2025 Theses Doctoral

Intracellular GPCR signaling platforms involved in pain

Teng, Shavonne Lee

Chronic pain is a debilitating condition and a serious public health concern. However, the detailed mechanisms underlying chronic pain are not well understood; and current treatments are inadequate and plagued with serious side effects. The recent opioid crisis highlights the need for improved pain therapies. Pain signaling is initiated by cell membrane receptors such as G protein-coupled receptors (GPCRs), which mediate many physiological and pathophysiological processes. GPCRs can be internalized into endosomes, which was conventionally thought to arrest plasma membrane signaling. Now, GPCRs have been shown to have sustained activation and signaling within endosomes and other subcellar compartments.

However, the precise cellular and molecular mechanisms for how intracellular GPCR signaling contributes to pain are not fully elucidated. This thesis focuses on GPCR signaling within intracellular compartments of protease-activated receptors (PARs), calcitonin-like receptor (CLR), and neurokinin-1 receptor (NK1R). My work mainly focuses on PAR2, a GPCR that mediates inflammatory pain. We hypothesized that these pronociceptive GPCRs form signaling complexes and continue to signal within multiple intracellular compartments including endosomes and the Golgi, and that nanoparticles loaded with antagonist can block these signals and mitigate inflammatory pain. To test our hypothesis, we first generated a genetically modified mouse line to localize the receptor in unstimulated and stimulated conditions. Resonance energy transfer assays were then employed to study compartmentalized receptor signaling, and nanoparticles containing selective antagonists were administered in preclinical mouse models of inflammatory bowel disease (IBD) to therapeutically block GPCR intracellular signaling.

To better understand GPCR cellular localization after injured and disease states in vivo, a knock-in mouse expressing PAR2 fused to monomeric ultrastable green fluorescent protein (muGFP) was generated and characterized by real-time PCR, immunofluorescence, and confocal microscopy. PAR2-muGFP was localized to endosomes in agonist-induced and colitis disease states. Resonance energy transfer (RET)-based assays were performed to examine signaling and kinetic differences of GPCRs PAR2 (Chapter 2), PAR4/PAR1 (Chapter 3), as well as CLR and NK1R (Chapter 4) within multiple subcellular compartments.

We then investigated whether compartmentalized signaling of pronociceptive GPCRs can be inhibited using targeted nanoparticle-based therapeutics. Specifically, the therapeutic potential of inhibiting PAR2 was tested using two nanoparticle formulations encapsulating PAR2 antagonist, AZ3451. To assess the efficacy of these nanoparticles in suppressing intracellular PAR2 signaling, RET-based studies were employed comparing nanoparticle-delivered AZ3451 with the free antagonist. Given the established role of PAR2 in colonic diseases, the therapeutic efficacy of nanoparticles containing AZ3451 was further evaluated in two preclinical mouse models of irritable bowel disease.
Agonist-induced and IBD mouse models demonstrated redistribution of PAR2 from the basolateral membrane of colonocytes to early endosomes. Agonists (proteases and neuropeptides) induced assembly of PAR2, PAR4/PAR1, CLR and NK1R signaling complexes (or signalosomes) at the plasma membrane, endosomes, and the Golgi. Two different types of nanoparticles, dendrimer and core-shell polymeric nanoparticles, loaded with AZ3451 inhibited PAR2 signaling complex formations in endosomes and the Golgi more effectively than unencapsulated AZ3451. Additionally, nanoparticles encapsulating AZ3451 inhibited G protein activity in subcellular compartments and blocked extracellular signal regulated kinase (ERK) activity in the cytosol and nucleus better than free antagonist. Finally, both nanoparticle formulations containing AZ3451 provided superior long-lasting analgesia and normalized aberrant behavior compared to unencapsulated drugs in two preclinical models of inflammatory bowel disease.

Overall, GPCRs generate sustained intracellular signals that play critical roles in mediating pain. Nanoparticles loaded with antagonists more effectively inhibited GPCR signaling within intracellular compartments compared to unencapsulated antagonist. In preclinical models of IBD, nanoparticles delivering PAR2 antagonist also provided greater therapeutic efficacy. These findings highlight the involvement of intracellular GPCR signaling in pain and demonstrate the therapeutic potential of targeting these intracellular signals to treat chronic diseases. Furthermore, nanoparticles are promising tools that enhance the delivery of therapeutic antagonists for more effective disease treatment. This thesis provides key evidence for the role of GPCR intracellular signaling in pain and establishes the foundation for future strategies to modulate subcellular signaling for the treatment of chronic diseases.