Oral cancer pain is a prevalent, debilitating, and chronic condition that disrupts patients’ ability to eat and speak. Patients develop tolerance from continuous use of opioids to treat cancer pain and require escalating doses to achieve relief. Opioids also produce severe side effects, including respiratory depression, addiction, and sedation. Alternative non-opioid strategies are necessary. However, the pathobiology of oral cancer pain is currently not well understood. My research proposal explores the mechanisms by which G-protein coupled receptors (GPCRs) and transient receptor potential (TRP) ion channels regulate oral cancer pain. Oral cancers and immune cells such as macrophages secrete proteases that cleave and activate GPCRs at the cell surface. GPCR activation and G protein coupling triggers downstream signaling cascades, which can sensitize TRP channels. Specifically, the GPCR protease-activated receptor-2 (PAR2) mediates oral cancer mechanical allodynia and leads to transient receptor potential vanilloid 4 (TRPV4) activation and hyperexcitability of nociceptors. However, GPCR signaling from the plasma membrane is transient. Beta arrestins desensitize GPCRs and couple the receptors for clathrin-mediated endocytosis, which together terminate plasma membrane signaling. While endosomes were traditionally thought of as a conduit for receptor recycling and degradation, recent work from my lab demonstrated that GPCRs in endosomes continue to generate sustained signals that mediate pain transmission. However, whether PAR2 signals from endosomes to sensitize TRPV4 and evoke hyperexcitability has not been established. The purpose of this research is to identify the mechanisms of endosomal PAR2 signaling and its effect on TRPV4 sensitization in oral cancer. pH-stimulus responsive nanoparticles that deliver PAR2 antagonists to endosomes of pain-sensing trigeminal neurons will be developed to examine this pathway. Aim 1 will develop pH-stimulus responsive nanoparticles and characterize the biophysical properties of the nanoparticles loaded with PAR2 antagonist. Aim 2 will explore the mechanisms of endosomal PAR2 signaling and its role in TRPV4 sensitization via genetically-encoded biosensors and calcium indicators. Aim 3 seeks to assess mechanisms of PAR2 signaling in pre-clinical mice models of oral cancer. Investigating endosomal PAR2 signaling via pH-stimulus nanoparticle will elucidate whether endosomal GPCRs are valid therapeutic targets for oral cancer pain. In completing the aims and training plan outlined in this proposal, the graduate student, Shavonne Teng, will gain a deep understanding of GPCR signaling relevant to chronic pain. She will learn bioengineering approaches to develop nanomedicines, learn biophysical and cell biology approaches to study GPCR and TRP regulation, and learn how to study orofacial pain in mouse models. This research training will expand on her past experience and also prepare her for a research career as an ind...