Gi Alpha Research Articles

A Region of the Third Intracellular Loop of the Short Form of the D2 Dopamine Receptor Dictates Gi Coupling Specificity

The D2 dopamine receptor has two isoforms, the short form (D2s receptor) and the long form (D2l receptor), which differ by the presence of a 29-amino acid insert in the third cytoplasmic loop. Both the D2s and D2l receptors have been shown to couple to members of the G alpha(i) family of G proteins, but whether each isoform couples to specific G alpha(i) protein(s) remains controversial. In previous studies using G alpha(i) mutants resistant to modification by pertussis toxin (G alpha(i)PT), we demonstrated that the D2s receptor couples selectively to G alpha(i2)PT and that the D2l receptor couples selectively to G alpha(i3)PT (Senogles, S. E. (1994) J. Biol. Chem. 269, 23120-23127). In this study, two point mutations of the D2s receptor were created by random mutagenesis (R233G and A234T). The two mutant D2s receptors demonstrated pharmacological characteristics comparable with those of the wild-type D2s receptor, with similar agonist and antagonist binding affinities. We used human embryonic kidney 293 cells stably transfected with G alpha(i1)PT, G alpha(i2)PT, or G alpha(i3)PT to measure agonist-mediated inhibition of forskolin-stimulated cAMP accumulation before and after pertussis toxin treatment. The two mutant D2s receptors demonstrated a change in G(i) coupling specificity compared with the wild-type D2s receptor. Whereas the wild-type D2s receptor coupled predominantly to G alpha(i2)PT, mutant R233G coupled preferentially to G alpha(i3)PT, and mutant A234T coupled preferentially to G alpha(i1)PT. These results suggest that this region of the third cytoplasmic loop is crucial for determining G(i) protein coupling specificity.

Purification of recombinant G protein alpha subunits from Escherichia coli.

The purification of recombinant G protein a subunits expressed in Escherichia coli (E. coli) is a convenient and inexpensive method to obtain homogeneous preparations of protein for biochemical and biophysical analyses. Wild-type and mutant forms of G alpha are easily produced for analysis of their intrinsic biochemical properties, as well as for reconstitution with receptors, effectors, regulators, and G protein beta gamma subunits. Methods are described for the expression of Gi alpha and Gs alpha proteins in E. coli. Protocols are provided for the purification of untagged G protein a subunits using conventional chromatography and histidine (His)-tagged subunits using metal chelate chromatography. Modification of G alpha with myristate can be recapitulated in E. coli by expressing N-myristoyltransferase (NMT) with its G protein substrate. Protocols for the production and purification of myristoylated G alpha are presented.

Thermodynamic Characterization of the Binding of Activator of G Protein Signaling 3 (AGS3) and Peptides Derived from AGS3 with Gαi1

Activator of G protein signaling 3 (AGS3) is a guanine nucleotide dissociation inhibitor (GDI) that contains four G protein regulatory (GPR) or GoLoco motifs in its C-terminal domain. The entire C-terminal domain (AGS3-C) as well as certain peptides corresponding to individual GPR motifs of AGS3 bound to G alpha i1 and inhibited the binding of GTP by stabilizing the GDP-bound conformation of G alpha i1. The stoichiometry, free energy, enthalpy, and dissociation constant for binding of AGS3-C to G alpha i1 were determined using isothermal titration calorimetry. AGS3-C possesses two apparent high affinity (Kd approximately 20 nm) and two apparent low affinity (Kd approximately 300 nm) binding sites for G alpha i1. Upon deletion of the C-terminal GPR motif from AGS3-C, the remaining sites were approximately equivalent with respect to their affinity (Kd approximately 400 nm) for G alpha i1. Peptides corresponding to each of the four GPR motifs of AGS3 (referred to as GPR1, GPR2, GPR3, and GPR4, respectively, going from N to C terminus) bound to G alpha i1 with Kd values in the range of 1-8 microm. Although GPR1, GPR2, and GPR4 inhibited the binding of the fluorescent GTP analog BODIPY-FL-guanosine 5'-3-O-(thio)triphosphate to G alpha i1, GPR3 did not. However, addition of N- and C-terminal flanking residues to the GPR3 GoLoco core increased its affinity for G alpha i1 and conferred GDI activity similar to that of AGS3-C itself. Similar increases were observed for extended GPR2 and extended GPR1 peptides. Thus, while the tertiary structure of AGS3 may affect the affinity and activity of the GPR motifs contained within its sequence, residues outside of the GPR motifs strongly potentiate their binding and GDI activity toward G alpha i1 even though the amino acid sequences of these residues are not conserved among the GPR repeats.

2′(3′)-O-(N-Methylanthraniloyl)-substituted GTP Analogs: A Novel Class of Potent Competitive Adenylyl Cyclase Inhibitors

2'(3')-O-(N-Methylanthraniloyl)-(MANT)-substituted nucleotides are fluorescent and widely used for the kinetic analysis of enzymes and signaling proteins. We studied the effects of MANT-guanosine 5'-[gamma-thio]triphosphate (MANT-GTP gamma S) and MANT-guanosine 5'-[beta,gamma-imido]triphosphate (MANT-GppNHp) on G alpha(s)- and G alpha(i)-protein-mediated signaling. MANT-GTP gamma S/MANT-GppNHp had lower affinities for G alpha(s) and G alpha(i) than GTP gamma S/GppNHp as assessed by inhibition of GTP hydrolysis of receptor-G alpha fusion proteins. MANT-GTP gamma S was much less effective than GTP gamma S at disrupting the ternary complex between the formyl peptide receptor and G alpha(i2). MANT-GTP gamma S/MANT-GppNHp non-competitively inhibited GTP gamma S/GppNHp-, AlF(4)(-)-, beta(2)-adrenoceptor plus GTP-, cholera toxin plus GTP-, and forskolin-stimulated adenylyl cyclase (AC) in G alpha(s)-expressing Sf9 insect cell membranes and S49 wild-type lymphoma cell membranes. AC inhibition by MANT-GTP gamma S/MANT-GppNHp was not due to G alpha(s) inhibition because it was also observed in G alpha(s)-deficient S49 cyc(-) lymphoma cell membranes. Mn(2+) blocked AC inhibition by GTP gamma S/GppNHp in S49 cyc(-) membranes but enhanced the potency of MANT-GTP gamma S/MANT-GppNHp at inhibiting AC by approximately 4-8-fold. MANT-GTP gamma S and MANT-GppNHp competitively inhibited forskolin/Mn(2+)-stimulated AC in S49 cyc(-) membranes with K(i) values of 53 and 160 nm, respectively. The K(i) value for MANT-GppNHp at insect cell AC was 155 nm. Collectively, MANT-GTP gamma S/MANT-GppNHp bind to G alpha(s)- and G alpha(i)-proteins with low affinity and are ineffective at activating G alpha. Instead, MANT-GTP gamma S/MANT-GppNHp constitute a novel class of potent competitive AC inhibitors.

Functional coupling of the human dopamine D2 receptor with G alpha i1, G alpha i2, G alpha i3 and G alpha o G proteins: evidence for agonist regulation of G protein selectivity.

(1) The human dopamine D(2long) (D(2L)) receptor was expressed with four different G proteins in Sf9 cells using the baculovirus expression system. When co-expressed with G(i)/G(o) G proteins (G(i1)alpha, G(i2)alpha, G(i3)alpha, or G(o)alpha, plus Gbeta(1) and Ggamma(2)), the receptor displayed a high-affinity binding site for the agonists (dopamine and NPA), which was sensitive to GTP (100 micro M), demonstrating interaction between the receptor and the different G proteins. (2) The receptor to G protein ratio (R : G ratio) was evaluated using [(3)H]-spiperone saturation binding (R) and [(35)S]-GTPgammaS saturation binding (G). R : G ratios of 1 : 12, 1 : 3, 1 : 14 and 1 : 5 were found for G(i1), G(i2), G(i3), and G(o) preparations, respectively. However, when R : G ratios of 1 : 2 and 1 : 12 were compared for G(i2) and G(o), no difference was found for the stimulation of [(35)S]-GTPgammaS binding. (3) Several agonists were tested for their ability to stimulate [(35)S]-GTPgammaS binding to membranes co-expressing the receptor and various G proteins. All the compounds tested showed agonist activity in preparations expressing G(i3) and G(o). However, for G(i2) and G(i1) preparations, compounds such as S-(-)-3-PPP and p-tyramine were unable to stimulate [(35)S]-GTPgammaS binding. (4) Most of the compounds showed higher relative efficacies (compared to dopamine) and higher potencies in the preparation expressing G(o). Comparison of the effects of different agonists in the different preparations showed that each agonist differentially activates the four G proteins. (5) We conclude that the degree of selectivity of G protein activation by the D(2L) receptor can depend on the conformation of the receptor stabilised by an agonist.

Regulators of G-protein Signaling (RGS) 4, Insertion into Model Membranes and Inhibition of Activity by Phosphatidic Acid

Regulators of G-protein signaling (RGS) proteins are critical for attenuating G protein-coupled signaling pathways. The membrane association of RGS4 has been reported to be crucial for its regulatory activity in reconstituted vesicles and physiological roles in vivo. In this study, we report that RGS4 initially binds onto the surface of anionic phospholipid vesicles and subsequently inserts into, but not through, the membrane bilayer. Phosphatidic acid, one of anionic phospholipids, could dramatically inhibit the ability of RGS4 to accelerate GTPase activity in vitro. Phosphatidic acid is an effective and potent inhibitor of RGS4 in a G alpha(i1)-[gamma-(32)P]GTP single turnover assay with an IC(50) approximately 4 microm and maximum inhibition of over 90%. Furthermore, phosphatidic acid was the only phospholipid tested that inhibited RGS4 activity in a receptor-mediated, steady-state GTP hydrolysis assay. When phosphatidic acid (10 mol %) was incorporated into m1 acetylcholine receptor-G alpha(q) vesicles, RGS4 GAP activity was markedly inhibited by more than 70% and the EC(50) of RGS4 was increased from 1.5 to 7 nm. Phosphatidic acid also induced a conformational change in the RGS domain of RGS4 measured by acrylamide-quenching experiments. Truncation of the N terminus of RGS4 (residues 1-57) resulted in the loss of both phosphatidic acid binding and lipid-mediated functional inhibition. A single point mutation in RGS4 (Lys(20) to Glu) permitted its binding to phosphatidic acid-containing vesicles but prevented lipid-induced conformational changes in the RGS domain and abolished the inhibition of its GAP activity. We speculate that the activation of phospholipase D or diacylglycerol kinase via G protein-mediated signaling cascades will increase the local concentration of phosphatidic acid, which in turn block RGS4 GAP activity in vivo. Thus, RGS4 may represent a novel effector of phosphatidic acid, and this phospholipid may function as a feedback regulator in G protein-mediated signaling pathways.

An agonist-induced switch in G protein coupling of the gonadotropin-releasing hormone receptor regulates pulsatile neuropeptide secretion.

The pulsatile secretion of gonadotropin-releasing hormone (GnRH) from normal and immortalized hypothalamic GnRH neurons is highly calcium-dependent and is stimulated by cAMP. It is also influenced by agonist activation of the endogenous GnRH receptor (GnRH-R), which couples to G(q/11) as indicated by release of membrane-bound alpha(q/11) subunits and increased inositol phosphate/Ca(2+) signaling. Conversely, GnRH antagonists increase membrane-associated alpha(q/11) subunits and abolish pulsatile GnRH secretion. GnRH also stimulates cAMP production but at high concentrations has a pertussis toxin-sensitive inhibitory effect, indicative of receptor coupling to G(i). Coupling of the agonist-activated GnRH-R to both G(s) and G(i) proteins was demonstrated by the ability of nanomolar GnRH concentrations to reduce membrane-associated alpha(s) and alpha(i3) levels and of higher concentrations to diminish alpha(i3) levels. Conversely, alpha(i3) was increased during GnRH antagonist and pertussis toxin treatment, with concomitant loss of pulsatile GnRH secretion. In cholera toxin-treated GnRH neurons, decreases in alpha(s) immunoreactivity and increases in cAMP production paralleled the responses to nanomolar GnRH concentrations. Treatment with cholera toxin and 8-bromo-cAMP amplified episodic GnRH pulses but did not affect their frequency. These findings suggest that an agonist concentration-dependent switch in coupling of the GnRH-R between specific G proteins modulates neuronal Ca(2+) signaling via G(s)-cAMP stimulatory and G(i)-cAMP inhibitory mechanisms. Activation of G(i) may also inhibit GnRH neuronal function and episodic secretion by regulating membrane ion currents. This autocrine mechanism could serve as a timer to determine the frequency of pulsatile GnRH release by regulating Ca(2+)- and cAMP-dependent signaling and GnRH neuronal firing.

Angiotensin-II-induced enhanced expression of Gi proteins is attenuated by losartan in A10 vascular smooth muscle cells: role of AT1 receptors.

We have previously shown that treatment of A10 vascular smooth muscle cells (VSMCs) with angiotensin II (Ang II) enhanced the expression of inhibitory guanine nucleotide regulatory proteins (Gi alpha2 and Gi alpha3). In the present studies, we have investigated the role of type 1 angiotensin receptors (AT1) in the Ang-II-induced enhanced expression of Gi alpha proteins and their functions in A10 SMCs. Ang II enhanced the levels of Gi alpha2 and Gi alpha3 proteins and their mRNA, as determined by Western and Northern blot analysis, respectively; losartan treatment attenuated the enhanced expression of Gi alpha2 and Gi alpha3 proteins and their mRNA in a concentration-dependent manner. In addition, the inhibition of adenylyl cyclase induced by Ang II and des(Glu18,Ser19,Glu20,Leu21,Gly22)ANP(4-23)-NH2 (C-ANP(4-23)), which was attenuated by Ang-II treatment, was partially restored by losartan treatment. Similarly, losartan was also able to restore the Ang-II-induced stimulatory responses of isoproterenol and N-ethylcarboxamide adenosine (NECA) on adenylyl cyclase activity. These results suggest a role for AT1 receptors in Ang-II-evoked increases in Gi alpha protein expression and Gs-mediated stimulation in VSMCs.

Fluorescence analysis of receptor-G protein interactions in cell membranes.

The dynamics of G protein heterotrimer complex formation and disassembly in response to nucleotide binding and receptor activation govern the rate of responses to external stimuli. We use a novel flow cytometry approach to study the effects of lipid modification, isoform specificity, lipid environment, and receptor stimulation on the affinity and kinetics of G protein subunit binding. Fluorescein-labeled myristoylated Galpha(i1) (F-alpha(i1)) was used as the ligand bound to Gbetagamma in competition binding studies with differently modified Galpha subunit isoforms. In detergent solutions, the binding affinity of Galpha(i) to betagamma was 2 orders of magnitude higher than for Galpha(o) and Galpha(s) (IC50 of 0.2 nM vs 17 and 27 nM, respectively), while in reconstituted bovine brain lipid vesicles, binding was slightly weaker. The effects of receptor on the G protein complex were assessed in alpha(2A)AR receptor expressing CHO cell membranes into which purified betagamma subunits and F-alpha(i1) were reconstituted. These cell membrane studies led to the following observations: (1) binding of alpha subunit to the betagamma was not enhanced by receptor in the presence or absence of agonist, indicating that betagamma contributed essentially all of the binding energy for alpha(i1) interaction with the membrane; (2) activation of the receptor facilitated GTPgammaS-stimulated detachment of F-alpha(i1) from betagamma and the membrane. Thus flow cytometry permits quantiatitive and real-time assessments of protein-protein interactions in complex membrane environments.

Recent progress in the neurobiology of the vomeronasal organ.

In many terrestrial tetrapodes, a pair of vomeronasal organs (VNOs), which are chemosensory apparatuses, are situated at the base of the nasal septum in the anterior nasal cavity. The purposes of this review are to summarize comparative neuroanatomy and to introduce recent progress in neurobiological studies of the VNO. Five types of VNOs can be identifiable in terms of anatomical organization; snakes possess the most complex one. Sensory cells in the VNO, vomeronasal receptor neurons (VRNs), are located in its neuroepithelium, vomeronassal sensory epithelium. The VRNs retain the characteristic of epithelial cells in that they are born continuously from progenitor cells. They contain two prominent subcellular structures: microvilli and extraordinarily large amounts of smooth endoplasmic reticulum and a few unique glycoconjugates. The VRNs express two types of G-protein -subunits: Gi(alpha2) and Go(alpha) and each of them is coupled with putative pheromone receptors, V1Rs and V2Rs, respectively. Recent physiological and biochemical studies have demonstrated that pheromones depolarize the V1R-Gi(alpha2) and V2R-Go(alpha) VRNs via IP(3)-mediated mechanisms. The VRNs do not show adaptation and are ultrasensitive to putative pheromones. Other than being a chemosensory organ, the VNO and its primordium might play important roles for brain development; hypothalamic neurons that produce gonadotropin-releasing hormone are born in the VNO primordium and a few other neuron-like cells may be born in the VNO primordium and VNO. In human fetuses, anatomical findings strongly suggest that their VNOs contain a neuroepithelium. By contrast, it is unlikely that adult human VNO serves as a chemosensory organ.

Agonists activate Gi1 alpha or Gi2 alpha fused to the human mu opioid receptor differently.

As preferential coupling of opioid receptor to various inhibitory Galpha subunits is still under debate, we have investigated the selectivity of the human mu opioid receptor fused to a pertussis toxin insensitive C351I Gi1 alpha or C352I Gi2 alpha in stably transfected HEK 293 cells. Overall agonist binding affinities were increased for both fusion constructs when compared to the wild type receptor. [35 S]GTPgammaS binding was performed on pertussis toxin treated cells to monitor coupling efficiency of the fusion constructs. Upon agonist addition hMOR-C351I Gi1 a exhibited an activation profile similar to the non-fused receptor while hMOR-C352I Gi2 alpha was poorly activated. Interestingly no correlation could be drawn between agonist binding affinity and efficacy. Upon agonist addition, forskolin-stimulated cAMP production, as measured using a reporter gene assay, was inhibited by signals transduced via the fused Gi1 alpha and Gi2 alpha mainly. In contrast both fusion constructs were able to initiate ERK-MAPK phosphorylation via coupling to endogenous G proteins only. In conclusion our data indicate that hMOR couples more efficiently to Gi1 alpha than Gi2 alpha and that the coupling efficacy is clearly agonist-dependent.

The complex of Arl2-GTP and PDE delta: from structure to function.

Arf-like (Arl) proteins are close relatives of the Arf regulators of vesicular transport, but their function is unknown. Here, we present the crystal structure of full-length Arl2-GTP in complex with its effector PDE delta solved in two crystal forms (Protein Data Bank codes 1KSG, 1KSH and 1KSJ). Arl2 shows a dramatic conformational change from the GDP-bound form, which suggests that it is reversibly membrane associated. PDE delta is structurally closely related to RhoGDI and contains a deep empty hydrophobic pocket. Further experiments show that H-Ras, Rheb, Rho6 and G alpha(i1) interact with PDE delta and that, at least for H-Ras, the intact C-terminus is required. We suggest PDE delta to be a specific soluble transport factor for certain prenylated proteins and Arl2-GTP a regulator of PDE delta-mediated transport.

Phosphatidylinositol 4,5-Bisphosphate (PIP2) Stimulates Epithelial Sodium Channel Activity in A6 Cells

Phosphatidylinositol 4,5-bisphosphate (PIP(2)) is a membrane lipid found in all eukaryotic cells, which regulates many important cellular processes, including ion channel activity. In this study, we used inside-out patch clamp technique, immunoprecipitation, and Western blot analysis to investigate the effect of PIP(2) on epithelial sodium channel activity in A6 cells. A6 cells were cultured in media supplemented with 1.5 microm aldosterone. Single sodium channel activity in excised, inside-out patches was increased by perfusion of the bath solution with 30 microm PIP(2) plus 100 microm GTP (NP(o) = 1.34 +/- 0.14) compared with the paired control (NP(o) = 0.09 +/- 0.02). However, neither 30 microm PIP(2) (NP(o) = 0.11 +/- 0.02) nor 100 microm GTP (NP(o) = 0.10 +/- 0.02) alone stimulated the sodium channels. The PIP(2)-stimulated channel activity was abolished by application of 10 nm G protein betagamma subunits (NP(o) = 0.14 +/- 0.05). However, 10 nm Galpha(i-3) + 30 microm PIP(2) increased both NP(o) and P(o). The stimulating effect of 10 nm Galpha(i-3) + 30 microm PIP(2) is similar to that of 30 microm PIP(2) plus 100 microm GTP. Immunoprecipitation and Western blot analysis show that both Gi(alpha-3) and PIP(2) bind beta and gamma epithelial Na(+) channels (ENaC), but not alpha ENaC. These results indicate that PIP(2) increases ENaC activity by direct interaction with beta or gamma xENaC in the presence of Galpha(i-3).

Evidence for a direct negative coupling between dopamine-D2 receptors and PLC by heterotrimeric Gi1/2 proteins in rat anterior pituitary cell membranes.

Dopamine (DA) is known to inhibit basal and hormone TRH- or angiotensin II (AngII)-stimulated PRL secretion and inositol phosphate accumulation in rat pituitary cells in primary culture. This inhibition persists when cells are incubated in a calcium-free medium (a condition in which DA could not inhibit PLC activities by blocking calcium influx) and is abolished by a Pertussis toxin treatment. These data suggest that DA receptor could be negatively coupled to PLC by a direct mechanism involving a Pertussis toxin-sensitive G protein. To demonstrate this hypothesis, we measured PLC activities on crude plasma membranes obtained from rat pituitary cells in primary culture grown in the presence of tritiated myo-inositol. We showed that 1) DA and quinpirole or RU24926 (specific D2 agonists) inhibited both basal and TRH- or AngII-stimulated membrane PLC activities. 2) Such inhibitions were completely prevented by sulpiride (specific D2 antagonist). 3) Heterotrimeric Gi1/2 proteins coupled the DA receptors to PLC because DA inhibitions were completely reversed by preincubation either with Pertussis toxin or with a specific G(alpha)i1/(alpha)i2 antibody. Such data are in favor of the existence of a direct negative coupling between DA-D2 receptor and PLC on a native physiological plasma membrane model.

Characterization of subcellular localization and stability of a splice variant of G alphai2

BackgroundAlternative mRNA splicing of αi2, a heterotrimeric G protein α subunit, has been shown to produce an additional protein, termed sαi2. In the sαi2 splice variant, 35 novel amino acids replace the normal C-terminal 24 amino acids of αi2. Whereas αi2 is found predominantly at cellular plasma membranes, sαi2 has been localized to intracellular Golgi membranes, and the unique 35 amino acids of sαi2 have been suggested to constitute a specific targeting signal.ResultsThis paper proposes and examines an alternative hypothesis: disruption of the normal C-terminus of αi2 produces an unstable protein that fails to localize to plasma membranes. sαi2 is poorly expressed upon transfection of cultured cells; however, radiolabeling indicated that αi2 and sαi2 undergo myristoylation, a co-translational modification, equally well suggesting that protein stability rather than translation is affected. Indeed, pulse-chase analysis indicates that sαi2 is more rapidly degraded compared to αi2. Co-expression of βγ rescues PM localization and increases expression of sαi2. In addition, αi2A327S, a mutant previously shown to be unstable and defective in guanine-nucleotide binding, and αi2(1–331), in which the C-terminal 24 amino acids of αi2 are deleted, show a similar pattern of subcellular localization as sαi2 (i.e., intracellular membranes rather than plasma membranes). Finally, sαi2 displays a propensity to localize to potential aggresome-like structures.ConclusionsThus, instead of the novel C-terminus of sαi2 functioning as a specific Golgi targeting signal, the results presented here indicate that the disruption of the normal C-terminus of αi2 causes mislocalization and rapid degradation of sαi2.

Expression, purification, and assay of cytosolic (catalytic) domains of membrane-bound mammalian adenylyl cyclases

The identification and isolation of the soluble catalytic domains of adenylyl cyclase have provided investigators with useful reagents for the study of these enzymes. They have permitted detailed mechanistic investigation of the actions of forskolin, Gs alpha, and the inhibitory G protein, Gi alpha. They have served as critical reagents for the development of plausible models of the catalytic mechanism of the enzyme. They have enabled X-ray crystallographic analysis of adenylyl cyclase; this technique was not approachable with the small quantities of the membrane-bound enzyme available previously. The information obtained by using the soluble domains of adenylyl cyclase has provided templates for description of the behavior of many forms of purine nucleotide cyclases from a variety of species. We now appreciate both adenylyl cyclases and guanylyl cyclases as dimeric enzymes with a 2-fold symmetrical domain arrangement (or pseudosymmetrical in the case of heterodimerization). The active sites are located at the interface between the two domains, both of which contribute binding surfaces.

Antibody response to dietary and autoantigens in G alpha i2-deficient mice.

Mice with a targeted mutation in the G protein subunit G alpha i2 gene develop a colonic mucosal inflammation, with a highly activated B-cell response. We wanted to investigate whether this increased B-cell activity was directed against dietary antigens and/or various self tissues. The level of antibodies specific for dietary (gliadin, soya and fish meal) antigens was measured by ELISA. Reactivity against self antigens was measured by immunohistochemistry on cryo-sectioned mouse and rat tissue. Sera and intestinal lavages were analysed from G alpha i2-/- mice before and after development of colitis and in age-matched wild type litter mates. Titres of antibodies against dietary antigens were significantly enhanced both in serum and in large intestinal lavages from G alpha i2-/- mice with ongoing colitis but not prior to disease, as compared to wild type mice. The autoreactivity to self tissues was significantly increased in G alpha i2-/- mice both before and after development of colitis as compared to litter mate control animals. Self tissue reactivity was directed not only against epithelial cells of the colon, small intestine and gastric glands, but also against smooth muscle cells, hepatocytes, bile duct cells, renal tubule and collecting tubule cells of the kidney. In analogy to human ulcerative colitis, autoantibodies against epithelial cells, bile duct epithelium and neutrophil granulocytes were found. Earlier increase in levels of autoantibodies (before onset of colitis) than of food antibodies (after onset of colitis) suggests the latter response to be a secondary phenomenon to e.g. a destroyed barrier function.

Gαq/11 Coupled to Mammalian Phospholipase C β3-like Enzyme Mediates the Ginsenoside Effect on Ca2+-activated Cl− Current in theXenopus Oocyte

Recently we demonstrated that ginsenosides, the active ingredients of Panax ginseng, enhanced Ca(2+)-activated Cl(-) current in the Xenopus oocyte through a signal transduction mechanism involving the activation of pertussis toxin-insensitive G protein and phospholipase C (PLC). However, it has not yet been determined precisely which G protein subunit(s) and which PLC isoform(s) participate in the ginsenoside signaling. To provide answers to these questions, we investigated the changes in ginsenoside effect on the Cl(-) current after intraoocyte injections of the cRNAs coding various G protein subunits, a regulator of G protein signaling (RGS2), and G beta gamma-binding proteins. In addition, we examined which of mammalian PLC beta 1-3 antibodies injected into the oocyte inhibited the action of ginsenosides on the Cl(-) current. Injection of G alpha(q) or G alpha(11) cRNA increased the basal Cl(-) current recorded 48 h after, and it further prevented ginsenosides from enhancing the Cl(-) current, whereas G alpha(i2) and G alpha(oA) cRNA injection had no significant effect. The changes following G alpha(q) cRNA injection were prevented when G beta(1)gamma(2) and G alpha(q) subunits were co-expressed by simultaneous injection of the cRNAs coding these subunits. Injection of cRNA coding G alpha(q)Q209L, a constitutively active mutant that does not bind to G beta gamma, produced effects similar to those of G alpha(q) cRNA injection. The effects of G alpha(q)Q209L cRNA injection, however, were not prevented by co-injection of G beta(1)gamma(2) cRNA. Injection of the cRNA coding RGS2, which interacts most selectively with G alpha(q/11) among various identified RGS isoforms and stimulates the hydrolysis of GTP to GDP in active GTP-bound G alpha subunit, resulted in a severe attenuation of ginsenoside effect on the Cl(-) current. Finally, antibodies against PLC beta 3, but not -beta 1 and -beta 2, markedly attenuated the ginsenoside effect examined at 3-h postinjection. These results suggest that G alpha(q/11) coupled to mammalian PLC beta 3-like enzyme mediates ginsenoside effect on Ca(2+)-activated Cl(-) current in the Xenopus oocyte.

RGS12 and RGS14 GoLoco Motifs Are GαiInteraction Sites with Guanine Nucleotide Dissociation Inhibitor Activity

The regulators of G-protein signaling (RGS) proteins accelerate the intrinsic guanosine triphosphatase activity of heterotrimeric G-protein alpha subunits and are thus recognized as key modulators of G-protein-coupled receptor signaling. RGS12 and RGS14 contain not only the hallmark RGS box responsible for GTPase-accelerating activity but also a single G alpha(i/o)-Loco (GoLoco) motif predicted to represent a second G alpha interaction site. Here, we describe functional characterization of the GoLoco motif regions of RGS12 and RGS14. Both regions interact exclusively with G alpha(i1), G alpha(i2), and G alpha(i3) in their GDP-bound forms. In GTP gamma S binding assays, both regions exhibit guanine nucleotide dissociation inhibitor (GDI) activity, inhibiting the rate of exchange of GDP for GTP by G alpha(i1). Both regions also stabilize G alpha(i1) in its GDP-bound form, inhibiting the increase in intrinsic tryptophan fluorescence stimulated by AlF(4)(-). Our results indicate that both RGS12 and RGS14 harbor two distinctly different G alpha interaction sites: a previously recognized N-terminal RGS box possessing G alpha(i/o) GAP activity and a C-terminal GoLoco region exhibiting G alpha(i) GDI activity. The presence of two, independent G alpha interaction sites suggests that RGS12 and RGS14 participate in a complex coordination of G-protein signaling beyond simple G alpha GAP activity.

Sodium Butyrate Induces Transcription from the Gαi2Gene Promoter through Multiple Sp1 Sites in the Promoter and by Activating the MEK-ERK Signal Transduction Pathway

Sodium butyrate, an erythroid differentiation inducer and a histone deacetylase inhibitor, increases G alpha(i2) levels in differentiating K562 cells. Here we show that sodium butyrate induces G alpha(i2) gene transcription via sequences at -50/-36 and -92/-85 in the G alpha(i2) gene promoter. Both sequences contain core sequence motif for Sp1 binding; electrophoretic mobility shift as well as supershift assays confirmed binding to Sp1. Transcription from the G alpha(i2) gene promoter was also activated by two other histone deacetylase inhibitors, trichostatin A and Helminthsporium carbonium toxin (HC toxin), which also induce erythroblastic differentiation in K562 cells. However, hydroxyurea, a potent erythroid differentiation inducer in these cells, did not activate transcription from this gene promoter, indicating that promoter activation is inducer-specific. Mutations within the Sp1 sites at -50/-36 and -92/-85 in the G alpha(i2) gene promoter substantially decreased transcriptional activation by sodium butyrate, trichostatin A, or HC toxin. Transfection with constitutively activated ERKs indicated that this promoter can be activated through the MEK-ERK signal transduction pathway. Inhibition of the MEK-ERK pathway with U0126 or reduction in the expression of endogenous ERK with an antisense oligonucleotide to ERK significantly inhibited sodium butyrate- and HC toxin-induced transcription but had no effect on trichostatin A-induced transcription. Inhibition of the JNK and p38 MAPKs, using selective inhibitors, had no effect on sodium butyrate-induced transcription. In cells in which sodium butyrate induction of promoter activation had been inhibited by various concentrations of U0126, constitutively activated ERK2 reversed this inhibition. These results show that the MEK-ERK signal transduction pathway is important in butyrate signaling, which eventually converges in the cell nucleus.