Armando, N. G. | Search bioRxiv

bioRxiv2023

Role of canonical and non-canonical cAMP Sources in CRHR2α-dependent Signaling

Hippocampal neurons exhibit activation of both the conventional transmembrane adenylyl cyclases (tmACs) and the non-canonical soluble adenylyl cyclase (sAC) as sources of cyclic AMP (cAMP). These two cAMP sources play crucial roles in mediating signaling pathways downstream of CRHR1 in neuronal and neuroendocrine contexts. In this study, we investigate the involvement of both cAMP sources in the molecular mechanisms triggered by CRHR2. Here we provide evidence demonstrating that UCN1 and UCN3 exert a neuritogenic effect on HT22-CRHR2 cells, which is solely dependent on the cAMP pool generated by sAC and PKA activity but independent of ERK1/2 activation. Through the characterization of the effectors implicated in neurite elongation, we found that CREB phosphorylation and c-Fos induction rely on PKA activity and ERK1/2 phosphorylation, underscoring the critical role of signaling pathway regulation. These findings strengthen the concept that localized cAMP microdomains actively participate in the regulation of these signaling processes.

molecular biology↗

bioRxiv2022

CRHR1-mediated Akt activation involves endocytosis and soluble adenylyl cyclase activity

Corticotropin-releasing hormone receptor 1 (CRHR1) signaling initiates at the cell membrane and continues after receptor internalization. A sustained cAMP generation after ligand-activated CRHR1 endocytosis supported by the soluble adenylyl cyclase (sAC) is required for the G protein-independent phase of ERK1/2 phosphorylation in neuronal cells. Here, we report that Akt is activated by CRHR1 stimulation in a mechanism dependent on the endosomal cAMP production by sAC activity. Moreover, AktS473 phosphorylation profile was distinct to that of phospho-ERK1/2 revealing a crosstalk between these pathways. We found that the CRHR1 activation of PI3K/Akt pathway is required for a full ERK1/2 activation but negatively regulates the cAMP response and CREB phosphorylation downstream CRHR1 stimulation. Immunofluorescence colocalization analysis revealed that activated CRHR1 transits to early (Rab5) and recycling (Rab11) endosomes. Additionally, CRHR1 colocalized with two Rab5 effectors, APPL1 and EEA1. shRNA mediated knockdown uncovered a role of these proteins in CRHR1 signaling. APPL1 silencing led to a decrease in pAktS473 levels without affecting ERK1/2 activation, whereas EEA1 depletion had the opposite effect, suggesting that CRHR1 intracellular signaling diversity may be achieved through different signaling platforms.

cell biology↗