Study of signaling and trafficking of CRH receptors: the use of a new Aza-bodipy fluorescent marker

ARMANDO, N.G.; SZALAI, A.M.; DOS SANTOS CLARO, P.; SENIN, S.; CAVASOTTO, CLAUDIO N.; ARAMENDÍA, P.F.; SILBERSTEIN, S.

Buenos Aires

Congreso; Frontiers in Bioscience 3; 2018

The corticotropin-releasing hormone (CRH) system, its ligands (CRH, urocortins 1-3) and receptors (CRHRs) orchestrates the response to stress, acting on the hypothalamic-pituitary-adrenal axis and on specific brain regions. CRH and related peptides operate through the GPCRs CRHR1 and CRHR2, which display different localization and ligand affinity. Dysregulation of this system is linked to psychiatric stress-mediated disorders. Our goal is to identify cellular mechanisms involved in CRH/UCNs signaling downstream of its CRHRs which will be instrumental for understanding their pathophysiological function in the stress response. CRHRs tracking in live neuronal contexts and real time measurement of the signaling responses are our major tools. We are exploring signaling pathways activated by CRHRs using a mouse hippocampal neuronal HT22 cells as a model, stably expressing CRHRs (HT22-CRHR1 and HT22-CRHR2α cells). We obtained a fluorescent probe for CRHR1 based on the structure of the commercial antagonist CP- 376395 that was co-crystalized with CRHR1. This new probe (ABP-09) is a small fluorescent molecule that allowed us to perform stochastic optical reconstruction microscopy (STORM) and proved colocalization with CRHR1 at 23 nM of resolution. We demonstrate antagonist effects of ABP-09 for CRHR1 in similar concentrations as the well-established antagonist CP-376395. Also, our results suggest that the commercial antagonist used to design ABP-09 inhibits CRHstimulated CRHR1 internalization.On the other hand, using fluorescent imaging and flow cytometry we appreciated a difference in trafficking between CRHRs: while the amount of CRHR2α at the plasma membrane increases after 6 minutes UCN1 stimulation, the amount of CRHR1 decreases shortly after CRH stimulation and remains in internalcompartments for at least 30 min.