scholarly journals Characterization, Neurosteroid Binding and Brain Distribution of Human Membrane Progesterone Receptors δ and ϵ (mPRδ and mPRϵ) and mPRδ Involvement in Neurosteroid Inhibition of Apoptosis

Endocrinology ◽  
2013 ◽  
Vol 154 (1) ◽  
pp. 283-295 ◽  
Author(s):  
Yefei Pang ◽  
Jing Dong ◽  
Peter Thomas
Keyword(s):  
G Protein ◽  
G Proteins ◽  
Mammalian Cells ◽  
Plasma Membranes ◽  
Dissociation Constants ◽  
Second Messengers ◽  
Progesterone Receptors ◽  
Transfected Cells ◽  
Stimulatory G Protein ◽  
Equilibrium Dissociation Constants

Three members of the progestin and adipoQ receptor (PAQR) family, PAQR-7, PAQR-8, and PAQR-5 [membrane progesterone (P4) receptor (PR) (mPR)α, mPRβ, and mPRγ], function as plasma mPRs coupled to G proteins in mammalian cells, but the characteristics of two other members, PAQR6 and PAQR9 (mPRδ and mPRϵ), remain unclear, because they have only been investigated in yeast expression systems. Here, we show that recombinant human mPRδ and mPRϵ expressed in MDA-MB-231 breast cancer cells display specific, saturable, high-affinity [3H]-P4 binding on the plasma membranes of transfected cells with equilibrium dissociation constants (Kds) of 2.71 and 2.85 nm, respectively, and low affinity for R5020, characteristics typical of mPRs. P4 treatment increased cAMP production as well as [35S]-guanosine 5′-triphosphate (GTP)γS binding to transfected cell membranes, which was immunoprecipitated with a stimulatory G protein antibody, suggesting both mPRδ and mPRϵ activate a stimulatory G protein (Gs), unlike other mPRs, which activate an inhibitory G protein (Gi). All five mPR mRNAs were detected in different regions of the human brain, but mPRδ showed greatest expression in many regions, including the forebrain, hypothalamus, amygdala, corpus callosum, and spinal cord, whereas mPRϵ was abundant in the pituitary gland and hypothalamus. Allopregnanolone and other neurosteroids bound to mPRδ and other mPRs and acted as agonists, activating second messengers and decreased starvation-induced cell death and apoptosis in mPRδ-transfected cells and in hippocampal neuronal cells at low nanomolar concentrations. The results suggest that mPRδ and mPRϵ function as mPRs coupled to G proteins and are potential intermediaries of nonclassical antiapoptotic actions of neurosteroids in the central nervous system.

scholarly journals Exocytosis in mast cells by basic secretagogues: evidence for direct activation of GTP-binding proteins.

1990 ◽  
Vol 111 (3) ◽  
pp. 909-917 ◽  
Author(s):  
M Aridor ◽  
L M Traub ◽  
R Sagi-Eisenberg
Keyword(s):  
Mast Cells ◽  
G Protein ◽  
G Proteins ◽  
Independent Manner ◽  
Histamine Secretion ◽  
Free System ◽  
Stimulatory G Protein ◽  
A Cell ◽  
Secretion Inhibition ◽  
Gamma S

Histamine release induced by the introduction of a nonhydrolyzable analogue of GTP, GTP-gamma-S, into ATP-permeabilized mast cells, is associated with phosphoinositide breakdown, as evidenced by the production of phosphatidic acid (PA) in a neomycin-sensitive process. The dependency of both PA formation and histamine secretion on GTP-gamma-S concentrations is bell shaped. Whereas concentrations of up to 0.1 mM GTP-gamma-S stimulate both processes, at higher concentrations the cells' responsiveness is inhibited. At a concentration of 1 mM, GTP-gamma-S self-inhibits both PA formation and histamine secretion. Inhibition of secretion can, however, be overcome by the basic secretagogues compound 48/80 and mastoparan that in suboptimal doses synergize with 1 mM GTP-gamma-S to potentiate secretion. Secretion under these conditions is not accompanied by PA formation and is resistant both to depletion of Ca2+ from internal stores and to pertussis toxin (PtX) treatment. In addition, 48/80, like mastoparan, is capable of directly stimulating the GTPase activity of G-proteins in a cell-free system. Together, our results are consistent with a model in which the continuous activation of a phosphoinositide-hydrolyzing phospholipase C (PLC) by a stimulatory G-protein suffices to trigger histamine secretion. Basic secretagogues of mast cells, such as compound 48/80 and mastoparan, are capable of inducing secretion in a mechanism that bypasses PLC by directly activating a G-protein that is presumably located downstream from PLC (GE). Thereby, these secretagogues induce histamine secretion in a receptor-independent manner.

Abscisic acid induction of GTP hydrolysis in maize coleoptile plasma membranes

1998 ◽  
Vol 25 (5) ◽  
pp. 539 ◽  
Author(s):  
Helen R. Irving
Keyword(s):  
Abscisic Acid ◽  
G Protein ◽  
G Proteins ◽  
Pertussis Toxin ◽  
Plasma Membranes ◽  
Dependent Manner ◽  
Gtpase Activity ◽  
Heterotrimeric G Proteins ◽  
Gtp Hydrolysis ◽  
Maize Coleoptile

Since receptor-coupled G proteins increase GTP hydrolysis (GTPase) activity upon ligands binding to the receptor, a study was undertaken to determine if abscisic acid (ABA) induced such an effect. Plasma membranes isolated from etiolated maize (Zea mays L.) coleoptiles were enriched in GTPase activity relative to microsomal fractions. Vanadate was included in the assay to inhibit the high levels of vanadate sensitive low affinity GTPases present. Under these conditions, GTPase activity was enhanced by Mg2+, stimulated by mastoparan, and inhibited by GTPγS indicating the presence of either monomeric or heterotrimeric G proteins. The combination of NaF and AlCl3 is expected to inhibit heterotrimeric G protein activity but had little effect on GTPase activity in maize coleoptile membranes. Cholera toxin enhanced basal GTPase activity, confirming the presence of heterotrimeric G proteins in maize plasma membranes. Pertussis toxin also slightly enhanced basal GTPase activity in maize membranes. Abscisic acid enhanced GTPase activity optimally at 5 mmol/L Mg2+ in a concentration dependent manner by 1.5-fold at 10 µmol/L and up to three-fold at 100 µmol/L ABA. Abscisic acid induced GTPase activity was inhibited by GTPγS, the combination of NaF and AlCl3, and pertussis toxin. Overall, these results are typical of a receptor-coupled G protein responding to its ligand.

Specific coupling of a cation-sensing receptor to G protein alpha-subunits in MDCK cells

1997 ◽  
Vol 273 (1) ◽  
pp. F129-F135 ◽  
Author(s):  
J. M. Arthur ◽  
G. P. Collinsworth ◽  
T. W. Gettys ◽  
L. D. Quarles ◽  
J. R. Raymond
Keyword(s):  
G Protein ◽  
G Proteins ◽  
Plasma Membranes ◽  
Mdck Cells ◽  
Dependent Manner ◽  
Concentration Dependent Manner ◽  
Cellular Activation ◽  
Protein Activation ◽  
A Cell ◽  
Cation Sensing

Extracellular cations such as Ca2+ stimulate a G protein-coupled, cation-sensing receptor (CaR). We used microphysiometry to determine whether an extracellular cation-sensing mechanism exists in Madin-Darby canine kidney (MDCK) cells. The CaR agonists Ca2+ and Gd3+ caused cellular activation in a concentration-dependent manner. mRNA for the CaR was identified by reverse transcription and polymerase chain reaction (PCR) using nested CaR-specific primers, identification of an appropriately located restriction site, and sequencing of the subcloned fragment obtained by PCR. G protein activation was evaluated using the GTP photoaffinity label [alpha-32P]GTP azidoanalide (AA-GTP). After stimulation with Gd3+ and cross-linking, plasma membranes were solubilized and immunoprecipitated with antisera specific for Gq/11 alpha and Gi alpha family members. Gd3+ increased incorporation of AA-GTP into Gq/11 alpha precipitates by 146 +/- 48% and into G alpha i-2 and G alpha i-3 to a lesser extent but not into G alpha i-1. Direct effects of Gd3+ on the G proteins were ruled out using partially purified mammalian G proteins expressed in Escherichia coli or Sf9 cells. We conclude that MDCK cells possess a cell-surface CaR that activates Gq/11 alpha, G alpha i-2, and G alpha i-3 but not G alpha i-1.

Role of G proteins and KCa channels in the muscarinic and beta-adrenergic regulation of airway smooth muscle

1995 ◽  
Vol 268 (2) ◽  
pp. L221-L229 ◽  
Author(s):  
H. Kume ◽  
K. Mikawa ◽  
K. Takagi ◽  
M. I. Kotlikoff
Keyword(s):  
Smooth Muscle ◽  
Adenylyl Cyclase ◽  
G Protein ◽  
Muscarinic Receptor ◽  
G Proteins ◽  
Isometric Tension ◽  
Guanine Nucleotide Binding Protein ◽  
Kca Channels ◽  
Stimulatory G Protein ◽  
Guanine Nucleotide Binding

We have examined the functional consequences of G protein coupling to calcium-activated potassium (KCa) channels using isometric tension records from guinea pig tracheal smooth muscle. After incubation with 1 microgram/ml pertussis toxin (PTX) for 6 h, the contraction response to 1 microM methacholine (MCh) was suppressed by 31.7 +/- 5.0% (n = 10). Similarly, the contraction was inhibited by 29.1 +/- 5.0% (n = 6) after application of 0.1 microM AF-DX 116, an M2-selective muscarinic receptor antagonist. Cholera toxin (CTX, 2.0 micrograms/ml for 6 h), which activates the stimulatory G protein of adenylyl cyclase (Gs), also suppressed contraction by 43.9 +/- 3.3% (n = 11). The inhibitory effects of PTX, AF-DX 116, or CTX were reversed in the presence of 100 nM charybdotoxin (ChTX), a selective KCa channel inhibitor. These findings suggest that disruption of inhibitory coupling between muscarinic receptor and KCa channels mediated by PTX-sensitive G proteins, or KCa channel activation induced by Gs/adenylyl cyclase-linked processes, antagonizes muscarinic contraction. The isoproterenol concentration-inhibition curves for precontracted trachea (1 microM MCh) were shifted to the left after perfusion with PTX or AF-DX 116, and the leftward shift of the curve was blocked by ChTX. Thus direct or indirect regulation of KCa channels mediated by the inhibitory guanine nucleotide binding protein (Gi) and Gs may play a functionally important role in the mechanical antagonism by the two receptor agonists.

scholarly journals Probing the mutational landscape of regulators of G protein signaling proteins in cancer

2020 ◽  
Vol 13 (617) ◽  
pp. eaax8620 ◽  
Author(s):  
Vincent DiGiacomo ◽  
Marcin Maziarz ◽  
Alex Luebbers ◽  
Jillian M. Norris ◽  
Pandu Laksono ◽  
...  
Keyword(s):  
G Protein ◽  
G Proteins ◽  
Mammalian Cells ◽  
G Protein Signaling ◽  
Rgs Proteins ◽  
Protein Signaling ◽  
Loss Of Function ◽  
Gpcr Signaling ◽  
Mutational Landscape ◽  
Binding Interface

The advent of deep-sequencing techniques has revealed that mutations in G protein–coupled receptor (GPCR) signaling pathways in cancer are more prominent than was previously appreciated. An emergent theme is that cancer-associated mutations tend to cause enhanced GPCR pathway activation to favor oncogenicity. Regulators of G protein signaling (RGS) proteins are critical modulators of GPCR signaling that dampen the activity of heterotrimeric G proteins through their GTPase-accelerating protein (GAP) activity, which is conferred by a conserved domain dubbed the “RGS-box.” Here, we developed an experimental pipeline to systematically assess the mutational landscape of RGS GAPs in cancer. A pan-cancer bioinformatics analysis of the 20 RGS domains with GAP activity revealed hundreds of low-frequency mutations spread throughout the conserved RGS domain structure with a slight enrichment at positions that interface with G proteins. We empirically tested multiple mutations representing all RGS GAP subfamilies and sampling both G protein interface and noninterface positions with a scalable, yeast-based assay. Last, a subset of mutants was validated using G protein activity biosensors in mammalian cells. Our findings reveal that a sizable fraction of RGS protein mutations leads to a loss of function through various mechanisms, including disruption of the G protein–binding interface, loss of protein stability, or allosteric effects on G protein coupling. Moreover, our results also validate a scalable pipeline for the rapid characterization of cancer-associated mutations in RGS proteins.

Ion channel regulation by G proteins

1995 ◽  
Vol 75 (4) ◽  
pp. 865-885 ◽  
Author(s):  
K. Wickman ◽  
D. E. Clapham
Keyword(s):  
Ion Channels ◽  
Ion Channel ◽  
G Protein ◽  
G Proteins ◽  
Second Messengers ◽  
Channel Activity ◽  
Ion Channel Regulation ◽  
Protein Subunits ◽  
Channel Regulation ◽  
Intracellular Cascades

Ion channels are poised uniquely to initiate, mediate, or regulate such distinct cellular activities as action potential propagation, secretion, and gene transcription. In retrospect, it is not surprising that studies of ion channels have revealed considerable diversities in their primary structures, regulation, and expression. From a functional standpoint, the various mechanisms coopted by cells to regulate channel activity are particularly fascinating. Extracellular ligands, membrane potential, phosphorylation, ions themselves, and diffusible second messengers are all well-established regulators of ion channel activity. Heterotrimeric GTP-binding proteins (G proteins) mediate many of these types of ion channel regulation by stimulating or inhibiting phosphorylation pathways, initiating intracellular cascades leading to elevation of cytosolic Ca2+ or adenosine 3',5'-cyclic monophosphate levels, or by generating various lipid-derived compounds. In some cases, it seems that activated G protein subunits can interact directly with ion channels to elicit regulation. Although there is currently little direct biochemical evidence to support such a mechanism, it is the working hypothesis for the most-studied G protein-regulated ion channels.

Diacylglycerol and ceramide formation induced by dopamine D2S receptors via Gβγ-subunits in Balb/c-3T3 cells

2003 ◽  
Vol 284 (3) ◽  
pp. C640-C648 ◽  
Author(s):  
Gele Liu ◽  
Mohammad H. Ghahremani ◽  
Behzad Banihashemi ◽  
Paul R. Albert
Keyword(s):  
Cell Growth ◽  
Phospholipase C ◽  
G Protein ◽  
G Proteins ◽  
Second Messengers ◽  
Mitogen Activated Protein Kinase ◽  
Fibroblast Cells ◽  
Induced Responses ◽  
3T3 Cells ◽  
Mitogen Activated Protein

Diacylglycerol (DAG) and ceramide are important second messengers affecting cell growth, differentiation, and apoptosis. Balb/c-3T3 fibroblast cells expressing dopamine-D2S (short) receptors (Balb-D2S cells) provide a model of G protein-mediated cell growth and transformation. In Balb-D2S cells, apomorphine (EC50= 10 nM) stimulated DAG and ceramide formation by 5.6- and 4.3-fold, respectively, maximal at 1 h and persisting over 6 h. These actions were blocked by pretreatment with pertussis toxin (PTX), implicating Gi/Goproteins. To address which G proteins are involved, Balb-D2S clones expressing individual PTX-insensitive Gαiproteins were treated with PTX and tested for apomorphine-induced responses. Neither PTX-insensitive Gαi2nor Gαi3rescued D2S-induced DAG or ceramide formation. Both D2S-induced DAG and ceramide signals required Gβγ-subunits and were blocked by inhibitors of phospholipase C [1-(6-[([17β]-3-methoxyestra-1,2,3[10]-trien- 17yl)amino]hexyl)-1H-pyrrole-2,5-dione (U-73122) and partially by D609]. The similar G protein specificity of D2S-induced calcium mobilization, DAG, and ceramide formation indicates a common Gβγ-dependent phospholipase C-mediated pathway. Both D2 agonists and ceramide specifically induced mitogen-activated protein kinase (ERK1/2), suggesting that ceramide mediates a novel pathway of D2S-induced ERK1/2 activation, leading to cell growth.

Effect of GTP gamma S on insulin binding and tyrosine phosphorylation in liver membranes and L6 muscle cells

1990 ◽  
Vol 258 (1) ◽  
pp. C99-C108 ◽  
Author(s):  
E. Burdett ◽  
G. B. Mills ◽  
A. Klip
Keyword(s):  
Insulin Receptor ◽  
Tyrosine Phosphorylation ◽  
G Protein ◽  
G Proteins ◽  
Plasma Membranes ◽  
Kinase Activity ◽  
Muscle Cells ◽  
Permeabilized Cells ◽  
L6 Muscle Cells ◽  
Gamma S

Guanosine 5'-O-(3-thiotriphosphate) (GTP gamma S), a specific activator of G proteins, did not change the Kd nor total binding of [125I]insulin in plasma membranes from rat liver. Insulin did not alter GTP gamma 35S binding nor polypeptide ADP ribosylation in crude and plasma membranes catalyzed either intrinsically or by cholera toxin. In L6 muscle cells, insulin caused tyrosine phosphorylation of a polypeptide of Mr 160,000. Cell electroporation enabled testing of G protein action in this cellular system. Phosphorylation of the Mr 160,000 polypeptide in these permeabilized cells was insulin and ATP dependent but other small molecules or ionic gradients were not essential. The reaction could not be mimicked by the G protein agonist GTP gamma S nor inhibited by the G protein antagonist guanosine 5'-O-(2-thiodiphosphate) (GDP beta S). However, GTP gamma S effectively decreased insulin-mediated phosphorylation of this polypeptide. This suggests that the tyrosine kinase activity of the insulin receptor can be modulated by G protein agonists. It is concluded that cross talk between the insulin receptor and G proteins could not be demonstrated in isolated membranes by strategies that detect interactions between beta-adrenergic receptors and G proteins. In contrast, in permeabilized cells, G protein-mediated regulation of the insulin receptor kinase activity could be detected.

scholarly journals Lifetime of muscarinic receptor–G-protein complexes determines coupling efficiency and G-protein subtype selectivity

2018 ◽  
Vol 115 (19) ◽  
pp. 5016-5021 ◽  
Author(s):  
Olga S. Ilyaskina ◽  
Horst Lemoine ◽  
Moritz Bünemann
Keyword(s):  
G Protein ◽  
G Proteins ◽  
Mammalian Cells ◽  
Protein Complexes ◽  
Coupling Efficiency ◽  
Dissociation Kinetics ◽  
Intact Cells ◽  
Permeabilized Cells ◽  
Subtype Selectivity ◽  
Intracellular Signals

G-protein–coupled receptors (GPCRs) are essential for the detection of extracellular stimuli by cells and transfer the encoded information via the activation of functionally distinct subsets of heterotrimeric G proteins into intracellular signals. Despite enormous achievements toward understanding GPCR structures, major aspects of the GPCR–G-protein selectivity mechanism remain unresolved. As this can be attributed to the lack of suitable and broadly applicable assays, we set out to develop a quantitative FRET-based assay to study kinetics and affinities of G protein binding to activated GPCRs in membranes of permeabilized cells in the absence of nucleotides. We measured the association and dissociation kinetics of agonist-induced binding of Gi/o, Gq/11, Gs, and G12/13 proteins to muscarinic M1, M2, and M3 receptors in the absence of nucleotides between fluorescently labeled G proteins and receptors expressed in mammalian cells. Our results show a strong quantitative correlation between not the on-rates of G-protein–M3–R interactions but rather the affinities of Gq and Go proteins to M3–Rs, their GPCR–G-protein lifetime and their coupling efficiencies determined in intact cells, suggesting that the G-protein subtype-specific affinity to the activated receptor in the absence of nucleotides is, in fact, a major determinant of the coupling efficiency. Our broadly applicable FRET-based assay represents a fast and reliable method to quantify the intrinsic affinity and relative coupling selectivity of GPCRs toward all G-protein subtypes.

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