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

• Published in: Proceedings of the National Academy of Sciences - PNAS Vol. 119; no. 6; p. 1
• Main Authors: Latorre, Rocco, Hegron, Alan, Peach, Chloe J, Teng, Shavonne, Tonello, Raquel, Retamal, Jeffri S, Klein-Cloud, Rafael, Bok, Diana, Jensen, Dane D, Gottesman-Katz, Lena, Rientjes, Jeanette, Veldhuis, Nicholas A, Poole, Daniel P, Schmidt, Brian L, Pothoulakis, Charalabos H, Rankin, Carl, Xie, Ying, Koon, Wai, Bunnett, Nigel W
• Format: Journal Article
• Language: English
• Published: Washington National Academy of Sciences 08.02.2022
• Subjects: Agonists; Antibodies; Arrestin; Biological Sciences; Colitis; Colon; Cytokines; DNA probes; Dorsal root ganglia; Dynamin; Endocytosis; Endosomes; Epithelial cells; Epithelium; Fluorescence; Ganglia; Green fluorescent protein; Hybridization; Inflammation; Inflammatory bowel disease; Intestine; Large intestine; Membranes; mRNA; Pain; Pain perception; Permeability; Protease; Proteins; Receptors; Signaling; Therapeutic applications; Therapeutic targets
• ISSN: 0027-8424; 1091-6490
• DOI: 10.1073/pnas.2112059119

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 β-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.