Molecular mechanisms of CRH/UCNs signaling and trafficking mediated by CRHR2α in a neuronal cell context

NATALIA G. ARMANDO; PAULA A. DOS SANTOS CLARO; SUSANA SILBERSTEIN

Simposio; 22nd EMBL PhD Symposium: The Roaring 20s - A New Decade for Life Sciences; 2020

EMBL- Heidelberg

The corticotropin-releasing hormone (CRH) system, its ligands (CRH and urocortins 1-3) and receptors (CRHR1 and CRHR2) orchestrate the response and adaptation to stress. Dysregulation of the CRH system is causally linked to psychiatric disorders as anxiety and depression. The CRHR2α splice variant, the main isoform in the mouse brain, has an uncleavable signal peptide which gives this receptor special trafficking and signaling characteristics. Little is known about the molecular mechanisms of CRHR2α action in a neuronal context. Our aim is to characterize the UCNs/CRHR2α signaling and trafficking in a neuronal context using the mouse hippocampal cell line HT22 stably expressing the receptor (HT22-CRHR2α). We also used MIN6 cells that express CRHRs endogenously and mouse primary cell cultures from cortex and hippocampus. ERK1/2, CREB and Akt are activated downstream CRHR2α in both cell types. In HT22-CRHR2α, activation was dependent on cyclic AMP (cAMP) generated by the soluble adenylyl cyclase (sAC) and transmembrane adenylyl cyclases, while in MIN6 was independent of this second messenger as CRHRs stimulation did not evoke an increase in cAMP. UCN-stimulated CRHR2α led to PKA activation that was involved in effector activation. c-fos transcriptional induction was dependent on the ligand and the cell type. Upon stimulation, HT22-CRHR2α cells undergo morphological changes that required sAC-generated cAMP and PKA activation. We observed an unusual trafficking for the CRHR2α where the fraction of the receptor in the plasma membrane increased 6 min after stimulation returning to basal levels after 30 min. To evaluate the role of endocytosis in CRHR2α signaling we measured effectors activation with pharmacological inhibitors and found that pERK1/2 and pAkt levels depend on this process in a opposite manner.These results highlight the importance of the cellular context and gives relevant information for understanding the role of the UCNs and the CRHR2α in the CRH system.