Creating Tools to Study the Signaling and Function of the Adhesion Family of GPCRs

Victor M. Mirka, The University of Western Ontario

Abstract

Adhesion GPCRs (aGPCRs) are difficult to study because they are activated by mechanical force. aGPCRs are autoproteolytically cleaved into N-terminal and C-terminal fragments. Mechanical force removes the N-terminal fragment revealing a tethered ligand activating the receptor. Proteinase Activated Receptors (PARs) are N-terminally cleaved by proteinases revealing a tethered ligand activating the receptor. We hypothesized the tethered ligand of aGPCRs could be revealed by replacing the N-terminal fragment with a PAR N-terminus. We fused the PAR2 N-terminus to the C-terminal fragments of four aGPCRs: CD97, EMR2, GPR56, and BAI1. PAR2-aGPCR chimeric receptors dose dependently recruited G-proteins and β-arrestins, supporting our hypothesis. Peptides made to mimic the tethered ligand, are sufficient to activate receptors. We developed a method for predicting aGPCR tethered ligand mimicking peptides and tested the approach for CD97. We found SSFAILMAH-NH₂ to be a potent CD97 activating peptide. In this thesis we developed two novel methods for studying the illusive aGPCRs.