Galpha Research Articles

Molecular Organization of the Complex between the Muscarinic M3 Receptor and the Regulator of G Protein Signaling, Gβ5−RGS7

The complex of the regulator of G protein signaling (RGS), Gbeta(5)-RGS7, can inhibit signal transduction via the M3 muscarinic acetylcholine receptor (M3R). RGS7 consists of three distinct structural entities: the DEP domain and its extension DHEX, the Ggamma-like (GGL) domain, which is permanently bound to Gbeta subunit Gbeta(5), and the RGS domain responsible for the interaction with Galpha subunits. Inhibition of the M3R by Gbeta(5)-RGS7 is independent of the RGS domain but requires binding of the DEP domain to the third intracellular loop of the receptor. Recent studies identified the dynamic intramolecular interaction between the Gbeta(5) and DEP domains, which suggested that the Gbeta(5)-RGS7 dimer could alternate between the "open" and "closed" conformations. Here, we identified point mutations that weaken DEP-Gbeta(5) binding, presumably stabilizing the open state, and tested their effects on the interaction of Gbeta(5)-RGS7 with the M3R. We found that these mutations facilitated binding of Gbeta(5)-RGS7 to the recombinant third intracellular loop of the M3R but did not enhance its ability to inhibit M3R-mediated Ca(2+) mobilization. This led us to the idea that the M3R can effectively induce the Gbeta(5)-RGS7 dimer to open; such a mechanism would require a region of the receptor distinct from the third loop. Indeed, we found that the C-terminus of M3R interacts with Gbeta(5)-RGS7. Truncation of the C-terminus rendered the M3R insensitive to inhibition by wild-type Gbeta(5)-RGS7; however, the open mutant of Gbeta(5)-RGS7 was able to inhibit signaling by the truncated M3R. The GST fusion of the M3R C-tail could not bind to wild-type Gbeta(5)-RGS7 but could associate with its open mutant as well as with the separated recombinant DEP domain or Gbeta(5). Taken together, our data are consistent with the following model: interaction of the M3R with Gbeta(5)-RGS7 causes the DEP domain and Gbeta(5) to dissociate from each other and bind to the C-tail, and the DEP domain also binds to the third loop, thereby inhibiting M3R-mediated signaling.

Multi-component signaling complexes of the δ-opioid receptor with STAT5B and G proteins

Besides mediating opioid responses in the nervous system and the peripheral tissues, opioid receptors are implicated in signaling mechanisms shared by cytokine receptors. Recent observations have shown that the Signal Transducer and Activator of Transcription 5A (STAT5A) interacts with the mu-opioid receptor (mu-OR) and is phosphorylated upon mu-OR stimulation (Mazarakou and Georgoussi, 2005). In the present study we demonstrate that another member of the STAT family, STAT5B, associates constitutively with the C-terminal tail of the delta-opioid receptor (delta-CT). [D-Ser(2), Leu(5), Thr(6)]-enkephalin-exposure of HEK293 cells, expressing stably the delta-opioid receptor (delta-OR), leads to receptor-dependent STAT5B tyrosine phosphorylation and transcriptional activation. This phosphorylation occurs in a G protein-dependent manner and is carried out by a c-Src kinase. Co-immunoprecipitation studies indicate that STAT5B forms pairs with selective Galpha and Gbetagamma subunits of G proteins and activated c-Src kinase in HEK293 cells. These interactions are formed either constitutively, or upon receptor stimulation. We also demonstrate that the delta-CT serves as a platform for the formation of a multi-component signaling complex (signalosome), consisting of STAT5B, c-Src and selective G protein members. We can thus conclude that STAT5B signaling can be modulated by its coupling with a specific subset of G protein subunits, revealing a novel signaling mechanism for the transcriptional regulation of STAT5B-dependent genes.

Neural tube defects and impaired neural progenitor cell proliferation in Gβ1‐deficient mice

Heterotrimeric G proteins are well known for their roles in signal transduction downstream of G protein-coupled receptors (GPCRs), and both Galpha subunits and tightly associated Gbetagamma subunits regulate downstream effector molecules. Compared to Galpha subunits, the physiological roles of individual Gbeta and Ggamma subunits are poorly understood. In this study, we generated mice deficient in the Gbeta1 gene and found that Gbeta1 is required for neural tube closure, neural progenitor cell proliferation, and neonatal development. About 40% Gbeta1(-/-) embryos developed neural tube defects (NTDs) and abnormal actin organization was observed in the basal side of neuroepithelium. In addition, Gbeta1(-/-) embryos without NTDs showed microencephaly and died within 2 days after birth. GPCR agonist-induced ERK phosphorylation, cell proliferation, and cell spreading, which were all found to be regulated by Galphai and Gbetagamma signaling, were abnormal in Gbeta1(-/-) neural progenitor cells. These data indicate that Gbeta1 is required for normal embryonic neurogenesis.

Thymoquinone from nutraceutical black cumin oil activates Neu4 sialidase in live macrophage, dendritic, and normal and type I sialidosis human fibroblast cells via GPCR Gαi proteins and matrix metalloproteinase-9

Anti-inflammatory activities of thymoquinone (TQ) have been demonstrated in in vitro and in vivo studies. However, the precise mechanism(s) of TQ in these anti-inflammatory activities is not well understood. Using a newly developed assay to detect sialidase activity in live macrophage cells (Glycoconj J doi: 10.1007/s10719-009-9239-8 ), here we show that TQ has no inhibitory effect on endotoxin lipopolysaccharide (LPS) induced sialidase activity in live BMC-2 macrophage cells. In contrast, the parent black seed oil (BSO) and another constituent of BSO para-cymene (p-CY) completely block LPS induced sialidase activity. All of these compounds had no effect on cell viability. On the other hand, TQ induces a vigorous sialidase activity in live BMC-2 macrophage cells in a dose dependent manner as well in live DC-2.4 dendritic cells, HEK-TLR4/MD2, HEK293, SP1 mammary adenocarcinoma cells, human WT and 1140F01 and WG0544 type I sialidosis fibroblast cells. Tamiflu (oseltamivir phosphate) inhibits TQ-induced sialidase activity in live BMC-2 cells with an IC(50) of 0.0194 microM compared to an IC(50) of 19.1 microM for neuraminidase inhibitor DANA (2-deoxy-2,3-dehydro-N-acetylneuraminic acid). Anti-Neu1, -2 and -3 antibodies have no inhibition of TQ-induced sialidase activity in live BMC-2 and human THP-1 macrophage cells but anti-Neu4 antibodies completely block this activity. There is a vigorous sialidase activity associated with TQ treated live primary bone marrow (BM) macrophage cells derived from WT and hypomorphic cathepsin A mice with a secondary Neu1 deficiency (NeuI KD), but not from Neu4 knockout (Neu4 KO) mice. Pertussis toxin (PTX), a specific inhibitor of Galphai proteins of G-protein coupled receptor (GPCR) and the broad range inhibitors of matrix metalloproteinase (MMP) galardin and piperazine applied to live BMC-2, THP-1 and primary BM macrophage cells completely block TQ-induced sialidase activity. These same inhibitory effects are not observed with the GM1 ganglioside specific cholera toxin subunit B (CTXB) as well as with CTX, tyrosine kinase inhibitor K252a, and the broad range GPCR inhibitor suramin. The specific inhibitor of MMP-9, anti-MMP-9 antibody and anti-Neu4 antibody, but not the specific inhibitor of MMP-3 completely block TQ-induced sialidase activity in live THP-1 cells, which express Neu4 and MMP-9 on the cell surface. Neu4 sialidase activity in cell lysates from TQ-treated live THP-1 cells desialylates natural gangliosides and mucin substrates. RT-PCR and western blot analyses reveal no correlation between mRNA and protein values for Neu3 and Neu4 in human monocytic THP-1 cells, suggesting for the first time a varied post-transcriptional mechanism for these two mammalian sialidases independent of TQ activation. Our findings establish an unprecedented activation of Neu4 sialidase on the cell surface by thymoquinone, which is derived from the nutraceutical black cumin oil. The potentiation of GPCR-signaling by TQ via membrane targeting of Galphai subunit proteins and matrix metalloproteinase-9 activation may be involved in the activation process of Neu4 sialidase on the cell surface.

G Proteins in Reverse Mode: RECEPTOR-MEDIATED GTP RELEASE INHIBITS G PROTEIN AND EFFECTOR FUNCTION

Active G protein-coupled receptors activate heterotrimeric Galphabetagamma proteins by catalyzing the exchange of GDP by GTP at the Galpha subunit. A paradoxical attenuation of G protein-activated inwardly rectifying potassium channels (GIRK) upon stimulation of native cells with high concentrations of agonist is known. However, a deactivation of activated G proteins by active receptors has not been experimentally studied in intact cells. We monitored GIRK currents and G(o) protein activation by means of fluorescence resonance energy transfer (FRET) in parallel. The results suggested that GIRK currents were paradoxically attenuated due to an inactivation of G(o) proteins by active alpha(2A)-adrenergic receptors. To study the mechanisms, G protein activation and receptor-G protein interactions were analyzed as a function of nucleotide type and nucleotide concentrations by means of FRET, while controlling intracellular nucleotides upon permeabilization of the cell membrane. Results suggested a receptor-catalyzed dissociation of GTP from activated heterotrimeric Galphabetagamma. Consequently, nucleotide-free G proteins were sequestrated in heterotrimeric conformation at the active receptor, thus attenuating downstream signaling in an agonist-dependent manner.

MoRic8 Is a Novel Component of G-Protein Signaling During Plant Infection by the Rice Blast FungusMagnaporthe oryzae

An insertional mutagenesis screen was used to investigate the biology of plant infection by the devastating rice blast pathogen, Magnaporthe oryzae. Here, we report the identification of a new mutant, LY-130, which is defective in multiple steps during infection-related morphogenesis and pathogenicity. Analysis of the mutation confirmed an insertion into gene MoRIC8, which encodes a 480-amino-acid protein that is a putative homologue of the Ric8 regulator of GTP-binding protein (G-protein) signaling, previously described in animals. Targeted gene deletion mutants of MoRIC8 were nonpathogenic and impaired in cellular differentiation associated with sporulation, sexual development, and plant infection. MoRic8 physically interacts with the Galpha subunit MagB in yeast two-hybrid assays and appears to act upstream of the cyclic AMP response pathway that is necessary for appressorium morphogenesis. Taken together, our results indicate that MoRic8 may act as a novel regulator of the G-protein signaling during infection-related development of rice blast fungus M. oryzae.

Gβγ Signaling Promotes Breast Cancer Cell Migration and Invasion

Signaling through G protein-coupled receptors (GPCRs) promotes breast cancer metastasis. G proteins convey GPCR signals by dissociating into Galpha and Gbetagamma subunits. The aim of the present study was to determine whether blockade of Gbetagamma signaling suppresses breast cancer cell migration and invasion, which are critical components of metastasis. Conditioned media (CM) of NIH-3T3 fibroblasts are widely used as chemoattractants in in vitro cancer metastasis studies. Expression of a Gbetagamma scavenger peptide attenuated NIH-3T3 CM-induced migration and invasion of both metastatic breast cancer MDA-MB-231 and MDA-MB-436 cells by 40 to 50% without effects on cell viability. Migration and invasion of cells in response to NIH-3T3 CM were also blocked by 8-(4,5,6-trihydroxy-3-oxo-3H-xanthen-9-yl)-1-naph-thalene-carboxylic acid) (M119K), a Gbetagamma inhibitor, with maximum inhibition exceeding 80% and half-maximal inhibitory concentration (IC50) values of 1 to 2 microM. M119K also attenuated Rac-dependent formation of lamellipodia, a key structure required for metastasis. Constitutively active Rac1 rescued Gbetagamma blockade-mediated inhibition of breast cancer cell migration, whereas dominant negative Rac1 inhibited cell migration similar to Gbetagamma blockade. Furthermore, M119K suppressed Gi protein-coupled CXC chemokine receptor 4 (CXCR4)-dependent MDA-MB-231 cell migration by 80% with an IC50 value of 1 microM, whereas tyrosine kinase receptor-dependent cell migration was significantly less inhibited. However, CXCR4-dependent inhibition of adenylyl cyclase, a Gialpha-mediated response in MDA-MB-231 cells, was not blocked by M119K but was blocked by pertussis toxin, which selectively inactivates Gialpha. This report is the first to directly demonstrate the role of Gbetagamma in cancer cell migration and invasion and suggests that targeting Gbetagamma signaling pathways may provide a novel strategy for suppressing breast cancer metastasis.

The beta subunit of the heterotrimeric G protein triggers the Kluyveromyces lactis pheromone response pathway in the absence of the gamma subunit.

The Kluyveromyces lactis heterotrimeric G protein is a canonical Galphabetagamma complex; however, in contrast to Saccharomyces cerevisiae, where the Ggamma subunit is essential for mating, disruption of the KlGgamma gene yielded cells with almost intact mating capacity. Expression of a nonfarnesylated Ggamma, which behaves as a dominant-negative in S. cerevisiae, did not affect mating in wild-type and DeltaGgamma cells of K. lactis. In contrast to the moderate sterility shown by the single DeltaKlGalpha, the double DeltaKlGalpha DeltaKlGgamma mutant displayed full sterility. A partial sterile phenotype of the DeltaKlGgamma mutant was obtained in conditions where the KlGbeta subunit interacted defectively with the Galpha subunit. The addition of a CCAAX motif to the C-end of KlGbeta, partially suppressed the lack of both KlGalpha and KlGgamma subunits. In cells lacking KlGgamma, the KlGbeta subunit cofractionated with KlGalpha in the plasma membrane, but in the DeltaKlGalpha DeltaKlGgamma strain was located in the cytosol. When the KlGbeta-KlGalpha interaction was affected in the DeltaKlGgamma mutant, most KlGbeta fractionated to the cytosol. In contrast to the generic model of G-protein function, the Gbeta subunit of K. lactis has the capacity to attach to the membrane and to activate mating effectors in absence of the Ggamma subunit.

Altered Endothelin-1 Signaling in Production of Thromboxane A2 in Kupffer Cells from Bile Duct Ligated Rats

Kupffer cells (KCs), the liver resident macrophages accounting for 80-90% of the total population of fixed tissue macrophages in the body, not only play a key role in host defense via removing particulate materials from the portal circulation, but may also contribute to the pathogenesis of various liver diseases. We have previously demonstrated that KCs play an important role in controlling portal hypertension and hepatocellular injury via releasing thromboxane A2 (TXA2) in early fibrosis induced by one-week bile duct ligation (BDL). Production of TXA2 is controlled by cytosolic phospholipase A2 (cPLA2) that is activated by the interaction of entothelin-1 (ET-1) with its G-protein coupled ET receptor B (ETBR). However, the signaling pathways that contribute to the ET-1-induced activation of cPLA2 and production of TXA2 in KCs in the normal healthy or injured livers are not yet clear, which are investigated in the present study using isolated KCs from one-week BDL or sham rats. The pharmacological inhibition of cPLA2 or chelation of intracellular calcium abrogated the ET-1 induction of TXA2 from KCs. Compared to those from sham rats, KCs from BDL animals displayed a significantly enhanced responsiveness of p38 MAPK to ET-1, increased ETBR and Galphai subunit but decreased Galphaq and Galpha11 expression. Inhibition of ERK1/2 or Gq signaling abrogated significantly the ET-1 induction of TXA2 in sham KCs but only slightly in BDL KCs. In contrast, inhibition of p38 MAPK and Gi signaling markedly attenuated the ET-1 induction of TXA2 in BDL KCs but had no effect in sham KCs. Lastly, inhibition of PLC or PKC abrogated ET-1 induction of TXA2 in KCs from both sham and BDL groups. The hepatic stress (such as BDL) induces significant modifications in the receptor and intermediates of ET-1 signaling in KC and subsequently alters ET-1 signaling mechanisms, particularly a shift from Gq induced signaling to Gi induced signaling, in the activation of cPLA2 and production of TXA2 in response to ET-1.

Structure and Function of Heterotrimeric G Protein-Regulated Rho Guanine Nucleotide Exchange Factors

Activation of certain classes of G protein-coupled receptors (GPCRs) can lead to alterations in the actin cytoskeleton, gene transcription, cell transformation, and other processes that are known to be regulated by Rho family small-molecular-weight GTPases. Although these responses can occur indirectly via cross-talk from canonical heterotrimeric G protein cascades, it has recently been demonstrated that Dbl family Rho guanine nucleotide exchange factors (RhoGEFs) can serve as the direct downstream effectors of heterotrimeric G proteins. Heterotrimeric Galpha(12/13), Galpha(q), and Gbetagamma subunits are each now known to directly bind and regulate RhoGEFs. Atomic structures have recently been determined for several of these RhoGEFs and their G protein complexes, providing fresh insight into the molecular mechanisms of signal transduction between GPCRs and small molecular weight G proteins. This review covers what is currently known about the structure, function, and regulation of these recently recognized effectors of heterotrimeric G proteins.

Heterogeneous Distribution of G Protein Alpha Subunits in the Main Olfactory and Vomeronasal Systems ofRhinella(Bufo)arenarumTadpoles

We evaluated the presence of G protein subtypes Galpha(o), Galpha(i2), and Galpha(olf) in the main olfactory system (MOS) and accessory or vomeronasal system (VNS) of Rhinella (Bufo) arenarum tadpoles, and here describe the fine structure of the sensory cells in the olfactory epithelium (OE) and vomeronasal organ (VNO). The OE shows olfactory receptor neurons (ORNs) with cilia in the apical surface, and the vomeronasal receptor neurons (VRNs) of the VNO are covered with microvilli. Immunohistochemistry detected the presence of at least two segregated populations of ORNs throughout the OE, coupled to Galpha(olf) and Galpha(o). An antiserum against Galpha(i2) was ineffective in staining the ORNs. In the VNO, Galpha(o) neurons stained strongly but lacked immunoreactivity to any other Galpha subunit in all larval stages analyzed. Western blot analyses and preabsorption experiments confirmed the specificity of the commercial antisera used. The functional significance of the heterogeneous G-protein distribution in R. arenarum tadpoles is not clear, but the study of G- protein distributions in various amphibian species is important, since this vertebrate group played a key role in the evolution of tetrapods. A more complete knowledge of the amphibian MOS and VNS would help to understand the functional organization and evolution of vertebrate chemosensory systems. This work demonstrates, for the first time, the existence of a segregated distribution of G-proteins in the OE of R. arenarum tadpoles.

Common Variants in the G Protein β3 Subunit Gene and Thyroid Disorders in a Formerly Iodine-Deficient Population

Heterotrimeric G proteins are key mediators of signals from membrane receptors-including the thyroid-stimulating hormone (TSH) receptor-to cellular effectors. Gain-of-function mutations in the TSH receptor and the Galpha(S) subunit occur frequently in hyperfunctioning thyroid nodules and differentiated thyroid carcinomas, whereby the T allele of a common polymorphism (825C>T, rs5443) in the G protein beta3 subunit gene (GNB3) is associated with increased G protein-mediated signal transduction and a complex phenotype. The aim of this study was to investigate whether this common polymorphism affects key parameters of thyroid function and morphology and influences the pathogenesis of thyroid diseases in the general population. The population-based cross-sectional Study of Health in Pomerania is a general health survey with focus on thyroid diseases in northeast Germany, a formerly iodine-deficient area. Data from 3428 subjects (1800 men and 1628 women) were analyzed for an association of the GNB3 genotype with TSH, free triiodothyronine and thyroxine levels, urine iodine and thiocyanate excretion, and thyroid ultrasound morphology including thyroid volume, presence of goiter, and thyroid nodules. There was no association between GNB3 genotype status and the functional or morphological thyroid parameters investigated, neither in crude analyses nor upon multivariable analyses including known confounders of thyroid disorders. Based on the data from this large population-based survey, we conclude that the GNB3 825C>T polymorphism does not affect key parameters of thyroid function and morphology in the general population of a formerly iodine-deficient area.

Purinergic regulation of the epithelial Na+ channel

1. The epithelial Na(+) channel (ENaC) is a major conductive pathway that transports Na(+) across the apical membrane of the distal nephron, the respiratory tract, the distal colon and the ducts of exocrine glands. The ENaC is regulated by hormonal and humoral factors, including extracellular nucleotides that are available from the epithelial cells themselves. 2. Extracellular nucleotides, via the P2Y2 receptors (P2Y2Rs) at the basolateral and apical membrane of the epithelia, trigger signalling systems that inhibit the activity of the ENaC and activate Ca(2+) -dependent Cl(-) secretion. 3. Recent data from our laboratory suggest that stimulation of the P2Y2Rs at the basolateral membrane inhibits ENaC activity by a signalling mechanism that involves G beta gamma subunits freed from a pertussis toxin (PTX)-sensitive G-protein and phospholipase C (PLC) beta 4. A similar signalling mechanism is also partially responsible for inhibition of the ENaC during activation of apical P2Y2Rs. 4. Stimulation of apical P2Y2Rs also activates an additional signalling mechanism that inhibits the ENaC and involves the activated Galpha subunit of a PTX-insensitive G-protein and activation of an unidentified PLC. The effect of this PTX-insensitive system requires the activity of the basolateral Na(+)/K(+)/2Cl(-) cotransporter.

Heterotrimeric G Protein Signaling Is Required for Epidermal Cell Death in Rice

In rice (Oryza sativa) adventitious root primordia are formed at the nodes as part of normal development. Upon submergence of rice plants, adventitious roots emerge from the nodes preceded by death of epidermal cells above the root primordia. Cell death is induced by ethylene and mediated by hydrogen peroxide (H(2)O(2)). Pharmacological experiments indicated that epidermal cell death was dependent on signaling through G proteins. Treatment with GTP-gamma-S induced epidermal cell death, whereas GDP-beta-S partially inhibited ethylene-induced cell death. The dwarf1 (d1) mutant of rice has repressed expression of the Galpha subunit RGA1 of heterotrimeric G protein. In d1 plants, cell death in response to ethylene and H(2)O(2) was nearly completely abolished, indicating that signaling through Galpha is essential. Ethylene and H(2)O(2) were previously shown to alter gene expression in epidermal cells that undergo cell death. Transcriptional regulation was not generally affected in the d1 mutant, indicating that altered gene expression is not sufficient to trigger cell death in the absence of Galpha. Analysis of genes encoding proteins related to G protein signaling revealed that four small GTPase genes, two GTPase-activating protein genes, and one GDP dissociation inhibitor gene but not RGA1 were differentially expressed in epidermal cells above adventitious roots, indicating that Galpha activity is regulated posttranscriptionally.

The high light response in Arabidopsis involves ABA signaling between vascular and bundle sheath cells.

Previously, it has been shown that Arabidopsis thaliana leaves exposed to high light accumulate hydrogen peroxide (H2O2) in bundle sheath cell (BSC) chloroplasts as part of a retrograde signaling network that induces ASCORBATE PEROXIDASE2 (APX2). Abscisic acid (ABA) signaling has been postulated to be involved in this network. To investigate the proposed role of ABA, a combination of physiological, pharmacological, bioinformatic, and molecular genetic approaches was used. ABA biosynthesis is initiated in vascular parenchyma and activates a signaling network in neighboring BSCs. This signaling network includes the Galpha subunit of the heterotrimeric G protein complex, the OPEN STOMATA1 protein kinase, and extracellular H2O2, which together coordinate with a redox-retrograde signal from BSC chloroplasts to activate APX2 expression. High light-responsive genes expressed in other leaf tissues are subject to a coordination of chloroplast retrograde signaling and transcellular signaling activated by ABA synthesized in vascular cells. ABA is necessary for the successful adjustment of the leaf to repeated episodes of high light. This process involves maintenance of photochemical quenching, which is required for dissipation of excess excitation energy.

Modulation of α2-Adrenoceptor Functions by Heterotrimeric Gαi Protein Isoforms

Subtype diversity of heterotrimeric G proteins and G protein-coupled receptors enables a wide spectrum of signal transduction. However, the significance of isoforms within receptor or G protein subfamilies has not been fully elucidated. In the present study, we have tested whether alpha(2)-adrenoceptors require specific Galpha isoforms for their function in vivo. In particular, we analyzed the role of the highly homologous Galpha(i) isoforms, Galpha(i1), Galpha(i2), and Galpha(i3), in typical alpha(2)-adrenoceptor-controlled functions. Mice with targeted deletions in the genes encoding Galpha(i1), Galpha(i2), or Galpha(i3) were used to test the effects of alpha(2)-adrenoceptor stimulation by the agonist medetomidine. The alpha(2)-adrenoceptor agonist medetomidine inhibited [(3)H]norepinephrine release from isolated prefrontal brain cortex or cardiac atria tissue specimens with similar potency and efficacy in tissues from wild-type or Galpha(i)-deficient mice. In vivo, bradycardia, hypotension, induction of sleep, antinociception, and hypothermia induced by alpha(2)-adrenoceptor activation did not differ between wild-type and Galpha(i)-knockout mice. However, the effects of the alpha(2)-agonists medetomidine or 5-bromo-6-(2-imidazolin-2-ylamino)quin-oxaline tartrate (UK14,304) on spontaneous locomotor activity or anesthetic sparing were reduced or absent, respectively, in mice lacking Galpha(i2). In microdissected locus coeruleus neurons or postganglionic sympathetic neurons from stellate ganglia, all three Galpha(i) subunits were expressed as determined by quantitative reverse transcription-polymerase chain reaction, with Galpha(i1) and Galpha(i2) dominating over Galpha(i3). Functional redundancy of the highly homologous Galpha(i) isoforms may predominate over specificity to regulate distinct intracellular pathways downstream of alpha(2)-adrenoceptors in vivo. In contrast, inhibition of locomotor activity and anesthetic sparing may be elicited by a specific coupling of alpha(2A)-adrenoceptors via the Galpha(i2) isoform to intracellular pathways.

Functional Selectivity of Natural and Synthetic Prostaglandin EP4Receptor Ligands

Classically, the prostaglandin E(2) (PGE(2)) receptor EP(4) has been classified as coupling to the Galpha(s) subunit, leading to intracellular cAMP increases. However, EP(4) signaling has been revealed to be more complex and also involves coupling to pertussis toxin-sensitive Galpha(i) proteins and beta-arrestin-mediated effects. There are now many examples of selective activation of independent pathways by G protein-coupled receptor (GPCR) ligands, a concept referred to as functional selectivity. Because most EP(4) ligands had thus far only been functionally characterized by their ability to stimulate cAMP production, we systematically determined the potencies and efficacies of a panel of EP(4) ligands for activation of Galpha(s), Galpha(i), and beta-arrestin relative to the endogenous ligand PGE(2). For this purpose, we adapted three bioluminescence resonance energy transfer (BRET) assays to evaluate the respective pathways in living cells. Our results suggest considerable functional selectivity among the tested, structurally related agonists. PGE(2) was the most selective in activating Galpha(s), whereas PGF(2alpha) and PGE(1) alcohol were the most biased for activating Galpha(i1) and beta-arrestin, respectively. We observed reversal in order of potencies between beta-arrestin 2 and Galpha(i1) functional assays comparing PGE(1) alcohol and either PGF(2alpha), PGD(2), or 7-[(1R,2R)-2-[(E,3R)-3-hydroxy-4-(phenoxy)but-1-enyl]-5-oxocyclopentyl]heptanoic acid (M&B28767). Most ligands were full agonists for the three pathways tested. Our results have implications for the use of PGE(2) analogs in experimental and possibly clinical settings, because their activity spectra on EP(4) differ from that of the native agonist. The BRET-based methodology used for this first systematic assessment of a set of EP(4) agonists should be applicable for the study of other GPCRs.

Structural Determinants of G-protein α Subunit Selectivity by Regulator of G-protein Signaling 2 (RGS2)

"Regulator of G-protein signaling" (RGS) proteins facilitate the termination of G protein-coupled receptor (GPCR) signaling via their ability to increase the intrinsic GTP hydrolysis rate of Galpha subunits (known as GTPase-accelerating protein or "GAP" activity). RGS2 is unique in its in vitro potency and selectivity as a GAP for Galpha(q) subunits. As many vasoconstrictive hormones signal via G(q) heterotrimer-coupled receptors, it is perhaps not surprising that RGS2-deficient mice exhibit constitutive hypertension. However, to date the particular structural features within RGS2 determining its selectivity for Galpha(q) over Galpha(i/o) substrates have not been completely characterized. Here, we examine a trio of point mutations to RGS2 that elicits Galpha(i)-directed binding and GAP activities without perturbing its association with Galpha(q). Using x-ray crystallography, we determined a model of the triple mutant RGS2 in complex with a transition state mimetic form of Galpha(i) at 2.8-A resolution. Structural comparison with unliganded, wild type RGS2 and of other RGS domain/Galpha complexes highlighted the roles of these residues in wild type RGS2 that weaken Galpha(i) subunit association. Moreover, these three amino acids are seen to be evolutionarily conserved among organisms with modern cardiovascular systems, suggesting that RGS2 arose from the R4-subfamily of RGS proteins to have specialized activity as a potent and selective Galpha(q) GAP that modulates cardiovascular function.

Heterotrimeric G-protein signaling and light response

Almost all creatures have invented sophisticated mechanisms to adjust their developmental and metabolic processes to the changing light intensities in day and night. Recent findings suggest that one such mechanism is signaling via heterotrimeric G-proteins.1 The Trichoderma reesei (anamorph of Hypocrea jecorina) G-alpha subunit gene gna3 was found to be responsive to light and influenced by the light regulatory protein ENVOY.2 GNA3 significantly impacts regulation of cellulase gene expression only in light.1 While the exact mechanism of this regulation remains to be determined, first hints point to a regulation at the transcriptional level, since we observed light induced complex formation within the gna3 promotor. At least some of the components of this putatively regulatory protein complex also bind to the env1- promotor. These data indicate that the signal related by GNA3 is of light-dependent significance for H. jecorina and that the pathway of heterotrimeric G-protein signaling may be a target of the light perception machinery in fungi.

Functional Expression of Chemoreceptors with the Help of a Guanine Nucleotide Exchange Factor

Odorant receptors and other chemoreceptors are usually poorly expressed in the plasma membrane of heterologous cells. A key point of regulation in G protein-mediated signaling is the interconversion between the active GTP-bound and inactive GDP-bound states of the Galpha subunit, which regulatory proteins, such as guanine nucleotide exchange factors (GEFs), can control. GEFs stimulate formation of the GTP-bound state of Galpha and therefore are considered to work as positive regulators of G protein-coupled receptor signaling. Ric-8B, a GEF that is specifically expressed in olfactory sensory neurons, promotes functional expression of odorant receptors in HEK293T cells because it amplifies the initially low receptor signaling through Galphaolf. This same strategy could be used to functionally express other types of chemoreceptors.