Experimental Validation\nof the Neurotrophic Factor-α1\nBinding Site on the Serotonin Receptor 1E (HTR1E) Responsible for\nβ‑Arrestin Activation and Subsequent Neuroprotection

Stress, such as neuroexcitotoxicity\nand oxidative stress, as well\nas traumatic brain injury, will result in neurodegeneration. Deciphering\nthe mechanisms underlying neuronal cell death will facilitate the\ndevelopment of drugs that can promote neuronal survival and repair\nthrough neurogenesis. Many growth and trophic factors, including transforming\ngrowth factors (TGFs), insulin-like growth factors (IGFs), epidermal\ngrowth factor (EGF), fibroblast growth factor 2 (FGF2), and brain-derived\nneurotrophic factor (BDNF), are known to play a role in neuroprotection\nand neurogenesis. Neurotrophic factor-α1 (NF-α1), also\nknown as carboxypeptidase E (CPE), has been shown experimentally to\nhave neuroprotective activity, acting extracellularly, independent\nof its intracellular enzymatic function in prohormone processing.\nWe previously reported experiments and molecular dynamics (MD) simulations\nshowing that a 200 amino acid segment of NF-α1/CPE interacts\nwith the serotonin receptor 1E (HTR1E) to protect human neurons against\noxidative and neuroexcitotoxic stress via β-arrestin and extracellular\nsignal-regulated kinase (ERK) signaling. We report here validation\nof our previously predicted binding site with a series of 16 carboxypeptidase\nE (CPE) mutants, identifying 3 mutants that substantially decrease\nthe binding to HTR1E. We then carried out pERK studies to show that\nthese 3 mutants also dramatically reduce β-arrestin activation.\nThis was followed by MD simulations of 8 selected mutants, finding\nthat the same 3 most dramatically reduced binding of the mutated CPE\nto 5-HTR1E. Then, we examined the binding of β-arrestin to these\n3 (after phosphorylating the intracellular Ser and Thr) and found\nthat the predicted binding decreased dramatically. Then, we examined\nthe predicted activation of the β-arrestin by these 3 and found\na dramatic decrease, just as in the pERK experiments. We consider\nthat these experiments and simulations fully validate the predicted\nbinding site for CPE, identifying the key amino acid residues critical\nfor binding and biological activity. This provides the target for\nexperiments and <i>in silico</i> computational screening\nto identify small molecules to replace the CPE protein as novel drugs\nto protect human neurons against oxidative/neuroexcitotoxic stress\nvia β-arrestin/ERK signaling.