EXPLORING CRHR2alpha SIGNALING AND TRAFFICKING IN A HIPPOCAMPAL CELLULAR CONTEXT

DOS SANTOS CLARO, P.; INDA, C.; SILBERSTEIN, S.; ARMANDO, N.G.; SENIN, S.

Buenos Aires

Congreso; Reunión conjunta de las Sociedades de Biociencias 2017, LXII Reunión Anual de la Sociedad Argentina de Investigacion Clínica. Buenos Aires, 13-17 de noviembre de 2017.; 2017

SAIC

The corticotropin-releasing hormone (CRH) system orchestrates the response and adaptation to stress, acting on the hypothalamic-pituitary-adrenal axis and in different brain regions. A large body of evidence points to dysregulation of CRH system signaling as causally linked to anxiety and depressive disorders. There are two different G-protein-coupled receptors (GPCRs), CRHR1 and CRHR2, encoded by different genes which display different localization and ligand preferences. We are particularly interested in the alpha splicing isoform (CRHR2α) given that it is the most important in the brain. CRHR2α has a pseudo signal peptide which gives this receptor specific trafficking and signaling characteristics. We are exploring the signaling pathways activated by CRHRs in order to identify mechanisms involved in CRH and its related peptides action in the brain. Given that previous results showed specific CRH actions in vivo in particular limbic structures such as hippocampus, we perform our studies in HT22 cells, a mouse hippocampal neuronal cell line widely used as a neuronal model. We generated stable clones expressing CRHR1 and CRHR2α in HT22 cells to explore the mechanisms involved in the signaling cascade and trafficking of each receptor. We used CRH and UCNs as ligands for each receptor to assess whether different signaling pathways are activated depending on the ligand. The stimulation of both CRHR1 and CRHR2α led to an increase of intracellular cAMP measured with FRET biosensors. We compared similarities and differences of the activation of CREB, ERK1/2 and AKT by western blot. We have previously reported that CRH-dependent ERK1/2 activation downstream of CRHR1 is biphasic, being dependent on G protein and receptor endocytosis mechanisms. Remarkably, the same pattern was observed when UCN1 was used as a CRHR1 ligand. However, the kinetics of ERK1/2 activation downstream of CRHR2α were different, either when CRH or UCNs were used for stimulation.