RESUMEN
Impaired functioning of pancreatic beta cells is a key hallmark of type 2 diabetes. beta cell function is modulated by the actions of different classes of heterotrimeric G proteins. The functional consequences of activating specific beta cell G protein signaling pathways in vivo are not well understood at present, primarily due to the fact that beta cell G protein-coupled receptors (GPCRs) are also expressed by many other tissues. To circumvent these difficulties, we developed a chemical-genetic approach that allows for the conditional and selective activation of specific beta cell G proteins in intact animals. Specifically, we created two lines of transgenic mice each of which expressed a specific designer GPCR in beta cells only. Importantly, the two designer receptors differed in their G protein-coupling properties (G(q/11) versus G(s)). They were unable to bind endogenous ligand(s), but could be efficiently activated by an otherwise pharmacologically inert compound (clozapine-N-oxide), leading to the conditional activation of either beta cell G(q/11) or G(s) G proteins. Here we report the findings that conditional and selective activation of beta cell G(q/11) signaling in vivo leads to striking increases in both first- and second-phase insulin release, greatly improved glucose tolerance in obese, insulin-resistant mice, and elevated beta cell mass, associated with pathway-specific alterations in islet gene expression levels. Selective stimulation of beta cell G(s) triggered qualitatively similar in vivo metabolic effects. Thus, this developed chemical-genetic strategy represents a powerful approach to study G protein regulation of beta cell function in vivo.
Asunto(s)
Diabetes Mellitus Tipo 2/metabolismo , Proteínas de Unión al GTP/metabolismo , Regulación de la Expresión Génica/fisiología , Células Secretoras de Insulina/metabolismo , Islotes Pancreáticos/anatomía & histología , Receptores Acoplados a Proteínas G/metabolismo , Transducción de Señal/fisiología , Animales , Células COS , Chlorocebus aethiops , Clozapina/análogos & derivados , Clozapina/farmacología , Femenino , Regulación de la Expresión Génica/efectos de los fármacos , Prueba de Tolerancia a la Glucosa , Insulina/metabolismo , Secreción de Insulina , Islotes Pancreáticos/metabolismo , Ratones , Ratones Transgénicos , Ensayo de Unión Radioligante , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Transducción de Señal/efectos de los fármacosRESUMEN
Examining the behavioral consequences of selective CNS neuronal activation is a powerful tool for elucidating mammalian brain function in health and disease. Newly developed genetic, pharmacological, and optical tools allow activation of neurons with exquisite spatiotemporal resolution; however, the inaccessibility to light of widely distributed neuronal populations and the invasiveness required for activation by light or infused ligands limit the utility of these methods. To overcome these barriers, we created transgenic mice expressing an evolved G protein-coupled receptor (hM3Dq) selectively activated by the pharmacologically inert, orally bioavailable drug clozapine-N-oxide (CNO). Here, we expressed hM3Dq in forebrain principal neurons. Local field potential and single-neuron recordings revealed that peripheral administration of CNO activated hippocampal neurons selectively in hM3Dq-expressing mice. Behavioral correlates of neuronal activation included increased locomotion, stereotypy, and limbic seizures. These results demonstrate a powerful chemical-genetic tool for remotely controlling the activity of discrete populations of neurons in vivo.
Asunto(s)
Evolución Molecular , Subunidades alfa de la Proteína de Unión al GTP Gq-G11/metabolismo , Expresión Génica/genética , Neuronas/fisiología , Receptores Acoplados a Proteínas G/metabolismo , Animales , Conducta Animal/efectos de los fármacos , Encéfalo/citología , Encéfalo/metabolismo , Clozapina/análogos & derivados , Clozapina/farmacología , Relación Dosis-Respuesta a Droga , Doxiciclina/farmacología , Potenciales Evocados/efectos de los fármacos , Potenciales Evocados/genética , Subunidades alfa de la Proteína de Unión al GTP Gq-G11/genética , Hipocampo/citología , Humanos , Técnicas In Vitro , Locomoción/efectos de los fármacos , Locomoción/genética , Potenciales de la Membrana/efectos de los fármacos , Potenciales de la Membrana/genética , Ratones , Ratones Transgénicos , Neuronas/efectos de los fármacos , Técnicas de Placa-Clamp/métodos , Receptores Acoplados a Proteínas G/genética , Conducta Estereotipada/efectos de los fármacos , Conducta Estereotipada/fisiología , Factores de TiempoRESUMEN
The M4 muscarinic acetylcholine (ACh) receptor (mAChR) is a potential therapeutic target but characterized by a lack of subtype-selective ligands. We recently generated "designer receptors exclusively activated by a designer drug" (DREADDs), which contained mutations of two conserved orthosteric-site residues (Y113C/A203G in the M4 mAChR) that caused a loss of ACh activity but a gain in responsiveness to clozapine-N-oxide (CNO). The current study characterized the interactions of the wild type and the M4 DREADD with a range of agonists, antagonists, and the recently discovered M4 mAChR allosteric potentiator, 3-amino-5-chloro-6-methoxy-4-methyl-thieno[2,3-b]pyridine-2-carboxylic acid cyclopropylamide (LY2033298). LY2033298 displayed positive binding cooperativity with ACh, neutral cooperativity with the antagonist, [3H]quinuclidinyl benzilate, and agonism for activation of phosphorylated extracellular signal-regulated kinase (ERK) 1/2 at the wild-type M4 mAChR. LY2033298's cooperativity with clozapine or CNO was weakly positive with respect to binding but profoundly negative with respect to LY2033298 signaling. Although the DREADD mutations increased the binding and function of clozapine-like compounds, all other agonists lost the ability to activate the mutant; for the orthosteric agonists ACh and pilocarpine, this was due partly to a reduced affinity, whereas the affinity of LY2033298 or the atypical agonist 4-I-[3-chlorophenyl]carbamoyloxy)-2-butynyltrimethylammnonium chloride was unaltered. The interaction between LY2033298 and clozapine-like compounds reverted to neutral cooperativity on the DREADD, whereas LY2033298 caused a striking functional rescue of ACh potency and efficacy at the DREADD. These results provide conclusive evidence for the retention of a functional allosteric site on the M4 DREADD and highlight a role for residues Tyr113 and Ala203 in the transmission of cooperativity.
Asunto(s)
Ácidos Nicotínicos/metabolismo , Ácidos Nicotínicos/farmacología , Receptor Muscarínico M4/fisiología , Tiofenos/metabolismo , Tiofenos/farmacología , Acetilcolina/química , Acetilcolina/metabolismo , Acetilcolina/farmacología , Regulación Alostérica/fisiología , Sitio Alostérico/fisiología , Animales , Células CHO , Clozapina/análogos & derivados , Clozapina/química , Clozapina/metabolismo , Clozapina/farmacología , Cricetinae , Cricetulus , Relación Dosis-Respuesta a Droga , Activación Enzimática/efectos de los fármacos , Proteína Quinasa 1 Activada por Mitógenos/metabolismo , Proteína Quinasa 3 Activada por Mitógenos/metabolismo , Modelos Biológicos , Estructura Molecular , Mutación , Ácidos Nicotínicos/química , Ácidos Nicotínicos/genética , Fosforilación/efectos de los fármacos , Quinuclidinil Bencilato/metabolismo , Quinuclidinil Bencilato/farmacología , Ensayo de Unión Radioligante , Receptor Muscarínico M4/agonistas , Receptor Muscarínico M4/antagonistas & inhibidores , Transducción de Señal , Tiofenos/químicaRESUMEN
The mammalian protein POT1 binds to telomeric single-stranded DNA (ssDNA), protecting chromosome ends from being detected as sites of DNA damage. POT1 is composed of an N-terminal ssDNA-binding domain and a C-terminal protein interaction domain. With regard to the latter, POT1 heterodimerizes with the protein TPP1 to foster binding to telomeric ssDNA in vitro and binds the telomeric double-stranded-DNA-binding protein TRF2. We sought to determine which of these functions-ssDNA, TPP1, or TRF2 binding-was required to protect chromosome ends from being detected as DNA damage. Using separation-of-function POT1 mutants deficient in one of these three activities, we found that binding to TRF2 is dispensable for protecting telomeres but fosters robust loading of POT1 onto telomeric chromatin. Furthermore, we found that the telomeric ssDNA-binding activity and binding to TPP1 are required in cis for POT1 to protect telomeres. Mechanistically, binding of POT1 to telomeric ssDNA and association with TPP1 inhibit the localization of RPA, which can function as a DNA damage sensor, to telomeres.
Asunto(s)
Proteínas de Unión a Telómeros/metabolismo , Telómero/metabolismo , Línea Celular , Daño del ADN , ADN de Cadena Simple/genética , ADN de Cadena Simple/metabolismo , Humanos , Modelos Biológicos , Proteínas Mutantes/metabolismo , Mutación/genética , Unión Proteica , Transporte de Proteínas , Proteína de Replicación A/metabolismo , Complejo Shelterina , Telómero/genética , Proteína 2 de Unión a Repeticiones Teloméricas/metabolismoRESUMEN
Lipid-derived inositol phosphates (IPs) are a complex group of second messengers generated by the sequential phosphorylation of inositol 1,4,5-trisphosphate (IP(3)). Synthetic pathways leading from IP(3) to the formation of inositol tetrakisphosphate IP(4), inositol pentakisphosphate IP(5), inositol hexakisphosphate IP(6), and inositol pyrophosphates PP-IPs have been elucidated in eukaryotes from yeast to human. Studies have attributed a variety of cellular functions to IPs, highlighting the importance of understanding how the pathways for their synthesis are regulated. This chapter summarizes experimental techniques for the biochemical characterization of the key inositol phosphate kinases IPKs necessary for producing the diverse array of IP species.
Asunto(s)
Inositol 1,4,5-Trifosfato/metabolismo , Fosfatos de Inositol/metabolismo , Fosfotransferasas/metabolismo , Arabidopsis/enzimología , Proteínas de Arabidopsis/genética , Proteínas de Arabidopsis/metabolismo , Cromatografía Líquida de Alta Presión/métodos , Cromatografía en Capa Delgada/métodos , Inositol 1,4,5-Trifosfato/química , Inositol 1,4,5-Trifosfato/genética , Espectroscopía de Resonancia Magnética/métodos , Fosforilación , Fosfotransferasas/aislamiento & purificación , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo , Especificidad por SustratoRESUMEN
We evolved muscarinic receptors in yeast to generate a family of G protein-coupled receptors (GPCRs) that are activated solely by a pharmacologically inert drug-like and bioavailable compound (clozapine-N-oxide). Subsequent screening in human cell lines facilitated the creation of a family of muscarinic acetylcholine GPCRs suitable for in vitro and in situ studies. We subsequently created lines of telomerase-immortalized human pulmonary artery smooth muscle cells stably expressing all five family members and found that each one faithfully recapitulated the signaling phenotype of the parent receptor. We also expressed a G(i)-coupled designer receptor in hippocampal neurons (hM(4)D) and demonstrated its ability to induce membrane hyperpolarization and neuronal silencing. We have thus devised a facile approach for designing families of GPCRs with engineered ligand specificities. Such reverse-engineered GPCRs will prove to be powerful tools for selectively modulating signal-transduction pathways in vitro and in vivo.
Asunto(s)
Evolución Molecular , Modelos Moleculares , Receptores Acoplados a Proteínas G/metabolismo , Animales , Línea Celular Transformada , Clozapina/análogos & derivados , Clozapina/farmacología , Drogas de Diseño , Epítopos , Canales de Potasio Rectificados Internamente Asociados a la Proteína G/metabolismo , Hipocampo/efectos de los fármacos , Humanos , Hidrólisis/efectos de los fármacos , Ligandos , Proteínas Mutantes/metabolismo , Miocitos del Músculo Liso/efectos de los fármacos , Neuronas/efectos de los fármacos , Fosfatidilinositoles/metabolismo , Ingeniería de Proteínas , Arteria Pulmonar/citología , Arteria Pulmonar/efectos de los fármacos , Ratas , Receptor Muscarínico M3/metabolismo , Receptor Muscarínico M4/metabolismo , Saccharomyces cerevisiaeRESUMEN
The identification of protein function based on biological information is an area of intense research. Here we consider a complementary technique that quantitatively groups and relates proteins based on the chemical similarity of their ligands. We began with 65,000 ligands annotated into sets for hundreds of drug targets. The similarity score between each set was calculated using ligand topology. A statistical model was developed to rank the significance of the resulting similarity scores, which are expressed as a minimum spanning tree to map the sets together. Although these maps are connected solely by chemical similarity, biologically sensible clusters nevertheless emerged. Links among unexpected targets also emerged, among them that methadone, emetine and loperamide (Imodium) may antagonize muscarinic M3, alpha2 adrenergic and neurokinin NK2 receptors, respectively. These predictions were subsequently confirmed experimentally. Relating receptors by ligand chemistry organizes biology to reveal unexpected relationships that may be assayed using the ligands themselves.
Asunto(s)
Sistemas de Liberación de Medicamentos/métodos , Ligandos , Preparaciones Farmacéuticas/química , Mapeo de Interacción de Proteínas/métodos , Proteínas/química , Alineación de Secuencia/métodos , Análisis de Secuencia de Proteína/métodos , Sitios de Unión , Bases de Datos de Proteínas , Diseño de Fármacos , Unión ProteicaRESUMEN
Activation of phospholipase C-dependent inositol polyphosphate signaling pathways generates distinct messengers derived from inositol 1,4,5-trisphosphate that control gene expression and mRNA export. Here we report the regulation of telomere length by production of a diphosphorylinositol tetrakisphosphate, PP-IP4, synthesized by the KCS1 gene product. Loss of PP-IP4 production results in lengthening of telomeres, whereas overproduction leads to their shortening. This effect requires the presence of Tel1, the yeast homologue of ATM, the protein mutated in the human disease ataxia telangiectasia. Our data provide in vivo evidence of a regulatory link between inositol polyphosphate signaling and the checkpoint kinase family and describe a third nuclear process modulated by phospholipase C activation.
Asunto(s)
Fosfatos de Inositol/metabolismo , Proteínas de Saccharomyces cerevisiae/fisiología , Transducción de Señal , Telómero/ultraestructura , Proteínas de la Ataxia Telangiectasia Mutada , Transporte Biológico , Southern Blotting , Proteínas de Ciclo Celular/metabolismo , Proteínas de Unión al ADN/metabolismo , Activación Enzimática , Regulación de la Expresión Génica , Prueba de Complementación Genética , Hidrólisis , Modelos Biológicos , Mutación , Sistemas de Lectura Abierta , Fosforilación , Fosfotransferasas (Aceptor del Grupo Fosfato) , Plásmidos/metabolismo , Proteínas Serina-Treonina Quinasas/metabolismo , ARN Mensajero/metabolismo , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/biosíntesis , Factores de Tiempo , Proteínas Supresoras de Tumor/metabolismo , Fosfolipasas de Tipo C/química , Fosfolipasas de Tipo C/metabolismoRESUMEN
The protein hPot1 shares homology with telomere-binding proteins in lower eukaryotes and associates with single-stranded telomeric DNA in vitro as well as colocalizing with telomere-binding proteins in vivo. We now show that hPot1 is coimmunoprecipitated with telomeric DNA and that stable expression of this protein in telomerase-positive cells results in telomere elongation, supporting the idea that hPot1 is a bona fide mammalian telomere-binding protein. We previously found that mutations in the N-terminal DAT domain of the hTERT catalytic subunit of telomerase rendered the enzyme catalytically active but unable to elongate telomeres in vivo. This phenotype could be partially rescued by fusion with the double-stranded telomeric protein hTRF2. Given that hPot1 binds to single-stranded DNA in vitro (at the same site that hTERT binds to in vivo), we addressed whether fusion of hPot1 can rescue the DAT mutations more efficiently than that of hTRF2. We now report that a DAT mutant of hTERT is indeed efficiently rescued upon fusion to hPot1. However, this rescue depended on the ability of hPot1 to localize to telomeres rather than binding to DNA per se. These data support a model whereby the DAT domain of hTERT is implicated in telomere-telomerase associations.
Asunto(s)
Mutación , Telomerasa/genética , Telomerasa/metabolismo , Proteínas de Unión a Telómeros/metabolismo , Telómero/metabolismo , Secuencia de Aminoácidos , Animales , Línea Celular , ADN/metabolismo , Proteínas de Unión al ADN , Humanos , Datos de Secuencia Molecular , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/metabolismo , Alineación de Secuencia , Complejo Shelterina , Proteínas de Unión a Telómeros/genéticaRESUMEN
PURPOSE: The most effective therapy for metastatic prostate cancer is androgen deprivation. Genes activated directly or possibly even indirectly by this steroid hormone represent potential targets for anticancer therapy. One such gene may be hTERT, which encodes the catalytic subunit of telomerase. In prostate cancer cells telomerase is activated, permitting sustained proliferation. Therefore, we tested whether hTERT gene expression is modulated in prostate cancer cells in vitro and in vivo by androgens. MATERIALS AND METHODS: Transcriptional activation of hTERT during androgen stimulation was assayed by luciferase assays using the hTERT promoter fused to the luciferase gene and by reverse transcriptase-polymerase chain reaction to detect endogenous hTERT mRNA in LNCaP cells. hTERT mRNA levels and telomerase activity were also measured in CWR22 prostate cancer cells implanted in mice that were subsequently castrated and left untreated or administered androgen. RESULTS: We report that the endogenous hTERT promoter is activated during the administration of androgen to androgen sensitive LNCaP prostate cancer cells. However, this effect was indirect since an hTERT promoter construct was not activated by androgens and transcription of the endogenous gene was not stimulated early enough in cultured cells to be considered a direct target of this steroid hormone. Importantly in an in vivo model of human prostate cancer androgen deprivation led to a decrease in hTERT expression, followed by a decrease in telomerase activity, which was reversed by a single administration of androgen. CONCLUSIONS: The hTERT gene is regulated in human prostate cancer cells in vivo by androgens.
Asunto(s)
Andrógenos/fisiología , Regulación Neoplásica de la Expresión Génica , Neoplasias de la Próstata/metabolismo , Telomerasa/metabolismo , Andrógenos/farmacología , Animales , Proteínas de Unión al ADN , Genes Reporteros , Células HeLa , Humanos , Técnicas In Vitro , Luciferasas/genética , Masculino , Ratones , Ratones Desnudos , Trasplante de Neoplasias , Neoplasias Hormono-Dependientes/genética , Neoplasias Hormono-Dependientes/metabolismo , Orquiectomía , Regiones Promotoras Genéticas , Neoplasias de la Próstata/genética , ARN Mensajero/análisis , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Telomerasa/genética , Activación Transcripcional , Células Tumorales Cultivadas , Regulación hacia ArribaRESUMEN
Telomerase, the enzyme that elongates telomeres, is essential to maintain telomere length and to immortalize most cancer cells. However, little is known about the regulation of this enzyme in higher eukaryotes. We previously described a domain in the hTERT telomerase catalytic subunit that is essential for telomere elongation and cell immortalization in vivo but dispensable for catalytic activity in vitro. Here, we show that fusions of hTERT containing different mutations in this domain to the telomere binding protein hTRF2 redirected the mutated hTERT to telomeres and rescued its in vivo functions. We suggest that this domain posttranscriptionally regulates telomerase function by targeting the enzyme to telomeres.
Asunto(s)
Dominio Catalítico , Telomerasa/metabolismo , Telómero/metabolismo , Línea Celular Transformada , Células Cultivadas , ADN/metabolismo , Proteínas de Unión al ADN , Humanos , Mutación , Estructura Terciaria de Proteína/fisiología , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/metabolismo , Telomerasa/genética , Proteína 2 de Unión a Repeticiones Teloméricas/genética , Proteína 2 de Unión a Repeticiones Teloméricas/metabolismoRESUMEN
Telomerase is the enzyme essential to complete the replication of the terminal DNA of most eukaryotic chromosomes. In humans, this enzyme is composed of the telomerase reverse transcriptase (hTERT) and telomerase RNA (hTR) subunits. hTR has been found in the nucleolus, a site of assembly of ribosomes as well as other ribonucleoproteins (RNPs). We therefore tested whether the hTERT component is also found in the nucleolus, where it could complex with the hTR RNA to form a functional enzyme. We report here that hTERT does indeed localize to the nucleolus, and we mapped the domain responsible for this localization to the hTR-binding region of the protein by deletion analysis. Substitution mutations in two of the three conserved hTR-binding domains in this nucleolar localization domain (NoLD) abolished nucleolar localization. However, another mutation that impeded hTR binding did not alter this subcellular localization. Additionally, wild type hTERT was detected in the nucleolus of cells that failed to express hTR. Taken together, we propose that the nucleolar localization of hTERT involves more than just the association with the hTR subunit. Furthermore, the coincidental targeting of both the hTR and hTERT subunits to the nucleolus supports the premise that the assembly of telomerase occurs in the nucleolus.