Mice Expressing Fluorescent Protease-Activated Receptor-2 as a Novel Tool to Visualize Intracellular Receptor Trafficking

Protease‐activated receptor‐2 (PAR 2 ) is a G‐protein coupled receptor (GPCR) that plays a critical role in mediating the proinflammatory and pronociceptive actions of serine and cysteine proteases in many tissues. In common with a growing number of GPCRs, PAR 2 can signal from endosomes as well as the plasma membrane, and sustained endosomal signaling underlies disease‐relevant processes. Our knowledge of the role of PAR 2 in health and disease states is hampered by an inadequate understanding of its cellular and subcellular localization. Since antibodies to GPCRs often lack sensitivity and specificity, and are unsuitable for studying receptor trafficking in living cells, we used a knockin approach to generate mice expressing PAR 2 fused to monomeric ultrastable GFP (muGFP). To investigate whether mouse PAR 2 fused at the C‐terminus to muGFP was functional, we compared agonist‐evoked signaling and trafficking of PAR 2 ‐muGFP and PAR 2 with a C‐terminal HA11 epitope expressed in KNRK cells. Trypsin and the PAR 2 selective peptide agonist 2‐Furoyl‐LIGRLO‐NH 2 similarly stimulated calcium signals and PAR 2 endocytosis. Thus, muGFP does not disrupt PAR 2 signaling and trafficking. We then generated knockin mice expressing mouse PAR 2 fused at the C‐terminus to muGFP. RT‐PCR revealed similar levels of expression of PAR 2 in PAR 2 ‐muGFP and wild‐type mice. Western blotting of colonic extracts using a GFP antibody revealed expression of PAR 2 ‐muGFP at the protein level. We localized PAR 2 ‐muGFP by immunofluorescence and confocal microscopy using the GFP antibody. PAR 2 ‐muGFP was highly expressed in epithelial cells in the digestive tract and skin, and was detected at lower levels in the enteric nervous system and pancreatic islets. In the unstimulated state, PAR 2 ‐muGFP was principally localized to the plasma membrane of colonocytes. Exposure to trypsin or 2‐Furoyl‐LIGRLO‐NH 2 induced redistribution of PAR 2 ‐muGFP from the plasma membrane to endosomes. PAR 2 ‐muGFP mice provide a valuable tool to study the localization, regulation, and trafficking of PAR 2 in intact animals in healthy and diseased conditions. Support or Funding Information NIH