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.; PIAZZA, V.; SENIN, S.; CAVASOTTO, CLAUDIO N.; ARAMENDÍA, P.F.; SILBERSTEIN, S.

Bariloche

Simposio; South American Spring Symposium in Signal Transduction and Molecular Medicine (SISTAM 2018); 2018

Thecorticotropin-releasing hormone (CRH) system, its ligands and receptorsorchestrates the response and adaptation to stress, acting on the hypothalamic-pituitary-adrenalaxis and in brain regions. Dysregulation of CRH system is causally linked to psychiatricdisorders as anxiety/depression. There are two G-protein-coupled receptors(GPCRs), CRHR1 and CRHR2, encoded by different genes which display different brainlocalization and ligand preferences. CRHR1 is widely distributed in the brainand pituitary whereas CRHR2 displays a more restricted distribution. The CRHR2αsplicing variant, the main isoform of the mouse brain, has an uncleavablesignal peptide which is supposed to give this receptor specific trafficking andsignaling characteristics in comparison with CRHR1. Toexplore the mechanisms involved in the signaling cascades and trafficking ofeach receptor we generated stable clones expressing CRHR1 and CRHR2α in hippocampalneuronal cell line HT22. Here, we demonstrate that ligand-activated CRHRs exhibit remarkablydifferent cell surface expression. CRHR1 internalization is fast, remaining ininternal compartments after 30 min of CRH stimulation. The fraction of CRHR2α increasedin the plasma membrane after 6 min of stimulation returning to basal levelsafter 30 min of ligand activation. Owing to the lack of reliable specificantibodies for CRHRs, studying its distribution by fluorescence microscopyrequires a different labeling approach. In line with this, we designed astrategy to label the CRHR1 in live cells using a small fluorescent molecule(ABP-09) that enables the performance of stochastic optical reconstructionmicroscopy (STORM) with 23 nm resolution. In this work we show thecolocalization of this probe with CRHR1 using HT22-CRHR1 cells. Moreover, we demonstrateantagonistic effects of the aza-BODIPY probe at similar concentrations as thewell-established commercial antagonist CP-376395.