Kathleen H. Young

Regional distribution of regulators of G-protein signaling (RGS) 1, 2, 13, 14, 16, and GAIP messenger ribonucleic acids by in situ hybridization in rat brain

Regulators of G-protein signaling (RGS) proteins are a novel family of GTPase-activating proteins that interact with Galpha subunits of the Gi/o, Gz, Gq and G(12/13) subfamilies to dampen G-protein-coupled receptor (GPCR)-mediated signaling by accelerating intrinsic Galpha-GTPase activity. In the present study, we report on messenger ribonucleic acid (mRNA) localization in rat brain of six RGS genes by in situ hybridization. The distribution patterns of RGS2, RGS13, RGS14 and GAIP (Galpha interacting protein) overlapped in most brain regions examined. Highest regional expression was observed for RGS2 in the cerebral cortical layers, striatum, hippocampal formation, several thalamic and hypothalamic nuclei and hindbrain regions such as the pontine, interpeduncular and dorsal raphe nuclei. Levels of RGS14 mRNA closely paralleled those of RGS2 expression levels throughout most brain regions. RGS13 mRNA was enriched in the hippocampal formation, amygdala, mammillary nuclei as well as the pontine and interpeduncular nuclei. GAIP expression levels were highest in the hippocampal formation with moderate to low levels present in all other regions studied. Of the six RGS genes probed, RGS16 mRNA displayed a discrete localization predominantly in the thalamic midline/intralaminar and principal relay nuclei, and the hypothalamic suprachiasmatic nucleus. RGS1 mRNA signal was not detected in brain. In conclusion, the in situ hybridization studies for RGS2, RGS13, RGS14, RGS16 and GAIP mRNAs extend our knowledge of the distribution of RGS genes expressed in the rat central nervous system, and indicate overlapping RGS-enriched regions that may be indicative of functional diversification in GPCR signaling pathway modulation.

The Yeast G Protein α Subunit Gpa1 Transmits a Signal through an RNA Binding Effector Protein Scp160.

In yeast Saccharomyces cerevisiae the G protein betagamma subunits (Ste4/Ste18) have long been known to transmit the signal required for mating. Here we demonstrate that GTPase-deficient mutants of Galpha (Gpa1) directly activate the mating response pathway. We also show that signaling by activated Gpa1 requires direct coupling to an RNA binding protein Scp160. These findings suggest an additional role for Gpa1 and reveal Scp160 as a component of the mating response pathway in yeast.

Brain Region Specific Actions of Regulator of G Protein Signaling 4 Oppose Morphine Reward and Dependence but Promote Analgesia

BACKGROUND Regulator of G protein signaling 4 (RGS4) is one of the smaller members of the RGS family of proteins, which are known to control signaling amplitude and duration via interactions with G protein alpha subunits or other signaling molecules. Earlier evidence suggests dynamic regulation of RGS4 levels in neuronal networks mediating actions of opiates and other drugs of abuse, but the consequences of RGS4 actions in vivo are largely unknown. METHODS In this study, we use constitutive and nucleus accumbens-inducible RGS4 knockout mice as well as mice overexpressing RGS4 in the nucleus accumbens via viral mediated gene transfer, to examine the influence of RGS4 on behavioral responses to opiates. We also use electrophysiology and immunoprecipitation assays to further understand the mechanisms underlying the tissue-specific actions of RGS4. RESULTS Inducible knockout or selective overexpression of RGS4 in the nucleus accumbens reveals that, in this brain region, RGS4 acts as a negative regulator of morphine reward, whereas in the locus coeruleus RGS4 opposes morphine physical dependence. In contrast, we show that RGS4 does not affect morphine analgesia or tolerance but is a positive modulator of certain opiate analgesics, such as methadone and fentanyl. CONCLUSIONS These findings provide fundamentally novel information concerning the role of RGS4 in the cellular mechanisms underlying the diverse actions of opiate drugs in the nervous system.

Regulator of G Protein Signaling Z1 (RGSZ1) Interacts with Gαi Subunits and Regulates Gαi-mediated Cell Signaling

Regulator of G protein signaling (RGS) proteins constitute a family of over 20 proteins that negatively regulate heterotrimeric G protein-coupled receptor signaling pathways by enhancing endogenous GTPase activities of G protein α subunits. RGSZ1, one of the RGS proteins specifically localized to the brain, has been cloned previously and described as a selective GTPase accelerating protein for Gαz subunit. Here, we employed several methods to provide new evidence that RGSZ1 interacts not only with Gαz, but also with Gαi, as supported byin vitro binding assays and functional studies. Using glutathione S-transferase fusion protein pull-down assays, glutathione S-transferase-RGSZ1 protein was shown to bind35S-labeled Gαi1 protein in anAlF 4 − dependent manner. The interaction between RGSZ1 and Gαi was confirmed further by co-immunoprecipitation studies and yeast two-hybrid experiments using a quantitative luciferase reporter gene. Extending these observations to functional studies, RGSZ1 accelerated endogenous GTPase activity of Gαi1 in single-turnover GTPase assays. Human RGSZ1 functionally regulated GPA1 (a yeast Gαi-like protein)-mediated yeast pheromone response when expressed in a SST2 (yeast RGS protein) knockout strain. In PC12 cells, transfected RGSZ1 blocked mitogen-activated protein kinase activity induced by UK14304, an α2-adrenergic receptor agonist. Furthermore, RGSZ1 attenuated D2 dopamine receptor agonist-induced serum response element reporter gene activity in Chinese hamster ovary cells. In summary, these data suggest that RGSZ1 serves as a GTPase accelerating protein for Gαi and regulates Gαi-mediated signaling, thus expanding the potential role of RGSZ1 in G protein-mediated cellular activities.

Snapin interacts with the N-terminus of regulator of G protein signaling 7

The N-terminus of regulator of G protein signaling 7 (RGS7) contains a dishevelled/egl-10/pleckstrin (DEP) domain of unknown function. To gain insight into its function, we used yeast two-hybrid analysis to screen a human whole brain cDNA library in order to identify proteins that interact specifically with the N-terminus of human RGS7 (amino acid residues 1-248). From this analysis, we identified snapin, a protein associated with the SNARE complex in neurons, as an interactor with the N-terminus of RGS7. Deletion mutation analysis in yeast demonstrated that the interaction between RGS7 and snapin is specific and is mediated primarily by amino acid residues 1-69 of RGS7 (which contains the proximal portion of the DEP domain). The interaction between RGS7 and snapin was also demonstrated in mammalian cells by coimmunoprecipitation and pull-down assays. Our results suggest that RGS7 could play a role in synaptic vesicle exocytosis through its interaction with snapin.

A Dual Luciferase Multiplexed High-Throughput Screening Platform for Protein-Protein Interactions

To study the biology of regulators of G-protein signaling (RGS) proteins and to facilitate the identification of small molecule modulators of RGS proteins, the authors recently developed an advanced yeast 2-hybrid (YTH) assay format for GαZand RGS-Z1. Moreover, they describe the development of a multiplexed luciferase-based assay that has been successfully adapted to screen large numbers of small molecule modulators of protein-protein interactions. They generated and evaluated 2 different luciferase reporter gene systems for YTH interactions, a Gal4 responsive firefly luciferase reporter gene and a Gal4 responsive Renilla luciferase reporter gene. Both the firefly and Renilla luciferase reporter genes demonstrated a 40-to 50-fold increase in luminescence in strains expressing interacting YTH fusion proteins versus negative control strains. Because the firefly and Renilla luciferase proteins have different substrate specificity, the assays were multiplexed. The multiplexed luciferase-based YTH platform adds speed, sensitivity, simplicity, quantification, and efficiency to YTH high-throughput applications and therefore greatly facilitates the identification of small molecule modulators of protein-protein interactions as tools or potential leads for drug discovery efforts.

Identification of Peptides That Inhibit Regulator of G Protein Signaling 4 Function

Regulators of G protein signaling (RGS) are a family of GTPase-activating proteins (GAP) that interact with heterotrimeric G proteins in the negative regulation of G-protein-coupled receptor (GPCR) signaling. RGS4, the first identified mammalian member of the RGS family, has been implicated in many GPCR signaling pathways involved in disease states. We report herein the identification of a 16-amino-acid peptide (P17) as an inhibitor of RGS4. The peptide was found by screening a random peptide library using RGS4 as ‘bait’ in a yeast two-hybrid system. This peptide inhibited RGS4 GAP activity on Gαi1 in a GTPase assay, and blocked the interaction between RGS4 and Gαi1 in a pull-down assay. The peptide displayed dose-dependent inhibition of RGS4 and Gα-interacting protein (GAIP) GAP activities, yet showed no substantial effect on RGS7. Electrophysiological studies in Xenopus oocytes demonstrated that P17 attenuates RGS4 modulation of M2 muscarinic receptor stimulation of GIRK (G-protein-mediated inwardly rectifying potassium) channels. Deletion of an arginine at the N terminus of P17 abolished its ability to inhibit RGS4 GAP activity, as did deletions of C-terminal residues. The P17 peptide showed no similarity to any known peptide sequence. Further investigation and optimization of the peptide may provide unique information for the development of RGS4 inhibitors for future therapeutic application.

Isogenic Human Cell Lines for Drug Discovery: Regulation of Target Gene Expression by Engineered Zinc-Finger Protein Transcription Factors

Isogenic cell lines differing only in the expression of the protein of interest provide the ideal platform for cell-based screening. However, related natural lines differentially expressing the therapeutic target of choice are rare. Here the authors report a strategy for drug screening employing isogenic human cell lines in which the expression of the target protein is regulated by a gene-specific engineered zinc-finger protein (ZFP) transcription factor (TF). To demonstrate this approach, a ZFP TF activator of the human parathyroid hormone receptor 1 (PTHR1) gene was identified and introduced into HEK293 cells (negative for PTHR1). Following induction of ZFP TF expression, this cell line produced functional PTHR1 protein, resulting in a robust and ligand-specific cyclic adenosine monophosphate (cAMP) response. Reciprocally, the natural expression of PTHR1 observed in SAOS2 cells was dramatically reduced by the introduction of the appropriate PTHR1-specific ZFP TF repressor. Moreover, this ZFP-driven PTHR1 repression selectively eliminated the functional cAMP response invoked by known ligands of PTHR1. These data establish ZFP TF–generated isogenic lines as a general approach for the identification of therapeutic agents specific for the target gene of interest.

Alterations in 5-HT2A receptor signaling in male and female transgenic rats over-expressing either Gq or RGS-insensitive Gq protein.

Serotonin 2A (5-HT2A) receptors are coupled to Galphaq and Galpha11 proteins to activate phospholipase C (PLC). Regulators of G-protein signaling proteins (RGS) modulate G-protein signaling by accelerating the intrinsic GTPase activity of Galphaq and Galpha11. This study investigated the effects of over-expression of wild-type Galphaq proteins (Gq-Tg) and over-expression of RGS-insensitive Galphaq proteins (G188S, RGSi-Tg) on 5-HT2A receptor mediated signaling in transgenic rats. Over-expression of wild-type Galphaq and RGS insensitive mutant Galphaq did not produce significant alterations in the levels of Galpha11, RGS2, RGS4, RGS7, RGS16 or 5-HT2A proteins. RGSi-Tg rats had higher oxytocin and corticosterone responses to (-)DOI, a 5-HT2A/2C receptor agonist, compared to Gq-Tg rats. RGSi-Tg and Gq-Tg rats had higher ACTH responses to (-)DOI compared to control rats. Similarly, 5-HT-stimulated PLC activity in the frontal cortex was higher in RGSi-Tg and Gq-Tg rats compared to control rats. In contrast, GTPgammaS-stimulated PLC activity was higher in Gq-Tg rats but not in RGSi-Tg rats compared to control rats. There was a small but statistically significant increase in the affinity of [125I]-DOI labeled 5-HT2A receptors in RGSi-Tg rats and Gq-Tg rats compared to controls. There were no significant differences in Bmax and Kd of [3H] ketanserin labeled 5-HT2A receptors among the three groups. These data suggest that the effect of RGS proteins on 5-HT2A receptor signaling is cell type specific. In transgenic rats over-expressing Galphaq, endogenous RGS proteins have a negative effect on 5-HT2A receptor-mediated oxytocin release. In contrast, endogenous RGS protein had no impact on 5-HT2A receptor-mediated ACTH release in transgenic rats.

Activation of Serum Response Element by D2 Dopamine Receptor Is Governed by Gβγ-Mediated MAPK and Rho Pathways and Regulated by RGS Proteins

In this study, we investigated the activation of the serum response element (SRE) by the D2 dopamine receptor (D2R) agonist quinpirole. Stimulation of CHO cells expressing the D2R by quinpirol evoked a dose-dependent SRE activation, which was completely blocked by overnight treatment of pertussis toxin or by co-expression of the β-adrenergic receptor kinase C-terminus, implicating the involvement of Gαi and Gβγ in the signal transduction. Furthermore, using MEK inhibitors and dominant negative mutants of RhoA, Rac1, and Cdc42, we showed that the Gβγ-mediated activation of the SRE in CHO cells utilizes both MAPK and Rho pathways. Expression of either regulator of G protein signaling 2 or 4 (RGS2 or RGS4) proteins significantly attenuated the quinpirole-induced SRE activation. These results delineate the signaling pathways which couple D2 receptor to the transcriptional activation of SRE and demonstrate a modulatory role for RGS proteins in these processes.