Investigation of How Receptor Localization and Endocytosis Regulate CXCR4 Signaling and Post-Translational Modification

Abstract

Due to their great specificity and regulatory role in nearly all cellular processes, G protein-coupled receptors (GPCR) are a common therapeutic target. Investigating the molecular mechanisms that regulate receptor activity is essential for understanding how cells sense and adapt to their environment. The traditional model for GPCR activation occurs at the plasma membrane. Upon agonist binding, receptors are quickly post-translationally modified and activate signal transduction cascades. Receptor post-translational modifications are believed to regulate signaling outcomes, and initiate receptor endocytosis and down regulation. Over the past decade, it has become increasingly apparent that GPCR localization is a master regulator of cell signaling. However, the molecular mechanisms involved in this process are poorly understood. Using CXC chemokine receptor 4 (CXCR4) as a model GPCR, my thesis work investigates how clathrin and clathrin-independent endocytic pathways and intracellular localization regulate CXCR4 signaling and post-translational modification. In Chapter 2, I present data that supports an expanded role for endocytosis in regulating CXCR4 signaling and post-translational modification. Clathrin heavy chain knockdown resulted in increased steady state and agonist-induced ERK1/2 phosphorylation. Interestingly, CXCR4 lipid raft localization has been implicated as a positive regulator of ERK1/2 signaling. In support of this hypothesis, we found that lipid rafts were necessary for CXCR4-dependent ERK1/2 phosphorylation and clathrin knockdown increased CXCR4 colocalization with caveolin-1. During this work, we also identified a new antibody to study CXCR4 post-translational modification and discovered that clathrin knockdown increases steady-state CXCR4 post-translational modification. Together, these results suggest that endocytic pathways compensate for one another and differentially modulate receptor signaling and post-translational modification. In Chapter 3, we shifted our attention from understanding how plasma membrane microdomains differentially regulate CXCR4 biology to investigate how intracellular GPCRs contribute to signaling. While receptor activation is traditionally thought to occur at the plasma membrane, there is growing evidence that intracellular GPCR activation also contributes to cell signaling. Through this work we discovered a new mechanism of intracellular GPCR activation. We found that upon agonist stimulation, both plasma membrane and intracellular pools of CXCR4 are post-translationally modified in a β-arrestin-1-dependent mechanism. Interestingly, intracellular CXCR4 activation increased CXCL12-dependent Egr1 transcription. Notably, these observations may explain that while CXCR4 overexpression is highly correlated with cancer metastasis and mortality, plasma membrane localization is not. Together these data support a model were a small initial pool of plasma membrane-localized GPCRs are capable of activating internal receptor-dependent signaling events. Identification of protein-protein interactions is one of the first steps in identifying a molecular mechanism. However, due to difficulty isolating GPCR signaling complexes, many of the molecular players involved in these processes remain unknown. In Chapter 4, I share our progress using spatially resolved proteomics to identify new CXCR4 interacting partners and investigate how the CXCR4 interactome changes during endocytic trafficking. Finally in Chapter 5, I use my work as an example to illustrate the importance of understanding the assumptions of biological measurements and highlight several cases where measurement assumptions influence result interpretation. In summary, the presented data support an increased role for receptor localization as a master regulator of cell signaling. By improving our understanding of receptor regulation, I hope that this research can lead to the identification of new molecular mechanisms that regulate cell signaling and potentially therapeutic strategies that target aberrant GPCR function.