G protein-coupled CRH receptors signaling in a neuronal hippocampal cellular context

PIAZZA, VERÓNICA; DOS SANTOS CLARO, PAULA A.; SILBERSTEIN, SUSANA; ARMANDO, NATALIA G.; INDA, CAROLINA

San Diego

Congreso; Neuroscience 2018; 2018

Society of Neuroscience

The corticotropin releasing hormone (CRH) system involves GPCRs and neuropeptides (CRH and urocortins 1-3) that act as neuromodulators to develop and integrate a coordinated response to stress. Previous work has shown that icv administration of CRH in mouse brain leads to ERK1/2 activation in the hippocampus and basoamygdalar complex making these areas suitable for the study of CRH neuromodulatory capability. The aim of our work is to characterize the signaling pathways activated by CRH receptors type 1 (CRHR1) and type 2 alpha (CRHR2α) using as a model the mouse hippocampal neuronal cell line HT22 stably expressing these receptors (HT22-CRHR1/HT22-CRHR2α). We have shown that HT22-CRHR1 cells recapitulate essential features of the cAMP response to CRH seen in primary cell cultures, making this an in vitro model useful to perform molecular and cellular experiments that would be more complex, difficult, and even unfeasible, in vivo. Furthermore, signaling mechanisms downstream CRHR1 have proven to be highly dependent on the cellular context, highlighting the importance of using neuronal derived cell lines like HT22.We use molecular and pharmacological tools to identify downstream effectors of CRHR1 and CRHR2α. The spatiotemporal features of signaling responses are assessed with Föster Resonance Energy Transfer (FRET)-based biosensors that allow real-time observation of signaling in live cells, and flow cytometric studies for quantitation of surface receptor. ERK1/2, CREB and Akt are effectors of these GPCRs and dependent on cAMP increase in HT22-CRHR1 cells. We report that activation profiles of these effectors depended on the CRHR and the ligand tested. Besides, CRHR1 and CRHR2α exhibit distinct intracellular distribution: CRH activation promoted a fast internalization of CRHR1, whereas CRHR2α remained intracellularly located until ligand activation. We discuss these findings in terms of different structural features of these GPCRs. We analyzed whether the Gβγ complex was involved in signaling pathways downstream of CRH-activated CRHR1 with a Gβγ scavenger (Gα-transducin) or a pharmacological inhibitor (gallein) finding reduced cAMP, ERK1/2, CREB and Akt responses in absence of the complex. Our preliminary results suggest that Gβγ may also play a role in CRHR1 intracellular trafficking, as complex blockage led to a decrease in CRH-induced receptor internalization. Thus, the molecular analysis of CRHRs signaling in a neuronal cell context provide clues to understand different functional roles of CRHR1 and CRHR2α in the central nervous system