GPCR-induced dissociation of G-protein subunits in early stage signal transduction

G-protein coupled receptors (GPCRs) form a ternary complex of agonist, receptor and G-proteins during primary signal transduction at the cell membrane. Downstream signalling is thought to be preceded by the process of dissociation of Galpha and Gbetagamma subunits, thus exposing new surfaces to interact with downstream effectors. We demonstrate here for the first time, the dissociation of heterotrimeric G-protein subunits (i.e., Galpha and Gbetagamma) following agonist-induced GPCR (alpha(2A)-adrenergic receptor; alpha(2A)-AR) activation in a cell-free assay system. alpha(2A)-AR membranes were reconstituted with the G-proteins (+/-hexahistidine-tagged) Galpha(i1) and Gbeta1gamma2 and functional signalling was determined following activation of the reconstituted receptor:G-protein complex with the potent agonist UK-14304, and [35S]GTPgammaS. In the presence of Ni(2+)-coated agarose beads, the activated his-tagged Galpha(i1)his-[35S]GTPgammaS complex was captured on the Ni(2+)-presenting surface. When his-tagged Gbeta1gamma2 (Gbeta1gamma2his) was used with Galpha(i1), the [35S]GTPgammaS-bound Galpha(i1) was not present on the Ni(2+)-coated beads, but rather, it was separated from the beta1gamma2(his)-beads, demonstrating receptor-induced dissociation of Galpha and Gbetagamma subunits. Treatment of the reconstituted alpha(2A)-AR membranes containing Gbeta1gamma2his:Galpha(i1) with imidazole confirmed the specificity for the Ni2+:G-protein surface dissociation of Galpha(i1) from Gbeta1gamma2his. These data demonstrate for the first time, the complete dissociation of the G-protein subunits and extend observations on the role of G-proteins in the assembly and disassembly of the ternary complex in the primary events of GPCR signalling.     

Gβγ interacts with mTOR and promotes its activation

Diverse G protein-coupled receptors depend on Gβγ heterodimers to promote cell polarization and survival via direct activation of PI3Kγ and potentially other effectors. These events involve full activation of AKT via its phosphorylation at Ser473, suggesting that mTORC2, the kinase that phosphorylates AKT at Ser473, is activated downstream of Gβγ. Thus, we tested the hypothesis that Gβγ directly contributes to mTOR signaling. Here, we demonstrate that endogenous mTOR interacts with Gβγ. Cell stimulation with serum modulates Gβγ interaction with mTOR. The carboxyl terminal region of mTOR, expressed as a GST-fusion protein, including the serine/threonine kinase domain, binds Gβγ heterodimers containing different Gβ subunits, except Gβ₄. Both, mTORC1 and mTORC2 complexes interact with Gβ₁γ₂ which promotes phosphorylation of their respective substrates, p70S6K and AKT. In addition, chronic treatment with rapamycin, a condition known to interfere with assembly of mTORC2, reduces the interaction between Gβγ and mTOR and the phosphorylation of AKT; whereas overexpression of Gαi interfered with the effect of Gβγ as promoter of p70S6K and AKT phosphorylation. Altogether, our results suggest that Gβγ positively regulates mTOR signaling via direct interactions and provide further support to emerging strategies based on the therapeutical potential of inhibiting different Gβγ signaling interfaces.     

Activation of a pertussis toxin-sensitive, inhibitory G-protein is necessary for steroid-mediated oocyte maturation in spotted seatrout

Oocyte maturation (OM) is initiated in lower vertebrates and echinoderms when maturation-inducing substances (MIS) bind oocyte membrane receptors. This study tested the hypothesis that activation of a G_i protein is necessary for MIS-mediated OM in spotted seatrout. Addition of MIS significantly decreased adenylyl cyclase activity in a steroid specific, pertussis toxin (PTX)-sensitive manner in oocyte membranes and microinjection of PTX into oocytes inhibited MIS-induced OM, suggesting the steroid activates a G_i protein. MIS significantly increased [³⁵S]GTPγS binding to ovarian membranes, confirming that MIS receptor binding activates a G-protein, and immunoprecipitation studies showed the increased [³⁵S]GTPγS binding was associated with Gα_i1-3 proteins. Radioligand binding studies in ovarian membranes using GTPγS and PTX demonstrated that the MIS binds a receptor coupled to a PTX-sensitive G-protein. This study provides the first direct evidence in a vertebrate model that MIS-induced activation of a G_i protein is necessary for OM. These results support a mechanism of MIS action involving binding to a novel, G-protein coupled receptor and activation of an inhibitory G-protein, the most comprehensive and plausible model of MIS initiation of OM proposed to date.     

Dynamics of apelin receptor/G protein coupling in living cells

During our research on apelin receptor (APJ) signalling in living cells with BRET and FRET, we demonstrated that apelin-13 stimulation can lead to the activation of Gαi₂ or Gαi₃ through undergoing a molecular rearrangement rather than dissociation in HEK293 cells expressing APJ. Furthermore, Gαo and Gαq also showed involvement in APJ activation through a classical dissociation model. However, both FRET signal and BRET ratio between fluorescent Gαi₁ subunit and Gβγ subunits demonstrated little change after apelin-13 stimulation. These results demonstrated that stimulation of APJ with apelin-13 causes activation of Gαi₂, Gαi₃, Gαo, Gαq; among which Gαi₂, Gαi₃ were activated through a novel rearrangement process. These results provide helpful data for understanding APJ mediated G-protein signalling.     

Intracellularly Truncated Human α2B-Adrenoceptors: Stable and Functional GPCRs for Structural Studies

All three α₂-adrenoceptor subtypes have a long third intracellular loop (3i), which is conserved by overall size and charge-hydrophobic properties but not by amino acid sequence similarity. These properties must be relevant for function and structure, because they have been preserved during hundreds of millions of years of evolution. The contribution of different loop portions to agonist/antagonist binding properties and G protein coupling of the human α_2B-adrenoceptor (α_2B-AR) was investigated with a series of 3i truncated constructs (Δ 3i). We used a variety of agonists/antagonists in competition binding assays. We stimulated α_2B-AR Δ3i with various agonists and measured [³⁵S]GTPγS binding in isolated cell membranes with or without antagonist inhibition. We also evaluated the ability of oligopeptides, analogous to the amino and carboxyl terminal parts of 3i, to promote G protein activation, monitored with the [³⁵S]GTPγS assay. Our results reveal that the carboxyl end residues of 3i, R360(6.24) to V372(6.36), are important for G_i/G_o protein activation. Deletions in regions from G206(5.72) to R245(5.110) altered the binding of some α_2B-AR agonists, indicating that agonist binding is dependent on the conformation of the 3i domain, possibly through the involvement of G protein interactions. The truncated receptor constructs may be more stable on purification and thus be useful for structural characterization of α_2B-AR.     

Modulation by Neuropeptide FF of the interaction of Mu-opioid (MOP) receptor with G-proteins

The Neuropeptide FF (NPFF) system is known to modulate the effects of opioids in vivo and in vitro. In the present study, we have investigated the effect of NPFF agonists on the coupling of the Mu-opioid (MOP) receptor to G-proteins in a model of SH-SY5Y cells transfected with NPFF₂ receptor, in which the neuronal anti-opioid activity of NPFF was previously reproduced. Activation of G-proteins was monitored by [³⁵S]GTPγS binding assay and analysis of G-protein subunits associated with MOP receptors was performed by Western blotting after immunoprecipitation of the receptor. The results demonstrate that concentrations of NPFF agonists that produce a cellular anti-opioid effect, did not affect the ability of the opioid agonist DAMGO to activate G-proteins. However, at saturating concentration of agonist or when expression of receptor was high, opioid and NPFF agonists did not stimulate [³⁵S]GTPγS binding in an additive manner, indicating that both receptors share a common fraction of a G-protein pool. In addition, stimulation of NPFF receptors in living cells modified the G-protein environment of MOP receptor by favoring its interaction with α_s, α_i2 and β subunits. This change in G-protein coupling to MOP receptor might participate in the mechanism by which NPFF agonists reduce the inhibitory activity of opioids.     

Bell-shaped agonist activation of 5-HT1A receptor-coupled Gαi3 G-proteins: Receptor density-dependent switch in receptor signaling

A previous study observed bell-shaped concentration-response isotherms for activation of Gα_i3 G-protein subunits by high efficacy 5-HT_1A receptor agonists in a Chinese hamster ovary (CHO) cell line expressing high levels of these receptors. This suggested that a signaling switch took place in that cell line (from Gα_i3 to activation of other G-proteins) but it was unclear if such effects are observed for 5-HT_1A receptors in other cellular environments.Here, using an antibody capture-based [³⁵S]GTPγS binding assay for Gα_i3 activation, we investigated whether efficacious 5-HT_1A receptor agonists (5-HT, F13714, befiradol, NLX-101), prototypical agonists ((+) and (−)8-OH-DPAT), and partial agonist, antagonists, inverse agonists (pindolol, WAY100635, spiperone) produced similar effects on 5 cell lines expressing different levels of human 5-HT_1A receptors.In membranes from cell lines (HeLa, C6-glia and CHO-low) expressing moderate receptor levels (between 1 and 4 pmol/mg of protein), 5-HT, F13714, befiradol and NLX-101 elicited classical sigmoid concentration-response isotherms. In contrast, in cell lines (CHO-high, HEK-293F) expressing high receptor levels (>9 pmol/mg) these agonists elicited bell-shaped concentration-response isotherms that peaked at nanomolar-range concentrations and then returned to baseline or below. Spiperone elicited inverse agonist inhibitory sigmoid isotherms in all membrane preparations while WAY100635 was mostly ‘silent’ for Gα_i3 activation. The other compounds elicited diverse responses in the different cell lines suggesting that other factors, in addition to receptor expression levels, could be influencing Gα_i3 activation.These data indicate that Gα_i3 G-protein activation by 5-HT_1A receptor ligands is highly dependent on receptor expression levels and on cellular background. Moreover, the induction of bell-shape concentration-response isotherms by 5-HT and other high-efficacy agonists is consistent with a switch in signaling to other G-protein-mediated signaling cascades, possibly elicited by receptor conformational changes.     

Development of inhibitors of heterotrimeric Gαi subunits

Heterotrimeric G-proteins are the immediate downstream effectors of G-protein coupled receptors (GPCRs). Endogenous protein guanine nucleotide dissociation inhibitors (GDIs) like AGS3/4 and RGS12/14 function through GPR/Goloco GDI domains. Extensive characterization of GPR domain peptides indicate they function as selective GDIs for Gα_i by competing for the GPCR and Gβγ and preventing GDP release. We modified a GPR consensus peptide by testing FGF and TAT leader sequences to make the peptide cell permeable. FGF modification inhibited GDI activity while TAT preserved GDI activity. TAT-GPR suppresses G-protein coupling to the receptor and completely blocked α₂-adrenoceptor (α₂AR) mediated decreases in cAMP in HEK293 cells at 100 nM. We then sought to discover selective small molecule inhibitors for Gα_i. Molecular docking was used to identify potential molecules that bind to and stabilize the Gα_i–GDP complex by directly interacting with both Gα_i and GDP. Gα_i–GTP and Gα_q–GDP were used as a computational counter screen and Gα_q–GDP was used as a biological counter screen. Thirty-seven molecules were tested using nucleotide exchange. STD NMR assays with compound 0990, a quinazoline derivative, showed direct interaction with Gα_i. Several compounds showed Gα_i specific inhibition and were able to block α₂AR mediated regulation of cAMP. In addition to being a pharmacologic tool, GDI inhibition of Gα subunits has the advantage of circumventing the upstream component of GPCR-related signaling in cases of overstimulation by agonists, mutations, polymorphisms, and expression-related defects often seen in disease.     

Binding of Gβγ subunits to cRaf1 downregulates G-protein-coupled receptor signalling

Receptors of the seven transmembrane domain family are coupled to heterotrimeric G proteins [1]. Binding of ligand to these receptors induces dissociation of the heterotrimeric complex into free GTP–Gα and Gβγ subunits, which then interact with their respective effector molecules to stimulate specific cellular responses. In some cases, these cellular responses involve mitogenic signalling [2]. The mitogen-activated protein (MAP) kinase cascade is initiated by the protein kinase cRaf1 and links growth factor receptor signalling to cell growth and differentiation [3]. The main activator of cRaf1 is the small GTP-binding protein Ras [4], and the binding of cRaf1 to GTP–Ras translocates cRaf1 to the plasma membrane, where it is activated [5]. It has been reported that cRaf1 associates directly with the β subunit of heterotrimeric G proteins in vitro, and with the βγ subunit complex in vivo[6], but the role of this association is not yet understood. Here, we show that cRaf1 associates with Gβ1γ2, and that this association in mammalian cells is significantly enhanced when active p21^Ras is present or when cRaf1 is otherwise targeted to the membrane. Association with Gβ1γ2 has no effect on the kinase activity of cRaf1, but cRaf1 can affect Gβγ-mediated signalling events. Thus, membrane-localised cRaf1 inhibits G-protein-coupled receptor (GPCR)-stimulated activation of phospholipase Cβ (PLCβ) by sequestration of Gβγ subunits, an effect also observed with endogenous levels of cRaf1. Our data suggest that cRaf1 may be an important regulator of signalling by Gβγ, particularly in those GPCR systems that stimulate the MAP kinase cascade through the activation of p21^Ras.     

Competitive interaction of 5-HT1A receptors with G-protein subtypes in CHO cells demonstrated by RNA interference

Following agonist action, G-protein-coupled receptors may exhibit differential coupling to G-proteins or second messenger pathways, supporting the notion of agonist-directed trafficking. To explore these mechanisms, we have designed and transfected synthetic siRNA duplexes to knockdown different G_α subunits in Chinese hamster ovary (CHO) cells expressing human (h)5-hydroxytryptamine 1A receptors (CHO-h5-HT_1A). siRNAs against G_αi2 and G_αi3 transfected alone or in combination caused a large decrease in the corresponding mRNA level (64-80%) and also at the protein level for G_αi3 (60-70%), whereas a non-specific siRNA showed no effect. In membranes of CHO-h5-HT_1A, 5-HT stimulated guanosine-5′-O-(3-[³⁵S]thio)-triphosphate ([³⁵S]GTPγS) binding was differentially affected by transfection of siRNAs against G_αi protein, siRNAs against G_αi2 inducing a more important decrease in the efficacy of 5-HT than transfection of siRNAs against G_αi3. The high potency component was abolished after transfection of siRNAs against G_αi3 and the lower potency component was suppressed after transfection of siRNAs against G_αi2. To directly investigate G_αi3 activation we used an antibody-capture/scintillation proximity assay. (+)8-OH-DPAT yielded bell-shaped curves for G_αi3 activation, a response that was abolished after transfection of siRNAs against G_αi3 protein. Interestingly, (+)8-OH-DPAT yielded a sigmoidal response when only G_αi3 protein was expressed. These data suggest that when efficacious agonists attain a high level of occupation of h5-HT_1A receptors, a change occurs that induces coupling to G_αi2 protein and suppresses signalling through G_αi3 subunits.     

Selective Increases in Phosphoinositide Signaling Activity and G Protein Levels in Postmortem Brain from Subjects with Schizophrenia or Alcohol Dependence

Abstract: Comparisons of the activity of the G protein-mediated phosphoinositide signal transduction system and of G protein levels were made in two regions of frontal cortex from eight schizophrenic, alcohol-dependent, and control subjects. G protein-mediated phosphoinositide hydrolysis was measured by stimulating cortical membranes incubated with [³H]phosphatidylinositol with 0.3–10 µM guanosine 5′-O-(3-thio)triphosphate (GTPγS). In frontal cortex areas 8/9, GTPγS-induced phosphoinositide hydrolysis was 50% greater in schizophrenic than control or alcohol-dependent subjects, whereas there were no differences among these groups of subjects in the response to GTPγS in frontal cortex area 10. Agonists for dopaminergic, cholinergic, purinergic, serotonergic, histaminergic, and glutamatergic receptors coupled to the phosphoinositide signaling system increased [³H]phosphatidylinositol hydrolysis in a GTPγS-dependent manner. Responses to most agonists were similar in all three subject groups in both cortical regions, with the largest difference being a 40% greater response to dopaminergic receptor stimulation in frontal cortex 8/9 from schizophrenic subjects. Measurements of the levels of phospholipase C-β, and of α-subunits of G_q, G_o, G_i1, G_i2, and G_s, made by immunoblot analyses revealed no differences among the groups of subjects except for increased Gα_o in schizophrenic subjects and increased Gα_o and Gα_i1 in alcohol-dependent subjects. These results demonstrate that schizophrenia is associated with increased activity of the phosphoinositide signal transduction system and increased levels of Gα_o, whereas the phosphoinositide system was unaltered in alcohol dependence, but Gα_o and Gα_i1 were increased.     

A Self-Scaffolding Model for G Protein Signaling

Activation of heterotrimeric G proteins is generally believed to induce dissociation of Gα and Gβγ subunits, which are then free to bind to and change the catalytic activity of a variety of intracellular enzymes. We have previously found that in cells, Gαq subunits remain complexed with its major effector, phospholipase Cβ1, through the activation cycle. To determine whether this behavior may be operative in other systems, we carried out Förster resonance energy transfer studies and found that eYFP-Gαi and eCFP-Gβγ remain associated after stimulation in HEK293 cells. We also found that the level of Forster resonance energy transfer between Alexa546-phospholipase Cβ2 and eGFP-Gβγ is significant and unchanged upon activation in HEK293 cells, thus showing that these proteins can localize into stable signaling complexes. To understand the basis for this stabilization, we carried out in vitro studies using a series of single-Cys mutants labeled with fluorescence tags and monitored their interaction with Gβγ subunits and changes in their fluorescence properties and accessibility upon activation and Gβγ binding. Our studies suggest a significant change in the orientation between G protein subunits upon activation that allows the G proteins to remain complexed while activating effectors.     

Palmitoylation participates in G protein coupled signal transduction by affecting its oligomerization

Much in vivo and in vitro evidence has shown that the α subunits of heterotrimeric GTP-binding proteins (G proteins) exist as oligomers in their base state and disaggregate when being activated. In this article, the influence of palmitoylation modification of Gα_o on its oligomerization was explored extensively. Gα_o protein was expressed and purified from Escherichia coli strain JM109 cotransformed with pQE60(Gα_o) and pBB131(N-myristoyltransferase). Non-denaturing gel electrophoresis analysis revealed that Gα_o existed to a small extent as monomers but mostly as oligomers including dimers, trimers, tetramers and pentamers which could disaggregate completely into monomers by GTPγS stimulation. Palmitoylated Gα_o, on the other hand, only present as oligomers that were difficult to disaggregate into monomers. The effect of palmitoylation on oligomerization of Gα_o was further investigated by several other biochemical and biophysical methods including gel filtration chromatography, analytical ultracentrifugation and atomic force microscopy analysis. The results consistently demonstrated that palmitoylation facilitated oligomerization of the Gα_o protein. Autoradiography indicated that [¹⁴C]-palmitoylated Gα_o would in no case disaggregate into monomers after treatment with GTPγS. [³⁵S]-GTPγS binding activity assay showed that palmitoylated Gα_o was saturated at only 7.8 nmol/mg compared to 21.8 nmol/mg for non-palmitoylated Gα_o. Fluorescent quenching studies using BODIPY FL-GTPγS as a probe showed that the conformation of GTP-binding domain of Gα_o tended to become more compact after palmitoylation. These results implied that palmitoylation may regulate the GDP/GTP exchange of Gα_o by influencing the oligomerization state of Gα_o and thereby modulate the on-off switch of the G protein in G protein-coupled signal transduction.     

Guanosine 5′-(γ-[35S]Thio)triphosphate Autoradiography Allows Selective Detection of Histamine H3 Receptor-Dependent G Protein Activation in Rat Brain Tissue Sections

Abstract: Histamine elicits its biological effects via three distinct G protein-coupled receptors, termed H₁, H₂, and H₃. We have used guanosine 5′-(γ-[³⁵S]thio)triphosphate (GTPγ[³⁵S]) autoradiography to localize histamine receptor-dependent G protein activation in rat brain tissue sections. Initial studies revealed that in basal conditions, adenosine was present in tissue sections in sufficient concentrations to generate an adenosine A₁ receptor-dependent GTPγ[³⁵S] signal in several brain regions. All further incubations therefore contained 8-cyclopentyl-1,3-dipropylxanthine (10 µM), a selective A₁ receptor antagonist. Histamine elicited dose-dependent increments in GTPγ[³⁵S] binding to discrete anatomical structures, most notably the caudate putamen, cerebral cortex, and substantia nigra. The overall anatomical pattern of the histamine-evoked binding response closely reflects the known distribution of H₃ binding sites and was faithfully mimicked by N^α-methylhistamine, (R)-α-methylhistamine, and immepip, three H₃-selective agonists. In all regions examined, the GTPγ[³⁵S] signal was reversed with thioperamide and clobenpropit, two potent H₃-selective antagonists, whereas mepyramine, a specific H₁ antagonist, and cimetidine, a prototypic H₂ antagonist, proved ineffective. These data indicate that in rat brain tissue sections, GTPγ[³⁵S] autoradiography selectively detects H₃ receptor-dependent signaling in response to histamine stimulation. As the existing evidence suggests that GTPγ[³⁵S] autoradiography preferentially reveals responses to G_i/o-coupled receptors, our data indicate that most, if not all, central H₃ binding sites represent functional receptors coupling to G_i/o, the inhibitory class of G proteins. Besides allowing more detailed studies on H₃ receptor signaling within anatomically restricted regions of the CNS, GTPγ[³⁵S] autoradiography offers a novel approach for functional in vitro screening of H₃ ligands.     

Dopamine-induced functional activation of Gαq mediated by dopamine D1-like receptor in rat cerebral cortical membranes

Although multiple roles of dopamine through D₁-like (D₁ and D₅) and D₂-like (D₂, D₃, and D₄) receptors are initiated primarily through stimulation or inhibition of adenylyl cyclase via G_s/olf or G_i/o, respectively, there have been many reports indicating diverse signaling mechanisms that involve alternative G protein coupling. In this study, dopamine-induced Gα_q activation in rat brain membranes was investigated. Agonist-induced Gα_q activation was assessed by increase in guanosine-5′-O-(3-[³⁵S]thio)triphosphate ([³⁵S]GTPγS) binding to Gα_q determined by [³⁵S]GTPγS binding/immunoprecipitation assay in rat brain membranes. Dopamine-stimulated Gα_q functionality was highest in cortex as compared to hippocampus or striatum. In cerebral cortical membranes, this effect was mimicked by benzazepine derivatives with agonist properties at dopamine D₁-like receptors, that is, SKF83959, SKF83822, R(+)-SKF81297, R(+)-SKF38393, and SKF82958, but not by the compounds with dopamine D₂-like receptor agonist properties except for aripiprazole. Against expectation, stimulatory effects were also induced by SKF83566, R(+)-SCH23390, and pergolide. The pharmacological profiling by using a series of antagonists indicated that dopamine-induced response was mediated through dopamine D₁-like receptor, which was distinct from the receptor involved in 5-HT-induced response (5-HT_2A receptor). Conversely, the responses induced by SKF83566, R(+)-SCH23390, and pergolide were most likely mediated by 5-HT_2A receptor, but not by dopamine D₁-like receptor. Caution should be paid when interpreting the experimental data, especially in behavioral pharmacological research, in which SKF83566 or R(+)-SCH23390 is used as a standard selective dopamine D₁-like receptor antagonist. Also, possible clinical implications of the agonistic effects of pergolide on 5-HT_2A receptor has been mentioned.     

Autoradiography of Serotonin 5-HT1A Receptor-Activated G Proteins in Guinea Pig Brain Sections by Agonist-Stimulated [35S]GTPγS Binding

Abstract: G protein activation mediated by serotonin 5-HT_1A and 5-HT_1B/D receptors in guinea pig brain was investigated by using quantitative autoradiography of agonist-stimulated [³⁵S]GTPγS binding to brain sections. [³⁵S]GTPγS binding was stimulated by the mixed 5-HT_1A/5-HT_1B/D agonist L694247 in brain structures enriched in 5-HT_1A binding sites, i.e., hippocampus (+140 ± 14%), dorsal raphe (+70 ± 8%), lateral septum (+52 ± 12%), cingulate (+36 ± 8%), and entorhinal cortex (+34 ± 5%). L694247 caused little or no stimulation of [³⁵S]GTPγS binding in brain regions with high densities of 5-HT_1B/D binding sites (e.g., substantia nigra, striatum, central gray, and dorsal subiculum). The [³⁵S]GTPγS binding response was antagonized by WAY100635 (10 µM) and methiothepin (10 µM). In contrast, the 5-HT_1B inverse agonist SB224289 (10 µM) did not affect the L694247-mediated [³⁵S]GTPγS binding response, and the mixed 5-HT_1B/D antagonist GR127935 (10 µM) yielded a partial blockade. The distribution pattern of the [³⁵S]GTPγS binding response and the antagonist profile suggest the L694247-mediated response in guinea pig brain to be mediated by 5-HT_1A receptors. In addition to L694247, 8-hydroxy-2-(di-n-propylamino)tetralin, and flesinoxan also stimulated [³⁵S]GTPγS binding; their maximal responses varied between 46 and 52% compared with L694247, irrespective of the brain structure being considered. Sumatriptan, rizatriptan, and zolmitriptan (10 µM) stimulated [³⁵S]GTPγS binding in the hippocampus by 20–50%. Naratriptan, CP122638, and dihydroergotamine stimulated [³⁵S]GTPγS binding to a similar level as L694247 in hippocampus, lateral septum, and dorsal raphe. It appears that under the present experimental conditions, G protein activation through 5-HT_1A but not 5-HT_1B/D receptors can be measured in guinea pig brain sections.     

Expression of the μ-Opioid Receptor in CHO Cells: Ability of μ-Opioid Ligands to Promote α-Azidoanilido[32P]GTP Labeling of Multiple G Protein α Subunits

Abstract: The identities of heterotrimeric G proteins that can interact with the μ-opioid receptor were investigated by α-azidoanilido[³²P]GTP labeling of α subunits in the presence of opioid agonists in Chinese hamster ovary (CHO)-MORIVA3 cells, a CHO clone that stably expressed μ-opioid receptor cDNA (MOR-1). This clone expressed 1.01 × 10⁶μ-opioid receptors per cell and had higher binding affinity and potency to inhibit adenylyl cyclase for the μ-opioid-selective ligands [d -Ala²,N-MePhe⁴,Gly-ol]-enkephalin and [N-MePhe³,d -Pro⁴]-morphiceptin, relative to the δ-selective opioid agonist [d -Pen²,d -Pen⁵]-enkephalin or the κ-selective opioid agonist U-50,488H. μ-Opioid ligands induced an increase in α-azidoanilido[³²P]GTP photoaffinity labeling of four Gα subunits in this clone, three of which were identified as G_i3α, G_i2α, and G_o2α. The same pattern of simultaneous interaction of the μ-opioid receptor with multiple Gα subunits was also observed in two other clones, one expressing about three times more and the other 10-fold fewer receptors as those expressed in CHO-MORIVA3 cells. The opioid-induced increase of labeling of these G proteins was agonist specific, concentration dependent, and blocked by naloxone and by pretreatment of these cells with pertussis toxin. A greater agonist-induced increase of α-azidoanilido[³²P]GTP incorporation into G_i2α (160–280%) and G_o2α (110–220%) than for an unknown Gα (G_?α) (60%) or G_i3α (40%) was produced by three different μ-opioid ligands tested. In addition, slight differences were also found between the ability of various μ-opioid agonists to produce half-maximal labeling (ED₅₀) of any given Gα subunit, with a rank order of G_i3α > G_o2α > G_i2α = G_?α. In any case, these results suggest that the activated μ-opioid receptor couples to four distinct G protein α subunits simultaneously.     

Backbone resonance assignments for G protein αi3 subunit in the GTP-bound state

Guanine-nucleotide binding proteins (G proteins) act as molecular switches in signaling pathways, by coupling the activation of G protein-coupled receptors (GPCRs) at the cell surface to intracellular responses. In the resting state, G protein forms a heterotrimer, consisting of GDP-bound form of the G protein α subunit (Gα(GDP)) and G protein βγ subunit (Gβγ). Ligand binding to GPCRs promotes the GDP-GTP exchange on Gα, leading to the dissociation of the GTP-bound form of Gα (Gα(GTP)) and Gβγ. Then, Gα(GTP) and Gβγ bind to their downstream effector enzymes or ion channels and regulate their activities, leading to a variety of cellular responses. Finally, Gα hydrolyzes the bound GTP to GDP and returns to the resting state by re-associating with Gβγ. G proteins are classified with four major families based on the amino acid sequences of Gα: i/o, s, q/11, and 12/13. Each family transduces the signaling from different GPCRs to the specific effectors. Here, we established the backbone resonance assignments of human Gα_i3, a member of the i/o family, with a molecular weight of 41 K in complex with a GTP analogue, GTPγS.     

Gβ3 forms distinct dimers with specific Gγ subunits and preferentially activates the β3 isoform of phospholipase C

Heterotrimeric G proteins regulate multiple effectors of which some are mediated via the Gβγ dimers. There is evidence to suggest that the functions of Gβγ dimers are not shared by all possible permutations of Gβγ complexes. Here, we report our efforts in defining the formation of distinct Gβγ dimers and their functional differences in activating phospholipase Cβ (PLCβ) isoforms. Co-immunoprecipitation assays using Cos-7 cells transiently expressing 48 different combinations of Gβ_(1–4) and Gγ_{(1–5, 7–13)} subunits showed that Gβ₁ and Gβ₄ could form dimers with all known Gγ subunits, whereas several dimers could not be observed for Gβ₂ and Gβ₃. All Gβ₁γ and Gβ₂γ dimers significantly stimulated PLCβ isoforms (PLCβ₂ ≥ PLCβ₃ > PLCβ₁), but Gβ₃γ and Gβ₄γ dimers were poor activators of PLCβ₁ and exhibited preference for PLCβ₃ and PLCβ₂, respectively. All Gβ subunits revealed to date contain the previously identified PLCβ₂-interacting residues, but their neighboring residues in the proposed 3-D structures are different. To test if differences in these neighboring residues affect the interactions with PLCβ isoforms, we generated several Gβ₃ mutants by replacing one or more of these residues with their Gβ₁ counterparts. One of these mutants (M120I, S140A and A141G triple mutant) acquired enhanced PLCβ₂-activating functions when co-expressed with different Gγ subunits, while the corresponding stimulation on PLCβ₃ was not altered. Taken together, our results show that the exact composition of a Gβγ dimer can determine its selectivity for activating PLCβ isoforms, and certain residues in Gβ₃ may account for the preferential stimulation of PLCβ₃ by Gβ₃γ dimers.     

Backbone resonance assignments for G protein αi3 subunit in the GDP-bound state

Guanine-nucleotide binding proteins (G proteins) serve as molecular switches in signaling pathways, by coupling the activation of G protein-coupled receptors (GPCRs) at the cell surface to intracellular responses. In the resting state, G protein forms a heterotrimer, consisting of the G protein α subunit with GDP (Gα·GDP) and the G protein βγ subunit (Gβγ). Ligand binding to GPCRs promotes the GDP–GTP exchange on Gα, leading to the dissociation of the GTP-bound form of Gα (Gα·GTP) and Gβγ. Then, Gα·GTP and Gβγ bind to their downstream effector enzymes or ion channels and regulate their activities, leading to a variety of cellular responses. Finally, Gα hydrolyzes the bound GTP to GDP and returns to the resting state by re-associating with Gβγ. The G proteins are classified with four major families based on the amino acid sequences of Gα: i/o, s, q/11, and 12/13. Here, we established the backbone resonance assignments of human Gα_i3, a member of the i/o family with a molecular weight of 41 K, in complex with GDP. The chemical shifts were compared with those of Gα_i3 in complex with a GTP-analogue, GTPγS, which we recently reported, indicating that the residues with significant chemical shift differences are mostly consistent with the regions with the structural differences between the GDP- and GTPγS-bound states, as indicated in the crystal structures. The assignments of Gα_i3·GDP would be useful for the analyses of the dynamics of Gα_i3 and its interactions with various target molecules.