Regulators Of G Protein Signaling Proteins Research Articles

Characterizations and functions of regulator of G protein signaling (RGS) in fungi

Proteins that serve as regulator of G protein signaling (RGS) primarily function as GTPase accelerators that promote GTP hydrolysis by the Gα subunits, thereby inactivating the G protein and rapidly switching off G protein-coupled signaling pathways. Since the first RGS protein was identified from the budding yeast Saccharomyces cerevisiae, more than 30 RGS and RGS-like proteins have been characterized from several model fungi, such as Aspergillus nidulans, Beauveria bassiana, Candida albicans, Fusarium verticillioides, Magnaporthe oryzae, and Metarhizium anisopliae. In this review, the partial biochemical properties and functional domains of RGS and RGS-like proteins were predicted and compared, and the roles of RGS and RGS-like proteins in different fungi were summarized. Moreover, the phylogenetic relationship among RGS and RGS-like proteins from various fungi was analyzed and discussed.

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

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.

Comparative proteomic analyses reveal that FlbA down-regulates gliT expression and SOD activity in Aspergillus fumigatus

FlbA is a regulator of G-protein signaling protein that plays a central role in attenuating heterotrimeric G-protein mediated vegetative growth signaling in Aspergillus. The deletion of flbA (∆flbA) in the opportunistic human pathogen Aspergillus fumigatus results in accelerated cell death and autolysis in submerged culture. To further investigate the effects of ∆flbA on intracellular protein levels we carried out 2-D proteome analyses of 2-day old submerged cultures of ∆flbA and wild type (WT) strains and observed 160 differentially expressed proteins. Via nano-LC-ESI-MS/MS analyses, we revealed the identity of 10 and 2 proteins exhibiting high and low level accumulation, respectively, in ∆flbA strain. Notably, the GliT protein is accumulated at about 1800-fold higher levels in ∆flbA than WT. Moreover, GliT is secreted at high levels from ∆flbA strain, whereas Sod1 (superoxide dismutase) is secreted at a higher level in WT. Northern blot analyses reveal that ∆flbA results in elevated accumulation of gliT mRNA. Consequently, ∆flbA strain exhibits enhanced tolerance to gliotoxin toxicity. Finally, ∆flbA strain displayed enhanced SOD activity and elevated resistance to menadione and paraquat. In summary, FlbA-mediated signaling control negatively affects cellular responses associated with detoxification of reactive oxygen species and of exogenous gliotoxin in A. fumigatus. Regulator of G protein Signaling (RGS) proteins play crucial roles in fundamental biological processes in filamentous fungi. FlbA is the first studied filamentous fungal RGS protein, yet much remains to be understood about its roles in the opportunistic human pathogen Aspergillus fumigatus. In the present study, we examined the effects of the deletion of flbA using comprehensive analyses of the intra- and extracellular proteomes of A. fumigatus wild type and the flbA deletion mutant. Via MS analyses, we identified 10 proteins exhibiting high level accumulation in the flbA deletion mutant and 8 proteins differentially secreted in wild type and the flbA mutant. Based on proteomic analyses, we further examined the role of FlbA and found that FlbA down-regulates gliT expression and SOD activity. Our results proposed that FlbA-mediated signaling control negatively affects cellular responses associated with detoxification of reactive oxygen species and exogenous gliotoxin in A. fumigatus.

A High Throughput Screen for RGS Proteins Using Steady State Monitoring of Free Phosphate Formation

G-protein coupled receptors are a diverse group that are the target of over 50% of marketed drugs. Activation of these receptors results in the exchange of bound GDP for GTP in the Gα subunit of the heterotrimeric G-protein. The Gα subunit dissociates from the β/γ subunits and both proceed to affect downstream signaling targets. The signal terminates by the hydrolysis of GTP to GDP and is temporally regulated by Regulators of G-protein Signaling (RGS) proteins that act as GTPase Activating Proteins (GAPs). This makes RGS proteins potentially desirable targets for “tuning” the effects of current therapies as well as developing novel pharmacotherapies. Current methods for evaluating RGS activity depend on laborious and/or expensive techniques. In this study we developed a simple and inexpensive assay for the steady state analysis of RGS protein GAP activity, using RGS4, RGS8 and RGS17 as models. Additionally, we report the use of RGS4 as a model for high throughput assay development. After initial setup, this assay can be conducted in a highly parallel fashion with a read time of less than 8 minutes for a 1536-well plate. The assay exhibited a robust Z-factor of 0.6 in a 1536-well plate. We conducted a pilot screen for inhibitors using a small, 2320 compound library. From this screen, 13 compounds were identified as compounds for further analysis. The successful development of this assay for high-throughput screening provides a low cost, high speed, simple method for assessing RGS protein activity.

Regulator of G-Protein Signaling – 5 (RGS5) Is a Novel Repressor of Hedgehog Signaling

Hedgehog (Hh) signaling plays fundamental roles in morphogenesis, tissue repair, and human disease. Initiation of Hh signaling is controlled by the interaction of two multipass membrane proteins, patched (Ptc) and smoothened (Smo). Recent studies identify Smo as a G-protein coupled receptor (GPCR)-like protein that signals through large G-protein complexes which contain the Gαi subunit. We hypothesize Regulator of G-Protein Signaling (RGS) proteins, and specifically RGS5, are endogenous repressors of Hh signaling via their ability to act as GTPase activating proteins (GAPs) for GTP-bound Gαi, downstream of Smo. In support of this hypothesis, we demonstrate that RGS5 over-expression inhibits sonic hedgehog (Shh)-mediated signaling and osteogenesis in C3H10T1/2 cells. Conversely, signaling is potentiated by siRNA-mediated knock-down of RGS5 expression, but not RGS4 expression. Furthermore, using immuohistochemical analysis and co-immunoprecipitation (Co-IP), we demonstrate that RGS5 is present with Smo in primary cilia. This organelle is required for canonical Hh signaling in mammalian cells, and RGS5 is found in a physical complex with Smo in these cells. We therefore conclude that RGS5 is an endogenous regulator of Hh-mediated signaling and that RGS proteins are potential targets for novel therapeutics in Hh-mediated diseases.

The Ras‐binding domain region of RGS14 regulates its functional interactions with heterotrimeric G proteins

RGS14 is a 60 kDa protein that contains a regulator of G protein signaling (RGS) domain near its N-terminus, a central region containing a pair of tandem Ras-binding domains (RBD), and a GPSM (G protein signaling modulator) domain (a.k.a. Gi/o-Loco binding [GoLoco] motif) near its C-terminus. The RGS domain of RGS14 exhibits GTPase accelerating protein (GAP) activity toward Gαi/o proteins, while its GPSM domain acts as a guanine nucleotide dissociation inhibitor (GDI) on Gαi1 and Gαi3. In the current study, we investigate the contribution of different domains of RGS14 to its biochemical functions. Here we show that the full-length protein has a greater GTPase activating activity but a weaker inhibition of nucleotide dissociation relative to its isolated RGS and GPSM regions, respectively. Our data suggest that these differences may be attributable to an inter-domain interaction within RGS14 that promotes the activity of the RGS domain, but simultaneously inhibits the activity of the GPSM domain. The RBD region seems to play an essential role in this regulatory activity. Moreover, this region of RGS14 is also able to bind to members of the B/R4 subfamily of RGS proteins and enhance their effects on GPCR-activated Gi/o proteins. Overall, our results suggest a mechanism wherein the RBD region associates with the RGS domain region, producing an intramolecular interaction within RGS14 that enhances the GTPase activating function of its RGS domain while disfavoring the negative effect of its GPSM domain on nucleotide dissociation.

Atrial Fibrillation Promotion by Endurance Exercise: Demonstration and Mechanistic Exploration in an Animal Model

The goal of this study was to assess mechanisms underlying atrial fibrillation (AF) promotion by exercise training inan animal model. High-level exercise training promotes AF, but the underlying mechanisms are unclear. AF susceptibility was assessed by programmed stimulation in rats after 8 (Ex8) and 16 (Ex16) weeks of daily 1-h treadmill training, along with 4 and 8 weeks after exercise cessation and time-matched sedentary (Sed) controls. Structural remodeling was evaluated by using serial echocardiography and histopathology, autonomic nervous system with pharmacological tools, acetylcholine-regulated potassium current (IKACh) with patch clamp recording, messenger ribonucleic acid expression with quantitative polymerase chain reaction, and regulators of G protein-signaling (RGS) 4 function in knockout mice. AF inducibility increased after 16 weeks of training (e.g., AF >30 s in 64% of Ex16 rats vs 15% of Sed rats; p< 0.01) and rapidly returned to baseline levels with detraining. Atropine restored sinus rhythm in 5 of 5 Ex rats with AF sustained >15 min. Atrial dilation and fibrosis developed after 16 weeks of training and failed to fully recover with exercise cessation. Parasympathetic tone was increased in Ex16 rats and normalized within 4 weeks of detraining. Baroreflex heart rate responses to phenylephrine-induced blood pressure elevation and IKACh sensitivity to carbachol were enhanced in Ex16 rats, implicating both central and end-organ mechanisms in vagal enhancement. Ex rats showed unchanged cardiac adrenergic and cholinergic receptor and IKACh-subunit gene expression, but significant messenger ribonucleic acid downregulation of IKACh-inhibiting RGS proteins was present at 16 weeks. RGS4 knockout mice showed significantly enhanced sensitivity to AF induction in the presence of carbachol. Chronic endurance exercise increased AF susceptibility in rats, with autonomic changes, atrial dilation, and fibrosis identified as potential mechanistic contributors. Vagal promotion is particularly important and occurs via augmented baroreflex responsiveness and increased cardiomyocyte sensitivity to cholinergic stimulation, possibly due to RGSprotein downregulation.

TARGETING THE REGULATOR OF G PROTEIN SIGNALING 17 (RGS17) FOR LUNG AND PROSTATE CANCER TREATMENTS

G‐Protein Coupled Receptors (GPCR) are one of the most important target in disease treatment with over 50% of all drugs on the market targeting these receptors. Regulator of G‐Protein Signaling (RGS) proteins are known to regulate the complex signaling pathways activated by GPCRs. Recent studies have implicated RGS‐proteins in the development and progression of pathologies, including some cancers. RGS17, the most‐recently identified family member of the RZ family of RGS proteins has been implicated in the growth, proliferation, metastasis and migration of prostate cancer cells as well as small‐cell and non‐small cell lung cancers. RGS17 is up‐regulated in lung and prostate tumors as much as 13 fold. RGS17 knockdown inhibits colony formation and decreases tumorigenesis in nude mice. Our studies implemented a high‐throughput screen campaign to determine the first Gαo: RGS17 protein: protein interaction (ppi) inhibitors. Immediate goals include determining the effectiveness of these compounds on inhibiting the GTPase acceleration activity of RGS17 in a steady‐state GTPase assay. The goal of this study is to establish lead compounds and develop a pharmacophore model for future avenues in cancer treatment. Research is supported by American Foundation for Pharmaceutical Education (AFPE), University of Iowa′s Center for Biocatalysis and Bioprocessing and The National Institutes of Health (R01CA160470).

Dynamic control of Allosteric Inhibitor Specificity for RGS4

G‐protein coupled receptors (GPCRs) are critically regulated through downstream protein‐protein interactions including Regulator of G‐protein signaling (RGS)‐proteins. They suppress GPCR output by enhancing the rate of GTP hydrolysis on the Gα subunit. A nM potency inhibitor (CCG‐50014) is several hundred‐fold selective for RGS4 over other RGS proteins. Like previous RGS inhibitors, CCG‐50014 depends on covalent cysteine modification for activity. Two key cysteines‐(95 and 148) are buried in an allosteric site. We hypothesized that inhibitor interaction at cys 95 or 148 alters the packing of alpha helices 4, 5, 6 and 7 to disrupt G‐protein binding. Consistent with this hypothesis, HSQC‐NMR experiments using RGS4 containing only Cys 95, in the presence of CCG‐50014, shows perturbations clustering in the helix bundle and in the G‐protein binding site. Molecular dynamics simulations predict motions that would provide inhibitor access to the buried allosteric cysteines, and open a novel cryptic pocket into which small molecules could be targeted. Binding of CCG‐50014 likely stabilizes a more open confirmation resulting in disruption of the G‐protein binding interface. Differences among RGS proteins involving these conformational transitions may drive the observed inhibitor selectivity for RGS4. (Supported by NIH T32 GM007767 and R01DA023253)

Structural Studies of RGS Inhibitors

Regulators of G‐protein signaling (RGS) proteins regulate G‐protein coupled receptor (GPCR) signal transduction cascades by acting as GTPase accelerating proteins upon binding activated Gα subunits. RGS proteins express in cell‐type specific manners, regulate specific GPCRs, and are overexpressed in pathologies from cancer to Parkinson's disease. As such, they are attractive drug targets. Small‐molecule RGS4 inhibitor screens have yielded a number of covalent thiol modifiers that act allosterically at Cys148, far from the RGS/Gα interface. Thus far, the structural basis of RGS4 inhibition is unknown, so in the current study we investigate the structural consequence of RGS4's covalent modification that leads to inhibition of Gα binding. We purified recombinant RGS4 from Escherichia coli and obtained nuclear magnetic resonance spectra for free and modified RGS4. Overlaid 15N‐1H HSQC spectra show significant changes in RGS4 structure upon small molecule modification. Data from circular dichroism, dynamic light scattering, and other techniques provide further evidence that the pocket may be amenable for rational drug design. Project funded by 5T32GM067795–08 and pre‐doctoral fellowship from AFPE.

Interactions of the α-subunits of heterotrimeric G-proteins with GPCRs, effectors and RGS proteins: A critical review and analysis of interacting surfaces, conformational shifts, structural diversity and electrostatic potentials

G-protein coupled receptors (GPCRs) are one of the largest families of membrane receptors in eukaryotes. Heterotrimeric G-proteins, composed of α, β and γ subunits, are important molecular switches in the mediation of GPCR signaling. Receptor stimulation after the binding of a suitable ligand leads to G-protein heterotrimer activation and dissociation into the Gα subunit and Gβγ heterodimer. These subunits then interact with a large number of effectors, leading to several cell responses. We studied the interactions between Gα subunits and their binding partners, using information from structural, mutagenesis and Bioinformatics studies, and conducted a series of comparisons of sequence, structure, electrostatic properties and intermolecular energies among different Gα families and subfamilies. We identified a number of Gα surfaces that may, in several occasions, participate in interactions with receptors as well as effectors. The study of Gα interacting surfaces in terms of sequence, structure and electrostatic potential reveals features that may account for the Gα subunit's behavior towards its interacting partners. The electrostatic properties of the Gα subunits, which in some cases differ greatly not only between families but also between subfamilies, as well as the G-protein interacting surfaces of effectors and regulators of G-protein signaling (RGS) suggest that electrostatic complementarity may be an important factor in G-protein interactions. Energy calculations also support this notion. This information may be useful in future studies of G-protein interactions with GPCRs and effectors.

RGS expression in cancer: oncomining the cancer microarray data

Heterotrimeric G proteins mediate myriads of cell functions including control of cancer cell proliferation and migration. The family of the Regulators of G protein Signaling (RGS) proteins, in turn, controls the activity of G proteins through the acceleration of GTPase activity of the alpha subunits of G proteins. Increasing evidence suggest that the expression of certain RGS proteins is changed dramatically in various cancers, and in some instances, the control of cancer cell proliferation or migration by RGS proteins has been demonstrated. We assessed if common trends might exist in the expression of various RGS proteins in several types of cancer by examining microarray data using the Oncomine database. We focused on the largest R4 sub-family of RGS proteins, containing RGS1, RGS2, RGS3, RGS4, RGS5, RGS8, RGS13, RGS16 and RGS18. This analysis suggests that a number (up to 6) of RGS transcripts are exclusively downregulated in certain cancers, while being exclusively upregulated in other cancer types. Furthermore, significant changes in the expression of certain RGS proteins trended toward the same direction across various cancers. To illustrate, RGS1 is largely upregulated, whereas RGS2 is downregulated in the majority of solid tumors, whereas RGS5 transcripts are greatly increased in eight subtypes of lymphoma with no reports of downregulation in hematological malignancies. Together, these data suggest that (i) RGS proteins may have a combined and cell-specific role in a control of cancer cell function, and (ii) a given RGS protein may regulate the progression of various cancers through a common mechanism.

Differential Control of Opioid Antinociception to Thermal Stimuli in a Knock-In Mouse Expressing Regulator of G-Protein Signaling-Insensitive GαoProtein

Regulator of G-protein signaling (RGS) proteins classically function as negative modulators of G-protein-coupled receptor signaling. In vitro, RGS proteins have been shown to inhibit signaling by agonists at the μ-opioid receptor, including morphine. The goal of the present study was to evaluate the contribution of endogenous RGS proteins to the antinociceptive effects of morphine and other opioid agonists. To do this, a knock-in mouse that expresses an RGS-insensitive (RGSi) mutant Gαo protein, Gαo(G184S) (Gαo RGSi), was evaluated for morphine or methadone antinociception in response to noxious thermal stimuli. Mice expressing Gαo RGSi subunits exhibited a naltrexone-sensitive enhancement of baseline latency in both the hot-plate and warm-water tail-withdrawal tests. In the hot-plate test, a measure of supraspinal nociception, morphine antinociception was increased, and this was associated with an increased ability of opioids to inhibit presynaptic GABA neurotransmission in the periaqueductal gray. In contrast, antinociception produced by either morphine or methadone was reduced in the tail-withdrawal test, a measure of spinal nociception. In whole-brain and spinal cord homogenates from mice expressing Gαo RGSi subunits, there was a small loss of Gαo expression and an accompanying decrease in basal G-protein activity. Our results strongly support a role for RGS proteins as negative regulators of opioid supraspinal antinociception and also reveal a potential novel function of RGS proteins as positive regulators of opioid spinal antinociceptive pathways.

Expressed sequence tag analysis of the emu (Dromaius novaehollandiae) pituitary by 454 GS Junior pyrosequencing

Emus (Dromaius novaehollandiae) are farmed for their oil for pharmaceutical and cosmetic uses. This emu pituitary expressed sequence tag study was undertaken to identify novel transcripts in the emu pituitary to propel their identification and functional studies. By mapping reads derived from the Roche 454 GS Junior pyrosequencer to 8 reference species (human, mouse, chicken, zebra finch, fruit fly, turkey, round worm, and Carolina anole lizard) from the UniGene database, a total of 81,788 reads (53,312 mapped reads) were obtained and assembled with Reference Sequence (RefSeq). We annotated 6,676 potential emu genes by referencing 7 species (excluding lizard) and identified 1,232 potential genes common among 3 species (human, mouse, and chicken) with complete available reference genomes. Gene Ontology analysis revealed 376 Gene Ontology terms showing, with the highest counts, their involvements in biological processes, metabolism, and cellular components. These potential genes were detected to associate with 20 pathways including mitogen-activated protein kinase, insulin, neurotrophin signaling pathways, and carbohydrate digestion and absorption pathway. We also revealed a panel of tissue-specific genes including regulator of G-protein signaling protein (RGS), glucagon-like peptide receptor (GLPR), and growth hormone-inducible transmembrane protein (GHITM). Additionally, fatty acid binding protein (FABP), fatty acid desaturase (FAS), and stearoyl-coenzyme A desaturase (SCD), key enzyme genes in fat metabolism, were found to be also expressed in emu pituitary. This expressed sequence tag study represents the first step in functional characterization of emu pituitary gene expression and SNP identification for the improvement of fat production in the emu.

Crystallization and preliminary X-ray diffraction studies ofDrosophila melanogasterGαo-subunit of heterotrimeric G protein in complex with the RGS domain of CG5036

Regulator of G-protein signalling (RGS) proteins negatively regulate heterotrimeric G-protein signalling through their conserved RGS domains. RGS domains act as GTPase-activating proteins, accelerating the GTP hydrolysis rate of the activated form of Gα-subunits. Although omnipresent in eukaryotes, RGS proteins have not been adequately analysed in non-mammalian organisms. The Drosophila melanogaster Gαo-subunit and the RGS domain of its interacting partner CG5036 have been overproduced and purified; the crystallization of the complex of the two proteins using PEG 4000 as a crystallizing agent and preliminary X-ray crystallographic analysis are reported. Diffraction data were collected to 2.0 Å resolution using a synchrotron-radiation source.

Modulation of μ-opioid receptor signaling by RGS19 in SH-SY5Y cells.

Regulator of G-protein signaling protein 19 (RGS19), also known as Gα-interacting protein (GAIP), acts as a GTPase accelerating protein for Gαz as well as Gαi/o subunits. Interactions with GAIP-interacting protein N-terminus and GAIP-interacting protein C-terminus (GIPC) link RGS19 to a variety of intracellular proteins. Here we show that RGS19 is abundantly expressed in human neuroblastoma SH-SY5Y cells that also express µ- and δ- opioid receptors (MORs and DORs, respectively) and nociceptin receptors (NOPRs). Lentiviral delivery of short hairpin RNA specifically targeted to RGS19 reduced RGS19 protein levels by 69%, with a similar reduction in GIPC. In RGS19-depleted cells, there was an increase in the ability of MOR (morphine) but not of DOR [(4-[(R)-[(2S,5R)-4-allyl-2,5-dimethylpiperazin-1-yl](3-methoxyphenyl)methyl]-N,N-diethylbenzamide (SNC80)] or NOPR (nociceptin) agonists to inhibit forskolin-stimulated adenylyl cyclase and increase mitogen-activated protein kinase (MAPK) activity. Overnight treatment with either MOR [D-Ala, N-Me-Phe, Gly-ol(5)-enkephalin (DAMGO) or morphine] or DOR (D-Pen(5)-enkephalin or SNC80) agonists increased RGS19 and GIPC protein levels in a time- and concentration-dependent manner. The MOR-induced increase in RGS19 protein was prevented by pretreatment with pertussis toxin or the opioid antagonist naloxone. Protein kinase C (PKC) activation alone increased the level of RGS19 and inhibitors of PKC 5,6,7,13-tetrahydro-13-methyl-5-oxo-12H-indolo[2,3-a]pyrrolo[3,4-c]carbazole-12-propanenitrile and mitogen-activated protein kinase kinase 1 2-(2-amino-3-methoxyphenyl)-4H-chromen-4-one, but not protein kinase A (H89), completely blocked DAMGO-induced RGS19 protein accumulation. The findings show that RGS19 and GIPC are jointly regulated, that RGS19 is a GTPase accelerating protein for MOR with selectivity over DOR and NOPR, and that chronic MOR or DOR agonist treatment increases RGS19 levels by a PKC and the MAPK pathway-dependent mechanism.

Gonadotrophin‐Releasing Hormone Signalling Downstream of Calmodulin

Gonadotrophin-releasing hormone (GnRH) regulates reproduction via binding a G-protein coupled receptor on the surface of the gonadotroph, through which it transmits signals, mostly via the mitogen-activated protein (MAPK) cascade, to increase synthesis of the gonadotrophin hormones: luteinising hormone (LH) and follicle-stimulating hormone (FSH). Activation of the MAPK cascade requires an elevation in cytosolic Ca(2+) levels, which is a result of both calcium influx and mobilisation from intracellular stores. However, Ca(2+) also transmits signals via an MAPK-independent pathway, through binding calmodulin (CaM), which is then able to bind a number of proteins to impart diverse downstream effects. Although the ability of GnRH to activate CaM was recognised over 20years ago, only recently have some of the downstream effects been elucidated. GnRH was shown to activate the CaM-dependent phosphatase, calcineurin, which targets gonadotrophin gene expression both directly and indirectly via transcription factors such as nuclear factor of activated T-cells and Nur77, the Transducer of Regulated CREB (TORC) co-activators and also the prolyl isomerase, Pin1. Gonadotrophin gene expression is also regulated by GnRH-induced CaM-dependent kinases (CaMKs); CaMKI is able to derepress the histone deacetylase-inhibition of β-subunit gene expression, whereas CaMKII appears to be essential for the GnRH-activation of all three subunit genes. Asides from activating gonadotrophin gene expression, GnRH also exerts additional effects on gonadotroph function, some of which clearly occur via CaM, including the proliferation of immature gonadotrophs, which is dependent on calcineurin. In this review, we summarise these pathways, and discuss the additional functions that have been proposed for CaM with respect to modifying GnRH-induced signalling pathways via the regulation of the small GTP-binding protein, Gem, and/or the regulator of G-protein signalling protein 2.

GIRK channel modulation by assembly with allosterically regulated RGS proteins

G-protein-activated inward-rectifying K(+) (GIRK) channels hyperpolarize neurons to inhibit synaptic transmission throughout the nervous system. By accelerating G-protein deactivation kinetics, the regulator of G-protein signaling (RGS) protein family modulates the timing of GIRK activity. Despite many investigations, whether RGS proteins modulate GIRK activity in neurons by mechanisms involving kinetic coupling, collision coupling, or macromolecular complex formation has remained unknown. Here we show that GIRK modulation occurs by channel assembly with R7-RGS/Gβ5 complexes under allosteric control of R7 RGS-binding protein (R7BP). Elimination of R7BP occludes the Gβ5 subunit that interacts with GIRK channels. R7BP-bound R7-RGS/Gβ5 complexes and Gβγ dimers interact noncompetitively with the intracellular domain of GIRK channels to facilitate rapid activation and deactivation of GIRK currents. By disrupting this allosterically regulated assembly mechanism, R7BP ablation augments GIRK activity. This enhanced GIRK activity increases the drug effects of agonists acting at G-protein-coupled receptors that signal via GIRK channels, as indicated by greater antinociceptive effects of GABA(B) or μ-opioid receptor agonists. These findings show that GIRK current modulation in vivo requires channel assembly with allosterically regulated RGS protein complexes, which provide a target for modulating GIRK activity in neurological disorders in which these channels have crucial roles, including pain, epilepsy, Parkinson's disease and Down syndrome.

Interaction between bradykinin and natriuretic peptides via RGS protein activation in HEK-293 cells

In this study, the interaction of natriuretic peptides (NP) and bradykinin (BK) signaling pathways was identified by measuring membrane potential (V(m)) and intracellular Ca(2+) using the patch-clamp technique and flow cytometry in HEK-293 cells. BK and NP receptor mRNA was identified using RT-PCR. BK (100 nM) depolarized cells activating bradykinin receptor type 2 (B(2)R) and Ca(2+)-dependent Cl(-) channels inhibitable by 5-nitro-2-(3-phenylpropylamino)benzoic acid (NPPB; 10 μM). The BK-induced Ca(2+) signal was blocked by the B(2)R inhibitor HOE 140. [Des-Arg(9)]-bradykinin, an activator of B(1)R, had no effect on intracellular Ca(2+). NP [atrial natriuretic peptide (ANP), brain natriuretic peptide (BNP), C-type natriuretic peptide (CNP), and urodilatin] depolarized HEK-293 cells inhibiting K(+) channels. ANP, urodilatin, BNP [binding to natriuretic peptide receptor (NPR)-A] and 8-bromo-(8-Br)-cGMP inhibited the BK-induced depolarization while CNP (binding to NPR-Bi) failed to do so. The inhibitory effect on BK-triggered depolarization could be reversed by blocking PKG using the specific inhibitor KT 5823. BK-stimulated depolarization as well as Ca(2+) signaling was completely blocked by the phospholipase C (PLC) inhibitor U-73122 (10 nM). The inositol 1,4,5-trisphosphate receptor blocker 2-aminoethoxydiphenyl borate (2-APB; 50 μM) completely inhibited the BK-induced Ca(2+) signaling. UTP, another activator of the PLC-mediated Ca(2+) signaling pathway, was blocked by U-73122 as well but not by 8-Br-cGMP, indicating an intermediate regulatory step for NP via PKG in BK signaling such as regulators of G-protein signaling (RGS) proteins. When RGS proteins were inhibited by CCG-63802 in the presence of BK and 8-Br-cGMP, cells started to depolarize again. In conclusion, as natural antagonists of the B(2)R signaling pathway, NP may also positively interact in pathological conditions caused by BK.

Amino-terminal Cysteine Residues Differentially Influence RGS4 Protein Plasma Membrane Targeting, Intracellular Trafficking, and Function

Regulator of G-protein signaling (RGS) proteins are potent inhibitors of heterotrimeric G-protein signaling. RGS4 attenuates G-protein activity in several tissues. Previous work demonstrated that cysteine palmitoylation on residues in the amino-terminal (Cys-2 and Cys-12) and core domains (Cys-95) of RGS4 is important for protein stability, plasma membrane targeting, and GTPase activating function. To date Cys-2 has been the priority target for RGS4 regulation by palmitoylation based on its putative role in stabilizing the RGS4 protein. Here, we investigate differences in the contribution of Cys-2 and Cys-12 to the intracellular localization and function of RGS4. Inhibition of RGS4 palmitoylation with 2-bromopalmitate dramatically reduced its localization to the plasma membrane. Similarly, mutation of the RGS4 amphipathic helix (L23D) prevented membrane localization and its G(q) inhibitory function. Together, these data suggest that both RGS4 palmitoylation and the amphipathic helix domain are required for optimal plasma membrane targeting and function of RGS4. Mutation of Cys-12 decreased RGS4 membrane targeting to a similar extent as 2-bromopalmitate, resulting in complete loss of its G(q) inhibitory function. Mutation of Cys-2 did not impair plasma membrane targeting but did partially impair its function as a G(q) inhibitor. Comparison of the endosomal distribution pattern of wild type and mutant RGS4 proteins with TGN38 indicated that palmitoylation of these two cysteines contributes differentially to the intracellular trafficking of RGS4. These data show for the first time that Cys-2 and Cys-12 play markedly different roles in the regulation of RGS4 membrane localization, intracellular trafficking, and G(q) inhibitory function via mechanisms that are unrelated to RGS4 protein stabilization.