Browsing by Subject "Arrestin"

β-Adrenergic receptors (βAR) are one of the key modulators of cardio-pulmonary functions and belong to a large family of membrane proteins, termed as G-protein coupled receptors (GPCRs). β-blockers (βAR antagonists) and βAR agonists are the mainstay treatments for heart failure and asthma respectively, which reflects the significance of βARs as therapeutic targets. The binding of catecholamines (e.g. adrenaline) to βARs activates intracellular transducer proteins such as hetero trimeric GTP binding proteins (G-proteins) or β-arrestins (βarr), which results in the regulation of cardiac output and bronchodilation.

The bifurcated signaling pathways initiated by G-protein and β-arrestin downstream of βAR, as well as other members in the GPCR family can be selectively activated, a phenomenon termed as ‘biased agonism’. Biased ligands, which can pharmacologically separate these pathways, are of major therapeutic interest due to their potential for improving the specificity of drug actions. For βAR, biased agonism towards β-arrestin is expected to render cardo-protective benefits, while selective activation of G proteins is hypothesized to subdue major side effects from current asthma therapy. Therefore, elucidation of how βARs can preferentially interact with their transducers is at the core of developing the next generation therapeutics, beyond conventional β-blockers and agonists.

Thus far, the exact mechanism behind GPCR biased agonism remains obscure. The leading hypothesis in the field is that GPCRs adopt distinct conformations that preferentially couple to G proteins or β-arrestins. In order to test this hypothesis, we developed and established a G protein biased mutant β2AR (Chapter 2), since efficacious biased ligands for this receptor are yet to be found. Subsequent assessment of GPCR kinase (GRK)-mediated phosphorylation states of this mutant receptor and phosphorylation rescue experiments revealed unexpected findings that contradict the initial hypothesis (Chapter 3). Next, we initiated a biophysical characterization of this mutant β2AR (Chapter 4) to comprehend the conformational and structural basis for its apparent biased phenotype. The cumulative insight gained from experiments described in chapters 2-4 highlight the underappreciated role of GRKs in determining GPCR biased agonism – the mutant β2AR is biased towards G protein due to conformational selection against GRKs, rather than β-arrestins. Furthermore, to obtain a comprehensive understanding of biased agonism, we devised a strategy to map the interface between β2AR-β-arrestin, which can also be used to form stable complexes for further biophysical characterizations (Chapter 5). In summary, this dissertation improves the current understanding of the molecular mechanism behind biased agonism at the prototypical GPCR, β2AR.

My thesis work has focused on characterizing mechanisms by which human fungal pathogens regulate their adaptive cellular responses in order to survive and cause disease in the human host. Unlike most microbial fungi found in the environment, Cryptococcus neoformans has become a successful human pathogen due to two intrinsic abilities: 1) to survive and grow at human body temperature and 2) to employ virulence factors to combat host immune defenses. Over the past two decades, the fungal pathogenesis field has made enormous progress in identifying and characterizing C. neoformans proteins responsible for these adaptive cellular responses with a particular focus on enzymes, like those involved in cell cycle progression or those responsible for synthesizing components of the fungal cell surface. Although we know a substantial amount about the functions of these enzymes and their implications on fungal pathogenesis, the mechanisms by which these enzymes are regulated are less clear. I have attempted to address this gap in knowledge by focusing my thesis work on the identification and characterization of C. neoformans non-enzymatic proteins that regulate enzymes important for adaptive cellular responses. I have identified and characterized the C. neoformans arrestin proteins as regulators of enzyme ubiquitination, and likely enzyme function, in response to specific extracellular stressors (Chapters 2 & 3). I have also characterized a Cryptococcus-specific protein, Mar1, as an important modulator of host-fungal interactions due to its regulation of cell surface remodeling through maintenance of mitochondrial metabolic activity and homeostasis in response to cellular stress (Chapters 4 & 5). Furthermore, I also performed a comprehensive comparative analysis of different RNA enrichment methods for RNA sequencing applications and long non-coding RNA identification in C. neoformans, which can help researchers select appropriate tools for studying adaptive cellular responses from the RNA level (Chapter 6). These studies collectively have demonstrated that non-enzymatic proteins are important “cellular coordinators” in human fungal pathogens; they regulate the activity of many different enzymes in response to distinct extracellular signals, and as a result are required for both fungal growth and virulence factor employment in response to host-relevant stressors.

Chemokine receptors are members of the seven transmembrane receptor (7TMR) superfamily and are regulated by the G-protein coupled Receptor Kinase (GRK)/ b-arrestin system. CCL19 and CCL21 are endogenous agonists for the chemokine receptor CCR7. They are known to be equipotent in promoting Gi/o mediated calcium mobilization, chemotaxis and inhibition of adenylyl cyclase activity. Here we test the hypothesis that these ligands are biased agonists that differentially activate the G-protein coupled Receptor Kinase (GRK)/ b-arrestin system.

In order to test whether these ligands have distinct activity, murine T lymphocytes were used to compare the effects of CCL19 and CCL21 activation of CCR7 at endogenous expression levels. While each ligand stimulates similar chemotactic responses, we also find that CCR7 ligands lead to differential signaling. For instance, CCL19 is markedly more efficacious than CCL21 for the activation of ERK and JNK, but not AKT in these cells. Furthermore, ERK activation and chemotaxis are maintained as separate pathways, also distinguishable by their dependency upon PKC and PI3 kinase, respectively. Thus, CCL19 and CCL21 stimulate equal activation of PI3 kinase, AKT, and chemotaxis, but are in fact biased agonists leading to differential activation of MAP kinase in murine T lymphocytes.

To determine the mechanism of CCR7 ligand bias, we used HEK-293 cells expressing CCR7 to compare the proximate signaling events following CCL19 and CCL21 activation. We found striking differences in the activation of the GRK/ b-arrestin system. CCL19 leads to robust CCR7 phosphorylation and b-arrestin2 recruitment catalyzed by both GRK3 and GRK6 while CCL21 activates GRK6 alone. This differential GRK activation leads to distinct functional consequences. Only CCL19 leads to the recruitment of b-arrestin2-GFP into endocytic vesicles and classical receptor desensitization. In contrast, each agonist is fully capable of signaling to MAP kinase through b-arrestin2 in a GRK6 dependent fashion.

Therefore, CCR7 and its ligands represent a natural example of ligand bias whose mechanism involves differential GRK isoform utilization by CCL19 and CCL21 despite similar G-protein signaling. This study suggests that the GRK signatures of 7TMRs can determine the function of discrete pools of b-arrestin and thus guide its cellular effects.

G protein-coupled receptors (GPCRs) are the most common transmembrane receptors in the human genome and the target of approximately one-third of all approved drugs. GPCRs interact with many transducers like G proteins and β-arrestins. Some GPCRs preferentially activate specific signaling transducers over others, leading to unique signaling profiles – a phenomenon called biased signaling. The chemokine system, a subfamily of GPCRs, serves as an endogenous example of biased signaling where over 50 different chemokines and 20 receptors interact promiscuously. While previous research has shown that chemokines which activate the same receptor can produce different physiologic responses, the mechanisms underlying these findings remain unclear. Using the three endogenous chemokines of the chemokine receptor CXCR3, we investigated two mechanisms underlying biased signaling at GPCRs. First, using mass spectrometry and cell-based assays, we determined that the chemokines induce different amounts and patterns of GPCR phosphorylation which direct CXCR3 engagement with different transducers. Second, we determined that biased signaling is dependent on the specific location of CXCR3, and subcellular signaling regulates inflammation in a mouse model of contact hypersensitivity. Together, we conclude that differential receptor phosphorylation and subcellular signaling are two mechanisms underlying the biased signaling observed at GPCRs.

G protein-coupled receptors (GPCRs) are targets for almost a third of all FDA-approved drugs. Canonically, GPCRs are known to signal through both heterotrimeric G proteins and β-arrestins. Traditionally these pathways were viewed as largely separable since G proteins primarily initiate downstream signaling while β-arrestins function to regulate receptor desensitization and trafficking in addition to modulating their own signaling. However more recent studies have provided evidence for an integrated role of G protein and β-arrestins in GPCRs signaling through the formation of G protein:β-arrestin:Receptor “megaplexes”. Additionally, G proteins and βarrs were found to associate across a panel of receptors. This association was surprisingly specific for inhibitory G protein (Gαi), even among receptors which do not classically signal through Gαi and suggested that this may be a novel non-canonical method of GPCR signaling. The mechanism required for such interaction and the biochemical and physiological significance remains unknown. To elucidate how this novel complex forms downstream of GPCRs, this dissertation focuses on uncovering mechanisms associated with Gαi: β-arrestin association. Utilizing biochemical and biosensor-based approaches, we show a large portion of selectivity for Gαi in these complexes lies within the N-terminal helical domain. Additionally, we detail the requirements for complex formation where β-arrestin translocation to the plasma membrane is sufficient for complex assembly and, surprisingly, G protein activation and recruitment via GPCRs is dispensable. These findings delineate a novel mechanism for GPCR signaling through β-arrestin recruitment to the plasma membrane that results in complex formation and signaling through novel signaling cascades.

β-arrestins (βarrs) interact with G protein-coupled receptors (GPCRs) to desensitize G protein signaling, initiate signaling on their own, and mediate receptor endocytosis.  Using a panel of GPCRs believed to couple differently to βarrs we demonstrate how distinct conformations of GPCR–βarr complexes are specialized to perform different subsets of these cellular functions. Our results thus provide a new signaling paradigm for the understanding of GPCRs, whereby a specific GPCR–βarr conformation mediates receptor desensitization, while another drives internalization and some forms of signaling.

In addition, some GPCRs activate G proteins from within internalized cellular compartments resulting in sustained signaling. We used a variety of biochemical, biophysical, and cell-based methods to demonstrate the existence, functionality, and architecture of internalized receptor “mega-complexes” composed of a single GPCR, βarr, and G protein. EM of purified ‘megaplexes’ reveals that a single receptor binds simultaneously through its core region with G protein and with βarr in the tail conformation. Thus, the two GPCR–βarr conformations carry out distinct cellular functions.