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

Abstract

Protease‐activated receptor‐2 (PAR2) 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, PAR2 can signal from endosomes as well as the plasma membrane, and sustained endosomal signaling underlies disease‐relevant processes. Our knowledge of the role of PAR2 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 PAR2 fused to monomeric ultrastable GFP (muGFP). To investigate whether mouse PAR2 fused at the C‐terminus to muGFP was functional, we compared agonist‐evoked signaling and trafficking of PAR2‐muGFP and PAR2 with a C‐terminal HA11 epitope expressed in KNRK cells. Trypsin and the PAR2 selective peptide agonist 2‐Furoyl‐LIGRLO‐NH2 similarly stimulated calcium signals and PAR2 endocytosis. Thus, muGFP does not disrupt PAR2 signaling and trafficking. We then generated knockin mice expressing mouse PAR2 fused at the C‐terminus to muGFP. RT‐PCR revealed similar levels of expression of PAR2 in PAR2‐muGFP and wild‐type mice. Western blotting of colonic extracts using a GFP antibody revealed expression of PAR2‐muGFP at the protein level. We localized PAR2‐muGFP by immunofluorescence and confocal microscopy using the GFP antibody. PAR2‐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, PAR2‐muGFP was principally localized to the plasma membrane of colonocytes. Exposure to trypsin or 2‐Furoyl‐LIGRLO‐NH2 induced redistribution of PAR2‐muGFP from the plasma membrane to endosomes. PAR2‐muGFP mice provide a valuable tool to study the localization, regulation, and trafficking of PAR2 in intact animals in healthy and diseased conditions.Support or Funding InformationNIH