STUDY OF THE Gbeta-gamma COMPLEX IN CRHR1 SIGNALING MECHANISMS

SENIN, S.; SILBERSTEIN, S.; DOS SANTOS CLARO, P. A.; INDA, C.

Congreso; Reunión conjunta de las Sociedades de Biociencias 2017, LXII Reunión Anual de la Sociedad Argentina de Investigacion Clínica. Buenos Aires, 13-17 de noviembre de 2017.; 2017

Corticotropin releasing hormone (CRH) plays a key role in the regulation of neuroendocrine, autonomic, and behavioral response to stress. The CRH/CRHR1 system dysregulation is linked to the onset and development of affective disorders, such as anxiety and depression. The aim of our work is to characterize the signaling pathways activated by CRH receptor type 1 (CRHR1). Being a GPCR, the canonical signaling pathway of CRHR1 is linked to the activation of heterotrimeric G proteins, which are composed of α, β and γ subunits. The most studied signal transduction cascade for GPCRs is the one downstream of Gα subunits.Using as a model the hippocampal neuronal cell line HT22 stably expressing CRHR1 (HT22-CRHR1), we analyzed whether the Gβγ complex was involved in signaling pathways downstream of CRH-activated CRHR1. Cells transfected with a Gβγ scavenger (Gα-transducin) or pretreated with a pharmacological inhibitor (gallein) showed reduced ERK1/2, Akt and CREB activation levels in response to CRH. In HT22-CRHR1 both ERK1/2 and CREB are dependent on cAMP increase. Using FRET-based biosensors we determined that the genetic and pharmacological blockage of the Gβγ complex diminished the CRH-mediated cAMP response. Having established a role for Gβγ, we performed a screening of transmembrane adenylyl cyclases (tmACs) in HT22-CRHR1 cells by RT-PCR finding that tmACs positively and negatively regulated by Gβγ are expressed in these cells. CRHR1 activates G protein?dependent and internalization-dependent signaling mechanisms, being able to signal from endosomal compartments. Our preliminary results suggest that the Gβγ dimer may also play a role in CRHR1 endocytosis, as the blockage of the complex led to a decrease in CRH-induced receptor internalization.These results indicate that Gβγ participates in CRH/CRHR1 signaling in a hippocampal neuronal context. The specific role of this pathway and the crosstalk between Gα- and Gβγ-activated cascades are currently being explored.