This thesis presents a comprehensive investigation into the structure and function of four orphan G protein-coupled receptors (GPCRs), GPR161 and the proton-sensing GPCRs, GPR4, GPR65, and GPR68. Orphan GPCRs, which lack known endogenous ligands, represent a challenging yet promising frontier in human biology and health due to their potential as novel therapeutic targets.

The first part of my thesis presents the structure-based deorphanization of GPR161, an orphan receptor involved in the Hedgehog signaling pathway. In this study, I and others study the activation mechanism of GPR161 to show a dual mode of activation that depends on both a self-interaction for stability and an allosteric cholesterol. While these features explain the cAMP signaling properties of GPR161, they unexpectedly did not explain the activity of GPR161 in the Hedgehog pathway. Our findings highlight the nuanced biology behind GPR161, and potentially other orphans, that cannot be known until a ligand is identified.

The second part of the thesis describes the activation mechanism for the proton-sensing GPCRs, GPR4, GPR65, and GPR68. Despite their physiological importance, the mechanism by which these receptors sense protons and transduce this signal into a cellular response has remained elusive. Our work identifies a distributed network of residues across the extracellular half of these receptors that contribute to activation, providing a new framework for understanding proton-sensing membrane proteins.

Collectively, these studies provide insights into the activation mechanisms of these GPCRs and pave the way for the development of novel modulators targeting these receptors. In addition to the specific scientific findings for these receptors, I hope these biochemical and biophysical techniques are applied to other orphan GPCRs so that their biology and therapeutic potential is realized.