Gbetagamma Research Articles

Larixol is not an inhibitor of Gαi containing G proteins and lacks effect on signaling mediated by human neutrophil expressed formyl peptide receptors

Neutrophils express several G protein-coupled receptors (GPCRs) connected to intracellular Gαi or Gαq containing G proteins for down-stream signaling. To dampen GPCR mediated inflammatory processes, several inhibitors targeting the receptors and/or their down-stream signals, have been developed. Potent and selective inhibitors for Gαq containing G proteins are available, but potent and specific inhibitors of Gαi containing G proteins are lacking. Recently, Larixol, a compound extracted from the root of Euphorbia formosana, was shown to abolish human neutrophil functions induced by N-formyl-methionyl-leucyl-phenylalanine (fMLF), an agonist recognized by formyl peptide receptor 1 (FPR1) which couple to Gαi containing G proteins. The inhibitory effect was suggested to be due to interference with/inhibition of signals transmitted by βγ complexes of the Gαi containing G proteins coupled to FPR1. In this study, we applied Larixol, obtained from two different commercial sources, to determine the receptor- and G protein- selectivity of this compound in human neutrophils. However, our data show that Larixol not only lacks inhibitory effect on neutrophil responses mediated through FPR1, but also on responses mediated through FPR2, a Gαi coupled GPCR closely related to FPR1. Furthermore, Larixol did not display any features as a selective inhibitor of neutrophil responses mediated through the Gαq coupled GPCRs for platelet activating factor and ATP. Hence, our results imply that the inhibitory effects described for the root extract of Euphorbia formosana are not mediated by Larixol and that the search for a selective inhibitor of G protein dependent signals generated by Gαi coupled neutrophil GPCRs must continue.

Larixol is not an inhibitor of Gαi containing G proteins and lacks effect on signaling mediated by human neutrophil expressed formyl peptide receptors

Neutrophils express several G protein-coupled receptors (GPCRs) connected to intracellular Gαi or Gαq containing G proteins for down-stream signaling. To dampen GPCR mediated inflammatory processes, several inhibitors targeting the receptors and/or their down-stream signals, have been developed. Potent and selective inhibitors for Gαq containing G proteins are available, but potent and specific inhibitors of Gαi containing G proteins are lacking. Recently, Larixol, a compound extracted from the root of Euphorbia formosana, was shown to abolish human neutrophil functions induced by N-formyl-methionyl-leucyl-phenylalanine (fMLF), an agonist recognized by formyl peptide receptor 1 (FPR1) which couple to Gαi containing G proteins. The inhibitory effect was suggested to be due to interference with/inhibition of signals transmitted by βγ complexes of the Gαi containing G proteins coupled to FPR1. In this study, we applied Larixol, obtained from two different commercial sources, to determine the receptor- and G protein- selectivity of this compound in human neutrophils. However, our data show that Larixol not only lacks inhibitory effect on neutrophil responses mediated through FPR1, but also on responses mediated through FPR2, a Gαi coupled GPCR closely related to FPR1. Furthermore, Larixol did not display any features as a selective inhibitor of neutrophil responses mediated through the Gαq coupled GPCRs for platelet activating factor and ATP. Hence, our results imply that the inhibitory effects described for the root extract of Euphorbia formosana are not mediated by Larixol and that the search for a selective inhibitor of G protein dependent signals generated by Gαi coupled neutrophil GPCRs must continue.

Presynaptic mechanisms underlying GABAB-receptor-mediated inhibition of spontaneous neurotransmitter release.

SUMMARYInhibition of neurotransmitter release by neurotransmitter substances constitutes a fundamental means of neuromodulation. In contrast to well-delineated mechanisms that underlie inhibition of evoked release via suppression of voltage-gated Ca2+ channels, processes that underlie neuromodulatory inhibition of spontaneous release remain unclear. Here, we interrogated inhibition of spontaneous glutamate and GABA release by presynaptic metabotropic GABAB receptors. Our findings show that this inhibition relies on Gβγ subunit action at the membrane, and it is largely independent of presynaptic Ca2+ signaling for both forms of release. In the case of spontaneous glutamate release, inhibition requires Gβγ interaction with the C terminus of the key fusion machinery component SNAP25, and it is modulated by synaptotagmin-1. Inhibition of spontaneous GABA release, on the other hand, is independent of these pathways and likely requires alternative Gβγ targets at the presynaptic terminal.

Computational Study of C-X-C Chemokine Receptor (CXCR)3 Binding with Its Natural Agonists Chemokine (C-X-C Motif) Ligand (CXCL)9, 10 and 11 and with Synthetic Antagonists: Insights of Receptor Activation towards Drug Design for Vitiligo.

Vitiligo is a hypopigmentary skin pathology resulting from the death of melanocytes due to the activity of CD8+ cytotoxic lymphocytes and overexpression of chemokines. These include CXCL9, CXCL10, and CXCL11 and its receptor CXCR3, both in peripheral cells of the immune system and in the skin of patients diagnosed with vitiligo. The three-dimensional structure of CXCR3 and CXCL9 has not been reported experimentally; thus, homology modeling and molecular dynamics could be useful for the study of this chemotaxis-promoter axis. In this work, a homology model of CXCR3 and CXCL9 and the structure of the CXCR3/Gαi/0βγ complex with post-translational modifications of CXCR3 are reported for the study of the interaction of chemokines with CXCR3 through all-atom (AA-MD) and coarse-grained molecular dynamics (CG-MD) simulations. AA-MD and CG-MD simulations showed the first activation step of the CXCR3 receptor with all chemokines and the second activation step in the CXCR3-CXCL10 complex through a decrease in the distance between the chemokine and the transmembrane region of CXCR3 and the separation of the βγ complex from the α subunit in the G-protein. Additionally, a general protein–ligand interaction model was calculated, based on known antagonists binding to CXCR3. These results contribute to understanding the activation mechanism of CXCR3 and the design of new molecules that inhibit chemokine binding or antagonize the receptor, provoking a decrease of chemotaxis caused by the CXCR3/chemokines axis.

G protein βγ translocation to the Golgi activates the mitogen‐activated protein kinases via phosphoinositide 3‐kinase γ

The activation of G protein‐coupled receptors (GPCRs) induces the translocation of G protein βγ complex from the plasma membrane (PM). However, its functions are not well known. Here we demonstrated that among the entire twelve Gγ family members, Gγ9 is the most translocatable Gγ subunit. More than 80% of Gγ9, in complex with Gβ1, relocated from the PM to the Golgi in response to CXCR4 activation by stromal cell‐derived factor 1α (SDF1α) and the half time of the translocation was about 5 seconds. To study the function of Gβγ translocation to the Golgi, we focused on the activation of the mitogen‐activated protein kinases (MAPK) ERK1/2. CRISPR‐Cas9‐mediated knockout of Gγ9 abolished ERK1/2 activation by SDF1α, whereas knockout of the least‐translocatable Gγ3 had no effect. To investigate if Gβγ‐mediated ERK1/2 activation indeed occurred on the Golgi, we employed an inducible system to specifically express Gβγ onto the Golgi. Interestingly, the targeted expression of Gγ2, Gγ3 and Gγ9, in complex with Gβ1, onto the Golgi each induced remarkable ERK1/2 activation. Such constitutive ERK1/2 activation was blocked by the expression of Gα subunits, the targeted expression of the Gβγ blocker GRK2ct at the Golgi, and chemical treatments to disrupt the Golgi structure. By contrast, the expression of individual Gγ subunits at the PM did not activate ERK1/2. To define the downstream effectors of Gβγ, we determined the effect of selective pharmacological inhibition of phosphoinositide 3‐kinase (PI3K), phospholipase C, PKC and PKD on ERK1/2 activation. This strategy identified PI3Kγ and PI3Kδ to be essential for Gβγ‐mediated ERK1/2 activation. Indeed, knockout of PI3Kγ by CRISPR‐Cas9 almost abolished ERK1/2 activation induced by both SDF1α and Gγ9 targeting to the Golgi. Similar to CXCR4, α2‐adrenergic receptor‐mediated ERK1/2 activation was also dramatically inhibited by Gγ9 and PI3Kγ knockout. We further showed that knockout of Gγ9 and PI3Kγ markedly suppressed the migration and invasion of prostate cancer PC3 cells in response to SDF1α stimulation in vitro. Gγ9 and PI3Kγ knockout also significantly reduced the bone metastasis of PC3 tumors following intra‐cardiac injection of PC3‐Luc cells in athymic nude mice. Overall, our data demonstrate a novel function for Gβγ translocation to the Golgi to spatiotemporally control the activation of the MAPK pathway by GPCRs and tumor progression.Support or Funding InformationNIH R01 GM118915

Characterization of G protein β subunit expression of human brain Glioma tumor.

Proteome indicates the protein content of a genome. Proteome analysis is effective in a new system formulation and prediction, prevention, and treatment based on protein. One of the purposes of proteomics researches is to know and understand the cancer mechanism. In this study, we separated the proteins by the Two-Dimensional Electrophorese method and analyzed and compared protein spots by statistical and software data. The spots were separated and identified by the proteins' Isoelectric PH differences, molecular weights, and data bank. In continuation, the protein profiles were clustered by MALDI-TOF-TOF and the main element was identified and confirmed. We have used site PhosphoSitePlus® to review post-translational modifications. The findings indicated that the G protein Beta subunit rate increased in the astrocytoma, oligodendroglia, and glioblastoma cerebral malignant tumors. The βγ complex formation may prevent and activates many paths of cellular growth. The βγ complex activity control of the changes after the conversion parallel to GTPase activity of this α subunit may be a formulation mechanism for the G signal path (Tab. 5, Fig. 4, Diagram 2, Ref. 29). Keywords: glioma, G protein β subunit, proteomics.

CAGE-seq analysis of Epstein-Barr virus lytic gene transcription: 3 kinetic classes from 2 mechanisms.

Epstein-Barr virus (EBV) lytic replication proceeds through an ordered cascade of gene expression that integrates lytic DNA amplification and late gene transcription. We and others previously demonstrated that 6 EBV proteins that have orthologs in β- and γ-, but not in α-herpesviruses, mediate late gene transcription in a lytic DNA replication-dependent manner. We proposed a model in which the βγ gene-encoded viral pre-initiation complex (vPIC) mediates transcription from newly replicated viral DNA. While this model explains the dependence of late gene transcription on lytic DNA replication, it does not account for this dependence in α-herpesviruses nor for recent reports that some EBV late genes are transcribed independently of vPIC. To rigorously define which transcription start sites (TSS) are dependent on viral lytic DNA replication or the βγ complex, we performed Cap Analysis of Gene Expression (CAGE)-seq on cells infected with wildtype EBV or EBV mutants defective for DNA replication, βγ function, or lacking an origin of lytic replication (OriLyt). This approach identified 16 true-late, 32 early, and 16 TSS that are active at low levels early and are further upregulated in a DNA replication-dependent manner (leaky late). Almost all late gene transcription is vPIC-dependent, with BCRF1 (vIL10), BDLF2, and BDLF3 transcripts being notable exceptions. We present evidence that leaky late transcription is not due to a distinct mechanism, but results from superimposition of the early and late transcription mechanisms at the same promoter. Our results represent the most comprehensive characterization of EBV lytic gene expression kinetics reported to date and suggest that most, but not all EBV late genes are vPIC-dependent.

Identifying a Phospholipase C Epsilon Region that Interacts with Gbeta Gamma

Many hormones and neurotransmitters function by activating isozymes of Phospholipase C (PLC) family through G protein coupled receptors (GPCRs). PLCs hydrolyze membrane phospholipids to generate downstream signaling molecules essential for normal function of the cell. Chronic PLC activation has been implicated in diseases that affect the body. The PLCɛ isoform has been shown by our laboratory to be scaffolded at the nuclear envelope yet responds to plasma membrane (PM) endothelin 1 (ET‐1) receptor stimulus. ET‐1 stimulates phosphatidyl inositol 4‐phosphate (PI4P) hydrolysis in the perinuclear Golgi apparatus generating diacyglycerol and activating a downstream nuclear pool of PKD in neonatal cardiac ventricular myocytes (NRVM). This signaling process leads to reactivation of the fetal gene program and pathologic myocyte hypertrophy (Zhang et al. Cell, 2013).Small GTPases and Heterotrimeric G protein activate PLCɛ. We have shown that the PM endothelin‐1 receptor stimulation of Golgi PI4P depletion is dependent on Gβγ dimer (Malik et al. Mol. Biol. Cell, 2015). Other groups have shown that PLCɛ co‐expressed with Gβγ and a select group of constitutively active small GTPases produce marked increases in inositol phosphate accumulation in COS7 cells. Domains of PLCɛ that interacts with small GTPases for its activation have previously been defined. However, it is not known whether PLCɛ interacts with Gβγ directly for its activation, or which region is required. Our laboratory has previously shown that Gβγ directly interacts with PLCβ2 in vitro in a region within its catalytic domain. We hypothesize that a region within the catalytic domain of PLCɛ interacts with Gβγ directly and propose to identify this region.We reexamined PLCβ2 and PLCɛ activation by Gβγ, Gq and small GTPases (hRasG12V) in COS7 cells co‐transfection assay and observed marked increases in inositol phosphate accumulation. We then identified a region of PLCɛ that aligns with the catalytic domain of PLCβ2. We are currently making mutations within the catalytic domain and other regions of PLCɛ which will be tested for Gβγ activation using the COS7 cell co‐transfection assay. We will also determine if purified Gβγ can interact with and activate PLCɛ in vitro.

Correction: Interaction of G-Protein βγ Complex with Chromatin Modulates GPCR-Dependent Gene Regulation.

[This corrects the article DOI: 10.1371/journal.pone.0052689.].

Inhibition of G-protein βγ signaling enhances T cell receptor-stimulated interleukin 2 transcription in CD4+ T helper cells.

G-protein-coupled receptor (GPCR) signaling modulates the expression of cytokines that are drug targets for immune disorders. However, although GPCRs are common targets for other diseases, there are few GPCR-based pharmaceuticals for inflammation. The purpose of this study was to determine whether targeting G-protein βγ (Gβγ) complexes could provide a useful new approach for modulating interleukin 2 (IL-2) levels in CD4+ T helper cells. Gallein, a small molecule inhibitor of Gβγ, increased levels of T cell receptor (TCR)-stimulated IL-2 mRNA in primary human naïve and memory CD4+ T helper cells and in Jurkat human CD4+ leukemia T cells. Gβ1 and Gβ2 mRNA accounted for >99% of Gβ mRNA, and small interfering RNA (siRNA)-mediated silencing of Gβ1 but not Gβ2 enhanced TCR-stimulated IL-2 mRNA increases. Blocking Gβγ enhanced TCR-stimulated increases in IL-2 transcription without affecting IL-2 mRNA stability. Blocking Gβγ also enhanced TCR-stimulated increases in nuclear localization of nuclear factor of activated T cells 1 (NFAT1), NFAT transcriptional activity, and levels of intracellular Ca2+. Potentiation of IL-2 transcription required continuous Gβγ inhibition during at least two days of TCR stimulation, suggesting that induction or repression of additional signaling proteins during T cell activation and differentiation might be involved. The potentiation of TCR-stimulated IL-2 transcription that results from blocking Gβγ in CD4+ T helper cells could have applications for autoimmune diseases.

Role of RKTG, a novel Golgi localized receptor, in Gβγ regulated transport of secretory proteins from the Golgi to the plasma membrane (604.4)

Recent studies have revealed a role for G protein betagamma signaling at the Golgi via protein kinase D (PKD) regulating fission of plasma membrane (PM) destined secretory vesicles from the trans Golgi network (TGN). The mechanism by which the Gbetagamma subunits are recruited to and activated at the Golgi in this process remains elusive. Recent studies reveal that a newly characterized Golgi localized, membrane protein belonging to the Progestin and AdipoQ Receptor (PAQR) family, RKTG (Raf Kinase Trapping to the Golgi), interacts with the Gbeta subunit and sequesters Gbetagamma to the Golgi thereby regulating the functions of Gbetagamma. Here we show that over expression of RKTG causes vesiculation of the Golgi, while a Gbeta binding mutant of RKTG does not cause vesiculation. Also, the C‐terminal fragment of GRK2 (GRK2ct), which interacts with, and inhibits Gbetagamma signaling, and Gallein, a small molecule inhibitor of Gbetagamma, were both able to inhibit Golgi vesiculation in the presence of RKTG. Furthermore, a dominant negative form of PKD (PKD‐DN) and a pharmacological inhibitor of PKD, Go6976, also inhibited RKTG mediated vesiculation of the Golgi. Collectively, these results reveal a novel role for the newly characterized, Golgi localized RKTG receptor, in regulating Gbetagamma at the Golgi, thus controlling Golgi to PM protein transport via the Gbetagamma‐PKD signaling pathway.Grant Funding Source: NIH GM056444

Biological roles of cAMP: variations on a theme in the different kingdoms of life

Cyclic AMP (cAMP) plays a key regulatory role in most types of cells; however, the pathways controlled by cAMP may present important differences between organisms and between tissues within a specific organism. Changes in cAMP levels are caused by multiple triggers, most affecting adenylyl cyclases, the enzymes that synthesize cAMP. Adenylyl cyclases form a large and diverse family including soluble forms and others with one or more transmembrane domains. Regulatory mechanisms for the soluble adenylyl cyclases involve either interaction with diverse proteins, as happens in Escherichia coli or yeasts, or with calcium or bicarbonate ions, as occurs in mammalian cells. The transmembrane cyclases can be regulated by a variety of proteins, among which the α subunit and the βγ complex from G proteins coupled to membrane receptors are prominent. cAMP levels also are controlled by the activity of phosphodiesterases, enzymes that hydrolyze cAMP. Phosphodiesterases can be regulated by cAMP, cGMP or calcium-calmodulin or by phosphorylation by different protein kinases. Regulation through cAMP depends on its binding to diverse proteins, its proximal targets, this in turn causing changes in a variety of distal targets. Specifically, binding of cAMP to regulatory subunits of cAMP-dependent protein kinases (PKAs) affects the activity of substrates of PKA, binding to exchange proteins directly activated by cAMP (Epac) regulates small GTPases, binding to transcription factors such as the cAMP receptor protein (CRP) or the virulence factor regulator (Vfr) modifies the rate of transcription of certain genes, while cAMP binding to ion channels modulates their activity directly. Further studies on cAMP signalling will have important implications, not only for advancing fundamental knowledge but also for identifying targets for the development of new therapeutic agents.

Interaction of G-Protein βγ Complex with Chromatin Modulates GPCR-Dependent Gene Regulation

Heterotrimeric G-protein signal transduction initiated by G-protein-coupled receptors (GPCRs) in the plasma membrane is thought to propagate through protein-protein interactions of subunits, Gα and Gβγ in the cytosol. In this study, we show novel nuclear functions of Gβγ through demonstrating interaction of Gβ2 with integral components of chromatin and effects of Gβ2 depletion on global gene expression. Agonist activation of several GPCRs including the angiotensin II type 1 receptor specifically augmented Gβ2 levels in the nucleus and Gβ2 interacted with specific nucleosome core histones and transcriptional modulators. Depletion of Gβ2 repressed the basal and angiotensin II-dependent transcriptional activities of myocyte enhancer factor 2. Gβ2 interacted with a sequence motif that was present in several transcription factors, whose genome-wide binding accounted for the Gβ2-dependent regulation of approximately 2% genes. These findings suggest a wide-ranging mechanism by which direct interaction of Gβγ with specific chromatin bound transcription factors regulates functional gene networks in response to GPCR activation in cells.

All G protein βγ complexes are capable of translocation on receptor activation

Heterotrimeric G proteins transduce signals sensed by transmembrane G protein coupled receptors (GPCRs). A subfamily of G protein βγ subunit types has been shown to selectively translocate from the plasma membrane to internal membranes on receptor activation. Using 4D imaging we show here that Gβγ translocation is not restricted to some subunit types but rather all 12 members of the family of mammalian γ subunits are capable of supporting βγ translocation. Translocation kinetics varies widely depending on the specific γ subunit type, with t1/2 ranging from 10s to many minutes. Using fluorescence complementation, we show that the β and γ subunits translocate as βγ dimers with kinetics determined by the γ subunit type. The expression patterns of endogenous γ subunit types in HeLa cells, hippocampal neurons and cardiomyocytes are distinctly different. Consistent with these differences, the βγ translocation rates vary widely. βγ translocation rates exhibit the same γ subunit dependent trends regardless of the specific receptor type or cell type showing that the translocation rates are intrinsic to the γ subunit types. βγ complexes with widely different rates of translocation had differential effects on muscarinic stimulation of GIRK channel activity. These results show that G protein βγ translocation is a general response to activation of GPCRs and may play a role in regulating signaling activity.

Hypercholesterolemia Induces Upregulation of KACh Cardiac Currents

Control of the heart rate is a complex process that integrates the function of multiple proteins. Among them, G protein regulated inwardly rectifying K+ (GIRK or Kir3) channels play a major role. Kir3 channels are important in regulating membrane excitability in cardiac, neuronal and endocrine cells. In the heart, the atrial KACh channels are heterotetrameric proteins that consist of two pore-forming subunits, Kir3.1 (GIRK1) and Kir3.4 (GIRK4). Activation of KACh by acetylcholine (ACh) via the muscarinic M2 receptor and pertussis toxin sensitive G proteins mediates the vagal negative chronotropic effect. Thus, activation of KACh channels can terminate paroxysmal supraventricular tachycardia, i.e., a rapid cardiac rhythm. On the other hand, vagal stimulation predisposes to atrial fibrillation, which can lead to thromboembolism and stroke. Thus, since hypercholesterolemia is a well-known risk factor for cardiovascular disease, we investigated the role of cholesterol on KACh currents (IK,ACh). We found that in rabbit atrial cardiomyocytes, cholesterol enrichment enhanced channel activity whereas cholesterol depletion suppressed IK,ACh. Moreover, a 20-24 week high-cholesterol diet resulted in up to 3-fold increase in IK,ACh in rodents. In accordance, elevated currents were observed in Xenopus oocytes expressing the Kir3.1/Kir3.4 heteromer. Interestingly, whereas similarly to the heteromer Kir3.1/Kir3.4, the homomeric pore mutant Kir3.4∗ (Kir3.4S143T) was also enhanced by cholesterol, Kir3.1∗ was suppressed by cholesterol. Furthermore, our data suggest that cholesterol affects IK,ACh via a mechanism which is independent of both PI(4,5)P2 and Gbeta gamma. Notably, the impact of cholesterol on IK,ACh is opposite to its impact on IK1 in atrial myocytes. The latter are suppressed by cholesterol enrichment and by high-cholesterol diet, and facilitated by cholesterol depletion. These findings indicate that cholesterol plays a critical role in modulating IK,ACh in atrial cardiomyocytes via a mechanism independent of the channel's major modulators.

Hβ2R–Gαs complex: prediction versus crystal structure—how valuable are predictions based on molecular modeling studies?

In 2010, we predicted two models for the hβ(2)R-Gα(s) complex by combining the technique of homology modeling with a potential energy surface scan, since a complete crystal structure of the hβ(2)R-Gα(s) complex was not available. The crystal structure of opsin co-crystallized with part of the C-terminus of Gα (3DQB) was used as a template to model the hβ(2)R, whereas the crystal structure of Gα (1AZT) was used as a template to model Gα(s). Utilizing a potential energy surface scan between hβ(2)R and Gα(s), a six-dimensional potential energy surface was obtained. Two significant minimum regions were located on this surface, and each was associated with a distinct hβ(2)R-Gα(s) complex, namely model I and model II [Straßer A, Wittmann H-J (2010) J Mol Model 16:1307-1318]. The crystal structure of the hβ(2)R-Gα(s)βγ complex has recently been published. Thus, the aim of the current study was, on the one hand, to compare our predicted structures with the true crystal structure, and on the other to discuss the question: how valuable are predictions based on molecular modeling studies?

Do Caveolae Have a Role in the Fidelity and Dynamics of Receptor Activation of G-protein-gated Inwardly Rectifying Potassium Channels?

In atrial and nodal cardiac myocytes, M2 muscarinic receptors activate inhibitory G-proteins (Gi/o), which in turn stimulate G-protein-gated inwardly rectifying K+ channels through direct binding of the Gβγ subunit. Despite also releasing Gβγ, Gs-coupled receptors such as the β-adrenergic receptor are not able to prominently activate this current. An appealing hypothesis would be if components were sequestered in membrane domains such as caveolae/rafts. Using biochemical fractionation followed by Western blotting and/or radioligand binding experiments, we examined the distribution of the components in stable HEK293 and HL-1 cells, which natively express the transduction cascade. The channel, M2 muscarinic, and A1 adenosine receptors were located in noncaveolar/nonraft fractions. Giα1/2 was enriched in both caveolar/raft and noncaveolar/nonraft fractions. In contrast, Gsα was only enriched in caveolar/raft fractions. We constructed YFP-tagged caveolin-2 (YFP-Cav2) and chimeras with the M2 (M2-YFP-Cav2) and A1 (A1-YFP-Cav2) receptors. Analysis of gradient fractions showed that these receptor chimeras were now localized to caveolae-enriched fractions. Microscopy showed that M2-YFP and A1-YFP had a diffuse homogenous membrane signal. YFP-Cav2, M2-YFP-Cav2, and A1-YFP-Cav2 revealed a more punctuate pattern. Finally, we looked at the consequences for signaling. Activation via M2-YFP-Cav2 or A1-YFP-Cav2 revealed substantially slower kinetics compared with M2-YFP or A1-YFP and was reversed by the addition of methyl-β-cyclodextrin. Thus the localization of the channel signal transduction cascade in non-cholesterol rich domains substantially enhances the speed of signaling. The presence of Gsα solely in caveolae may account for signaling selectivity between Gi/o and Gs-coupled receptors.

In vivo expression of G‐protein β1γ2 dimer in adult mouse skeletal muscle alters L‐type calcium current and excitation–contraction coupling

A number of G-protein-coupled receptors are expressed in skeletal muscle but their roles in muscle physiology and downstream effector systems remain poorly investigated. Here we explored the functional importance of the G-protein betagamma (Gbetagamma) signalling pathway on voltage-controlled Ca(2+) homeostasis in single isolated adult skeletal muscle fibres. A GFP-tagged Gbeta(1)gamma(2) dimer was expressed in vivo in mice muscle fibres. The GFP fluorescence pattern was consistent with a Gbeta(1)gamma(2) dimer localization in the transverse-tubule membrane. Membrane current and indo-1 fluorescence measurements performed under voltage-clamp conditions reveal a drastic reduction of both L-type Ca(2+) current density and of peak amplitude of the voltage-activated Ca(2+) transient in Gbeta(1)gamma(2)-expressing fibres. These effects were not observed upon expression of Gbeta(2)gamma(2), Gbeta(3)gamma(2) or Gbeta(4)gamma(2). Our data suggest that the G-protein beta(1)gamma(2) dimer may play an important regulatory role in skeletal muscle excitation-contraction coupling.

Establishment of recombinant cannabinoid receptor assays and characterization of several natural and synthetic ligands

Cannabinoid receptors (CBR) are important drug targets for the treatment of various inflammatory, metabolic and neurological diseases. Therefore, sensitive test systems for the assessment of ligands are needed. In this study, a steady-state GTPase assay for human CBR subtypes 1 and 2 was developed to characterize the pharmacological property of ligands at a very proximal point of the signal transduction cascade. Establishing these in vitro test sytems, we studied cell or tissue membranes heterogenously or endogenously expressing CBR, such as CBR-infected Human Embryonic Kidney (HEK) 293 cells, rat cerebellum and spleen cells. The lack of effects in the GTPase assay and in [(35)S]GTPgammaS binding experiments in these expression system, directed us to use Spodoptera frugiperda (Sf9) cells. Co-expressing CBR, different Galpha-subunits, Gbetagamma heterodimer, and RGS (Regulator of G-protein signaling)-proteins in Sf9 cell membranes greatly improved the sensitivity of the assay, with highest GTPase activation in the CBR + Galpha(i2) + Gbeta(1)gamma(2) + RGS4 system. We examined exogenous and endogenous standard ligands as well as secondary metabolites as Delta(9)-tetrahydrocannabinol (Delta(9)-THC), dodeca-2E,4E-dienoic acid isobutylamide, an alkylamide from Echinacea purpurea, and an E. purpurea hexane extract according their agonistic and antagonistic properties. The suitability of the assay for screening procedures was also proven by detecting the activity of Delta(9)-THC in a matrix of other less active compounds (Delta(9)-THC-free Cannabis sativa extract). In conclusion, we have developed highly sensitive test systems for the analysis of CBR ligands.

Synergistic Activation of Phospholipase C-β3 by Gαq and Gβγ Describes a Simple Two-State Coincidence Detector

Receptors that couple to G(i) and G(q) often interact synergistically in cells to elicit cytosolic Ca(2+) transients that are several-fold higher than the sum of those driven by each receptor alone. Such synergism is commonly assumed to be complex, requiring regulatory interaction between components, multiple pathways, or multiple states of the target protein. We show that cellular G(i)-G(q) synergism derives from direct supra-additive stimulation of phospholipase C-beta3 (PLC-beta3) by G protein subunits Gbetagamma and Galpha(q), the relevant components of the G(i) and G(q) signaling pathways. No additional pathway or proteins are required. Synergism is quantitatively explained by the classical and simple two-state (inactive<-->active) allosteric mechanism. We show generally that synergistic activation of a two-state enzyme reflects enhanced conversion to the active state when both ligands are bound, not merely the enhancement of ligand affinity predicted by positive cooperativity. The two-state mechanism also explains why synergism is unique to PLC-beta3 among the four PLC-beta isoforms and, in general, why one enzyme may respond synergistically to two activators while another does not. Expression of synergism demands that an enzyme display low basal activity in the absence of ligand and becomes significant only when basal activity is </= 0.1% of maximal. Synergism can be explained by a simple and general mechanism, and such a mechanism sets parameters for its occurrence. Any two-state enzyme is predicted to respond synergistically to multiple activating ligands if, but only if, its basal activity is strongly suppressed.