5HTR1E receptor interacts with Neurotrophic factor-alpha 1 and serotonin to activate two distinct signaling pathways

Neurotrophic factor-α1/Carboxypeptidase E (NFα1/CPE) is a prohormone processing enzyme intracellularly and as a neurotrophic factor, extracellularly. Previously we showed that extracellular treatment of hippocampal neurons with NFα1/CPE protected them from oxidative stress-induced cell death through ERK/BCL2 signaling. Based on these results, we hypothesized that NFα1/CPE activation of the ERK/BCL2 pathway to mediate neuroprotection should involve interaction with receptors at the cell membrane. We have identified 5-hydroxytryptamine receptor 1E (5HTR1E), a Gi coupled serotonin receptor, as an extracellular binding partner of NFα1/CPE. This interaction was confirmed by co-IP and pull-down experiments followed by radioligand binding studies in HEK293-5HTR1E overexpressing cells. Molecular modeling studies also predicted that NFα1/CPE interacted with HTR1E, not at the serotonin binding pocket, but via the extracellular loop 2 (ECL2) of 5HTR1E, forming 3 stable salt bridges with specific amino acid residues in NFα1/CPE. To identify the HTR1E downstream signaling pathways, we used HEK293 cells stably expressing 5HTR1E. First, we tested the dose dependent effect of both serotonin and NFα1/CPE on ERK, and cAMP signaling and found that both can increase ERK phosphorylation while cAMP pathway activation is serotonin dependent only. To further dissect the 5HTR1E mediated functions of both serotonin and NFα1/CPE we tested Gi, Gq, PKA, and PI3K inhibitors and observed that these proteins are not involved in NFα1/CPE mediated ERK phosphorylation, while serotonin-stimulated ERK activation was Gi-PKA dependent. We then explored the involvement of β-arrestin, an alternative coupling mechanism with GPCRs for ERK activation. Studies in 5HTR1E expressing-HEK293-β-arrestin KO cells revealed that NFα1/CPE-5HTR1E complex was unable to induce ERK signaling in the absence of β-arrestin proteins. Furthermore, molecular docking studies revealed a very strong coupling of b-arrestin to intracellular loop (ICL) 2 and ICL3 of HTR1E. Using LDH cytotoxicity assay we demonstrated that NF-α1/CPE was able to protect HEK-293-5HTR1E expressing cells against H O -induced cytotoxicity, via increasing pro-survival protein BCL2 expression; however, this protective effect of NFα1/CPE was diminished in the β-arrestin KO cells. Our study identified two ligands for HTR1E: NFα1/CPE which interacts with HTR1E to activate β-arrestin/ERK/BCL2 signaling pathway for protecting cells against oxidative stress; and serotonin which activated a Gi-cAMP-dependent pathway, the physiological role is under investigation.