Electronic Thesis and Dissertation Repository

Thesis Format

Integrated Article


Master of Science


Physiology and Pharmacology


Ramachandran, Rithwik


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.

Summary for Lay Audience

The various systems of our bodies are controlled through an intercellular communication network mediated by soluble chemical messengers. These chemical messengers relay information throughout our bodies by binding receptors expressed on the surface of our cells. Exogenous molecules, drugs, can be made to mimic these chemical messengers, which can then bind cell surface receptors to modulate bodily functions and treat disease. G-protein coupled receptors (GPCRs) are the largest targets of FDA approved drugs, with around 25-36% of all FDA approved drugs targeting GPCRs. Despite this fact, the second largest GPCR subfamily, the adhesion GPCRs (aGPCRs), are not targeted by a single FDA approved drug. These receptors are attractive drug targets as they have been shown to be involved in brain development, the immune system, inflammatory diseases, and many different cancers. Lack of drug development at these receptors stems from the fact that they are activated in a two-cell system by mechanical force. As their mechanism of action is very difficult to reproduce and control in a laboratory setting, in this thesis we set out to design tools to enable their study. We designed a method in where aGPCRs could be activated by proteolytic cleavage instead of mechanical force, allowing receptor activation to be controlled by adjusting the concentration of enzyme. Further, we determined a method of predicting aGPCR agonist peptides. We then tested our two new methods on four of the more well studied aGPCRs: CD97 (ADGRE5), EMR2 (ADGRE2), GPR56 (ADGRG1), and BAI1 (ADGRB1). Both methods proved to be very effective and based on our result we believe they can be applied to study the whole family of 33 aGPCRs.