TY - JOUR
T1 - Mice expressing fluorescent PAR2 reveal that endocytosis mediates colonic inflammation and pain
AU - Latorre, Rocco
AU - Hegron, Alan
AU - Peach, Chloe J.
AU - Teng, Shavonne
AU - Tonello, Raquel
AU - Retamal, Jeffri S.
AU - Klein-Cloud, Rafael
AU - Bok, Diana
AU - Jensen, Dane D.
AU - Gottesman-Katz, Lena
AU - Rientjes, Jeanette
AU - Veldhuis, Nicholas A.
AU - Poole, Daniel P.
AU - Schmidt, Brian L.
AU - Pothoulakis, Charalabos H.
AU - Rankin, Carl
AU - Xie, Ying
AU - Koon, Hon Wai
AU - Bunnett, Nigel W.
N1 - Publisher Copyright:
© This article is distributed under Creative Commons Attribution-NonCommercialNoDerivatives License 4.0 (CC BY-NC-ND).
PY - 2022/2/8
Y1 - 2022/2/8
N2 - 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.
AB - 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.
KW - Endocytosis
KW - Inflammation
KW - Proteases
KW - Receptors
KW - Signaling
KW - Receptor, PAR-2/metabolism
KW - Humans
KW - Mice, Inbred C57BL
KW - Arrestins/metabolism
KW - Inflammation/metabolism
KW - Endosomes/metabolism
KW - Ganglia, Spinal/metabolism
KW - Colon/metabolism
KW - Signal Transduction/physiology
KW - Animals
KW - Nociception/physiology
KW - Cell Membrane/metabolism
KW - Female
KW - Mice
KW - Fluorescent Dyes/metabolism
KW - Endocytosis/physiology
KW - Pain/metabolism
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U2 - 10.1073/pnas.2112059119
DO - 10.1073/pnas.2112059119
M3 - Article
C2 - 35110404
AN - SCOPUS:85123974780
SN - 0027-8424
VL - 119
JO - Proceedings of the National Academy of Sciences of the United States of America
JF - Proceedings of the National Academy of Sciences of the United States of America
IS - 6
M1 - e2112059119
ER -