Endosomal Platforms for Signaling Pain

PROJECT SUMMARY/ABSTRACT G protein-coupled receptors (GPCRs) and receptor tyrosine kinases (RTKs) are mediators of and therapeutic targets for diverse disorders, including chronic pain. This proposal challenges the dogmas that these receptors signal primarily from the plasma membrane and that cell surface receptors are the optimal therapeutic targets. Completed studies revealed that GPCR endosomal signaling controls pain and that endosomally-targeted antagonists provide more effective analgesia than conventional drugs. The renewal application extends these concepts to tropomyosin receptor kinase A (TrkA), a RTK for nerve growth factor (NGF). Although successful for the treatment of pain, NGF monoclonal antibodies have not been approved due to adverse outcomes of systemic antagonism. A deeper understanding of how NGF and TrkA signal pain is required. Anatomical and electrophysiological studies with mouse and human nociceptors, behavioral studies in mice, biophysical assays using recombinant proteins, model cells and nociceptors, and nanoparticle-encapsulated antagonists will be used to study NGF pain. Aim 1 hypothesizes that neuropilin 1 (NRP1) is required for NGF-induced sensitization of nociceptors and NGF-evoked nociception. Preliminary studies suggest that NRP1 and the scaffolding protein GAIP/RGS19-interacting protein (GIPC1) are necessary for NGF-induced nociception. Co- expression of TrkA, NRP1 and GIPC1 protein and mRNA in mouse and human nociceptors will be studied. Inhibitors of NRP1 and GIPC1 will be used to ascertain their contributions to NGF- induced sensitization of nociceptors and nociception. Aim 2 hypothesizes that NRP1 acts as an NGF coreceptor and TrkA chaperone to enhance NGF signaling of pain in nociceptors. Biophysical approaches will be used to study NGF association with NRP1, assembly of TrkA/NRP1 heteromers, TrkA surface expression, and NGF signaling in subcellular compartments of nociceptors. The role of GIPC1 as a scaffold for TrkA and NRP1 association will be studied. Aim 3 hypothesizes that endocytosis and endosomal NGF/TrkA/NRP1 signaling in nociceptors mediates sustained sensitization and nociception. The contribution of endocytosis to NGF-induced nociception will be studied using inhibitors of endocytosis and NGF/TrkA/NRP1 antagonists encapsulated into nanoparticles designed to deliver cargo to endosomes of nociceptors. NGF-induced endocytosis of TrkA/NRP1, compartmentalized signaling, nociceptor sensitization and nociception will be studied. A deep understanding of TrkA trafficking-dependent signaling will provide insights into the mechanisms and treatment of chronic pain, with implications for other RTK-mediated pathologies (e.g., cancer).