METHODS: We have studied the effect of five different albumins in plate antibody capture binding assay. For IGF-IR studies the IGF-IR specific antibody 24-31 was used and for IR/IGF-IR hybrid receptors the IR specific antibody 83-7 was used. Binding to IGF-IR was studied by displacement of (125)I-IGF-I with IGF-I in the absence or presence of 0.1%, 0.5% or 1% (w/v) albumin. The IR/IGF-IR hybrid receptors were studied in the presence of 0.5% (w/v) of HSA A-1887 and BSA A-7888 and with IGF-I or insulin displacement of (125)I-IGF-I. The albumins used were purchased from Sigma-Aldrich. Two batches of albumins from each catalog number were tested. The albumins were: HSA A-1887, BSA A-4503, BSA A-6003, BSA A-7030, and BSA A-7888. Contaminants in the albumins were characterized as proteins with IGF-I binding properties by cross-linking to (125)I-IGF-I and SDS-page analysis. RESULTS: BSA A-4503, A-7030 and A-7888 from Sigma-Aldrich contain proteins with IGF-I binding properties. These contaminants increased the determined EC50 for displacement of (125)I-IGF-I from IGF-IR up to 40-fold in a BSA dependent manner. The presence of BSA-7888 in binding experiments increased the determined EC50 for IR/IGF-IR hybrid receptors 8-16-fold. CONCLUSIONS: When IGF-I is characterized with respect to the effect on living cells and on binding to potential receptors unspecific binding to surfaces is often prevented by the addition of albumin in the assay. Here we report that when binding to the classical IGF-IR and IR/IGF-IR hybrid receptors are studied the measured EC50 values can be albumin dependent if it is contaminated with proteins with IGF-I binding properties. The free IGF-I concentration will be lower than estimated. Thus, the contaminated BSA preparations result in artifacts leading to misinterpretations and underestimation of the effect of IGF-I. Our results provide one possible explanation as to why different laboratories report different EC50 values for IGF-I.
Asunto(s)
Contaminación de Medicamentos , Factor I del Crecimiento Similar a la Insulina/metabolismo , Receptor IGF Tipo 1/metabolismo , Albúmina Sérica Bovina/farmacología , Animales , Unión Competitiva/efectos de los fármacos , Proteínas Portadoras/metabolismo , Células Cultivadas , Cricetinae , Humanos , Radioisótopos de Yodo/análisis , Radioisótopos de Yodo/metabolismo , Concentración Osmolar , Unión Proteica/efectos de los fármacos , Ensayo de Unión Radioligante/métodos , Albúmina Sérica Bovina/químicaRESUMEN
Insulin receptor (IR) and insulin-like growth factor I receptor (IGF-IR) are both from the same subgroup of receptor tyrosine kinases that exist as covalently bound receptor dimers at the cell surface. For both IR and IGF-IR, the most described forms are homodimer receptors. However, hybrid receptors consisting of one-half IR and one-half IGF-IR are also present at the cell surface. Two splice variants of IR are expressed that enable formation of two isoforms of the IGF-IR/IR hybrid receptor. In this study, these two splice variants of hybrid receptors were studied with respect to binding affinities of insulin, insulin-like growth factor I (IGF-I), and insulin-like growth factor II (IGF-II). Unlike previously published data, in which semipurified receptors have been studied, we found that the two hybrid receptor splice variants had similar binding characteristics with respect to insulin, IGF-I, and IGF-II binding. We studied both semipurified and purified hybrid receptors. In all cases we found that IGF-I had at least 50-fold higher affinity than insulin, irrespective of the splice variant. The binding characteristics of insulin and IGF-I to both splice variants of the hybrid receptors were similar to classical homodimer IGF-IR.
Asunto(s)
Empalme Alternativo , Factor I del Crecimiento Similar a la Insulina/metabolismo , Insulina/metabolismo , Isoformas de Proteínas/metabolismo , Receptor IGF Tipo 1/metabolismo , Receptor de Insulina/metabolismo , Proteínas Recombinantes de Fusión/metabolismo , Animales , Línea Celular , Cricetinae , Exones , Humanos , Unión Proteica , Isoformas de Proteínas/genética , Receptor IGF Tipo 1/genética , Receptor IGF Tipo 1/aislamiento & purificación , Receptor de Insulina/genética , Receptor de Insulina/aislamiento & purificación , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/aislamiento & purificaciónRESUMEN
The human UV-damaged DNA-binding protein Ddb1 associates with cullin 4 ubiquitin ligases implicated in nucleotide excision repair (NER). These complexes also contain the signalosome (CSN), but NER-relevant ubiquitination targets have not yet been identified. We report that fission yeast Ddb1, Cullin 4 (Pcu4), and CSN subunits Csn1 and Csn2 are required for degradation of the ribonucleotide reductase (RNR) inhibitor protein Spd1. Ddb1-deficient cells have >20-fold increased spontaneous mutation rate. This is partly dependent on the error-prone translesion DNA polymerases. Spd1 deletion substantially reduced the mutation rate, suggesting that insufficient RNR activity accounts for approximately 50% of observed mutations. Epistasis analysis indicated that Ddb1 contributed to mutation avoidance and tolerance to DNA damage in a pathway distinct from NER. Finally, we show that Ddb1/Csn1/Cullin 4-mediated Spd1 degradation becomes essential when cells differentiate into meiosis. These results suggest that Ddb1, along with Cullin 4 and the signalosome, constitute a major pathway controlling genome stability, repair, and differentiation via RNR regulation.
Asunto(s)
Proteínas de Unión al ADN/metabolismo , Inestabilidad Genómica/fisiología , Meiosis/fisiología , Proteínas de Schizosaccharomyces pombe/metabolismo , Schizosaccharomyces/metabolismo , Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/metabolismo , Proteínas Cullin/metabolismo , Proteínas de Unión al ADN/genética , Proteínas Fúngicas/genética , Proteínas Fúngicas/metabolismo , Proteína 2 Homóloga a MutS , Mutación , Schizosaccharomyces/genética , Proteínas de Schizosaccharomyces pombe/genéticaRESUMEN
To prepare reagents for a study of the interactions of prolactin (PRL) and growth hormone (GH) receptors (Rs) with suppressor of cytokine signaling (SOCS) proteins in living cells by fluorescence resonance energy transfer methodology, the respective proteins were tagged with cyan (CFP) or yellow (YFP) fluorescent protein. Constructs encoding ovine (o)PRLR-YFP, oPRLR-CFP, oGHR-YFP, and oGHR-CFP tagged downstream of the receptor DNA were prepared in the plasmid pcDNA plasmid and tested for biological activity in HEK 293T cells transiently cotransfected with those constructs and the reporter gene encoding luciferase. All four constructs were biologically active and as potent as their untagged counterparts. Cells transfected with those proteins exhibited fluorescence in the cytoplasm and the membrane. Constructs encoding DNA tagged with YFP or CFP upstream of SOCS1, SOCS2, SOCS3, and SOCS6 were prepared in pECFP-C1 and pEYFP-C1 plasmids. The biological activities of SOCS1 and SOCS3 tagged at their amino termini were assayed by their ability to inhibit placental lactogen (PL)- or GH-induced activation of JAK2/STAT5-mediated luciferase transcription in HEK 293T cells; the activity of SOCS2 was assayed by its ability to abolish SOCS1-induced inhibition. The tagged proteins exhibited biological activity that was equal to or even more potent than their untagged counterparts. The biological activities of CFP-SOCS2 and YFP-SOCS2 were also assayed using GST-GHR binding assay. Their interaction with the cytosolic domain of GHR was equivalent to their respective untagged counterparts. The biological activity of the construct encoding SOCS6 was not tested because of lack of a suitable assay. Cells transfected with eight of these tagged constructs expressed the fluorescent proteins in both the nucleus and cytosol; the tagged SOCS2 was localized mostly in the latter compartment.