PHOSPHORYLATED SERINE AND THREONINE RESIDUES OF PAR2 C‐TERMINUS DIRECT β‐ARRESTIN 1/2 RECRUITMENT AND BINDING

Protease activated receptor 2 (PAR2)is a seven transmembrane receptor. Its activation involves tryptic cleavage of N terminus releasing a tethered ligand. PAR2 predominantly signals via Gαq pathway. Classic paradigm of GPCR downregulation requires phosphorylation of serine/threonine residues in C terminus by second messenger kinases and G protein coupled receptor kinases. This recruits β arrestin 1/2 which uncouples the receptor from G proteins and mediates receptor endocytosis. Role of phosphorylation of PAR2 C terminus on β arrestin recruitment and binding are yet to be investigated. PAR2 signaling by Gαq, generates Protein kinase C (PKC), which can phosphorylate specific sites in its C tail. Blocking the Gαq cascade with inhibitors, we show that PAR2 recruits β‐arrestin independent of PKC phosphorylation. PAR2S363/6A, with mutations in the PKC sites show slower rates of β arrestin 1/2 recruitment compared to PAR2WT. Bioluminescence Resonance Energy Transfer (BRET) assay indicates reduced affinity of PAR2S363/6a towards β arrestin 1/2. Experiments will be designed using a phosphorylation nil mutant to determine the specific role of PAR2 C terminal phosphorylation on β arrestin dependent signaling pathways.