Mice expressing fluorescent PAR2 reveal that endocytosis mediates colonic inflammation and pain.

G protein-coupled receptors (GPCRs) regulate many pathophysiological processes and are major therapeutic targets. The impact of disease on the subcellular distribution and function of GPCRs is poorly understood. We investigated trafficking and signaling of protease-activated receptor 2 (PAR2) in colitis. To localize PAR2 and assess redistribution during disease, we generated knockin mice expressing PAR2 fused to monomeric ultrastable green fluorescent protein (muGFP). PAR2-muGFP signaled and trafficked normally. PAR2 messenger RNA was detected at similar levels in Par2-mugfp and wild-type mice. Immunostaining with a GFP antibody and RNAScope in situ hybridization using F2rl1 (PAR2) and Gfp probes revealed that PAR2-muGFP was expressed in epithelial cells of the small and large intestine and in subsets of enteric and dorsal root ganglia neurons. In healthy mice, PAR2-muGFP was prominently localized to the basolateral membrane of colonocytes. In mice with colitis, PAR2-muGFP was depleted from the plasma membrane of colonocytes and redistributed to early endosomes, consistent with generation of proinflammatory proteases that activate PAR2 PAR2 agonists stimulated endocytosis of PAR2 and recruitment of Gαq, Gαi, and ß-arrestin to early endosomes of T84 colon carcinoma cells. PAR2 agonists increased paracellular permeability of colonic epithelial cells, induced colonic inflammation and hyperalgesia in mice, and stimulated proinflammatory cytokine release from segments of human colon. Knockdown of dynamin-2 (Dnm2), the major colonocyte isoform, and Dnm inhibition attenuated PAR2 endocytosis, signaling complex assembly and colonic inflammation and hyperalgesia. Thus, PAR2 endocytosis sustains protease-evoked inflammation and nociception and PAR2 in endosomes is a potential therapeutic target for colitis.

Targeting G protein-coupled receptors for the treatment of chronic pain in the digestive system.

Chronic pain is a hallmark of functional disorders, inflammatory diseases and cancer of the digestive system. The mechanisms that initiate and sustain chronic pain are incompletely understood, and available therapies are inadequate. This review highlights recent advances in the structure and function of pronociceptive and antinociceptive G protein-coupled receptors (GPCRs) that provide insights into the mechanisms and treatment of chronic pain. This knowledge, derived from studies of somatic pain, can guide research into visceral pain. Mediators from injured tissues transiently activate GPCRs at the plasma membrane of neurons, leading to sensitisation of ion channels and acute hyperexcitability and nociception. Sustained agonist release evokes GPCR redistribution to endosomes, where persistent signalling regulates activity of channels and genes that control chronic hyperexcitability and nociception. Endosomally targeted GPCR antagonists provide superior pain relief in preclinical models. Biased agonists stabilise GPCR conformations that favour signalling of beneficial actions at the expense of detrimental side effects. Biased agonists of µ-opioid receptors (MOPrs) can provide analgesia without addiction, respiratory depression and constipation. Opioids that preferentially bind to MOPrs in the acidic microenvironment of diseased tissues produce analgesia without side effects. Allosteric modulators of GPCRs fine-tune actions of endogenous ligands, offering the prospect of refined pain control. GPCR dimers might function as distinct therapeutic targets for nociception. The discovery that GPCRs that control itch also mediate irritant sensation in the colon has revealed new targets. A deeper understanding of GPCR structure and function in different microenvironments offers the potential of developing superior treatments for GI pain.