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1.  Exome Sequencing in 53 Sporadic Cases of Schizophrenia Identifies 18 Putative Candidate Genes 
PLoS ONE  2014;9(11):e112745.
Schizophrenia (SCZ) is a severe, debilitating mental illness which has a significant genetic component. The identification of genetic factors related to SCZ has been challenging and these factors remain largely unknown. To evaluate the contribution of de novo variants (DNVs) to SCZ, we sequenced the exomes of 53 individuals with sporadic SCZ and of their non-affected parents. We identified 49 DNVs, 18 of which were predicted to alter gene function, including 13 damaging missense mutations, 2 conserved splice site mutations, 2 nonsense mutations, and 1 frameshift deletion. The average number of exonic DNV per proband was 0.88, which corresponds to an exonic point mutation rate of 1.7×10−8 per nucleotide per generation. The non-synonymous-to-synonymous mutation ratio of 2.06 did not differ from neutral expectations. Overall, this study provides a list of 18 putative candidate genes for sporadic SCZ, and when combined with the results of similar reports, identifies a second proband carrying a non-synonymous DNV in the RGS12 gene.
doi:10.1371/journal.pone.0112745
PMCID: PMC4242613  PMID: 25420024
2.  G protein signaling modulator-3: a leukocyte regulator of inflammation in health and disease 
G protein signaling modulator-3 (GPSM3), also known as G18 or AGS4, is a member of a family of proteins containing one or more copies of a small regulatory motif known as the GoLoco (or GPR) motif. GPSM3 interacts directly with Gα and Gβ subunits of heterotrimeric G proteins to regulate downstream intracellular signals initiated by G protein coupled receptors (GPCRs) that are activated via binding to their cognate ligands. GPSM3 has a selective tissue distribution and is highly expressed in immune system cells; genome-wide association studies (GWAS) have recently revealed that single nucleotide polymorphisms (SNPs) in GPSM3 are associated with chronic inflammatory diseases. This review highlights the current knowledge of GPSM3 function in normal and pathologic immune-mediated conditions.
PMCID: PMC4138133  PMID: 25143870
GoLoco; G protein coupled receptor; GPSM3; chemokine; migration; rheumatoid arthritis
3.  Entamoeba histolytica Rho1 regulates actin polymerization through a divergent, Diaphanous-related formin 
Biochemistry  2012;51(44):8791-8801.
Entamoeba histolytica requires a dynamic actin cytoskeleton for intestinal and systemic pathogenicity. Diaphanous-related formins represent an important family of actin regulators that are activated by Rho GTPases. The E. histolytica genome encodes a large family of Rho GTPases and three diaphanous-related formins, of which EhFormin1 is known to regulate mitosis and cytokinesis in trophozoites. We demonstrate that EhFormin1 modulates actin polymerization through its formin homology 2 (FH2) domain. Despite a highly divergent diaphanous autoinhibitory domain, EhFormin1 is autoinhibited by an N- and C-terminal intramolecular interaction, but activated upon binding of EhRho1 to the N-terminal domain tandem. A crystal structure of the EhRho1·GTPγS/EhFormin1 complex illustrates an EhFormin1 conformation that diverges from mammalian mDia1 and lacks a secondary interaction with a Rho insert helix. The structural model also highlights residues required for specific recognition of the EhRho1 GTPase and suggests that the molecular mechanisms of EhFormin1 autoinhibition and activation differ from mammalian homologs.
doi:10.1021/bi300954g
PMCID: PMC3491106  PMID: 23050667
actin polymerization; guanine nucleotide binding protein
4.  G-protein signaling modulator-3, a gene linked to autoimmune diseases, regulates monocyte function and its deficiency protects from inflammatory arthritis 
Molecular immunology  2012;54(2):193-198.
Polymorphism at the GPSM3 gene locus is inversely associated with four systemic autoimmune diseases, including rheumatoid arthritis and ankylosing spondylitis. G-protein signaling modulator-3 (GPSM3) expression is most pronounced in myeloid cells, in which it targets heterotrimeric G-protein Gαi subunits of chemokine receptors, critical to immune function. To begin to explore the regulatory role of GPSM3 in monocytes, human THP-1 and primary mouse myeloid cells were cultured under stimulus conditions; GPSM3 was found by immunoblotting to be expressed at highest levels in the mature monocyte. To evaluate the effects of GPSM3 deficiency on a myeloid-dependent autoimmune disease, Collagen Antibody-Induced Arthritis (CAIA) was induced in Gpsm3−/− and control mice, which were then analyzed for clinical score, paw swelling, intra-articular proinflammatory markers, and histopathology. Mice lacking GPSM3 were protected from CAIA, and expression of monocyte-representative pro-inflammatory chemokine receptors and cytokines in paws of Gpsm3−/− mice were decreased. Flow cytometry, apoptosis, and transwell chemotaxis experiments were conducted to further characterize the effect of GPSM3 deficiency on survival and chemokine responsiveness of monocytes. GPSM3-deficient myeloid cells had reduced migration ex vivo to CCL2, CX3CL1, and chemerin and enhanced apoptosis in vitro. Our results suggest that GPSM3 is an important regulator of monocyte function involving mechanisms of differentiation, survival, and chemotaxis, and deficiency in GPSM3 expression is protective in acute inflammatory arthritis.
doi:10.1016/j.molimm.2012.12.001
PMCID: PMC3563835  PMID: 23280397
cell signaling molecules; collagen induced arthritis; autoimmune diseases; macrophages; rheumatoid arthritis
5.  RGS21, A Regulator of Taste and Mucociliary Clearance? 
The Laryngoscope  2013;124(3):E56-E63.
Objectives/Hypothesis:
Motile cilia of airway epithelial cells help to expel harmful inhaled material. Activation of bitterant-responsive G protein-coupled receptors (GPCRs) is believed to potentiate cilia beat frequency and mucociliary clearance. In this study, we investigated whether regulator of G protein signaling-21 (RGS21) has the potential to modulate signaling pathways connected to airway mucociliary clearance, given that RGS proteins modulate GPCR signaling by acting as GTPase-accelerating proteins (GAPs) for the Gα subunits of heterotrimeric G proteins.
Study Design:
This is a pilot investigation to determine if RGS21, a potential tastant specific RGS gene, is expressed in sinonasal mucosa, and to determine its specific Gα substrate using in vitro biochemical assays with purified proteins.
Methods:
Rgs21 expression in sinonasal mucosa was determined using quantitative, real-time PCR and a transgenic mouse expressing RFP from the Rgs21 promoter. Rgs21 was cloned, over-expressed, and purified using multistep protein chromatography. Biochemical and biophysical assays were used to determine if RGS21 could bind and accelerate the hydrolysis of GTP on heterotrimeric Gα subunits.
Results:
Rgs21 was expressed in sinonasal mucosa and lingual epithelium. Purified recombinant protein directly bound and accelerated GTP hydrolysis on Gα subunits.
Conclusions:
Rgs21 is expressed in sinonasal mucosa, is amenable to purification as a recombinant protein, and can bind to Gαi/o/q subunits. Furthermore, RGS21 can accelerate the hydrolysis rate of GTP on Gαi subunits. This provides evidence that RGS21 may be a negative regulator of bitterant responses. Future studies will be needed to determine the physiological role of this protein in mucociliary clearance.
doi:10.1002/lary.24326
PMCID: PMC3985343  PMID: 23908053
Regulators of G protein signaling (RGS); G proteins; gustation
6.  Evaluating Modulators of ‘Regulator of G-protein Signaling’ (RGS) Proteins 
Current Protocols in Pharmacology  2012;CHAPTER:Unit2.8.
‘Regulator of G-protein Signaling’ (RGS) proteins constitute a class of intracellular signaling regulators that accelerate GTP hydrolysis by heterotrimeric Gα subunits. In recent years, RGS proteins have emerged as potential drug targets for small molecule modulation. Described in this unit are high-throughput screening procedures for identifying modulators of RGS protein-mediated GTPase acceleration (‘GAP activity’), for assessment of RGS domain/Gα interactions (most avid in vitro when Gα is bound by aluminum tetrafluoride), and for validation of candidate GAP-modulatory molecules with the single turnover GTP hydrolysis assay.
doi:10.1002/0471141755.ph0208s56
PMCID: PMC3306263  PMID: 22382998
Regulator of G-protein Signaling (RGS) proteins; heterotrimeric G-protein α subunits; Förster resonance energy transfer (FRET); single-turnover GTP hydrolysis; fluorescence polarization (FP)
7.  Structural Determinants of RGS-RhoGEF Signaling Critical to Entamoeba histolytica Pathogenesis 
Summary
G-protein signaling pathways, as key components of physiologic responsiveness and timing, are frequent targets for pharmacologic intervention. Here, we identify an effector for heterotrimeric G-protein α subunit (EhGα1) signaling from Entamoeba histolytica, the causative agent of amoebic colitis. EhGα1 interacts with this effector and GTPase-accelerating protein (GAP), EhRGS-RhoGEF, in a nucleotide state-selective fashion. Co-expression of EhRGS-RhoGEF with constitutively active EhGα1 and EhRacC leads to Rac-dependent spreading in Drosophila S2 cells. EhRGS-RhoGEF overexpression in E. histolytica trophozoites leads to reduced migration toward serum and lower cysteine protease activity, as well as reduced attachment to, and killing of, host cells. A 2.3 Å crystal structure of the full-length EhRGS-RhoGEF reveals a putative inhibitory helix engaging the DH domain Rho-binding surface and the PH domain. Mutational analysis of the EhGα1/EhRGS-RhoGEF interface confirms a canonical RGS domain rather than a RhoGEF-RGS (“rgRGS”) domain, suggesting a convergent evolution toward heterotrimeric and small G-protein cross-talk.
doi:10.1016/j.str.2012.11.012
PMCID: PMC3545058  PMID: 23260656
8.  The mitochondrial proteins NLRX1 and TUFM form a complex that regulates type 1 interferon and autophagy 
Immunity  2012;36(6):933-946.
SUMMARY
The mitochondrial protein MAVS (also known as IPS-1, VISA, CARDIF) interacts with RLR (RIG-I-like receptors) to induce type 1 interferon (IFN-I) during viral infection. NLRX1 is a mitochondrial NLR (nucleotide-binding, leucine-rich repeats containing) protein that attenuates MAVS-RLR signaling. Using Nlrx1−/− cells we confirmed NLRX1 attenuated IFN-I production, but additionally promoted autophagy during viral infection. This dual function of NLRX1 paralleled the previously described functions of the autophagy-related proteins Atg5-Atg12, but NLRX1 did not associate with Atg5-Atg12. High throughput quantitative mass spectrometry and endogenous protein-protein interaction revealed an NLRX1-interacting partner, mitochondrial Tu translation elongation factor (TUFM). TUFM interacted with Atg5-Atg12 and Atg16L1, and has similar functions as NLRX1 by inhibiting RLR-induced IFN-I but promoting autophagy. In the absence of NLRX1, increased IFN-I and decreased autophagy provide an advantage for host defense against vesicular stomatitis virus. This study establishes a link between an NLR protein and the viral-induced autophagic machinery via an intermediary partner, TUFM.
doi:10.1016/j.immuni.2012.03.025
PMCID: PMC3397828  PMID: 22749352
9.  A Homogeneous Method to Measure Nucleotide Exchange by α-Subunits of Heterotrimeric G-Proteins Using Fluorescence Polarization 
Abstract
The mainstay of assessing guanosine diphosphate release by the α-subunit of a heterotrimeric G-protein is the [35S]guanosine 5′-O-(3-thiotriphosphate) (GTPγS) radionucleotide-binding assay. This assay requires separation of protein-bound GTPγS from free GTPγS at multiple time points followed by quantification via liquid scintillation. The arduous nature of this assay makes it difficult to quickly characterize multiple mutants, determine the effects of individual variables (e.g., temperature and Mg2+ concentration) on nucleotide exchange, or screen for small molecule modulators of Gα nucleotide binding/cycling properties. Here, we describe a robust, homogeneous, fluorescence polarization assay using a red-shifted fluorescent GTPγS probe that can rapidly determine the rate of GTPγS binding by Gα subunits.
doi:10.1089/adt.2010.0286
PMCID: PMC2957273  PMID: 20662737
10.  Inhibition of Dopamine Transporter Activity by G Protein βγ Subunits 
PLoS ONE  2013;8(3):e59788.
Uptake through the Dopamine Transporter (DAT) is the primary mechanism of terminating dopamine signaling within the brain, thus playing an essential role in neuronal homeostasis. Deregulation of DAT function has been linked to several neurological and psychiatric disorders including ADHD, schizophrenia, Parkinson’s disease, and drug addiction. Over the last 15 years, several studies have revealed a plethora of mechanisms influencing the activity and cellular distribution of DAT; suggesting that fine-tuning of dopamine homeostasis occurs via an elaborate interplay of multiple pathways. Here, we show for the first time that the βγ subunits of G proteins regulate DAT activity. In heterologous cells and brain tissue, a physical association between Gβγ subunits and DAT was demonstrated by co-immunoprecipitation. Furthermore, in vitro pull-down assays using purified proteins established that this association occurs via a direct interaction between the intracellular carboxy-terminus of DAT and Gβγ. Functional assays performed in the presence of the non-hydrolyzable GTP analog GTP-γ-S, Gβγ subunit overexpression, or the Gβγ activator mSIRK all resulted in rapid inhibition of DAT activity in heterologous systems. Gβγ activation by mSIRK also inhibited dopamine uptake in brain synaptosomes and dopamine clearance from mouse striatum as measured by high-speed chronoamperometry in vivo. Gβγ subunits are intracellular signaling molecules that regulate a multitude of physiological processes through interactions with enzymes and ion channels. Our findings add neurotransmitter transporters to the growing list of molecules regulated by G-proteins and suggest a novel role for Gβγ signaling in the control of dopamine homeostasis.
doi:10.1371/journal.pone.0059788
PMCID: PMC3608556  PMID: 23555781
11.  Two Gαi1 Rate-Modifying Mutations Act in Concert to Allow Receptor-Independent, Steady-State Measurements of RGS Protein Activity 
Journal of biomolecular screening  2009;14(10):1195-1206.
RGS proteins are critical modulators of G protein-coupled receptor (GPCR) signaling given their ability to deactivate Gα subunits via “GTPase-accelerating protein” (GAP) activity. Their selectivity for specific GPCRs makes them attractive therapeutic targets. However, measuring GAP activity is complicated by slow GDP release from Gα and lack of solution-phase assays for detecting free GDP in the presence of excess GTP. To overcome these hurdles, we developed a Gαi1 mutant with increased GDP dissociation and decreased GTP hydrolysis, enabling detection of GAP activity using steady-state GTP hydrolysis. Gαi1(R178M/A326S) GTPase activity was stimulated 6~12 fold by RGS proteins known to act on Gαi subunits, and not affected by those unable to act on Gαi, demonstrating that the Gα/RGS domain interaction selectivity was not altered by mutation. Gαi1(R178M/A326S) interacted with RGS proteins with expected binding specificity and affinities. To enable non-radioactive, homogenous detection of RGS protein effects on Gαi1(R178M/A326S), we developed a Transcreener® fluorescence polarization immunoassay based on a monoclonal antibody that recognizes GDP with greater than 100-fold selectivity over GTP. Combining Gαi1(R178M/A326S) with a homogenous, fluorescence-based GDP detection assay provides a facile means to explore the targeting of RGS proteins as a new approach for selective modulation of GPCR signaling.
doi:10.1177/1087057109347473
PMCID: PMC2795102  PMID: 19820068
Fluorescence polarization; GDP detection; regulators of G-protein signaling; surface plasmon resonance
12.  Heterotrimeric G-protein Signaling Is Critical to Pathogenic Processes in Entamoeba histolytica 
PLoS Pathogens  2012;8(11):e1003040.
Heterotrimeric G-protein signaling pathways are vital components of physiology, and many are amenable to pharmacologic manipulation. Here, we identify functional heterotrimeric G-protein subunits in Entamoeba histolytica, the causative agent of amoebic colitis. The E. histolytica Gα subunit EhGα1 exhibits conventional nucleotide cycling properties and is seen to interact with EhGβγ dimers and a candidate effector, EhRGS-RhoGEF, in typical, nucleotide-state-selective fashions. In contrast, a crystal structure of EhGα1 highlights unique features and classification outside of conventional mammalian Gα subfamilies. E. histolytica trophozoites overexpressing wildtype EhGα1 in an inducible manner exhibit an enhanced ability to kill host cells that may be wholly or partially due to enhanced host cell attachment. EhGα1-overexpressing trophozoites also display enhanced transmigration across a Matrigel barrier, an effect that may result from altered baseline migration. Inducible expression of a dominant negative EhGα1 variant engenders the converse phenotypes. Transcriptomic studies reveal that modulation of pathogenesis-related trophozoite behaviors by perturbed heterotrimeric G-protein expression includes transcriptional regulation of virulence factors and altered trafficking of cysteine proteases. Collectively, our studies suggest that E. histolytica possesses a divergent heterotrimeric G-protein signaling axis that modulates key aspects of cellular processes related to the pathogenesis of this infectious organism.
Author Summary
Entamoeba histolytica causes an estimated 50 million intestinal infections and 100,000 deaths per year worldwide. Here, we identify functional heterotrimeric G-protein subunits in Entamoeba histolytica, constituting a signaling pathway which, when perturbed, is seen to regulate multiple cellular processes required for pathogenesis. Like mammalian counterparts, EhGα1 forms a heterotrimer with EhGβγ that is dependent on guanine nucleotide exchange and hydrolysis. Despite engaging a classical G-protein effector, EhRGS-RhoGEF, EhGα1 diverges from mammalian Gα subunits and cannot be classified within mammalian Gα subfamilies, as highlighted by distinct structural features in our crystal structure of EhGα1 in the inactive conformation. To identify roles of G-protein signaling in pathogenesis-related cellular processes, we engineered trophozoites for inducible expression of EhGα1 or a dominant negative mutant, finding that G-protein signaling perturbation affects host cell attachment and the related process of contact-dependent killing, as well as trophozoite migration and Matrigel transmigration. A transcriptomic comparison of our engineered strains revealed differential expression of known virulence-associated genes, including amoebapores and cytotoxic cysteine proteases. The expression data suggested, and biochemical experiments confirmed, that cysteine protease secretion is altered upon G-protein overexpression, identifying a mechanism by which pathogenesis-related trophozoite behaviors are perturbed. In summary, E. histolytica encodes a vital heterotrimeric G-protein signaling pathway that is likely amenable to pharmacologic manipulation.
doi:10.1371/journal.ppat.1003040
PMCID: PMC3499586  PMID: 23166501
13.  The RGS protein inhibitor CCG-4986 is a covalent modifier of the RGS4 Gα-interaction face 
Biochimica et biophysica acta  2007;1774(9):1213-1220.
Summary
Regulator of G-protein signaling (RGS) proteins accelerate GTP hydrolysis by Gα subunits and are thus crucial to the timing of G protein-coupled receptor (GPCR) signaling. Small molecule inhibition of RGS proteins is an attractive therapeutic approach to diseases involving dysregulated GPCR signaling. Methyl-N-[(4-chlorophenyl)sulfonyl]-4-nitrobenzenesulfinimidoate (CCG-4986) was reported as a selective RGS4 inhibitor, but with an unknown mechanism of action (Roman et al., 2007 Mol Pharmacol. 71:169−75). Here, we describe its mechanism of action as covalent modification of RGS4. Mutant RGS4 proteins devoid of surface-exposed cysteine residues were characterized using surface plasmon resonance and FRET assays of Gα binding, as well as single-turnover GTP hydrolysis assays of RGS4 GAP activity, demonstrating that cysteine-132 within RGS4 is required for sensitivity to CCG-4986 inhibition. Sensitivity to CCG-4986 can be engendered within RGS8 by replacing the wildtype residue found in this position to cysteine. Mass spectrometry analysis identified a 153 dalton fragment of CCG-4986 as being covalently attached to the surface-exposed cysteines of the RGS4 RGS domain. We conclude that the mechanism of action of the RGS protein inhibitor CCG-4986 is via covalent modification of Cys-132 of RGS4, likely causing steric hinderance with the all-helical domain of the Gα substrate.
doi:10.1016/j.bbapap.2007.06.002
PMCID: PMC2084260  PMID: 17660054
CCG-4986; RGS4; RGS protein inhibitor; regulator of G-protein signaling; thiol adduct
14.  Computational Design of the Sequence and Structure of a Protein-Binding Peptide 
The de novo design of protein-binding peptides is challenging, because it requires identifying both a sequence and a backbone conformation favorable for binding. We used a computational strategy that iterates between structure and sequence optimization to redesign the C-terminal portion of the RGS14 GoLoco motif peptide so that it adopts a new conformation when bound to Gαi1. An X-ray crystal structure of the redesigned complex closely matches the computational model, with a backbone RMSD of 1.1 Å.
doi:10.1021/ja110296z
PMCID: PMC3081598  PMID: 21388199
15.  Structure of Gαi1 bound to a GDP-selective peptide provides insight into guanine nucleotide exchange 
Structure (London, England : 1993)  2005;13(7):1069-1080.
Heterotrimeric G-proteins are molecular switches that regulate numerous signaling pathways involved in cellular physiology. This characteristic is achieved by the adoption of two principal states: an inactive, GDP-bound and an active, GTP-bound state. Under basal conditions G-proteins exist in the inactive GDP-bound state, thus nucleotide exchange is crucial to the onset of signaling. Despite our understanding of G-protein signaling pathways, the mechanism of nucleotide exchange remains elusive. We employed phage display technology to identify nucleotide-state-dependent Gα binding peptides. Herein, we report a GDP-selective Gα-binding peptide, KB-752, that enhances spontaneous nucleotide exchange of Gαi subunits. Structural determination of the Gαi1/peptide complex reveals unique changes in the Gα switch regions predicted to enhance nucleotide exchange by creating a GDP dissociation route. Our results cast light onto a potential mechanism by which Gα subunits adopt a conformation suitable for nucleotide exchange.
doi:10.1016/j.str.2005.04.007
PMCID: PMC1405235  PMID: 16004878
16.  A bifunctional Gαi/Gαs modulatory peptide that attenuates adenylyl cyclase activity 
FEBS letters  2005;579(25):5746-5750.
Signaling via G-protein coupled receptors is initiated by receptor-catalyzed nucleotide exchange on Gα subunits normally bound to GDP and Gβγ. Activated Gα·GTP then regulates effectors such as adenylyl cyclase. Except for Gβγ, no known regulators bind the adenylyl cyclase-stimulatory subunit Gαs in its GDP-bound state. We recently described a peptide, KB-752, that binds and enhances the nucleotide exchange rate of the adenylyl cyclase-inhibitory subunit Gαi. Herein, we report that KB-752 binds Gαs·GDP yet slows its rate of nucleotide exchange. KB-752 inhibits GTPγS-stimulated adenylyl cyclase activity in cell membranes, reflecting its opposing effects on nucleotide exchange by Gαi and Gαs.
doi:10.1016/j.febslet.2005.09.059
PMCID: PMC1363735  PMID: 16225870
Adenylyl cyclase; Biosensors; G-proteins; Phage display; Signal transduction; Surface plasmon resonance
17.  A P-loop Mutation in Gα Subunits Prevents Transition to the Active State: Implications for G-protein Signaling in Fungal Pathogenesis 
PLoS Pathogens  2012;8(2):e1002553.
Heterotrimeric G-proteins are molecular switches integral to a panoply of different physiological responses that many organisms make to environmental cues. The switch from inactive to active Gαβγ heterotrimer relies on nucleotide cycling by the Gα subunit: exchange of GTP for GDP activates Gα, whereas its intrinsic enzymatic activity catalyzes GTP hydrolysis to GDP and inorganic phosphate, thereby reverting Gα to its inactive state. In several genetic studies of filamentous fungi, such as the rice blast fungus Magnaporthe oryzae, a G42R mutation in the phosphate-binding loop of Gα subunits is assumed to be GTPase-deficient and thus constitutively active. Here, we demonstrate that Gα(G42R) mutants are not GTPase deficient, but rather incapable of achieving the activated conformation. Two crystal structure models suggest that Arg-42 prevents a typical switch region conformational change upon Gαi1(G42R) binding to GDP·AlF4− or GTP, but rotameric flexibility at this locus allows for unperturbed GTP hydrolysis. Gα(G42R) mutants do not engage the active state-selective peptide KB-1753 nor RGS domains with high affinity, but instead favor interaction with Gβγ and GoLoco motifs in any nucleotide state. The corresponding Gαq(G48R) mutant is not constitutively active in cells and responds poorly to aluminum tetrafluoride activation. Comparative analyses of M. oryzae strains harboring either G42R or GTPase-deficient Q/L mutations in the Gα subunits MagA or MagB illustrate functional differences in environmental cue processing and intracellular signaling outcomes between these two Gα mutants, thus demonstrating the in vivo functional divergence of G42R and activating G-protein mutants.
Author Summary
Heterotrimeric G-proteins function as molecular switches to convey cellular signals. When a G-protein coupled receptor encounters its ligand at the cellular membrane, it catalyzes guanine nucleotide exchange on the Gα subunit, resulting in a shift from an inactive to an active conformation. G-protein signaling pathways are conserved from mammals to plants and fungi, including the rice blast fungus Magnaporthe oryzae. A mutation in the Gα subunit (G42R), previously thought to eliminate its GTPase activity, leading to constitutive activation, has been utilized to investigate roles of heterotrimeric G-protein signaling pathways in multiple species of filamentous fungi. Here, we demonstrate through structural, biochemical, and cellular approaches that G42R mutants are neither GTPase deficient nor constitutively active, but rather are unable to transition to the activated conformation. A direct comparison of M. oryzae fungal strains harboring either G42R or truly constitutively activating mutations in two Gα subunits, MagA and MagB, revealed markedly different phenotypes. Our results suggest that activation of MagB is critical for pathogenic development of M. oryzae in response to hydrophobic surfaces, such as plant leaves. Furthermore, the lack of constitutive activity by Gα(G42R) mutants prompts a re-evaluation of its use in previous genetic experiments in multiple fungal species.
doi:10.1371/journal.ppat.1002553
PMCID: PMC3285607  PMID: 22383884
18.  Integrating energy calculations with functional assays to decipher the specificity of G-protein inactivation by RGS proteins 
The diverse RGS protein family is responsible for the precise timing of G-protein signaling. To understand how RGS protein structure encodes their common ability to inactivate G-proteins and their selective G-protein recognition, we integrated structure-based energy calculations with biochemical measurements of RGS protein activity. We revealed that, in addition to previously identified conserved residues, RGS proteins contain another group of variable modulatory residues, which reside at the periphery of the RGS-domain–G-protein interface and fine-tune G-protein recognition. Mutations of modulatory residues in high-activity RGS proteins impaired RGS function, whereas redesign of low-activity RGS proteins in critical modulatory positions yielded complete gain-of-function. Therefore, RGS proteins combine a conserved core interface with peripheral modulatory residues to selectively optimize G-protein recognition and inactivation. Finally, we show that our quantitative framework for analyzing protein-protein interactions can be extended to analyze interaction specificity across other large protein families.
doi:10.1038/nsmb.2068
PMCID: PMC3130846  PMID: 21685921
19.  The GAPs, GEFs, and GDIs of heterotrimeric G-protein alpha subunits 
The heterotrimeric G-protein alpha subunit has long been considered a bimodal, GTP-hydrolyzing switch controlling the duration of signal transduction by seven-transmembrane domain (7TM) cell-surface receptors. In 1996, we and others identified a superfamily of “regulator of G-protein signaling” (RGS) proteins that accelerate the rate of GTP hydrolysis by Gα subunits (dubbed GTPase-accelerating protein or “GAP” activity). This discovery resolved the paradox between the rapid physiological timing seen for 7TM receptor signal transduction in vivo and the slow rates of GTP hydrolysis exhibited by purified Gα subunits in vitro. Here, we review more recent discoveries that have highlighted newly-appreciated roles for RGS proteins beyond mere negative regulators of 7TM signaling. These new roles include the RGS-box-containing, RhoA-specific guanine nucleotide exchange factors (RGS-RhoGEFs) that serve as Gα effectors to couple 7TM and semaphorin receptor signaling to RhoA activation, the potential for RGS12 to serve as a nexus for signaling from tyrosine kinases and G-proteins of both the Gα and Ras-superfamilies, the potential for R7-subfamily RGS proteins to couple Gα subunits to 7TM receptors in the absence of conventional Gβγ dimers, and the potential for the conjoint 7TM/RGS-box Arabidopsis protein AtRGS1 to serve as a ligand-operated GAP for the plant Gα AtGPA1. Moreover, we review the discovery of novel biochemical activities that also impinge on the guanine nucleotide binding and hydrolysis cycle of Gα subunits: namely, the guanine nucleotide dissociation inhibitor (GDI) activity of the GoLoco motif-containing proteins and the 7TM receptor-independent guanine nucleotide exchange factor (GEF) activity of Ric‑8/synembryn. Discovery of these novel GAP, GDI, and GEF activities have helped to illuminate a new role for Gα subunit GDP/GTP cycling required for microtubule force generation and mitotic spindle function in chromosomal segregation.
PMCID: PMC1142213  PMID: 15951850
asymmetric cell division; GoLoco motif; G-protein; RGS proteins; RIC-8
20.  A capture coupling method for the covalent immobilization of hexahistidine tagged proteins for surface plasmon resonance 
i. Summary
Surface Plasmon Resonance (SPR) is a robust method to detect and quantify macromolecular interactions; however, to measure binding interactions, one component must be immobilized on a sensor surface. This is typically achieved using covalent immobilization via free amines or thiols, or noncovalent immobilization using high affinity interactions such as biotin/streptavidin or antibody/antigen. In this Chapter we describe a robust method to covalently immobilize His6 fusion proteins on the sensor surface for SPR analysis.
doi:10.1007/978-1-60761-670-2_5
PMCID: PMC3031178  PMID: 20217615
Biacore; SPR; Capture Coupling; Immobilization; Hexahistidine; His-tag
21.  High affinity immobilization of proteins using biotin- and GST-based coupling strategies 
Surface Plasmon Resonance (SPR) is a highly sensitive method for the detection of molecular interactions. One interacting partner is immobilized on the sensor chip surface while the other is injected across the sensor surface. This chapter focuses on high affinity immobilization of protein substrates for affinity and kinetic analyses using biotin/streptavidin interaction and GST/anti-GST-antibody interaction.
doi:10.1007/978-1-60761-670-2_4
PMCID: PMC3025018  PMID: 20217614
BIAcore 3000; Biotin; Streptavidin; GST; High-Affinity Immobilization; SPR
22.  LGN regulates mitotic spindle orientation during epithelial morphogenesis 
The Journal of Cell Biology  2010;189(2):275-288.
Disrupting LGN’s function at lateral membrane domains displaces the axis of cell division in cyst-forming MDCK cells.
Coordinated cell polarization and mitotic spindle orientation are thought to be important for epithelial morphogenesis. Whether spindle orientation is indeed linked to epithelial morphogenesis and how it is controlled at the molecular level is still unknown. Here, we show that the NuMA- and Gα-binding protein LGN is required for directing spindle orientation during cystogenesis of MDCK cells. LGN localizes to the lateral cell cortex, and is excluded from the apical cell cortex of dividing cells. Depleting LGN, preventing its cortical localization, or disrupting its interaction with endogenous NuMA or Gα proteins all lead to spindle misorientation and abnormal cystogenesis. Moreover, artificial mistargeting of endogenous LGN to the apical membrane results in a near 90° rotation of the spindle axis and profound cystogenesis defects that are dependent on cell division. The normal apical exclusion of LGN during mitosis appears to be mediated by atypical PKC. Thus, cell polarization–mediated spatial restriction of spindle orientation determinants is critical for epithelial morphogenesis.
doi:10.1083/jcb.200910021
PMCID: PMC2856901  PMID: 20385777
23.  Helix dipole movement and conformational variability contribute to allosteric GDP release in Gαi subunits†‡ 
Biochemistry  2009;48(12):2630-2642.
Heterotrimeric G proteins (Gαβγ) transmit signals from activated G protein coupled receptors (GPCRs) to downstream effectors through a guanine nucleotide signaling cycle. Numerous studies indicate that the carboxy-terminal α5 helix of Gα subunits participate in Gα-receptor binding, and previous EPR studies suggest this receptor-mediated interaction induces a rotation and translation of the α5 helix of the Gα subunit [Oldham et al., Nat. Struct. Mol. Biol., 13: 772-7 (2006)]. Based on this result, an engineered disulfide bond was designed to constrain the α5 helix of Gαi1 into its EPR-measured receptor-associated conformation through the introduction of cysteines at positions 56 in the α1 helix and 333 in the α5 helix (I56C/Q333C Gαi1). A functional mimetic of the EPR-measured α5 helix dipole movement upon receptor association was additionally created by introduction of a positive charge at the amino-terminus of this helix, D328R Gαi1. Both proteins exhibit dramatically elevated basal nucleotide exchange. The 2.9 Å resolution crystal structure of the I56C/Q333C Gαi1 in complex with GDP-AlF4 − reveals the shift of the α5 helix toward the guanine nucleotide-binding site that is anticipated by EPR measurements. The structure of the I56C/Q333C Gαi1 subunit further revealed altered positions for the switch regions and throughout the Gαi1 subunit, accompanied by significantly elevated crystallographic temperature factors. Combined with previous evidence in the literature, the structural analysis supports the critical role of electrostatics of the α5 helix dipole and overall conformational variability during nucleotide release.
doi:10.1021/bi801853a
PMCID: PMC2736342  PMID: 19222191
24.  Structure of the parathyroid hormone receptor C-terminus bound to the G-protein dimer Gβ1γ2 
Structure (London, England : 1993)  2008;16(7):1086-1094.
Summary
A critical role of the Gβγ dimer in heterotrimeric G-protein signaling is to facilitate engagement and activation of the Gα subunit by cell-surface G protein-coupled receptors. However, high-resolution structural information of the connectivity between receptor and Gβγ has not previously been available. Here, we describe the structural determinants of Gβ1γ2 in complex with a C-terminal region of the parathyroid hormone receptor-1 (PTH1R) as obtained by x-ray crystallography. The structure reveals that several critical residues within PTH1R contact solely Gβ residues located within the outer edge of WD1 and WD7 repeat segments of the Gβ toroid structure. These regions encompass a predicted membrane-facing region of Gβ thought to be oriented in a fashion that is accessible to the membrane-spanning receptor. Mutation of key receptor contact residues on Gβ1 lead to a selective loss-of-function in receptor/heterotrimer coupling while preserving Gβ1γ2 activation of the effector phospholipase-C beta.
doi:10.1016/j.str.2008.04.010
PMCID: PMC2601695  PMID: 18611381
25.  Crystal structure of the multifunctional Gβ5–RGS9 complex 
Regulators of G-protein signaling (RGS) proteins enhance the intrinsic GTPase activity of G protein α (Gα) subunits and are vital for proper signaling kinetics downstream of G protein–coupled receptors (GPCRs). R7 subfamily RGS proteins specifically and obligately dimerize with the atypical G protein β5 (Gβ5) subunit through an internal G protein γ (Gγ)-subunit–like (GGL) domain. Here we present the 1.95-Å crystal structure of the Gβ5–RGS9 complex, which is essential for normal visual and neuronal signal transduction. This structure reveals a canonical RGS domain that is functionally integrated within a molecular complex that is poised for integration of multiple steps during G-protein activation and deactivation.
doi:10.1038/nsmb.1377
PMCID: PMC2702320  PMID: 18204463

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