Nikhil M. Urs

A dopamine D1 receptor-dependent β-arrestin signaling complex potentially regulates morphine-induced psychomotor activation but not reward in mice.

Morphine is a widely used analgesic in humans that is associated with multiple untoward effects, such as addiction and physical dependence. In rodent models, morphine also induces locomotor activity. These effects likely involve functionally selective mechanisms. Indeed, G protein-coupled receptor desensitization and adaptor protein β-arrestin 2 (βarr2) through its interaction with the μ-opioid receptor regulates the analgesic but not the rewarding properties of morphine. However, βarr2 is also required for morphine-induced locomotor activity in mice, but the exact cellular and molecular mechanisms that mediate this arrestin-dependent behavior are not understood. In this study, we show that βarr2 is required for morphine-induced locomotor activity in a dopamine D1 receptor (D1R)-dependent manner and that a βarr2/phospho-ERK (βarr2/pERK) signaling complex may mediate this behavior. Systemic administration of SL327, an MEK inhibitor, inhibits morphine-induced locomotion in wild-type mice in a dose-dependent manner. Acute morphine administration to mice promotes the formation of a βarr2/pERK signaling complex. Morphine-induced locomotor activity and formation of the βarr2/pERK signaling complex is blunted in D1R knockout (D1-KO) mice and is presumably independent of D2 dopamine receptors. However, D1Rs are not required for morphine-induced reward as D1-KO mice show the same conditioned place preference for morphine as do control mice. Taken together, these results suggest a potential role for a D1R-dependent βarr2/pERK signaling complex in selectively mediating the locomotor-stimulating but not the rewarding properties of morphine.

Deletion of GSK3β in D2R-expressing neurons reveals distinct roles for β-arrestin signaling in antipsychotic and lithium action

Several studies in rodent models have shown that glycogen synthase kinase 3 β (GSK3β) plays an important role in the actions of antispychotics and mood stabilizers. Recently it was demonstrated that GSK3β through a β-arrestin2/protein kinase B (PKB or Akt)/protein phosphatase 2A (PP2A) signaling complex regulates dopamine (DA)- and lithium-sensitive behaviors and is required to mediate endophenotypes of mania and depression in rodents. We have previously shown that atypical antipsychotics antagonize DA D2 receptor (D2R)/β-arrestin2 interactions more efficaciously than G-protein–dependent signaling, whereas typical antipsychotics inhibit both pathways with similar efficacy. To elucidate the site of action of GSK3β in regulating DA- or lithium-sensitive behaviors, we generated conditional knockouts of GSK3β, where GSK3β was deleted in either DA D1- or D2-receptor–expressing neurons. We analyzed these mice for behaviors commonly used to test antipsychotic efficacy or behaviors that are sensitive to lithium treatment. Mice with deletion of GSK3β in D2 (D2GSK3β−/−) but not D1 (D1GSK3β−/−) neurons mimic antipsychotic action. However, haloperidol (HAL)-induced catalepsy was unchanged in either D2GSK3β−/− or D1GSK3β−/− mice compared with control mice. Interestingly, genetic stabilization of β-catenin, a downstream target of GSK3β, in D2 neurons did not affect any of the behaviors tested. Moreover, D2GSK3β−/− or D1GSK3β−/− mice showed similar responses to controls in the tail suspension test (TST) and dark–light emergence test, behaviors which were previously shown to be β-arrestin2- and GSK3β-dependent and sensitive to lithium treatment. Taken together these studies suggest that selective deletion of GSK3β but not stabilization of β-catenin in D2 neurons mimics antipsychotic action without affecting signaling pathways involved in catalepsy or certain mood-related behaviors.

Elucidation of G-protein and β-arrestin functional selectivity at the dopamine D2 receptor

Significance The dopamine D2 receptor (D2R), a G protein-coupled receptor, can initiate signaling events through both activation of G proteins and interactions with β-arrestins. To begin to understand the contribution of these events in the physiology of the dopamine system, D2R was mutated to be functionally selective for each signaling pathway. The engineered receptors are functional in vitro and in vivo. Furthermore, both functions are essentially dissociable and mediate different physiological and pharmacological responses. These tools provide an additional but previously unavailable approach to elucidate the role of biased signaling in the multiple physiological actions of the dopamine system. 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 signal transduction for either the G-protein or β-arrestin interactions. These mutants were used to resolve the role of G proteins and β-arrestins in D2R signaling assays. The results show that D2R interactions with the two downstream effectors are dissociable and that G-protein signaling accounts for D2R canonical MAP kinase signaling cascade activation, whereas β-arrestin only activates elements of this cascade under certain conditions. Nevertheless, when expressed in mice in GABAergic medium spiny neurons of the striatum, the β-arrestin–biased D2R caused a significant potentiation of amphetamine-induced locomotion, whereas the G protein-biased D2R had minimal effects. The mutant receptors generated here provide a molecular tool set that should enable a better definition of the individual roles of G-protein and β-arrestin signaling pathways in D2R pharmacology, neurobiology, and associated pathologies.

Distinct cortical and striatal actions of a β-arrestin–biased dopamine D2 receptor ligand reveal unique antipsychotic-like properties

Significance Schizophrenia is a debilitating psychiatric disorder characterized by positive, negative, and cognitive symptoms. Current antipsychotic drugs, including D2 receptor (D2R) partial agonist aripiprazole, antagonize excess striatal dopamine (DA) neurotransmission and reverse positive symptoms but are not efficacious at reversing cortical-related cognitive symptoms. Here, we show using pharmacological, behavioral, and electrophysiological approaches that a β-arrestin2 (βarr2)-biased D2R ligand has opposite antagonist and agonist actions in the striatum and cortex, respectively. This phenomenon is regulated by differential expression levels of signal transducer proteins G protein-coupled receptor kinase 2 and βarr2. Thus, D2R-βarr2–biased ligands have the potential to simultaneously target excess striatal and deficient cortical DA neurotransmission and provide more broadly effective therapies for schizophrenia. The current dopamine (DA) hypothesis of schizophrenia postulates striatal hyperdopaminergia and cortical hypodopaminergia. Although partial agonists at DA D2 receptors (D2Rs), like aripiprazole, were developed to simultaneously target both phenomena, they do not effectively improve cortical dysfunction. In this study, we investigate the potential for newly developed β-arrestin2 (βarr2)-biased D2R partial agonists to simultaneously target hyper- and hypodopaminergia. Using neuron-specific βarr2-KO mice, we show that the antipsychotic-like effects of a βarr2-biased D2R ligand are driven through both striatal antagonism and cortical agonism of D2R-βarr2 signaling. Furthermore, βarr2-biased D2R agonism enhances firing of cortical fast-spiking interneurons. This enhanced cortical agonism of the biased ligand can be attributed to a lack of G-protein signaling and elevated expression of βarr2 and G protein-coupled receptor (GPCR) kinase 2 in the cortex versus the striatum. Therefore, we propose that βarr2-biased D2R ligands that exert region-selective actions could provide a path to develop more effective antipsychotic therapies.

G Protein and β-Arrestin Signaling Bias at the Ghrelin Receptor

Background: The G protein coupled receptor GHSR1a mediates feeding and addictive behaviors. Results: Mutagenesis of the second intracellular loop of GHSR1a generates biased receptors, favoring distinct signaling events. Conclusion: Receptor conformations that support signaling bias at the wild-type receptor should exist. Significance: Recapitulating signaling bias at GHSR1a may facilitate the identification of novel selective therapies to treat addiction. The G protein-coupled ghrelin receptor GHSR1a is a potential pharmacological target for treating obesity and addiction because of the critical role ghrelin plays in energy homeostasis and dopamine-dependent reward. GHSR1a enhances growth hormone release, appetite, and dopamine signaling through Gq/11, Gi/o, and G12/13 as well as β-arrestin-based scaffolds. However, the contribution of individual G protein and β-arrestin pathways to the diverse physiological responses mediated by ghrelin remains unknown. To characterize whether a signaling bias occurs for GHSR1a, we investigated ghrelin signaling in a number of cell-based assays, including Ca2+ mobilization, serum response factor response element, stress fiber formation, ERK1/2 phosphorylation, and β-arrestin translocation, utilizing intracellular second loop and C-tail mutants of GHSR1a. We observed that GHSR1a and β-arrestin rapidly form metastable plasma membrane complexes following exposure to an agonist, but replacement of the GHSR1a C-tail by the tail of the vasopressin 2 receptor greatly stabilizes them, producing complexes observable on the plasma membrane and also in endocytic vesicles. Mutations of the contiguous conserved amino acids Pro-148 and Leu-149 in the GHSR1a intracellular second loop generate receptors with a strong bias to G protein and β-arrestin, respectively, supporting a role for conformation-dependent signaling bias in the wild-type receptor. Our results demonstrate more balance in GHSR1a-mediated ERK signaling from G proteins and β-arrestin but uncover an important role for β-arrestin in RhoA activation and stress fiber formation. These findings suggest an avenue for modulating drug abuse-associated changes in synaptic plasticity via GHSR1a and indicate the development of GHSR1a-biased ligands as a promising strategy for selectively targeting downstream signaling events.

D1 Dopamine Receptor Coupling to PLCβ Regulates Forward Locomotion in Mice

Several studies have reported the coupling of dopamine signaling to phospholipase C β (PLCβ) both in vitro and in vivo. However, the precise physiological relevance of this signaling pathway in mediating dopamine behaviors is still unclear. Here we report that stimulation of dopamine receptor signaling in vivo with systemic administration of apomorphine, amphetamine, and cocaine leads to increased production of inositol triphosphate (IP3) in the mouse striatum. Using selective antagonists and dopamine D1 and D2 receptor knock-out animals, we show that the production of IP3 is mediated by the D1 receptor, but not the D2 receptor. A selective blocker of PLCβ, U73122, was used to assess the physiological relevance of D1-mediated IP3 production. We show that U73122 inhibits the locomotor-stimulating effects of apomorphine, amphetamine, cocaine, and SKF81297. Furthermore, U73122 also suppresses the spontaneous hyperactivity exhibited by dopamine transporter knock-out mice. Importantly, the effects of U73122 are selective to dopamine-mediated hyperactivity, as this compound does not affect hyperactivity induced by the glutamate NMDA receptor antagonist MK801. Finally, we present evidence showing that an imbalance of D1- and D2-mediated signaling following U73122 treatment modifies the locomotor output of animals from horizontal locomotor activity to vertical activity, further highlighting the importance of the PLCβ pathway in the regulation of forward locomotion via dopamine receptors.

Targeting β-arrestin2 in the treatment of l-DOPA–induced dyskinesia in Parkinson’s disease

Significance β-Arrestins are unique proteins that have multiple cellular functions such as G protein-coupled receptor signal desensitization, protein trafficking and signaling molecule scaffolding. Treatment of Parkinson’s disease (PD) motor symptoms by l-3,4-dihydroxyphenylalanine (l-DOPA) has been hampered by abnormal involuntary movements or dyskinetic side effects. The cause of these dyskinesias has been attributed to receptor supersensitivity and uncontrolled neuronal excitability. Here we demonstrate in multiple preclinical models of l-DOPA–induced dyskinesias and PD that expression levels of β-arrestin2 can alter manifestation of these dyskinesias by reducing receptor supersensitivity while maintaining the therapeutic effect of l-DOPA. Thus novel drugs that increase β-arrestin–dependent function at dopamine receptors may be useful in ameliorating PD motor symptoms without inducing dyskinesias. Parkinson’s disease (PD) is characterized by severe locomotor deficits and is commonly treated with the dopamine (DA) precursor l-3,4-dihydroxyphenylalanine (l-DOPA), but its prolonged use causes dyskinesias referred to as l-DOPA–induced dyskinesias (LIDs). Recent studies in animal models of PD have suggested that dyskinesias are associated with the overactivation of G protein-mediated signaling through DA receptors. β-Arrestins desensitize G protein signaling at DA receptors (D1R and D2R) in addition to activating their own G protein-independent signaling events, which have been shown to mediate locomotion. Therefore, targeting β-arrestins in PD l-DOPA therapy might prove to be a desirable approach. Here we show in a bilateral DA-depletion mouse model of Parkinson’s symptoms that genetic deletion of β-arrestin2 significantly limits the beneficial locomotor effects while markedly enhancing the dyskinesia-like effects of acute or chronic l-DOPA treatment. Viral rescue or overexpression of β-arrestin2 in knockout or control mice either reverses or protects against LIDs and its key biochemical markers. In other more conventional animal models of DA neuron loss and PD, such as 6-hydroxydopamine–treated mice or rats and 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine–treated nonhuman primates, β-arrestin2 overexpression significantly reduced dyskinesias while maintaining the therapeutic effect of l-DOPA. Considerable efforts are being spent in the pharmaceutical industry to identify therapeutic approaches to block LIDs in patients with PD. Our results point to a potential therapeutic approach, whereby development of either a genetic or pharmacological intervention to enhance β-arrestin2- or limit G protein-dependent D1/D2R signaling could represent a more mechanistically informed strategy.

New Concepts in Dopamine D2 Receptor Biased Signaling and Implications for Schizophrenia Therapy

The dopamine D2 receptor (D2R) is a G protein-coupled receptor that is a common target for antipsychotic drugs. Antagonism of D2R signaling in the striatum is thought to be the primary mode of action of antipsychotic drugs in alleviating psychotic symptoms. However, antipsychotic drugs are not clinically effective at reversing cortical-related symptoms, such as cognitive deficits in schizophrenia. While the exact mechanistic underpinnings of these cognitive deficits are largely unknown, deficits in cortical dopamine function likely play a contributing role. It is now recognized that similar to most G protein-coupled receptors, D2Rs signal not only through canonical G protein pathways but also through noncanonical beta-arrestin2-dependent pathways. We review the current mechanistic bases for this dual signaling mode of D2Rs and how these new concepts might be leveraged for therapeutic gain to target both cortical and striatal dysfunction in dopamine neurotransmission and hence have the potential to correct both positive and cognitive symptoms of schizophrenia.

Different Mechanisms Regulate Lysophosphatidic Acid (LPA)-dependent Versus Phorbol Ester-dependent Internalization of the LPA1 Receptor

Lysophosphatidic acid (LPA) stimulates cells by activation of five G-protein-coupled receptors, termed LPA1–5. The LPA1 receptor is the most widely expressed and is a major regulator of cell migration. In this study, we show that phorbol ester (PMA)-induced internalization of the LPA1 receptor requires clathrin AP-2 complexes, protein kinase C, and a distal dileucine motif (amino acids 352 and 353) in the cytoplasmic tail but not β-arrestin. Agonist-dependent internalization of LPA1, however, requires a cluster of serine residues (amino acids 341–347) located proximal to the dileucine motif, β-arrestin, and to a lesser extent clathrin AP-2. The serine cluster of LPA1 is required for β-arrestin2-GFP translocation to the plasma membrane and signal desensitization. In contrast, the dileucine motif (IL) is required for both basal and PMA-induced internalization. Evidence for the β-arrestin independence of PMA-induced internalization of LPA1 comes from the observations that β-arrestin2-GFP is not recruited to the plasma membrane upon PMA treatment and that LPA1 is readily internalized in β-arrestin1/2 knock-out mouse embryonic fibroblasts. These results indicate that distinct molecular mechanisms regulate agonist-dependent and PMA-dependent internalization of the LPA1 receptor.

Selective Deletion of GRK2 Alters Psychostimulant-Induced Behaviors and Dopamine Neurotransmission

GRK2 is a G protein-coupled receptor kinase (GRK) that is broadly expressed and is known to regulate diverse types of receptors. GRK2 null animals exhibit embryonic lethality due to a severe developmental heart defect, which has precluded the study of this kinase in the adult brain. To elucidate the specific role of GRK2 in the brain dopamine (DA) system, we used a conditional gene knockout approach to selectively delete GRK2 in DA D1 receptor (D1R)-, DA D2 receptor (D2R)-, adenosine 2A receptor (A2AR)-, or DA transporter (DAT)-expressing neurons. Here we show that select GRK2-deficient mice display hyperactivity, hyposensitivity, or hypersensitivity to the psychomotor effects of cocaine, altered striatal signaling, and DA release and uptake. Mice with GRK2 deficiency in D2R-expressing neurons also exhibited increased D2 autoreceptor activity. These findings reveal a cell-type-specific role for GRK2 in the regulation of normal motor behavior, sensitivity to psychostimulants, dopamine neurotransmission, and D2 autoreceptor function.