PROJECT SUMMARY/ABSTRACT Pharmacologic therapy for common forms of chronic pain is ineffective and plagued with side effects. The long- term goal of the parent grant is to reveal mechanisms of pain/nociceptive signaling and define drug targets. The grant examines the mechanisms by which G protein-coupled receptors (GPCRs) within endosomes (eGPCRs) generate sustained signals in subcellular compartments that underlie persistent neuronal hyperexcitability and chronic pain. The central hypothesis is that activation of pronociceptive eGPCRs produces nociceptive signaling and most forms of chronic pain; antagonists of eGPCRs block nociceptive signaling and are anti-nociceptive. Nanoparticles containing eGPCR amtagonists engineered to release cargo in the acidic endosomal environment are used to discover the mechanisms of eGPCR signaling and to validate eGPCRs as therapeutic targets for the treatment of chronic inflammatory, neuropathic and cancer pain. The administrative supplement builds on and extends the concepts of the parent grant to receptor tyrosine kinases (RTKs). RTKs are a family of cell surface receptors for growth factors. In addition to their well-known roles in proliferation and differentiation, certain RTKs also signal pain. Experiments will study endosomal signaling of tropomyosin receptor kinase A (TrkA), the high affinity receptor for nerve growth factor (NGF). NGF is produced by damaged tissues and tumors. NGF activates TrkA on nociceptors to mediate excitability and pain. Neuropilin 1 (NRP1) will also be studied, since preliminary results suggest that NRP1 is a previously unrecognized coreceptor for NGF with an important role in pain. NRP1 is transmembrane coreceptor for vascular endothelial growth factor, but its role in NGF signaling is unexplored. The Spefic Aim is to discover the mechanisms of eRTK signaling that regulate excitability of trigeminal nociceptors and validate eRTKs as a therapeutic target for chronic craniofacial pain. Biophysical and imaging approaches will be used to study the assembly and signaling of NGF/TrkA/NRP1 signalosomes in endosomes of mouse trigeminal nociceptors. Neuronal sensitization and activation of ion channels will be studied by electrophysiology and Ca2+ imaging. Preclinical models of trigeminal neuropathic pain and oral cancer pain will be studied in mice. NP-encapsulated antagonists and inhibitors of clathrin-mediated endocytosis will be used to discover the importance of endosomal signals for NGF-mediated hyperexcitability of nociceptors and to validate endosomal NGF/TrkA/NRP1 as therapeutic targets. The proposed pain mechanism is a novel explanation that resolves the enigma of widespread clinical trial failures of RTK-targeted drugs. Innovation in the proposal extends to the NP approach to probe the mechanism and validate the target. The proposal is clinically significant because it validates an eRTK-target that offers superior pain relief with fewer side-effects and is applicable to most pa...