Novel interaction between neurotrophic factor-α1/carboxypeptidase E and serotonin receptor, 5-HTR1E, protects human neurons against oxidative/neuroexcitotoxic stress via β-arrestin/ERK signaling

Protecting neurons from death during oxidative and neuroexcitotoxic stress is key for preventing cognitive dysfunction. We uncovered a novel neuroprotective mechanism involving interaction between neurotrophic factor-alpha 1 (NF-alpha 1/carboxypeptidase E, CPE) and human 5-HTR1E, a G protein-coupled serotonin receptor with no previously known neurological function. Co-immunoprecipitation and pull-down assays confirmed interaction between NF alpha 1/CPE and 5-HTR1E and I-125 NF-alpha 1/CPE-binding studies demonstrated saturable, high-affinity binding to 5-HTR1E in stably transfected HEK293 cells (Kd = 13.82 nM). Treatment of 5-HTR1E stable cells with NF-alpha 1/CPE increased pERK 1/2 and pCREB levels which prevented a decrease in pro-survival protein, BCL2, during H2O2-induced oxidative stress. Cell survival assay in beta-arrestin Knockout HEK293 cells showed that the NF-alpha 1/CPE-5-HTR1E-mediated protection against oxidative stress was beta-arrestin-dependent. Molecular dynamics studies revealed that NF-alpha 1/CPE interacts with 5-HTR1E via 3 salt bridges, stabilized by several hydrogen bonds, independent of the serotonin pocket. Furthermore, after phosphorylating the C-terminal tail and intracellular loop 3 (ICL3) of NF-alpha 1/CPE-5-HTR1E, it recruited beta-arrestin1 by forming numerous salt bridges and hydrogen bonds to ICL2 and ICL3, leading to activation of beta-arrestin1. Immunofluorescence studies showed 5-HTR1E and NF-alpha 1/CPE are highly expressed and co-localized on cell surface of human hippocampal neurons. Importantly, knock-down of 5-HTR1E in human primary neurons diminished the NF-alpha 1/CPE-mediated protection of these neurons against oxidative stress and glutamate neurotoxicity-induced cell death. Thus, NF-alpha 1/CPE uniquely interacts with serotonin receptor 5-HTR1E to activate the beta-arrestin/ERK/CREB/BCL2 pathway to mediate stress-induced neuroprotection.

Automated summary

Interaction between NF-alpha 1/CPE and 5-HTR1E activates the beta-arrestin/ERK/CREB/BCL2 pathway to protect neurons from oxidative stress and neuroexcitotoxicity, preventing cognitive dysfunction.