Browsing by Author "Caron, Marc G"

Schizophrenia is marked by significant disruptions to dopaminergic signaling across the mesolimbic and mesocortical circuits. Antipsychotic drugs have been largely unsuccessfully treating cognitive symptoms that debilitate the schizophrenia patient population. Dopamine 2 Receptor (D2R)- βeta arrestin 2 (βarr2) biased signaling, independent of the canonical G protein signaling, has emerged as a potential mechanism for antipsychotic drugs to restore dopaminergic signaling and improve treatment resistant cognitive symptoms. In the following experiments, I described gene editing tools to systematically investigate D2R signaling in a region or cell specific manner. Next, I evaluated the behavioral effects of two functionally selective D2-like βarr2 biased ligands against psychotomimetic challenge from phencyclidine or amphetamine. Then I employed chemogenetics to perform synthetic pharmacology experiments e.g. studying the signaling cascade of a drug without using the drug, to discover how D2- R βarr2 signaling produces antipsychotic effects in the prefrontal cortex. Lastly, I characterized the neurophysiological changes induced by phencyclidine and a D2R βarr2 biased ligand within relevant brain regions in the meso -limbic and -cortical circuits. Our results determined antipsychotic like activity is 1) regulated by excitation-inhibitory balance maintained by cortical GABA interneurons 2) dependent on βarr2.

The neuromodulator dopamine signals through the dopamine D2 receptor (D2R) to modulate central nervous system functions through diverse signal transduction pathways. D2R is a prominent target for drug treatments in disorders where dopamine function is aberrant, such as schizophrenia. D2R signals through distinct G protein and β-arrestin pathways and drugs that are functionally selective for these pathways could have improved therapeutic potential. How D2R signals through the two pathways is still not well defined, and efforts to elucidate these pathways have been hampered by the lack of adequate tools for assessing the contribution of each pathway independently. To address this, Evolutionary Trace was used to produce D2R mutants with strongly biased interactions for either G protein or β-arrestin. Additionally, various permutations of these mutants were used to identify critical determinants of D2R functional selectivity. D2R interactions with the two major downstream signal transducers were effectively dissociated and G protein signaling accounts for D2R canonical MAP kinase signaling cascade activation. Nevertheless, when expressed in mice, the β-arrestin biased D2R caused a significant potentiation of amphetamine-induced locomotion, while the G protein biased D2R had minimal effects. The mutant receptors generated here provide a new molecular tool set that enable a better definition of the individual roles of G protein and β-arrestin signaling in D2R pharmacology, neurobiology and associated pathologies.

G protein-coupled receptors (GPCRs) represent the largest and the most versatile family of cell surface receptors. Members of this receptor family translate diverse extracellular cues to intracellular responses, and are commonly targeted for medicinal therapeutics. In the current model of GPCR signaling, agonist binging not only initiates G protein-mediated signaling through generation of second messengers such as cyclic AMP and diacylglycerol, but also through the multifunctional adaptor protein beta-arrestin acting as a signal transducer. While some ligands have balanced agonist activity defined as equal efficacy for G protein and beta-arrestin-mediated pathways, other ligands stimulates GPCR signaling preferentially through beta-arrestin, a concept know as beta-arrestin-biased agonism.

The beta1 and beta2 adrenergic receptors (betaARs) are the predominant GPCR subtypes expressed in the heart, and play an important role in the pathophysiology of human heart disease. Of the four families of G alpha-proteins (G alpha s, G alpha i/o, G alpha q/11 and G alpha 12/13), beta1ARs are recognized as classical G alpha s-coupled receptors since agonist binding promotes coupling to heterotrimeric G protein (G alpha-beta-gamma) triggering dissociation of G alpha s from G beta-gamma. Here, we identify a new signaling mechanism unlike that previously for any known G alpha s-coupled receptor whereby the inhibitory G protein (G alpha i) is required for beta1AR-mediated beta-arrestin-biased signaling. Stimulation with the beta-arrestin-biased agonist carvedilol induces switching of the beta1AR from a classical G alpha s-coupled receptor to a G alpha i-coupled receptor and stabilizes a unique receptor conformation required for beta-arrestin-mediated signaling. Recruitment of G alpha i was not induced by any other betaAR agonist or antagonist screened, nor was it required for beta-arrestin-bias activated by the beta2AR subtype of the betaAR family.

We also found that G alpha i is involved in the membrane stretch-induced signaling activated by the angiotensin II type 1 receptor (AT1R), another GPCR mediating a variety of physiological responses and is commonly targeted for cardiac drug therapy. It is appreciated that the AT1R can function as a mechanical sensor. When activated by mechanical stretch, AT1Rs transmit signaling through beta-arrestin, rather than the G protein pathways. To date, the ligand-independent membrane stretch-induced AT1R conformation that triggers signaling is thought to be the same as that induced by a beta-arrestin-biased agonist, which can selectively engage beta-arrestin and prevent G protein coupling. Here we show that membrane stretch promotes a distinct biased conformation of the AT1R that couples to G alpha i. In contrast, recruitment of G alpha i was not induced by the balanced agonist angiotensin II, nor by the beta-arrestin-biased agonist TRV120023. The stretch-triggered G alpha i coupling induces the recruitment and a unique conformational change in beta-arrestin2 allowing for downstream signaling such as EGFR internalization and ERK phosphorylation.

Taken together, we identified a previously unrecognized role for G alpha i in beta-arrestin-biased GPCR signaling, and suggests that the concept of beta-arrestin-bias may need to be refined to incorporate the selective bias of receptors towards distinct G protein subtypes. We also demonstrate that different mechanisms for beta-arrestin bias may be operative between the signaling induced by distinct receptor activation modes, in the case of AT1R, the beta-arrestin-biased agonists and by the ligand-independent mechanical stretch.

These data underscore the complexity of beta-arrestin-biased agonism and have important implications when considering the development of new therapeutic ligands to selectively target beta-arrestin-biased signaling pathways.

Monoamines were first discovered at the end of the 19th century when William Bates identified epinephrine (EPI) and noted its hemostatic effects. During the 20th century, norepinephrine (NE), dopamine (DA), and serotonin (5HT) were discovered in both the periphery and the brain. Due, in part, to the implication of monoamines in the etiology of a wide range of dysfunctions, the examination of their physiological functions became the subject of a considerable volume of research. Much progress has been made in describing the function and endogenous regulation of these systems, as well as their response to pharmacological intervention. However, many aspects of these systems remain unexplored. For example, though the role of pharmacological agents in regulating monoamine transporter function has been widely studied, relatively little is known about basal regulation in terms of protein processing and targeting. Similarly, the role of phosphorylation has been well characterized in the regulation of tyrosine hydroxylase (TH), but little is known about the regulation of the closely related tryptophan hydroxylases. The recent discovery of the second isoform of tryptophan hydroxylase (TPH2) has brought renewed interest to this field as the majority of this second isoform is centrally expressed and it contains an additional 41 amino acids at the N-terminus compared to TPH1, the peripheral enzyme. To increase the understanding of these aspects of monoamine signaling, this study characterizes the regulatory role played by the extended N-terminus of TPH2 using mutagenesis and cell culture systems and identifies determinants of monoamine transporter targeting and processing using the dopamine transporter (DAT) as a model. In chapter 2, we demonstrate that TPH2 is synthesized less efficiently and is also less stable than TPH1 when expressed in cultured cells. Furthermore, we identify a region centered upon amino acids 10-20 in TPH2 that appears responsible for the bulk of this difference. We also demonstrate here that phosphorylation of S19 in TPH2 results in increased TPH2 stability, and a consequent increase in 5HT production. Because this domain is unique to TPH2, these data provide evidence for selective regulation of brain 5HT synthesis. Based on measured uptake capacity and both visual and biochemical markers of protein localization, the results presented in chapter 3 suggest that a conserved YAAY motif in the C-tail of the monoamine transporters is essential for normal levels of membrane expression. We also demonstrate that disruption of this sequence interferes to some extent with the previously described hDAT/Hic-5 interaction. Together, the data presented here contribute to the understanding of the physiological regulation of brain monoaminergic signaling.

Herein I describe studies I have undertaken that were aimed at understanding the mechanisms of achieving β-arrestin-biased signaling at the dopamine D2 receptor (D2R), methods for studying downstream mediators of β-arrestin-biased signaling, and the development of a mouse model of schizophrenia that could test the efficacy of β-arrestin-biased D2R ligands. Each of these studies will improve our understanding of how to better tailor drugs to treat schizophrenia. I have employed a wide variety of in vitro and in vivo methods ranging from bioluminescent resonance energy transfer (BRET) to adeno-associated viral delivery of neuronal actuators. Ultimately, I was able to demonstrate that the D2R can achieve β-arrestin biased signaling through its ability to directly recruit the G protein-coupled receptor kinase 2 (GRK2). Next, I developed a BRET-based approach to study interactions of GPCR-β-arrestin complexes and applied this to the D2R. Finally, I have laid the ground work for a mouse model of schizophrenia capable of generating dopamine circuit imbalances hypothesized to occur in schizophrenia as a means to test β-arrestin-biased D2R ligands.