RESUMEN
In contrast to most rhodopsin-like G protein-coupled receptors, the glycoprotein hormone receptors (GPHR) have a large extracellular N-terminus for hormone binding. The hormones do not directly activate the transmembrane domain but mediate their action via a, thus, far only partially known Tethered Agonistic LIgand (TALI). The existence of such an intramolecular agonist was initially indicated by site-directed mutation studies and activating peptides derived from the extracellular hinge region. It is still unknown precisely how TALI is involved in intramolecular signal transmission. We combined systematic mutagenesis studies at the luteinizing hormone receptor and the thyroid-stimulating hormone receptor (TSHR), stimulation with a drug-like agonist (E2) of the TSHR, and structural homology modeling to unravel the functional and structural properties defining the TALI region. Here, we report that TALI (a) is predisposed to constitutively activate GPHR, (b) can by itself rearrange GPHR into a fully active conformation, (c) stabilizes active GPHR conformation, and (d) is not involved in activation of the TSHR by E2. In the active state conformation, TALI forms specific interactions between the N-terminus and the transmembrane domain. We show that stabilization of an active state is dependent on TALI, including activation by hormones and constitutively activating mutations.
Asunto(s)
Glicoproteínas/metabolismo , Hormonas/metabolismo , Glicoproteínas/genética , Células HEK293 , Hormonas/genética , Humanos , Ligandos , Proteínas de la Membrana/genética , Proteínas de la Membrana/metabolismo , Mutagénesis/genética , Mutagénesis Sitio-Dirigida/métodos , Mutación/genética , Péptidos/genética , Péptidos/metabolismo , Unión Proteica/genética , Dominios Proteicos/genética , Dominios Proteicos/fisiología , Receptores de Tirotropina/genética , Receptores de Tirotropina/metabolismo , Transducción de Señal/genéticaRESUMEN
The thyrotropin (TSH) receptor (TSHR) signals via G proteins of all four classes and ß-arrestin 1. Stimulation of TSHR leads to increasing cAMP production that has been reported as a monotonic dose-response curve that plateaus at high TSH doses. In HEK 293 cells overexpressing TSHRs (HEK-TSHR cells), we found that TSHR activation exhibits an "inverted U-shaped dose-response curve" with increasing cAMP production at low doses of TSH and decreased cAMP production at high doses (>1 mU/ml). Since protein kinase A inhibition by H-89 and knockdown of ß-arrestin 1 or ß-arrestin 2 did not affect the decreased cAMP production at high TSH doses, we studied the roles of TSHR downregulation and of Gi/Go proteins. A high TSH dose (100 mU/ml) caused a 33% decrease in cell-surface TSHR. However, because inhibiting TSHR downregulation with combined expression of a dominant negative dynamin 1 and ß-arrestin 2 knockdown had no effect, we concluded that downregulation is not involved in the biphasic cAMP response. Pertussis toxin, which inhibits activation of Gi/Go, abolished the biphasic response with no statistically significant difference in cAMP levels at 1 and 100 mU/ml TSH. Concordantly, co-knockdown of Gi/Go proteins increased cAMP levels stimulated by 100 mU/ml TSH from 55% to 73% of the peak level. These data show that biphasic regulation of cAMP production is mediated by Gs and Gi/Go at low and high TSH doses, respectively, which may represent a mechanism to prevent overstimulation in TSHR-expressing cells. SIGNIFICANCE STATEMENT: We demonstrate biphasic regulation of TSH-mediated cAMP production involving coupling of the TSH receptor (TSHR) to Gs at low TSH doses and to Gi/o at high TSH doses. We suggest that this biphasic cAMP response allows the TSHR to mediate responses at lower levels of TSH and that decreased cAMP production at high doses may represent a mechanism to prevent overstimulation of TSHR-expressing cells. This mechanism could prevent chronic stimulation of thyroid gland function.
Asunto(s)
AMP Cíclico/metabolismo , Subunidades alfa de la Proteína de Unión al GTP Gi-Go/metabolismo , Receptores de Tirotropina/metabolismo , Transducción de Señal/efectos de los fármacos , Tirotropina/administración & dosificación , Relación Dosis-Respuesta a Droga , Regulación hacia Abajo , Dinamina I/genética , Dinamina I/metabolismo , Subunidades alfa de la Proteína de Unión al GTP Gi-Go/antagonistas & inhibidores , Subunidades alfa de la Proteína de Unión al GTP Gi-Go/genética , Técnicas de Silenciamiento del Gen , Células HEK293 , Humanos , Toxina del Pertussis/administración & dosificación , Receptores de Tirotropina/genética , Transducción de Señal/genética , Arrestina beta 2/genética , Arrestina beta 2/metabolismoRESUMEN
In this review, we summarize the evidence against direct stimulation of insulin-like growth factor 1 receptors (IGF1Rs) by autoantibodies in Graves' orbitopathy (GO) pathogenesis. We describe a model of thyroid-stimulating hormone (TSH) receptor (TSHR)/IGF1R crosstalk and present evidence that observations indicating IGF1R's role in GO could be explained by this mechanism. We evaluate the evidence for and against IGF1R as a direct target of stimulating IGF1R antibodies (IGF1RAbs) and conclude that GO pathogenesis does not involve directly stimulating IGF1RAbs. We further conclude that the preponderance of evidence supports TSHR as the direct and only target of stimulating autoantibodies in GO and maintain that the TSHR should remain a major target for further development of a medical therapy for GO in concert with drugs that target TSHR/IGF1R crosstalk.
Asunto(s)
Oftalmopatía de Graves/patología , Receptor IGF Tipo 1/inmunología , Receptores de Tirotropina/metabolismo , Autoanticuerpos/inmunología , Oftalmopatía de Graves/inmunología , Humanos , Ácido Hialurónico/metabolismo , Receptor Cross-Talk/inmunología , Receptor IGF Tipo 1/metabolismo , Receptores de Somatomedina , Receptores de Tirotropina/inmunologíaRESUMEN
The phosphoinositide kinase PIP5K6 has recently been identified as a target for the mitogen-activated protein kinase (MAPK) MPK6. Phosphorylation of PIP5K6 inhibited the production of phosphatidylinositol 4,5-bisphosphate (PtdIns(4,5)P2 ), impacting membrane trafficking and cell expansion in pollen tubes. Here, we analyzed whether MPK6 regulated PIP5K6 in vegetative Arabidopsis cells in response to the pathogen-associated molecular pattern (PAMP) flg22. Promoter-ß-glucuronidase analyses and quantitative real-time reverse transcription polymerase chain reaction data show PIP5K6 expressed throughout Arabidopsis tissues. Upon flg22 treatment of transgenic protoplasts, the PIP5K6 protein was phosphorylated, and this modification was reduced for a PIP5K6 variant lacking MPK6-targeted residues, or in protoplasts from mpk6 mutants. Upon flg22 treatment of Arabidopsis plants, phosphoinositide levels mildly decreased and a fluorescent reporter for PtdIns(4,5)P2 displayed reduced plasma membrane association, contrasting with phosphoinositide increases reported for abiotic stress responses. Flg22 treatment and chemical induction of the upstream MAPK kinase, MKK5, decreased phosphatidylinositol 4-phosphate 5-kinase activity in mesophyll protoplasts, indicating that the flg22-activated MAPK cascade limited PtdIns(4,5)P2 production. PIP5K6 expression or PIP5K6 protein abundance changed only marginally upon flg22 treatment, consistent with post-translational control of PIP5K6 activity. PtdIns(4,5)P2 -dependent endocytosis of FM 4-64, PIN2 and the NADPH-oxidase RbohD were reduced upon flg22 treatment or MKK5 induction. Reduced RbohD-endocytosis was correlated with enhanced ROS production. We conclude that MPK6-mediated phosphorylation of PIP5K6 limits the production of a functional PtdIns(4,5)P2 pool upon PAMP perception.
Asunto(s)
Proteínas de Arabidopsis/metabolismo , Arabidopsis/metabolismo , Sistema de Señalización de MAP Quinasas/efectos de los fármacos , Moléculas de Patrón Molecular Asociado a Patógenos/farmacología , Fosfatidilinositol 4,5-Difosfato/metabolismo , Fosfotransferasas (Aceptor de Grupo Alcohol)/metabolismo , Arabidopsis/genética , Proteínas de Arabidopsis/genética , Relación Dosis-Respuesta a Droga , Flagelina/química , Regulación Enzimológica de la Expresión Génica , Regulación de la Expresión Génica de las Plantas/fisiología , Sistema de Señalización de MAP Quinasas/fisiología , Moléculas de Patrón Molecular Asociado a Patógenos/administración & dosificación , Moléculas de Patrón Molecular Asociado a Patógenos/química , Fosfotransferasas (Aceptor de Grupo Alcohol)/genética , Protoplastos/metabolismoRESUMEN
The thyroid gland secretes primarily tetraiodothyronine (T4), and some triiodothyronine (T3). Under normal physiological circumstances, only one-fifth of circulating T3 is directly released by the thyroid, but in states of hyperactivation of thyroid-stimulating hormone receptors (TSHRs), patients develop a syndrome of relative T3 toxicosis. Thyroidal T4 production results from iodination of thyroglobulin (TG) at residues Tyr5 and Tyr130, whereas thyroidal T3 production may originate in several different ways. In this study, the data demonstrate that within the carboxyl-terminal portion of mouse TG, T3 is formed de novo independently of deiodination from T4 We found that upon iodination in vitro, de novo T3 formation in TG was decreased in mice lacking TSHRs. Conversely, de novo T3 that can be formed upon iodination of TG secreted from PCCL3 (rat thyrocyte) cells was augmented from cells previously exposed to increased TSH, a TSHR agonist, a cAMP analog, or a TSHR-stimulating antibody. We present data suggesting that TSH-stimulated TG phosphorylation contributes to enhanced de novo T3 formation. These effects were reversed within a few days after removal of the hyperstimulating conditions. Indeed, direct exposure of PCCL3 cells to human serum from two patients with Graves' disease, but not control sera, led to secretion of TG with an increased intrinsic ability to form T3 upon in vitro iodination. Furthermore, TG secreted from human thyrocyte cultures hyperstimulated with TSH also showed an increased intrinsic ability to form T3 Our data support the hypothesis that TG processing in the secretory pathway of TSHR-hyperstimulated thyrocytes alters the structure of the iodination substrate in a way that enhances de novo T3 formation, contributing to the relative T3 toxicosis of Graves' disease.
Asunto(s)
Procesamiento Proteico-Postraduccional , Receptores de Tirotropina/agonistas , Transducción de Señal , Tiroglobulina/metabolismo , Células Epiteliales Tiroideas/metabolismo , Tirotropina/metabolismo , Triyodotironina/biosíntesis , Animales , Proteínas de Unión al Calcio/agonistas , Proteínas de Unión al Calcio/genética , Proteínas de Unión al Calcio/metabolismo , Quinasa de la Caseína I/genética , Quinasa de la Caseína I/metabolismo , Línea Celular , Células Cultivadas , Proteínas de la Matriz Extracelular/agonistas , Proteínas de la Matriz Extracelular/genética , Proteínas de la Matriz Extracelular/metabolismo , Enfermedad de Graves/sangre , Enfermedad de Graves/metabolismo , Enfermedad de Graves/patología , Halogenación , Humanos , Ratones Endogámicos C57BL , Ratones Noqueados , Fosforilación , Proteínas Serina-Treonina Quinasas/química , Proteínas Serina-Treonina Quinasas/genética , Proteínas Serina-Treonina Quinasas/metabolismo , Ratas , Receptores de Tirotropina/genética , Receptores de Tirotropina/metabolismo , Células Epiteliales Tiroideas/citología , Células Epiteliales Tiroideas/patología , Tirosina/metabolismo , Regulación hacia ArribaRESUMEN
Recently, we showed that TSH-enhanced differentiation of a human preosteoblast-like cell model involved a ß-arrestin 1 (ß-Arr 1)-mediated pathway. To study this pathway in more detail, we sought to discover a small molecule ligand that was functionally selective toward human TSH receptor (TSHR) activation of ß-Arr 1. High-throughput screening using a cell line stably expressing mutated TSHRs and mutated ß-Arr 1 (DiscoverX1 cells) led to the discovery of agonists that stimulated translocation of ß-Arr 1 to the TSHR, but did not activate Gs-mediated signaling pathways, i.e., cAMP production. D3-ßArr (NCGC00379308) was selected. In DiscoverX1 cells, D3-ßArr stimulated ß-Arr 1 translocation with a 5.1-fold greater efficacy than TSH and therefore potentiated the effect of TSH in stimulating ß-Arr 1 translocation. In human U2OS-TSHR cells expressing wild-type TSHRs, which is a model of human preosteoblast-like cells, TSH upregulated the osteoblast-specific genes osteopontin (OPN) and alkaline phosphatase (ALPL). D3-ßArr alone had only a weak effect to upregulate these bone markers, but D3-ßArr potentiated TSH-induced upregulation of ALPL and OPN mRNA levels 1.6-fold and 5.5-fold, respectively, at the maximum dose of ligands. Furthermore, the positive allosteric modulator effect of D3-ßArr resulted in an increase of TSH-induced secretion of OPN protein. In summary, we have discovered the first small molecule positive allosteric modulator of TSHR. As D3-ßArr potentiates the effect of TSH to enhance differentiation of a human preosteoblast in an in vitro model, it will allow a novel experimental approach for probing the role of TSH-induced ß-Arr 1 signaling in osteoblast differentiation.
Asunto(s)
Diferenciación Celular/efectos de los fármacos , Descubrimiento de Drogas/métodos , Osteoblastos/efectos de los fármacos , Receptores de Tirotropina/agonistas , Tirotropina/farmacología , Regulación Alostérica/efectos de los fármacos , Regulación Alostérica/fisiología , Animales , Células CHO , Diferenciación Celular/fisiología , Línea Celular Tumoral , Células Cultivadas , Cricetinae , Cricetulus , Ensayos Analíticos de Alto Rendimiento/métodos , Humanos , Osteoblastos/fisiología , Receptores de Tirotropina/fisiología , Células Epiteliales Tiroideas/efectos de los fármacos , Células Epiteliales Tiroideas/metabolismo , Tirotropina/análogos & derivadosRESUMEN
BACKGROUND: Increased motor activity or social interactions through enriched environment are strong stimulators of grey and white matter plasticity in the adult rodent brain. In the present study we evaluated whether specific reaching training of the dominant forelimb (RT) and stimulation of unspecific motor activity through enriched environment (EE) influence the generation of distinct oligodendrocyte subpopulations in the sensorimotor cortex and corpus callosum of the adult rat brain. Animals were placed in three different housing conditions: one group was transferred to an EE, a second group received daily RT, whereas a third group remained in the standard cage. Bromodeoxyuridine (BrdU) was applied at days 2-6 after start of experiments and animals were allowed to survive for 10 and 42 days. RESULTS: Enriched environment and daily reaching training of the dominant forelimb significantly increased the number of newly differentiated GSTπ+ oligodendrocytes at day 10 and newly differentiated CNPase+ oligodendrocytes in the sensorimotor cortex at day 42. The myelin level as measured by CNPase expression was increased in the frontal cortex at day 42. Distribution of newly differentiated NG2+ subpopulations changed between 10 and 42 days with an increase of GSTπ+ subtypes and a decrease of NG2+ cells in the sensorimotor cortex and corpus callosum. Analysis of neuronal marker doublecortin (DCX) showed that more than half of NG2+ cells express DCX in the cortex. The number of new DCX+NG2+ cells was reduced by EE at day 10. CONCLUSIONS: Our results indicate for the first time that specific and unspecific motor training conditions differentially alter the process of differentiation from oligodendrocyte subpopulations, in particular NG2+DCX+ cells, in the sensorimotor cortex and corpus callosum.
Asunto(s)
Cuerpo Calloso/fisiología , Vivienda para Animales , Destreza Motora/fisiología , Oligodendroglía/fisiología , Práctica Psicológica , Corteza Sensoriomotora/fisiología , 2',3'-Nucleótido Cíclico Fosfodiesterasas/metabolismo , Animales , Antígenos/metabolismo , Bromodesoxiuridina , Cuerpo Calloso/citología , Proteínas de Dominio Doblecortina , Proteína Doblecortina , Miembro Anterior/fisiología , Lóbulo Frontal/citología , Lóbulo Frontal/fisiología , Masculino , Proteínas Asociadas a Microtúbulos/metabolismo , Modelos Animales , Neurogénesis/fisiología , Neuropéptidos/metabolismo , Oligodendroglía/citología , Proteoglicanos/metabolismo , Distribución Aleatoria , Ratas Wistar , Tiempo de Reacción , Corteza Sensoriomotora/citologíaAsunto(s)
Ciclofosfamida/uso terapéutico , Dexametasona/uso terapéutico , Enfermedad de Graves/tratamiento farmacológico , Inmunosupresores/uso terapéutico , Mixedema/tratamiento farmacológico , Rituximab/uso terapéutico , Enfermedad Aguda , Ciclofosfamida/administración & dosificación , Dexametasona/administración & dosificación , Esquema de Medicación , Quimioterapia Combinada , Enfermedad de Graves/complicaciones , Humanos , Inmunosupresores/administración & dosificación , Masculino , Persona de Mediana Edad , Mixedema/etiología , Rituximab/administración & dosificaciónRESUMEN
Thyrotropin (TSH) activation of the TSH receptor (TSHR), a 7-transmembrane-spanning receptor (7TMR), may have osteoprotective properties by direct effects on bone. TSHR activation by TSH phosphorylates protein kinases AKT1, p38α, and ERK1/2 in some cells. We found TSH-induced phosphorylation of these kinases in 2 cell lines engineered to express TSHRs, human embryonic kidney HEK-TSHR cells and human osteoblastic U2OS-TSHR cells. In U2OS-TSHR cells, TSH up-regulated pAKT1 (7.1±0.5-fold), p38α (2.9±0.4-fold), and pERK1/2 (3.1±0.2-fold), whereas small molecule TSHR agonist C2 had no or little effect on pAKT1 (1.8±0.08-fold), p38α (1.2±0.09-fold), and pERK1/2 (1.6±0.19-fold). Furthermore, TSH increased expression of osteoblast marker genes ALPL (8.2±4.6-fold), RANKL (21±5.9-fold), and osteopontin (OPN; 17±5.3-fold), whereas C2 had little effect (ALPL, 1.7±0.5-fold; RANKL, 1.3±0.6-fold; and OPN, 2.2±0.7-fold). ß-Arrestin-1 and -2 can mediate activatory signals by 7TMRs. TSH stimulated translocation of ß-arrestin-1 and -2 to TSHR, whereas C2 failed to translocate either ß-arrestin. Down-regulation of ß-arrestin-1 by siRNA inhibited TSH-stimulated phosphorylation of ERK1/2, p38α, and AKT1, whereas down-regulation of ß-arrestin-2 increased phosphorylation of AKT1 in both cell types and of ERK1/2 in HEK-TSHR cells. Knockdown of ß-arrestin-1 inhibited TSH-stimulated up-regulation of mRNAs for OPN by 87 ± 1.7% and RANKL by 73 ± 2.4%, and OPN secretion by 74 ± 10%. We conclude that TSH enhances osteoblast differentiation in U2OS cells that is, in part, caused by activatory signals mediated by ß-arrestin-1.
Asunto(s)
Arrestinas/fisiología , Osteoblastos/efectos de los fármacos , Tirotropina/fisiología , Neoplasias Óseas/patología , Línea Celular Tumoral , Regulación Neoplásica de la Expresión Génica , Células HEK293 , Humanos , Proteínas de Neoplasias/fisiología , Osteoblastos/citología , Osteopontina/metabolismo , Osteosarcoma/patología , Fosforilación , Proteínas Quinasas/metabolismo , Procesamiento Proteico-Postraduccional , Interferencia de ARN , ARN Interferente Pequeño/farmacología , Receptores de Tirotropina/fisiología , Proteínas Recombinantes de Fusión , Transducción de Señal/fisiología , Tirotropina/farmacología , beta-Arrestina 1 , Arrestina beta 2 , beta-ArrestinasRESUMEN
To study the network dynamics of the riboflavin biosynthesis pathway and to identify potential bottlenecks in the system, an ordinary differential equation-based model was constructed using available literature data for production strains. The results confirmed that the RibA protein is rate limiting in the pathway. Under the conditions investigated, we determined a potential limiting order of the remaining enzymes under increased RibA concentration (>0.102 mM) and therefore higher riboflavin production (>0.045 mmol g(CDW)(-1) h(-1) and 0.0035 mM s(-1), respectively). The reductase activity of RibG and lumazine synthase (RibH) might be the next most limiting steps. The computational minimization of the enzyme concentrations of the pathway suggested the need for a greater RibH concentration (0.251 mM) compared with the other enzymes (RibG: 0.188 mM, RibB: 0.023 mM).
Asunto(s)
Bacillus subtilis/metabolismo , Modelos Biológicos , Riboflavina/biosíntesis , Simulación por Computador , Cinética , Redes y Vías Metabólicas , Riboflavina/química , Riboflavina/metabolismoRESUMEN
CONTEXT: Thyroid-stimulating hormone (or thyrotropin) receptor (TSHR) could be a selective target for small molecule ligands to treat thyroid cancer (TC). OBJECTIVE: We report a novel, orally efficacious ligand for TSHR that exhibits proliferation inhibitory activity against human TC in vitro and in vivo, and inhibition of metastasis in vivo. METHODS: A35 (NCATS-SM4420; NCGC00241808) was selected from a sublibrary of >200 TSHR ligands. Cell proliferation assays including BrdU incorporation and WST-1, along with molecular docking studies were done. In vivo activity of A35 was assessed in TC cell-derived xenograft (CDX) models with immunocompromised (NSG) mice. Formalin-fixed, paraffin-embedded sections of tumor and lung tissues were observed for the extent of cell death and metastasis. RESULTS: A35 was shown to stimulate cAMP production in some cell types by activating TSHR but not in TC cells, MDA-T32, and MDA-T85. A35 inhibited proliferation of MDA-T32 and MDA-T85 in vitro and in vivo, and pulmonary metastasis of MDA-T85F1 in mice. In vitro, A35 inhibition of proliferation was reduced by a selective TSHR antagonist. Inhibition of CDX tumor growth without decreases in mouse weights and liver function showed A35 to be efficacious without apparent toxicity. Lastly, A35 reduced levels of Ki67 in the tumors and metastatic markers in lung tissues. CONCLUSION: We conclude that A35 is a TSHR-selective inhibitor of TC cell proliferation and metastasis, and suggest that A35 may be a promising lead drug candidate for the treatment of differentiated TC in humans.
Asunto(s)
Proliferación Celular , Receptores de Tirotropina , Neoplasias de la Tiroides , Ensayos Antitumor por Modelo de Xenoinjerto , Animales , Humanos , Ratones , Neoplasias de la Tiroides/tratamiento farmacológico , Neoplasias de la Tiroides/patología , Neoplasias de la Tiroides/metabolismo , Proliferación Celular/efectos de los fármacos , Receptores de Tirotropina/antagonistas & inhibidores , Receptores de Tirotropina/metabolismo , Ligandos , Línea Celular Tumoral , Administración Oral , Antineoplásicos/farmacología , Antineoplásicos/uso terapéutico , Antineoplásicos/administración & dosificación , Metástasis de la Neoplasia , Simulación del Acoplamiento Molecular , Neoplasias Pulmonares/tratamiento farmacológico , Neoplasias Pulmonares/secundario , Neoplasias Pulmonares/patología , Neoplasias Pulmonares/metabolismo , FemeninoRESUMEN
G-protein-coupled receptors with dissociable agonists for thyrotropin, parathyroid hormone, and sphingosine-1-phosphate were found to signal persistently hours after agonist withdrawal. Here we show that mouse thyrotropin-releasing hormone (TRH) receptors, subtypes 2 and 1(TRH-R2 and TRH-R1), can signal persistently in HEK-EM293 cells under appropriate conditions, but TRH-R2 exhibits higher persistent signaling activity. Both receptors couple primarily to Gα(q/11). To gain insight into the mechanism of persistent signaling, we compared proximal steps of inositolmonophosphate (IP1) signaling by TRH-Rs. Persistent signaling was not caused by slower dissociation of TRH from TRH-R2 (t(1/2)=77 ± 8.1 min) compared with TRH-R1 (t(1/2)=82 ± 12 min) and was independent of internalization, as inhibition of internalization did not affect persistent signaling (115% of control), but required continuously activated receptors, as an inverse agonist decreased persistent signaling by 60%. Gα(q/11) knockdown decreased persistent signaling by TRH-R2 by 82%, and overexpression of Gα(q/11) induced persistent signaling in cells expressing TRH-R1. Lastly, persistent signaling was induced in cells expressing high levels of TRH-R1. We suggest that persistent signaling by TRHRs is exhibited when sufficient levels of agonist/receptor/G-protein complexes are established and maintained and that TRH-R2 forms and maintains these complexes more efficiently than TRH-R1.
Asunto(s)
Subunidades alfa de la Proteína de Unión al GTP Gq-G11/fisiología , Receptores de Hormona Liberadora de Tirotropina/fisiología , Transducción de Señal/fisiología , Animales , Fosfatos de Inositol/biosíntesis , Ratones , Receptores Acoplados a Proteínas G , Receptores de Hormona Liberadora de Tirotropina/agonistas , Receptores de Hormona Liberadora de Tirotropina/genéticaRESUMEN
BACKGROUND: Astrocytes respond to injury and disease through a process known as reactive astrogliosis, of which inflammatory signaling is one subset. This inflammatory response is heterogeneous with respect to the inductive stimuli and the afflicted central nervous system region. This is of plausible importance in e.g. traumatic axonal injury (TAI), where lesions in the brainstem carries a particularly poor prognosis. In fact, astrogliotic forebrain astrocytes were recently suggested to cause neuronal death following axotomy. We therefore sought to assess if ventral brainstem- or rostroventral spinal astrocytes exert similar effects on motor neurons in vitro. METHODS: We derived brainstem/rostroventral spinal astrocyte-like cells (ES-astrocytes) and motor neurons using directed differentiation of mouse embryonic stem cells (ES). We activated the ES-astrocytes using the neurotoxicity-eliciting cytokines interleukin- (IL-) 1α and tumor necrosis factor-(TNF-)α and clinically relevant inflammatory mediators. In co-cultures with reactive ES-astrocytes and motor neurons, we assessed neurotoxic ES-astrocyte activity, similarly to what has previously been shown for other central nervous system (CNS) regions. RESULTS: We confirmed the brainstem/rostroventral ES-astrocyte identity using RNA-sequencing, immunocytochemistry, and by comparison with primary subventricular zone-astrocytes. Following cytokine stimulation, the c-Jun N-terminal kinase pathway down-stream product phosphorylated c-Jun was increased, thus demonstrating ES-astrocyte reactivity. These reactive ES-astrocytes conferred a contact-dependent neurotoxic effect upon co-culture with motor neurons. When exposed to IL-1ß and IL-6, two neuroinflammatory cytokines found in the cerebrospinal fluid and serum proteome following human severe traumatic brain injury (TBI), ES-astrocytes exerted similar effects on motor neurons. Activation of ES-astrocytes by these cytokines was associated with pathways relating to endoplasmic reticulum stress and altered regulation of MYC. CONCLUSIONS: Ventral brainstem and rostroventral spinal cord astrocytes differentiated from mouse ES can exert neurotoxic effects in vitro. This highlights how neuroinflammation following CNS lesions can exert region- and cell-specific effects. Our in vitro model system, which uniquely portrays astrocytes and neurons from one niche, allows for a detailed and translationally relevant model system for future studies on how to improve neuronal survival in particularly vulnerable CNS regions following e.g. TAI.
RESUMEN
The extracellular region of the thyrotropin receptor (TSHR) can be subdivided into the leucine-rich repeat domain (LRRD) and the hinge region. Both the LRRD and the hinge region interact with thyrotropin (TSH) or autoantibodies. Structural data for the TSHR LRRD were previously determined by crystallization (amino acids Glu(30)-Thr(257), 10 repeats), but the structure of the hinge region is still undefined. Of note, the amino acid sequence (Trp(258)-Tyr(279)) following the crystallized LRRD comprises a pattern typical for leucine-rich repeats with conserved hydrophobic side chains stabilizing the repeat fold. Moreover, functional data for amino acids between the LRRD and the transmembrane domain were fragmentary. We therefore investigated systematically these TSHR regions by mutagenesis to reveal insights into their functional contribution and potential structural features. We found that mutations of conserved hydrophobic residues between Thr(257) and Tyr(279) cause TSHR misfold, which supports a structural fold of this peptide, probably as an additional leucine-rich repeat. Furthermore, we identified several new mutations of hydrophilic amino acids in the entire hinge region leading to partial TSHR inactivation, indicating that these positions are important for intramolecular signal transduction. In summary, we provide new information regarding the structural features and functionalities of extracellular TSHR regions. Based on these insights and in context with previous results, we suggest an extracellular activation mechanism that supports an intramolecular agonistic unit as a central switch for activating effects at the extracellular region toward the serpentine domain.
Asunto(s)
Espacio Extracelular/metabolismo , Receptores de Tirotropina/química , Receptores de Tirotropina/metabolismo , Secuencia de Aminoácidos , Sustitución de Aminoácidos , Animales , Células COS , Bovinos , Chlorocebus aethiops , Secuencia Conservada , AMP Cíclico/metabolismo , Regulación de la Expresión Génica , Humanos , Interacciones Hidrofóbicas e Hidrofílicas , Fosfatos de Inositol/metabolismo , Modelos Moleculares , Datos de Secuencia Molecular , Mutagénesis Sitio-Dirigida , Mutación , Pliegue de Proteína , Estructura Secundaria de Proteína , Estructura Terciaria de Proteína , Receptores de Tirotropina/genética , Tirotropina/metabolismoRESUMEN
The thyroid-stimulating hormone (TSH) receptor signals via G(s) to produce cAMP and via G(q/11) to produce inositol-1,4,5-trisphosphate, which is degraded to inositol monophosphate (IP1; phosphoinositide signaling). The potency of TSH for cAMP signaling is higher than for phosphoinositide signaling, and it was suggested that there are "spare receptors" for cAMP signaling. In a human embryonic kidney macrophage scavenger receptor-expressing (HEK-EM) 293 model system, there are no spare receptors, but the cells still exhibited 100-fold differences in potencies. Dose responses for TSH-stimulated dissociation of prebound (125)I-TSH (negative cooperativity; EC(50)=70 mU/ml), which requires TSH binding to both sites of the TSH receptor (TSHR) homodimer, and TSH-stimulated IP1 production (EC(50)=50 mU/ml) were indistinguishable. Fluorescence resonance energy transfer (FRET) using tagged receptors showed that TSHR formed homodimers and heterodimers with two binding-deficient mutant TSHRs, L252P and C41S. When L252P or C41S was expressed with TSHR, that is, when TSHR/L252P or TSHR/C41S heterodimers could only bind one TSH, TSH-stimulated IP1 production was decreased relative to cAMP production. The slopes of linear regression analyses comparing fold stimulation by TSH of IP1 vs. cAMP production were 0.044 ± 0.0047, 0.0043 ± 0.0041, and 0.0059 ± 0.0014 for cells expressing TSHR alone, TSHR and L252P, or TSHR and C41S, respectively. We suggest that TSHR coupling to phosphoinositide signaling is dependent on binding 2 molecules of TSH to TSHR homodimer, causing a conformational change allowing coupling to G(q/11).
Asunto(s)
Fosfatidilinositoles/metabolismo , Receptores de Tirotropina/metabolismo , Transducción de Señal/fisiología , AMP Cíclico , Subunidades alfa de la Proteína de Unión al GTP Gq-G11 , Subunidades alfa de la Proteína de Unión al GTP Gs/metabolismo , Regulación de la Expresión Génica/fisiología , Células HEK293 , Humanos , Unión Proteica , Subunidades de Proteína , Receptores de Tirotropina/química , Tirotropina/metabolismoRESUMEN
The thyrotropin receptor (TSHR) exhibits elevated cAMP signaling in the basal state and becomes fully activated by thyrotropin. Previously we presented evidence that small-molecule ligands act allosterically within the transmembrane region in contrast to the orthosteric extracellular hormone-binding sites. Our goal in this study was to identify positions that surround the allosteric pocket and that are sensitive for inactivation of TSHR. Homology modeling combined with site-directed mutagenesis and functional characterization revealed seven mutants located in the allosteric binding site that led to a decrease of basal cAMP signaling activity. The majority of these silencing mutations, which constrain the TSHR in an inactive conformation, are found in two clusters when mapped onto the 3D structural model. We suggest that the amino acid positions identified herein are indicating locations where small-molecule antagonists, both neutral antagonists and inverse agonists, might interfere with active TSHR conformations.
Asunto(s)
Mutación , Receptores de Tirotropina/genética , Transducción de Señal/genética , Sitios de Unión , AMP Cíclico/metabolismo , Células HEK293 , Humanos , Ligandos , Modelos Moleculares , Estructura Terciaria de Proteína/genética , Receptores de Tirotropina/química , Receptores de Tirotropina/fisiologíaRESUMEN
Seven-transmembrane-spanning receptors (7TMRs) are prominent drug targets. However, small-molecule ligands for 7-transmembrane-spanning receptors for which the natural ligands are large, heterodimeric glycoprotein hormones, like thyroid-stimulating hormone (TSH; thyrotropin), have only recently been reported, and none are approved for human use. We have used quantitative high-throughput screening to identify a small-molecule TSH receptor (TSHR) agonist that was modified to produce a second agonist with increased potency. We show that these agonists are highly selective for human TSHR versus other glycoprotein hormone receptors and interact with the receptor's serpentine domain. A binding pocket within the transmembrane domain was defined by docking into a TSHR homology model and was supported by site-directed mutagenesis. In primary cultures of human thyrocytes, both TSH and the agonists increase mRNA levels for thyroglobulin, thyroperoxidase, sodium iodide symporter, and deiodinase type 2, and deiodinase type 2 enzyme activity. Moreover, oral administration of the agonist stimulated thyroid function in mice, resulting in increased serum thyroxine and thyroidal radioiodide uptake. Thus, we discovered a small molecule that activates human TSHR in vitro, is orally active in mice, and could be a lead for development of drugs to use in place of recombinant human TSH in patients with thyroid cancer.
Asunto(s)
Compuestos Orgánicos/farmacología , Receptores de Tirotropina/agonistas , Glándula Tiroides/efectos de los fármacos , Acetamidas/síntesis química , Acetamidas/química , Acetamidas/farmacología , Animales , Sitios de Unión , Línea Celular , Células Cultivadas , AMP Cíclico/metabolismo , Femenino , Expresión Génica/efectos de los fármacos , Humanos , Yoduro Peroxidasa/genética , Yoduro Peroxidasa/metabolismo , Ratones , Ratones Endogámicos C57BL , Modelos Moleculares , Estructura Molecular , Compuestos Orgánicos/síntesis química , Compuestos Orgánicos/química , Estructura Terciaria de Proteína , Quinazolinonas/síntesis química , Quinazolinonas/química , Quinazolinonas/farmacología , Receptores de Tirotropina/genética , Receptores de Tirotropina/metabolismo , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Tiroglobulina/genética , Tiroglobulina/metabolismo , Glándula Tiroides/citología , Glándula Tiroides/fisiología , Tirotropina/farmacología , TransfecciónRESUMEN
Proximity ligation assay (PLA) is a methodology that permits detection of protein-protein closeness, that is, proteins that are within 40 nanometers of each other, in cells or tissues at endogenous protein levels or after exogenous overexpression. It detects the protein(s) with high sensitivity and specificity because it employs a DNA hybridization step followed by DNA amplification. PLA has been used successfully with many types of proteins. In this methods paper, we will describe the workings of PLA and provide examples of its use to study TSH/IGF-1 receptor crosstalk in Graves' orbital fibroblasts (GOFs) and TSH receptor homodimerization in primary cultures of human thyrocytes.
Asunto(s)
Receptor IGF Tipo 1 , Receptores de Tirotropina , ADN , Humanos , Receptor IGF Tipo 1/genética , Receptor IGF Tipo 1/metabolismo , Receptores de Tirotropina/genética , Receptores de Tirotropina/metabolismo , Glándula Tiroides/metabolismo , TirotropinaRESUMEN
The TSH receptor (TSHR) is the major regulator of thyroid hormone biosynthesis in human thyrocytes by regulating the transcription of a number of genes including thyroglobulin (TG) and thyroperoxidase (TPO). Until recently, it was thought that TSHR initiated signal transduction pathways only at the cell-surface and that internalization was primarily involved in TSHR desensitization and downregulation. Studies primarily in mouse cells showed that TSHR internalization regulates gene transcription at an intracellular site also. However, this has not been shown for genes involved in thyroid hormone biosynthesis in human thyrocytes. We used human thyrocytes in primary culture. In these cells, the dose-response to TSH for gene expression is biphasic with low doses upregulating gene expression and higher doses decreasing gene expression. We used two approaches to inhibit internalization. In the first, we used inhibitors of dynamins, dynasore and dyngo-4a. Pretreatment with dynasore or dyngo-4a markedly inhibited TSH upregulation of TG and TPO mRNAs, as well as TG secretion. In the second, we used knockdown of dynamin 2, which is the most abundant dynamin in human thyrocytes. We showed that dynamin 2 knockdown inhibited TSHR internalization and decreased the TSH-stimulated levels of TG and TPO mRNAs and proteins. Lastly, we showed that the level of the activatory transcription factor phosphorylated cAMP response element binding protein (pCREB) in the cell nuclei was reduced by 68% when internalization was inhibited. We conclude that upregulation of genes involved in thyroid hormone synthesis in human thyrocytes is, in part, dependent on internalization leading to nuclear localization of an activated transcription factor(s).
Asunto(s)
Yoduro Peroxidasa , Tiroglobulina , Animales , Humanos , Yoduro Peroxidasa/genética , Yoduro Peroxidasa/metabolismo , Ratones , Receptores de Tirotropina/genética , Receptores de Tirotropina/metabolismo , Tiroglobulina/genética , Tiroglobulina/metabolismo , Tirotropina/genética , Tirotropina/farmacología , Transcripción GenéticaRESUMEN
CONTEXT: We previously presented evidence that TSH receptor (TSHR)-stimulating autoantibodies (TSAbs) bind to and activate TSHRs but do not bind to IGF1 receptors (IGF1Rs). Nevertheless, we showed that IGF1Rs were involved in thyroid eye disease (TED) pathogenesis because TSAbs activated crosstalk between TSHR and IGF1R. Teprotumumab, originally generated to inhibit IGF1 binding to IGF1R, was recently approved for the treatment of TED (Tepezza). OBJECTIVE: To investigate the role of TSHR/IGF1R crosstalk in teprotumumab treatment of TED. DESIGN: We used orbital fibroblasts from patients with TED (TEDOFs) and measured stimulated hyaluronan (HA) secretion as a measure of orbital fibroblast activation by TED immunoglobulins (TED-Igs) and monoclonal TSAb M22. We previously showed that M22, which does not bind to IGF1R, stimulated HA in a biphasic dose-response with the higher potency phase dependent on TSHR/IGF1R crosstalk and the lower potency phase independent of IGF1R. Stimulation by TED-Igs and M22 was measured in the absence or presence of teprotumumab biosimilar (Tepro) or K1-70, an antibody that inhibits TSHR. RESULTS: We show: (1) Tepro dose-dependently inhibits stimulation by TED-Igs; (2) Tepro does not bind to TSHRs; (3) Tepro inhibits IGF1R-dependent M22-induced HA production, which is mediated by TSHR/IGF1R crosstalk, but not IGF1R-independent M22 stimulation; and (4) ß-arrestin 1 knockdown, which blocks TSHR/IGF1R crosstalk and prevents Tepro inhibition of HA production by M22 and by a pool of TED-Igs. CONCLUSION: We conclude that Tepro inhibits HA production by TEDOFs by inhibiting TSHR/IGF1R crosstalk and suggest that inhibition of TSHR/IGF1R crosstalk is the mechanism of its action in treating TED.