Abstract
The biology of a protein cannot be fully understood in isolation from its structure or its physiological microenvironment. Reducing a protein to its linear sequence or studying it within arbitrary buffers yields an incomplete picture of its behavior. This thesis investigates the necessity of context-dependent inquiry across two distinct classes of membrane proteins: uncharacterized targets within the "dark proteome" and canonical G-protein coupled receptors (GPCRs) operating in complex physiological spaces.
A protein’s biological function can be preserved through its structure despite sequence divergence—a class we define as “superdark” proteins. Historically inaccessible to systematic study, they can now be identified by mining the proteome using structural informatics and recent advances like AlphaFold2. Applying this principle, we identify TMEM184C as a superdark G-protein coupled receptor (GPCR) that lacks sequence homology but exhibits striking similarity to the characteristic 7TM architecture. Although direct G-protein coupling remains unresolved, TMEM184C is highly evolutionarily conserved, recruits arrestins and GPCR kinases, and localizes to intracellular vesicles rather than the plasma membrane. These vesicles mediate intercellular cargo transfer across cellular projections and contribute to the formation of these projections. We further elucidate the mechanisms by which TM184C is involved in mTOR signaling and autophagy, and demonstrate the relevance of this regulation to cancer biology. Collectively, these findings reframe and validate a new discovery paradigm for uncharacterized proteins.
This same microenvironmental logic is applied to canonical GPCRs, specifically the adenosine A2A receptor (A2AR) and the opioid receptor family. Collectively, these findings reframe receptor pharmacology.
Across all chapters, this thesis establishes that structure-first, context-sensitive investigation is a broadly applicable and powerful framework for decoding protein biology.