B-Raf and CRHR1 internalization mediate biphasic ERK1/2 activation by CRH in hippocampal HT22 cells

• Autores: Bonfiglio, J.J.; Inda, C.; Senin, S.; Maccarrone, G.; Refojo, D.; Giacomini, D.; Turck, C.W.; Holsboer, F.; Arzt, E.; Silberstein, S.
• Año de publicación: 2013
• Idioma: inglés
• Tipo de recurso: artículo
• Fuente: Mol. Endocrinol. 2013;27(3):491-510
• Materia: B Raf kinase; beta arrestin 2; calcium; calcium calmodulin dependent protein kinase II; corticotropin releasing factor; corticotropin releasing factor receptor 1; cyclic AMP; cyclic AMP dependent protein kinase; dynamin; mitogen activated protein kinase 1; protein 14 3 3; Rap1 protein; vimentin; animal cell; article; basolateral amygdala; behavior; calcium cell level; calcium transport; cellular distribution; controlled study; enzyme activation; enzyme inactivation; hippocampus; internalization; learning; mass spectrometry; memory; mouse; nerve cell plasticity; nonhuman; physiological process; priority journal; screening; Adenylate Cyclase; Animals; Arrestins; Calcium; Calcium-Calmodulin-Dependent Protein Kinase Type 2; Corticotropin-Releasing Hormone; Cyclic AMP; Endocytosis; Enzyme Activation; Hippocampus; Humans; Mice; Mitogen-Activated Protein Kinase 1; Mitogen-Activated Protein Kinase 3; Models, Biological; Proto-Oncogene Proteins B-raf; Rats; Receptors, Corticotropin-Releasing Hormone; Signal Transduction; Subcellular Fractions; Time Factors; Vimentin
• Institución: Universidad Nacional de Buenos Aires. Facultad de Ciencias Exactas y Naturales

CRH is a key regulator of neuroendocrine, autonomic, and behavioral response to stress. CRHstimulated CRH receptor 1 (CRHR1) activates ERK1/2 depending on intracellular context. In a previous work, we demonstrated that CRH activates ERK1/2 in limbic areas of the mouse brain (hippocampus and basolateral amygdala). ERK1/2 is an essential mediator of hippocampal physiological processes including emotional behavior, synaptic plasticity, learning, and memory. To elucidate the molecular mechanisms by which CRH activates ERK1/2 in hippocampal neurons, we used the mouse hippocampal cell line HT22. We document for the first time that ERK1/2 activation in response to CRH is biphasic, involving a first cAMP- and B-Raf-dependent early phase and a second phase that critically depends on CRHR1 internalization and β-arrestin2. By means of mass-spectrometry-based screening, we identified B-Raf-associated proteins that coimmunoprecipitate with endogenous B-Raf after CRHR1 activation. Using molecular and pharmacological tools, the functional impact of selected B-Raf partners in CRH-dependent ERK1/2 activation was dissected. These results indicate that 14-3-3 proteins, protein kinase A, and Rap1, are essential for early CRH-induced ERK1/2 activation, whereas dynamin and vimentin are required for the CRHR1 internalization-dependent phase. Both phases of ERK1/2 activation depend on calcium influx and are affected by calcium/calmodulin-dependent protein kinase II inactivation. Thus, this report describes the dynamics and biphasic nature of ERK1/2 activation downstream neuronal CRHR1 and identifies several new critical components of the CRHR1 signaling machinery that selectively controls the early and late phases of ERK1/2 activation, thus providing new potential therapeutic targets for stress-related disorders. © 2013 by The Endocrine Society.
Fil:Bonfiglio, J.J. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales; Argentina.
Fil:Refojo, D. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales; Argentina.
Fil:Giacomini, D. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales; Argentina.
Fil:Silberstein, S. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales; Argentina.