Study of signaling and trafficking of CRH receptors: the use of a new aza-BODIPY fluorescent marker.

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

Bariloche

Congreso; The Fourth South American Symposium in Signal Transduction and Molecular Medicine (SISTAM 2018); 2018

The corticotropin-releasing hormone (CRH) system, its ligands and receptors orchestrates the response and adaptation to stress, acting on the hypothalamic-pituitary-adrenal axis and in brain regions. Dysregulation of CRH system is causally linked to psychiatric disorders as anxiety/depression. There are two G-protein-coupled receptors (GPCRs), CRHR1 and CRHR2, encoded by different genes which display different brain localization and ligand preferences. CRHR1 is widely distributed in the brain and pituitary whereas CRHR2 displays a more restricted distribution. The CRHR2α splicing variant, the main isoform of the mouse brain, has an uncleavable signal peptide which is supposed to give this receptor specific trafficking and signaling characteristics in comparison with CRHR1. To explore the mechanisms involved in the signaling cascades and trafficking of each receptor we generated stable clones expressing CRHR1 and CRHR2α in hippocampal neuronal cell line HT22. Here, we demonstrate that ligand-activated CRHRs exhibit remarkably different cell surface expression. CRHR1 internalization is fast, remaining in internal compartments after 30 min of CRH stimulation. The fraction of CRHR2α increased in the plasma membrane after 6 min of stimulation returning to basal levels after 30 min of ligand activation. Owing to the lack of reliable specific antibodies for CRHRs, studying its distribution by fluorescence microscopy requires a different labeling approach. In line with this, we designed a strategy to label the CRHR1 in live cells using a small fluorescent molecule (ABP-09) that enables the performance of stochastic optical reconstruction microscopy (STORM) with 23 nm resolution. In this work we show the colocalization of this probe with CRHR1 using HT22-CRHR1 cells. Moreover, we demonstrate antagonistic effects of the aza-BODIPY probe at similar concentrations as the well-established commercial antagonist CP-376395. Funded by ANPCyT, CONICET and FOCEM (COF 03/11).