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

ABSTRACT 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.