Asunto(s)
Hormona Liberadora de Hormona del Crecimiento/fisiología , Receptores de Neuropéptido/fisiología , Receptores de Hormona Reguladora de Hormona Hipofisaria/fisiología , Animales , Hormona Liberadora de Hormona del Crecimiento/química , Hormona Liberadora de Hormona del Crecimiento/genética , Hormona Liberadora de Hormona del Crecimiento/uso terapéutico , Humanos , Ligandos , Receptores de Neuropéptido/química , Receptores de Neuropéptido/genética , Receptores de Neuropéptido/aislamiento & purificación , Receptores de Hormona Reguladora de Hormona Hipofisaria/química , Receptores de Hormona Reguladora de Hormona Hipofisaria/genética , Receptores de Hormona Reguladora de Hormona Hipofisaria/aislamiento & purificación , Transducción de SeñalRESUMEN
Cell adhesion, migration, differentiation and survival or death is amongst a large spectrum of biological responses that can be elicited by ligation of extracellular matrix components to their corresponding receptors. As regards the physiology of the thymus, cell migration is a crucial event in the general process of T cell differentiation. Studies on the intrathymic distribution of ECM components revealed that fibronectin, laminin and type IV collagen, are not restrictedly located at typical basement membrane sites, also forming a thick network in the medullary region of the thymic lobules, whereas very thin ECM fibers are found within the cortex. These ECM components are essentially produced by thymic microenvironmental cells, which also drive thymocyte differentiation. Signals triggered by ECM are conveyed into thymocytes or microenvironmental cells through specific membrane receptors, and most of them belong to the integrin type, such as the VLA-3, VLA-4, VLA-5 and VLA-6. In vitro studies revealed that adhesion of thymocytes to thymic microenvironmental cells is mediated by extracellular matrix. Such an adhesion is preferentially done by immature thymocytes. Importantly, ECM-mediated interactions also govern the entrance and exit of thymocytes in the lymphoepithelial complexes named thymic nurse cells. Lastly, pathological conditions, including infectious and autoimmune diseases, in which changes of ECM ligands and receptors are observed, course with alterations in thymocyte migration and death. In conclusion, the fact that ECM can modulate traffic, differentiation, death and survival of normal thymocytes adds clues for understanding how ECM-mediated interactions behave in the thymus, not only in normal, but also in pathological conditions.
Asunto(s)
Matriz Extracelular/fisiología , Linfocitos T/fisiología , Animales , Adhesión Celular , Movimiento Celular , Enfermedad de Chagas/inmunología , Humanos , Receptores de Hialuranos/análisis , Laminina/análisis , Ratones , Ratones Endogámicos NOD , Proteoglicanos/análisis , Timo/citologíaRESUMEN
Hormones from the hypothalamus mediate interactions between the nervous and endocrine systems by controlling the activity of specific target cells in the anterior pituitary gland. The hypothalamic peptide, growth hormone-releasing hormone (GHRH), acts on pituitary somatotroph cells to stimulate their proliferation during development and to regulate their ability to produce and secrete growth hormone (GH). These actions are mediated by a recently identified receptor for GHRH that belongs to family B-III of the G protein-coupled receptor superfamily. The rat GHRH receptor is expressed predominantly in the pituitary gland and in somatotroph cells. To investigate this tissue- and cell-specific expression, the receptor gene has been cloned and characterized. The receptor gene promoter is selectively expressed in pituitary cells and is regulated by the pituitary-specific transcription factor Pit-1. There is a sexual dimorphism in GHRH receptor expression in the rat pituitary, suggesting regulation by gonadal steroids. In addition, glucocorticoids are potent positive regulators of GHRH receptor gene expression. Substantial evidence points to an important role for GHRH in regulating the proliferation and functional activity of the somatotroph cell. This is best observed in the dwarf little mouse, which harbors a mutation in the extracellular domain of the GHRH receptor that abolishes the receptor's hormone-binding and signaling properties, resulting in severe somatotroph hypoplasia. Complementary studies in transgenic mice overexpressing the ligand GHRH reveal corresponding somatotroph hyperplasia. Consistent with these observations, GHRH potently activates the MAP kinase pathway in pituitary somatotroph cells. To better understand the hormone-binding and signaling properties of the GHRH receptor, mutant and chimeric receptors have been analyzed to define domains important for GHRH interaction. The GHRH receptor signals predominantly through cAMP-dependent pathways; however, a variant form of the GHRH receptor with an insertion into the third intracellular domain, generated through alternative RNA processing, binds GHRH but fails to signal, suggesting potential modulation of receptor function at a post-transcriptional level. This chapter will integrate these basic investigations of GHRH and its receptor with current information on the involvement of the GHRH signaling system in human diseases of GH secretion and growth.
Asunto(s)
Hormona Liberadora de Hormona del Crecimiento/fisiología , Hipófisis/citología , Hipófisis/fisiología , Receptores de Neuropéptido/fisiología , Receptores de Hormona Reguladora de Hormona Hipofisaria/fisiología , Animales , División Celular , Femenino , Regulación de la Expresión Génica/efectos de los fármacos , Glucocorticoides/farmacología , Humanos , Masculino , Ratones , Proteínas Quinasas Activadas por Mitógenos/metabolismo , Sistemas Neurosecretores/fisiología , Ratas , Receptores de Neuropéptido/química , Receptores de Neuropéptido/genética , Receptores de Hormona Reguladora de Hormona Hipofisaria/química , Receptores de Hormona Reguladora de Hormona Hipofisaria/genética , Transducción de Señal , Esteroides/farmacologíaRESUMEN
The little mouse is a dwarf strain characterized by low levels of GH, pituitary hypoplasia, and an unresponsiveness to treatment with exogenous GHRH. The defect has been mapped to a missense mutation in the GHRH receptor gene that abolishes the function of the receptor, but the mechanism of this inactivation is unknown. Receptor function might be affected at the level of protein expression, maturation and processing, localization to the cell surface, ligand binding, or signaling. In this study, Western blots, using antiserum raised against the GHRH receptor and immunoprecipitation analysis of epitope-tagged receptors, demonstrate that both wild-type and mutant receptor proteins are expressed at equivalent levels in transfected cells. Immunofluorescence analysis of intact and permeabilized cells expressing the epitope-tagged receptors suggests that wild-type and little mouse receptors are similarly localized to the cell surface. A species homologous binding assay was developed and used to show that 125I-mouse GHRH binds with high affinity to the wild-type mouse receptor but not to the little mutant receptor. Consistent with this, the mutant receptor fails to stimulate intracellular cAMP accumulation. Our results demonstrate that the little mutation does not dramatically affect the expression level, glycosylation, or cellular localization of the receptor protein but that it blocks specific GHRH binding, and therefore, signaling does not take place.
Asunto(s)
Hormona Liberadora de Hormona del Crecimiento/metabolismo , Mutación Missense , Receptores de Neuropéptido/genética , Receptores de Neuropéptido/metabolismo , Receptores de Hormona Reguladora de Hormona Hipofisaria/genética , Receptores de Hormona Reguladora de Hormona Hipofisaria/metabolismo , Secuencia de Aminoácidos , Animales , Western Blotting , Membrana Celular/química , Membrana Celular/metabolismo , Enanismo Hipofisario/genética , Técnica del Anticuerpo Fluorescente Indirecta , Glicosilación , Humanos , Técnicas de Inmunoadsorción , Ratones , Ratones Mutantes , Datos de Secuencia Molecular , Receptores de Neuropéptido/análisis , Receptores de Hormona Reguladora de Hormona Hipofisaria/análisis , Transducción de Señal , Relación Estructura-ActividadRESUMEN
The interaction of GHRH with membrane-bound receptors on somatotroph cells of the anterior pituitary is an important step in the regulation of GH synthesis and secretion. The identification of a G protein-coupled receptor for GHRH has made it possible to investigate the pathway by which GHRH regulates pituitary somatotroph cell function. To initiate an analysis of the mechanisms regulating expression and function of the GHRH receptor, the structure of the gene and its promoter region were analyzed. The coding sequence of the rat GHRH receptor gene is contained within 14 exons spanning approximately 15 kb of genomic DNA. Four transcription start sites are located within 286 bp upstream of the initiation codon. The 5' flanking region of the GHRH receptor gene acts as a functional promoter in rat pituitary tumor GH3 cells, and basal promoter activity is enhanced in GH3 and COS7 cells by cotransfection of an expression construct encoding the pituitary-specific transcription factor Pit-1. The rat GHRH receptor gene is subject to at least 1 alternative RNA processing event that generates 2 receptor isoforms differing by 41 amino acids within the third intracellular loop (IL) of the protein. The short isoform of the GHRH receptor is predominant in pituitary cells. The MtT/S pituitary tumor cell line was found to express the GHRH receptor, and different populations of these cells produce predominantly the long or short isoforms of the receptor messenger RNA, suggesting that the alternative splicing can be regulated. Functional analysis of the two GHRH receptor isoforms demonstrates that both bind GHRH, but only the short isoform signals through a cAMP-mediated pathway. Neither receptor isoform is able to stimulate calcium mobilization from internal stores after GHRH treatment. Our findings indicate that the pituitary-specific transcription factor Pit-1 is involved in the somatotroph-specific expression of the GHRH receptor gene and that functionally distinct receptor proteins are generated by an alternative RNA processing mechanism.
Asunto(s)
Ratas/genética , Receptores de Neuropéptido/genética , Receptores de Hormona Reguladora de Hormona Hipofisaria/genética , Secuencia de Aminoácidos/genética , Animales , Secuencia de Bases/genética , Exones/genética , Regulación de la Expresión Génica/fisiología , Genoma , Masculino , Datos de Secuencia Molecular , Regiones Promotoras Genéticas/genética , Isoformas de Proteínas/fisiología , Ratas Sprague-Dawley , Receptores de Neuropéptido/fisiología , Receptores de Hormona Reguladora de Hormona Hipofisaria/fisiología , Transducción de Señal/fisiología , Transcripción Genética/fisiologíaRESUMEN
The hypothalamic peptide GH-releasing hormone (GHRH) stimulates the release of GH from the pituitary through binding and activation of the GHRH receptor, which belongs to the family of G protein-coupled receptors. The objective of this study was to identify regions of the receptor critical for interaction with the ligand by expressing and analyzing truncated and chimeric epitope-tagged GHRH receptors. Two truncated receptors, GHRHdeltaN, in which part of the N-terminal domain between the putative signal sequence and the first transmembrane domain was deleted, and GHRHdeltaC, which was truncated downstream of the first intracellular loop, were generated. Both the receptors were deficient in ligand binding, indicating that neither the N-terminal extracellular domain (N terminus) nor the membrane-spanning domains with the associated extracellular loops (C terminus) are alone sufficient for interaction with GHRH. In subsequent studies, chimeric proteins between the receptors for GHRH and vasoactive intestinal peptide (VIP) or secretin were generated, using the predicted start of the first transmembrane domain as the junction for the exchange of the N terminus between receptors. The chimeras having the N terminus of the GHRH receptor and the C terminus of either the VIP or secretin receptor (GNVC and GNSC) did not bind GHRH or activate adenylate cyclase after GHRH treatment. The reciprocal chimeras having the N terminus of either the VIP or secretin receptors and the C terminus of the GHRH receptor (VNGC and SNGC) bound GHRH and stimulated cAMP accumulation after GHRH treatment. These results suggest that although the N-terminal extracellular domain is essential for ligand binding, the transmembrane domains and associated extracellular loop regions of the GHRH receptor provide critical information necessary for specific interaction with GHRH.
Asunto(s)
Mutagénesis Sitio-Dirigida , Receptores de Neuropéptido/genética , Receptores de Neuropéptido/metabolismo , Receptores de Hormona Reguladora de Hormona Hipofisaria/genética , Receptores de Hormona Reguladora de Hormona Hipofisaria/metabolismo , Proteínas Recombinantes de Fusión/metabolismo , Antígenos Virales/genética , Sitios de Unión/genética , Epítopos/genética , Células HeLa , Glicoproteínas Hemaglutininas del Virus de la Influenza/genética , Humanos , Ligandos , Modelos Moleculares , Unión Proteica/genética , Estructura Terciaria de Proteína , Receptores Acoplados a Proteínas G , Receptores de la Hormona Gastrointestinal/análisis , Receptores de la Hormona Gastrointestinal/biosíntesis , Receptores de la Hormona Gastrointestinal/genética , Receptores de Neuropéptido/análisis , Receptores de Hormona Reguladora de Hormona Hipofisaria/análisis , Receptores de Péptido Intestinal Vasoactivo/análisis , Receptores de Péptido Intestinal Vasoactivo/biosíntesis , Receptores de Péptido Intestinal Vasoactivo/genética , Proteínas Recombinantes de Fusión/análisis , Proteínas Recombinantes de Fusión/biosíntesis , Secretina/metabolismoRESUMEN
We now summarize key issues that we have investigated and highlight additional areas that need to be addressed. We are interested in two basic aspects of the GHRH pathway, those occurring in the brain, involving the synthesis of GHRH, and those occurring in the pituitary, involving signaling by GHRH. We have a long-term interest in the activity and regulation of the hypothalamic neurosecretory cells that synthesize GHRH. With respect to human disease, it is interesting that, despite the primal role played by GHRH in growth-hormone secretion, no mutations in the GHRH gene have yet been identified in association with growth disorders. Focusing on the downstream signaling components of the GHRH pathway, we now know quite a lot about the structure of the GHRH receptor and about some aspects of the signal transduction pathways that mediate the actions of GHRH. With respect to human disease, we have found that in an animal model, the little mouse, a mutation of the GHRH receptor results in growth-hormone deficiency and a dwarf phenotype, and there are ongoing attempts in several laboratories to try to identify similar inactivating mutations in the GHRH receptor in patients with isolated growth-hormone deficiency. Conversely, there is also substantial interest in whether activating mutations in this receptor might be identified in patients with growth-hormone-secreting pituitary tumors. We are also interested in whether there are additional receptors that might mediate some of the extrapituitary actions of GHRH. Finally, a major direction we are taking in the laboratory at the present time is toward understanding the developmental, hormonal, and tissue-specific regulation of the GHRH receptor gene.
Asunto(s)
Expresión Génica , Hormona Liberadora de Hormona del Crecimiento/fisiología , Receptores de Neuropéptido/fisiología , Receptores de Hormona Reguladora de Hormona Hipofisaria/fisiología , Transducción de Señal , Secuencia de Aminoácidos , Animales , Encéfalo/fisiología , Enanismo/genética , Hormona de Crecimiento Humana/deficiencia , Humanos , Sistema Hipotálamo-Hipofisario/fisiología , Ratones , Ratones Mutantes , Ratones Transgénicos , Modelos Biológicos , Datos de Secuencia Molecular , Estructura Secundaria de Proteína , Receptores de Neuropéptido/biosíntesis , Receptores de Neuropéptido/química , Receptores de Hormona Reguladora de Hormona Hipofisaria/biosíntesis , Receptores de Hormona Reguladora de Hormona Hipofisaria/químicaRESUMEN
The ability to switch from nasal to oral breathing in response to nasal obstruction is crucial for survival, and has been suggested to be an important mechanism in preventing sudden infant death syndrome (SIDS). To know whether the ability to switch from nasal to oral breathing is uniformly present during the early neonatal period, we examined the effects of slow and fast nasal occlusions on the establishment of oral breathing in preterm infants. Slow occlusions were used to mimic more closely occlusions occurring spontaneously. We studied 17 healthy preterm infants [birth weight, 1830 +/- 27 g (mean +/- SE); study weight, 1800 +/- 109 g; gestational age, 32 +/- 1 weeks; postnatal age, 12 +/- 2 days]. We used a nosepiece with a nasal occluder and a flow-through system to measure ventilation. A CO2 sampling catheter at the mouth was used to detect oral breathing. Of 58 occlusions, 29 were slow [resistance increasing slowly from 0 to infinite (occlusion)], and 29 were fast (infinite elastance applied in < 1 sec). Oral breathing was always established following slow and fast occlusions. In 44% of the slow occlusions, oral breathing started before complete occlusion. Arousal was observed in 12/58 (17%) of all occlusions, occurring primarily after initiation of oral breathing. Oxygen saturation and respiratory rate decreased significantly following occlusions, from 96 +/- 0.6 to 87 +/- 1.2% and 49 +/- 2.8 to 38 +/- 2 breaths/min, respectively.(ABSTRACT TRUNCATED AT 250 WORDS)