Experimental Validation of the Neurotrophic Factor-α1 Binding Site on the Serotonin Receptor 1E (HTR1E) Responsible for β-Arrestin Activation and Subsequent Neuroprotection

Year: 2024 DOI: 10.1021/acsomega.4c05367
Stress, such as neuroexcitotoxicity and oxidative stress, as well as traumatic brain injury, will result in neurodegeneration. Deciphering the mechanisms underlying neuronal cell death will facilitate the development of drugs that can promote neuronal survival and repair through neurogenesis. Many growth and trophic factors, including transforming growth factors (TGFs), insulin-like growth factors (IGFs), epidermal growth factor (EGF), fibroblast growth factor 2 (FGF2), and brain-derived neurotrophic factor (BDNF), are known to play a role in neuroprotection and neurogenesis. Neurotrophic factor-α1 (NF-α1), also known as carboxypeptidase E (CPE), has been shown experimentally to have neuroprotective activity, acting extracellularly, independent of its intracellular enzymatic function in prohormone processing. We previously reported experiments and molecular dynamics (MD) simulations showing that a 200 amino acid segment of NF-α1/CPE interacts with the serotonin receptor 1E (HTR1E) to protect human neurons against oxidative and neuroexcitotoxic stress via β-arrestin and extracellular signal-regulated kinase (ERK) signaling. We report here validation of our previously predicted binding site with a series of 16 carboxypeptidase E (CPE) mutants, identifying 3 mutants that substantially decrease the binding to HTR1E. We then carried out pERK studies to show that these 3 mutants also dramatically reduce β-arrestin activation. This was followed by MD simulations of 8 selected mutants, finding that the same 3 most dramatically reduced binding of the mutated CPE to 5-HTR1E. Then, we examined the binding of β-arrestin to these 3 (after phosphorylating the intracellular Ser and Thr) and found that the predicted binding decreased dramatically. Then, we examined the predicted activation of the β-arrestin by these 3 and found a dramatic decrease, just as in the pERK experiments. We consider that these experiments and simulations fully validate the predicted binding site for CPE, identifying the key amino acid residues critical for binding and biological activity. This provides the target for experiments and in silico computational screening to identify small molecules to replace the CPE protein as novel drugs to protect human neurons against oxidative/neuroexcitotoxic stress via β-arrestin/ERK signaling.