A Functional and Mechanistic Exploration of G Proteincoupled Receptor Endocytic Signaling
Cellular responses to environmental signals are mediated by a host of signal transduction proteins known as receptors. G protein-coupled receptors (GPCRs) are a major class of these signaling proteins that, despite their name, couple to a variety of downstream signaling pathways. Different drugs acting at the same GPCR can produce vastly different responses through a mechanism that has yet to be completely elucidated. Recent advances have indicated that one mechanism by which different drugs can modulate receptor signaling is by changing how a receptor is trafficked subcellularly after drug binding. The following work explores two distinct types of trafficking that are affected differentially by distinct ligands. The first, endocytosis of receptors following drug binding, is explored in Chapters 1, 2, and 3. Chapter 1 summarizes current knowledge in the field about how GPCRs control the rate of their own endocytosis, how this affects receptor signaling, and how these effects are mediated on a molecular level. Chapter 2 is an in-depth investigation of how two ligands, morphine and the endogenous peptide endomorphin-2, act at the clinically relevant μ opioid receptor (MOR) to produce distinct endocytic patterns and signaling consequences. The work shows that morphine’s signaling can be mimicked by a mutant receptor, and signaling of the mutant receptor, as well as morphine at the wild-type receptor, can be rescued by changing the receptor’s endocytic rate. Chapter 3 then explores the molecular mechanisms by which these changes in endocytic trafficking are controlled, using the β2-adrenergic receptor (B2AR) as a model. The effect on endocytosis and signaling of three B2AR-interacting proteins is investigated with complex results. Chapter 4 focuses on a distinct trafficking phenomenon, namely that of receptor recycling. Analysis software used in Chapters 2 and 3 is adapted to quantitatively measure GPCR exocytic events following ligand addition. This automated analysis is then used to both confirm previously known characteristics of MOR recycling events, and to elucidate physiological differences between MOR and B2AR recycling. Overall, this work furthers current understanding both of the subtleties that underlie differential receptor trafficking and of how those differences modulate receptor signaling.