Ikuo Masuho

Mutations in GNAL cause primary torsion dystonia

Dystonia is a movement disorder characterized by repetitive twisting muscle contractions and postures. Its molecular pathophysiology is poorly understood, in part owing to limited knowledge of the genetic basis of the disorder. Only three genes for primary torsion dystonia (PTD), TOR1A (DYT1), THAP1 (DYT6) and CIZ1 (ref. 5), have been identified. Using exome sequencing in two families with PTD, we identified a new causative gene, GNAL, with a nonsense mutation encoding p.Ser293* resulting in a premature stop codon in one family and a missense mutation encoding p.Val137Met in the other. Screening of GNAL in 39 families with PTD identified 6 additional new mutations in this gene. Impaired function of several of the mutants was shown by bioluminescence resonance energy transfer (BRET) assays.

Retina-Specific GTPase Accelerator RGS11/Gβ5S/R9AP Is a Constitutive Heterotrimer Selectively Targeted to mGluR6 in ON-Bipolar Neurons

Members of the R7 family of the regulators of G-protein signaling (R7 RGS) proteins form multi-subunit complexes that play crucial roles in processing the light responses of retinal neurons. The disruption of these complexes has been shown to lead to the loss of temporal resolution in retinal photoreceptors and deficient synaptic transmission to downstream neurons. Despite the well established role of one member of this family, RGS9-1, in controlling vertebrate phototransduction, the roles and organizational principles of other members in the retina are poorly understood. Here we investigate the composition, localization, and function of complexes containing RGS11, the closest homolog of RGS9-1. We find that RGS11 forms a novel obligatory trimeric complex with the short splice isoform of the type 5 G-protein β subunit (Gβ5) and the RGS9 anchor protein (R9AP). The complex is expressed exclusively in the dendritic tips of ON-bipolar cells in which its localization is accomplished through a direct association with mGluR6, the glutamate receptor essential for the ON-bipolar light response. Although association with both R9AP and mGluR6 contributed to the proteolytic stabilization of the complex, postsynaptic targeting of RGS11 was not determined by its membrane anchor R9AP. Electrophysiological recordings of the light response in mouse rod ON-bipolar cells reveal that the genetic elimination of RGS11 has little effect on the deactivation of Gαo in dark-adapted cells or during adaptation to background light. These results suggest that the deactivation of mGluR6 cascade during the light response may require the contribution of multiple GTPase activating proteins.

GPR158/179 regulate G protein signaling by controlling localization and activity of the RGS7 complexes.

Interaction of RGS proteins with orphan GPCRs promotes signaling compartmentalization and specificity.

Distinct profiles of functional discrimination among G proteins determine the actions of G protein–coupled receptors

Screening G protein–coupling specificities reveals unexpectedly diverse signaling pathways for individual GPCRs and their ligands. Fingerprinting GPCRs G protein–coupled receptors (GPCRs) influence most aspects of physiology and are targeted by many clinically used drugs. The physiological functions of this large family of proteins are thought to be mediated by highly specific G protein interactions (see the Focus by Smrcka). Masuho et al. devised a bioluminescence-based assay in transfected cells to examine the G protein–coupling specificities of different GPCRs for 13 different G proteins, generating fingerprint-like profiles for each receptor. These assays revealed unexpected use of G proteins by certain GPCRs, biased G protein usage by a given GPCR in response to different agonists, and G protein activation by a ligand thought to be an antagonist, findings that were verified in relevant cells. Given the clinical importance of this family of receptors, assays such as this one aid in understanding the physiological effects of currently used drugs, as well as in designing better therapeutics and limiting their potential side effects. Members of the heterotrimeric guanine nucleotide–binding protein (G protein)–coupled receptor (GPCR) family play key roles in many physiological functions and are extensively exploited pharmacologically to treat diseases. Many of the diverse effects of individual GPCRs on cellular physiology are transduced by heterotrimeric G proteins, which are composed of α, β, and γ subunits. GPCRs interact with and stimulate the binding of guanosine triphosphate (GTP) to the α subunit to initiate signaling. Mammalian genomes encode 16 different G protein α subunits, each one of which has distinct properties. We developed a single-platform, optical strategy to monitor G protein activation in live cells. With this system, we profiled the coupling ability of individual GPCRs for different α subunits, simultaneously quantifying the magnitude of the signal and the rates at which the receptors activated the G proteins. We found that individual receptors engaged multiple G proteins with varying efficacy and kinetics, generating fingerprint-like profiles. Different classes of GPCR ligands, including full and partial agonists, allosteric modulators, and antagonists, distinctly affected these fingerprints to functionally bias GPCR signaling. Finally, we showed that intracellular signaling modulators further altered the G protein–coupling profiles of GPCRs, which suggests that their differential abundance may alter signaling outcomes in a cell-specific manner. These observations suggest that the diversity of the effects of GPCRs on cellular physiology may be determined by their differential engagement of multiple G proteins, coupling to which produces signals with varying signal magnitudes and activation kinetics, properties that may be exploited pharmacologically.

Mutations in GNAL: A Novel Cause of Craniocervical Dystonia

IMPORTANCE Mutations in the GNAL gene have recently been shown to cause primary torsion dystonia. The GNAL-encoded protein (Gαolf) is important for dopamine D1 receptor function and odorant signal transduction. We sequenced all 12 exons of GNAL in 461 patients from Germany, Serbia, and Japan, including 318 patients with dystonia (190 with cervical dystonia), 51 with hyposmia and Parkinson disease, and 92 with tardive dyskinesia or acute dystonic reactions. OBSERVATIONS We identified the following two novel heterozygous putative mutations in GNAL: p.Gly213Ser in a German patient and p.Ala353Thr in a Japanese patient. These variants were predicted to be pathogenic in silico, were absent in ethnically matched control individuals, and impaired Gαolf coupling to D1 receptors in a bioluminescence energy transfer (BRET) assay. Two additional variants appeared to be benign because they behaved like wild-type samples in the BRET assay (p.Ala311Thr) or were detected in ethnically matched controls (p.Thr92Ala). Both patients with likely pathogenic mutations had craniocervical dystonia with onset in the fifth decade of life. No pathogenic mutations were detected in the patients with hyposmia and Parkinson disease, tardive dyskinesias, or acute dystonic reactions. CONCLUSIONS AND RELEVANCE Mutations in GNAL can cause craniocervical dystonia in different ethnicities. The BRET assay may be a useful tool to support the pathogenicity of identified variants in the GNAL gene.

Interaction of Transducin with Uncoordinated 119 Protein (UNC119) IMPLICATIONS FOR THE MODEL OF TRANSDUCIN TRAFFICKING IN ROD PHOTORECEPTORS

The key visual G protein, transducin undergoes bi-directional translocations between the outer segment (OS) and inner compartments of rod photoreceptors in a light-dependent manner thereby contributing to adaptation and neuroprotection of rods. A mammalian uncoordinated 119 protein (UNC119), also known as Retina Gene 4 protein (RG4), has been recently implicated in transducin transport to the OS in the dark through its interaction with the N-acylated GTP-bound transducin-α subunit (Gαt1). Here, we demonstrate that the interaction of human UNC119 (HRG4) with transducin is dependent on the N-acylation, but does not require the GTP-bound form of Gαt1. The lipid specificity of UNC119 is unique: UNC119 bound the myristoylated N terminus of Gαt1 with much higher affinity than a prenylated substrate, whereas the homologous prenyl-binding protein PrBP/δ did not interact with the myristoylated peptide. UNC119 was capable of interacting with Gαt1GDP as well as with heterotrimeric transducin (Gt). This interaction of UNC119 with Gt led to displacement of Gβ1γ1 from the heterotrimer. Furthermore, UNC119 facilitated solubilization of Gt from dark-adapted rod OS membranes. Consistent with these observations, UNC119 inhibited rhodopsin-dependent activation of Gt, but had no effect on the GTP-hydrolysis by Gαt1. A model for the role of UNC119 in the IS→OS translocation of Gt is proposed based on the UNC119 ability to dissociate Gt subunits from each other and the membrane. We also found that UNC119 inhibited activation of Go by D2 dopamine receptor in cultured cells. Thus, UNC119 may play conserved inhibitory role in regulation of GPCR-G protein signaling in non-visual tissues.

RGS7/Gβ5/R7BP complex regulates synaptic plasticity and memory by modulating hippocampal GABABR-GIRK signaling.

In the hippocampus, the inhibitory neurotransmitter GABA shapes the activity of the output pyramidal neurons and plays important role in cognition. Most of its inhibitory effects are mediated by signaling from GABAB receptor to the G protein-gated Inwardly-rectifying K+ (GIRK) channels. Here, we show that RGS7, in cooperation with its binding partner R7BP, regulates GABABR-GIRK signaling in hippocampal pyramidal neurons. Deletion of RGS7 in mice dramatically sensitizes GIRK responses to GABAB receptor stimulation and markedly slows channel deactivation kinetics. Enhanced activity of this signaling pathway leads to decreased neuronal excitability and selective disruption of inhibitory forms of synaptic plasticity. As a result, mice lacking RGS7 exhibit deficits in learning and memory. We further report that RGS7 is selectively modulated by its membrane anchoring subunit R7BP, which sets the dynamic range of GIRK responses. Together, these results demonstrate a novel role of RGS7 in hippocampal synaptic plasticity and memory formation. DOI: http://dx.doi.org/10.7554/eLife.02053.001

The Complex of G Protein Regulator RGS9-2 and Gβ5 Controls Sensitization and Signaling Kinetics of Type 5 Adenylyl Cyclase in the Striatum

By suppressing cAMP production in the striatum, the RGS9-2/Gβ5 complex could affect the development of opioid addiction. Suppressing Drug Addiction? Signaling through dopamine and opioid G protein–coupled receptors (GPCRs) in the brain structure called the striatum is involved in behaviors caused by drug addiction. Type 5 adenylyl cyclase (AC5) is a downstream effector of dopamine and opioid GPCRs that produces the second messenger cyclic adenosine monophosphate (cAMP). Chronic opioid treatment causes sensitization of AC5 and, upon withdrawal of opioids, leads to increased cAMP production, a phenomenon called superactivation, which may contribute to the development of opiate tolerance and dependence. The detailed biochemical and cellular analyses performed by Xie et al. revealed that a complex consisting of RGS9-2 (regulator of G protein signaling 9-2) and the G protein β subunit Gβ5 bound to and suppressed the activity of AC5 by several different mechanisms. Accordingly, stimulation of the dopamine D1 receptor produced higher cAMP concentrations in striatal membranes from mice lacking Gβ5 than those from wild-type mice. In mice undergoing acute withdrawal from chronic morphine administration, the cAMP concentration was higher in striata of RGS9 knockout mice than in those of wild-type mice. Thus, these results suggest that targeting the interaction between RGS9-2 and Gβ5 could affect the development of drug addiction. Multiple neurotransmitter systems in the striatum converge to regulate the excitability of striatal neurons by activating several heterotrimeric guanine nucleotide–binding protein (G protein)–coupled receptors (GPCRs) that signal to the type 5 adenylyl cyclase (AC5), the key effector enzyme that produces the intracellular second messenger cyclic adenosine monophosphate (cAMP). Plasticity of cAMP signaling in the striatum is thought to play an essential role in the development of drug addiction. We showed that the complex of the ninth regulator of G protein signaling (RGS9-2) with the G protein β subunit (Gβ5) critically controlled signaling from dopamine and opioid GPCRs to AC5 in the striatum. RGS9-2/Gβ5 directly interacted with and suppressed the basal activity of AC5. In addition, the RGS9-2/Gβ5 complex attenuated the stimulatory action of Gβγ on AC5 by facilitating the GTPase (guanosine triphosphatase) activity of Gαo, thus promoting the formation of the inactive heterotrimer and inhibiting Gβγ. Furthermore, by increasing the deactivation rate of Gαi, RGS9-2/Gβ5 facilitated the recovery of AC5 from inhibition. Mice lacking RGS9 showed increased cAMP production and, upon withdrawal from opioid administration, enhanced sensitization of AC5. Our findings establish RGS9-2/Gβ5 complexes as regulators of three key aspects of cAMP signaling: basal activity, sensitization, and temporal kinetics of AC5, thus highlighting the role of this complex in regulating both inhibitory and stimulatory GPCRs that shape cAMP signaling in the striatum.

Essential Role of the m2R-RGS6-IKACh Pathway in Controlling Intrinsic Heart Rate Variability

Normal heart function requires generation of a regular rhythm by sinoatrial pacemaker cells and the alteration of this spontaneous heart rate by the autonomic input to match physiological demand. However, the molecular mechanisms that ensure consistent periodicity of cardiac contractions and fine tuning of this process by autonomic system are not completely understood. Here we examined the contribution of the m2R-IKACh intracellular signaling pathway, which mediates the negative chronotropic effect of parasympathetic stimulation, to the regulation of the cardiac pacemaking rhythm. Using isolated heart preparations and single-cell recordings we show that the m2R-IKACh signaling pathway controls the excitability and firing pattern of the sinoatrial cardiomyocytes and determines variability of cardiac rhythm in a manner independent from the autonomic input. Ablation of the major regulator of this pathway, Rgs6, in mice results in irregular cardiac rhythmicity and increases susceptibility to atrial fibrillation. We further identify several human subjects with variants in the RGS6 gene and show that the loss of function in RGS6 correlates with increased heart rate variability. These findings identify the essential role of the m2R-IKACh signaling pathway in the regulation of cardiac sinus rhythm and implicate RGS6 in arrhythmia pathogenesis.

Macromolecular Composition Dictates Receptor and G Protein Selectivity of Regulator of G Protein Signaling (RGS) 7 and 9-2 Protein Complexes in Living Cells

Background: RGS7 and RGS9-2 regulate G protein signaling in the striatum, but the selectivity of their action is largely unknown. Results: RGS protein complexes show distinct patterns of receptor and G protein selectivity. Conclusion: Macromolecular composition dictates receptor and G protein selectivity of the RGS7 and RGS9-2 protein complexes. Significance: These data demonstrate novel mechanisms contributing to the regulation of striatal G protein signaling. Regulator of G protein signaling (RGS) proteins play essential roles in the regulation of signaling via G protein-coupled receptors (GPCRs). With hundreds of GPCRs and dozens of G proteins, it is important to understand how RGS regulates selective GPCR-G protein signaling. In neurons of the striatum, two RGS proteins, RGS7 and RGS9-2, regulate signaling by μ-opioid receptor (MOR) and dopamine D2 receptor (D2R) and are implicated in drug addiction, movement disorders, and nociception. Both proteins form trimeric complexes with the atypical G protein β subunit Gβ5 and a membrane anchor, R7BP. In this study, we examined GTPase-accelerating protein (GAP) activity as well as Gα and GPCR selectivity of RGS7 and RGS9-2 complexes in live cells using a bioluminescence resonance energy transfer-based assay that monitors dissociation of G protein subunits. We showed that RGS9-2/Gβ5 regulated both Gi and Go with a bias toward Go, but RGS7/Gβ5 could serve as a GAP only for Go. Interestingly, R7BP enhanced GAP activity of RGS7 and RGS9-2 toward Go and Gi and enabled RGS7 to regulate Gi signaling. Neither RGS7 nor RGS9-2 had any activity toward Gz, Gs, or Gq in the absence or presence of R7BP. We also observed no effect of GPCRs (MOR and D2R) on the G protein bias of R7 RGS proteins. However, the GAP activity of RGS9-2 showed a strong receptor preference for D2R over MOR. Finally, RGS7 displayed an four times greater GAP activity relative to RGS9-2. These findings illustrate the principles involved in establishing G protein and GPCR selectivity of striatal RGS proteins.