Endosomal MrGPRX1 signaling sensitizes TRPV1 to enhance itch

G protein-coupled receptors (GPCRs) and TRPV (transient receptor potential vanilloid) channels are crucial for signal transduction in physiological processes, including neurotransmission, pain, and itch. Downstream effectors of GPCR signaling can directly stimulate TRPV channels or enhance their sensitivity to stimuli, a process known as TRPV sensitization. Traditionally, GPCRs are activated at the cell surface by extracellular agonists, triggering signaling cascades. Recent evidence suggests GPCRs continue to signal from intracellular organelles. The human Mas-related G-protein coupled receptor X1 (MrGPRX1) is a GPCR expressed in primary sensory neurons involved in nociception and pruritus. Recent studies demonstrated how intracellular GPCR signaling regulates neuronal activity. However, there is no evidence characterizing MrGPRX1 trafficking or intracellular signaling. Herein, we characterized MrGPRX1 signaling within the endosomal network and its role in sensitizing TRPV1 channels to enhance itch signaling. Utilizing subcellular targeted biosensors, we demonstrated MrGPRX1 can traffic and signal from endosomes. Immunofluorescence analysis showed that MrGPRX1 internalizes following BAM8-22 stimulation. BRET assays revealed that MrGPRX1 activation induces Gα _q and β-arrestin-1 recruitment to the plasma membrane and early endosomes. Inhibition of dynamin or clathrin blocked BAM8-22-induced MrGPRX1 endocytosis and decreased nuclear extracellular signal-regulated kinase (ERK) signaling. Calcium signaling confirmed that MrGPRX1-mediated TRPV1 sensitization is mediated by protein kinase C and ERK activation. Our findings reveal a novel role for MrGPRX1 endosomal signaling in TRPV1 sensitization. Understanding the mechanisms of MrGPRX1 signaling offers valuable insights into differentiating between pain and itch pathways, aiding in the development of targeted therapies for chronic pain and persistent itch.