El papel de la proteína de unión al factor liberador de corticotrofina en el tráfico de receptores de corticotrofina tipo 2
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2018
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es
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Universidad Andrés Bello
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Licencia CC
Resumen
La respuesta a estrés es regulada por el sistema del factor liberador de corticotrofina (CRF). Dentro de este sistema, el receptor de CRF tipo 2 (CRFR2) y la proteína de unión a CRF (CRF-BP) están involucrados en el establecimiento y recaída en la búsqueda de cocaína inducida por estrés. Recientemente, una investigación de nuestro laboratorio mostró que CRF-BP interactúa físicamente con la isoforma alfa de CRFR2 (CRFR2α) y se propuso que esta interacción depende de un pseudopéptido señal en la región N-terminal de CRFR2α. Anterior a esto, se observó que este mismo pseudopéptido está involucrado en la asociación entre CRFR2α y calnexina, una proteína chaperona del retículo endoplasmático (ER). La interacción entre CRFR2α y CRF-BP facilita la translocación de CRFR2α desde compartimentos calnexina positivos hacia la membrana plasmática en un modelo de expresión heteróloga, y de forma similar, se ha reportado que situaciones de estrés agudo aumentan la presencia de CRFR2α en la membrana plasmática de neuronas serotoninérgicas del núcleo dorsal del rafe (DRN). Estos antecedentes nos llevaron a hipotetizar que en situaciones de estrés agudo CRF-BP actúa reduciendo la interacción entre CRFR2α y calnexina favoreciendo la presencia de este receptor en la membrana plasmática de neuronas serotoninérgicas del DRN.
Para probar esta hipótesis, evaluamos la presencia de calnexina en la precipitación de CRFR2α con y sin la co-expresión de CRF-BP. Además, analizamos la precipitación y localización subcelular de estas proteínas en presencia de tapsigargina, una molécula que indirectamente reduce la afinidad de calnexina por sus sustratos. Los resultados obtenidos sugieren que CRF-BP disminuye la interacción entre CRFR2α y calnexina lo que a su vez podría favorecer la translocación de CRFR2α fuera de compartimientos calnexina positivos del ER. Además, se observó que tapsigargina aumenta tanto la expresión de CRFR2α cuando es co-expresado junto a CRF-BP como la interacción entre CRFR2α y CRF-BP. Adicionalmente, estos mecanismos podrían ser importantes en la regulación de la respuesta al estrés mediada por el DRN ya que se observó que CRF-BP se encuentra presente en esta área del cerebro de rata y que sus niveles podrían ser regulados por estrés. Este último resultado abre la oportunidad de estudiar estos mecanismos en un modelo animal.
The stress response is regulated by the corticotropin releasing factor (CRF) system. In this system, CRF type 2 receptor (CRFR2) and CRF binding protein (CRF-BP) are involved in the establishment and relapse to cocaine seeking induced by stressful stimuli. Recently, an investigation from our laboratory showed that CRF-BP physically interacts with the alpha isoform of CRFR2 (CRFR2α) and it was proposed that this interaction depends on the pseudo signal peptide located in the N-terminal region of CRFR2α. Previously, it was shown that this same pseudo signal peptide is also involved in the association between CRFR2α and calnexin, a chaperone protein of the endoplasmic reticulum (ER). The interaction between CRFR2α and CRF-BP facilitates CRFR2α translocation from calnexin positive compartments towards the plasmatic membrane in a heterologous expression model. Similarly, it has been reported that acute stress situations increase the presence of CRFR2α on the plasmatic membrane of serotoninergic neurons of the dorsal raphe nucleus (DRN). These evidences lead us to hypothesize that in acute stressful situations CRF-BP acts reducing the interaction between CRFR2α and calnexin favoring presence this receptor of this receptor on the plasmatic membrane of serotoninergic neurons of the DRN. To test this hypothesis, we evaluated the presence of calnexin on the precipitation of CRFR2α, with or without CRF-BP co-expression. Furthermore, we analyzed the precipitation and subcellular localization of these proteins in the presence of thapsigargin, a molecule that indirectly reduces calnexin affinity for its substrates. The obtained results suggest that CRF-BP decreases the interaction between CRFR2α and calnexin which in turns might favor CRFR2α translocation outside calnexin positive compartments of the ER. In addition, it was observed that thapsigargin increases CRFR2α levels when is co-expressed with CRF-BP as well as the interaction between CRFR2α and CRF-BP. Moreover, these mechanisms might be important in the regulation of DRN mediated stress responses due to the observation that CRF-BP is present in this region of the rat brain and its levels might be regulated by stress. This last result opens the opportunity to study this mechanisms in an animal model.
The stress response is regulated by the corticotropin releasing factor (CRF) system. In this system, CRF type 2 receptor (CRFR2) and CRF binding protein (CRF-BP) are involved in the establishment and relapse to cocaine seeking induced by stressful stimuli. Recently, an investigation from our laboratory showed that CRF-BP physically interacts with the alpha isoform of CRFR2 (CRFR2α) and it was proposed that this interaction depends on the pseudo signal peptide located in the N-terminal region of CRFR2α. Previously, it was shown that this same pseudo signal peptide is also involved in the association between CRFR2α and calnexin, a chaperone protein of the endoplasmic reticulum (ER). The interaction between CRFR2α and CRF-BP facilitates CRFR2α translocation from calnexin positive compartments towards the plasmatic membrane in a heterologous expression model. Similarly, it has been reported that acute stress situations increase the presence of CRFR2α on the plasmatic membrane of serotoninergic neurons of the dorsal raphe nucleus (DRN). These evidences lead us to hypothesize that in acute stressful situations CRF-BP acts reducing the interaction between CRFR2α and calnexin favoring presence this receptor of this receptor on the plasmatic membrane of serotoninergic neurons of the DRN. To test this hypothesis, we evaluated the presence of calnexin on the precipitation of CRFR2α, with or without CRF-BP co-expression. Furthermore, we analyzed the precipitation and subcellular localization of these proteins in the presence of thapsigargin, a molecule that indirectly reduces calnexin affinity for its substrates. The obtained results suggest that CRF-BP decreases the interaction between CRFR2α and calnexin which in turns might favor CRFR2α translocation outside calnexin positive compartments of the ER. In addition, it was observed that thapsigargin increases CRFR2α levels when is co-expressed with CRF-BP as well as the interaction between CRFR2α and CRF-BP. Moreover, these mechanisms might be important in the regulation of DRN mediated stress responses due to the observation that CRF-BP is present in this region of the rat brain and its levels might be regulated by stress. This last result opens the opportunity to study this mechanisms in an animal model.
Notas
Tesis (Magíster en Biotecnología)
Palabras clave
Corticotrofina, Proteínas