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.

Phospholipase D in rat myometrium: occurrence of a membrane-bound ARF6 (ADP-ribosylation factor 6)-regulated activity controlled by βγ subunits of heterotrimeric G-proteins

2000 ◽  
Vol 352 (2) ◽  
pp. 491-499 ◽  
Author(s):  
Hervé LE STUNFF ◽  
Lien DOKHAC ◽  
Sylvain BOURGOIN ◽  
Marie-France BADER ◽  
Simone HARBON
Keyword(s):  
G Protein ◽  
G Proteins ◽  
Ammonium Sulphate ◽  
Phospholipase D ◽  
Tyrosine Kinases ◽  
Glutathione S Transferase ◽  
Free System ◽  
Adp Ribosylation ◽  
A Cell ◽  
Membrane Bound

Both protein kinase C and protein tyrosine kinases have been shown to be involved in phospholipase D (PLD) activation in intact rat myometrium [Le Stunff, Dokhac and Harbon (2000) J. Pharmacol. Exp. Ther. 292, 629–637]. In this study we assessed the involvement of monomeric G-proteins in PLD activation in a cell-free system derived from myometrial tissue. Both the PLD1 and PLD2 isoforms were detected. Two forms of PLD activity, essentially membrane-bound, were found in myometrial preparations. One form was stimulated by oleate and insensitive to guanosine 5ƀ-[γ-thio] triphosphate (GTP[S]). The second required ammonium sulphate to be detected and was stimulated by GTP[S]. ADP-ribosylation factors (ARF1 and ARF6) and RhoA were immunodetected in myometrial preparations. ARF1 and RhoA were present in the membrane and cytosolic fractions whereas ARF6 was detected exclusively in the membrane fraction. A synthetic myristoylated peptide corresponding to the N-terminal domain of ARF6 [myrARF6(2–13)] totally abolished PLD activation in the presence of ammonium sulphate and GTP[S], whereas myrARF1(2–17) and the inhibitory GDP/GTP-exchange factor, Rho GDI, did not. These data are consistent with a membrane-bound ARF6-regulated PLD activity. Finally, the stimulation of PLD by ARF6 was inhibited by AlF-4 and this inhibition was counteracted by the fusion protein glutathione S-transferase-β-adrenergic receptor kinase 1 (495–689) and by the QEHA peptide (from adenylate cyclase ACII), which act as G-protein βγ-subunit scavengers. It is concluded that G-protein subunits βγ are involved in a pathway modulating PLD activation by ARF6, illustrating cross-talk between heterotrimeric and monomeric G-proteins.

scholarly journals Neomycin is a potent secretagogue of mast cells that directly activates a GTP-binding protein involved in exocytosis.

1990 ◽  
Vol 111 (6) ◽  
pp. 2885-2891 ◽  
Author(s):  
M Aridor ◽  
R Sagi-Eisenberg
Keyword(s):  
Mast Cells ◽  
Phosphatidic Acid ◽  
Rat Brain ◽  
G Protein ◽  
Gtp Binding Protein ◽  
Histamine Secretion ◽  
Gtp Binding ◽  
Rat Brain Membranes ◽  
Gamma S ◽  
Dose Dependent

When loaded alongside GTP-gamma-S into ATP-permeabilized cells, neomycin, at concentrations below 1 mM, inhibits GTP-gamma-S-induced histamine secretion and phosphatidic acid formation (Cockcroft, S., and B. D. Gomperts, 1985. Nature (Lond.). 314: 534-536; Aridor, M., L. M. Traub, and R. Sagi-Eisenberg. 1990. J. Cell Biol. 111:909-917). However, at higher concentrations internally applied neomycin induces histamine secretion in a process that is: (a) dose dependent; (b) dependent on the internal application of GTP; (c) independent of phosphoinositide breakdown; and (d) inhibited by pertussis toxin (PtX) treatment. These results indicate that neomycin can stimulate histamine secretion in a mechanism that bypasses phospholipase C (PLC) activation and yet involves a PtX-sensitive GTP-binding protein (G protein). Unlike its dual effects, when internally applied, neomycin induces histamine secretion from intact mast cells in a dose-dependent manner. Half-maximal and maximal effects are obtained at 0.5 and 1 mM neomycin, respectively. This process is rapid (approximately 30 s), is independent of external Ca2+, and is associated with phosphatidic acid formation, implying that neomycin can activate histamine secretion by a mechanism similar to that utilized by other basic secretagogues of mast cells. Neomycin stimulates fourfold the GTPase activity of cholate-solubilized rat brain membranes in a PtX-inhibitable manner. In addition neomycin, as well as the basic secretagogues of mast cells, compound 48/80, and mastoparan, significantly reduce (by approximately 80%) the ADP ribosylation of PtX substrates present in rat brain membranes. Taken together these data suggest that neomycin can stimulate secretion from mast cells by directly activating G proteins that play a role in stimulus-secretion coupling. When internally applied, neomycin presumably stimulates secretion by activating a G protein that is located downstream to PLC. This G protein serves as a substrate for PtX.

Mechanism of estrogen receptor-dependent transcription in a cell-free system

1990 ◽  
Vol 10 (12) ◽  
pp. 6607-6612
Author(s):  
J F Elliston ◽  
S E Fawell ◽  
L Klein-Hitpass ◽  
S Y Tsai ◽  
M J Tsai ◽  
...  
Keyword(s):  
Estrogen Receptor ◽  
Rna Polymerase Ii ◽  
Expression System ◽  
Steroid Receptor ◽  
Free Expression ◽  
Independent Manner ◽  
Free System ◽  
A Cell ◽  
Regulated Gene Expression ◽  
Cell Free Expression System

RNA synthesis was stimulated directly in a cell-free expression system by crude preparations of recombinant mouse estrogen receptor (ER). Receptor-stimulated transcription required the presence of estrogen response elements (EREs) in the test template and could be specifically inhibited by addition of competitor oligonucleotides containing EREs. Moreover, polyclonal antibodies directed against the DNA-binding region of ER inhibited ER-dependent transcription. In our cell-free expression system, hormone-free ER induced transcription in a hormone-independent manner. Evidence is presented suggesting that ER acts by facilitating the formation of a stable preinitiation complex at the target gene promoter and thus augments the initiation of transcription by RNA polymerase II. These observations lend support to our current understanding of the mechanism of steroid receptor-regulated gene expression and suggest strong conservation of function among members of the steroid receptor superfamily.

scholarly journals G Proteins and GPCRs in C. elegans Development: A Story of Mutual Infidelity

2018 ◽  
Vol 6 (4) ◽  
pp. 28 ◽  
Author(s):  
Daniel Matúš ◽  
Simone Prömel
Keyword(s):  
Signaling Pathways ◽  
G Protein ◽  
G Proteins ◽  
Cell Polarity ◽  
Protein Activity ◽  
Independent Manner ◽  
C Elegans ◽  
Downstream Effectors ◽  
Complex Signaling ◽  
G Protein Coupled

Many vital processes during C. elegans development, especially the establishment and maintenance of cell polarity in embryogenesis, are controlled by complex signaling pathways. G protein-coupled receptors (GPCRs), such as the four Frizzled family Wnt receptors, are linchpins in regulating and orchestrating several of these mechanisms. However, despite being GPCRs, which usually couple to G proteins, these receptors do not seem to activate classical heterotrimeric G protein-mediated signaling cascades. The view on signaling during embryogenesis is further complicated by the fact that heterotrimeric G proteins do play essential roles in cell polarity during embryogenesis, but their activity is modulated in a predominantly GPCR-independent manner via G protein regulators such as GEFs GAPs and GDIs. Further, the triggered downstream effectors are not typical. Only very few GPCR-dependent and G protein-mediated signaling pathways have been unambiguously defined in this context. This unusual and highly intriguing concept of separating GPCR function and G-protein activity, which is not restricted to embryogenesis in C. elegans but can also be found in other organisms, allows for essential and multi-faceted ways of regulating cellular communication and response. Although its relevance cannot be debated, its impact is still poorly discussed, and C. elegans is an ideal model to understand the underlying principles.

scholarly journals Regulation of Ca2+ current in frog ventricular cardiomyocytes by guanosine 5'-triphosphate analogues and isoproterenol.

1993 ◽  
Vol 102 (3) ◽  
pp. 525-549 ◽  
Author(s):  
T D Parsons ◽  
H C Hartzell
Keyword(s):  
G Protein ◽  
G Proteins ◽  
Ventricular Myocytes ◽  
Calcium Currents ◽  
Internal Perfusion ◽  
High Concentration ◽  
Patch Clamp Technique ◽  
Intact Cells ◽  
Molecule Interactions ◽  
Gamma S

Calcium currents (ICa) were measured in frog ventricular myocytes using the whole-cell patch clamp technique and a perfused pipette. To gain insight into the role of G proteins in the regulation of ICa in intact cells, the effect of internal perfusion with hydrolysis-resistant GTP analogues, guanylyl 5'-imidodiphosphate (GppNHp) or guanosine 5'-thiotriphosphate (GTP gamma S), on ICa stimulated by isoproterenol (Iso) or forskolin (Forsk) was examined. Significant differences were observed between the effects of the two GTP analogues. Internal perfusion of GppNHp resulted in a near-complete (approximately 80%) and irreversible inhibition of Iso-stimulated ICa. In contrast, internal perfusion with GTP gamma S resulted in only a partial (approximately 40%) inhibition of Iso- or Forsk-stimulated ICa. The fraction of the current not inhibited by GTP gamma S remained persistently elevated after the washout of Iso but declined to basal levels upon washout of Forsk. Excess internal GTP or GppNHp did not reduce the persistent ICa. Internal adenosine 5'-thiotriphosphate (ATP gamma S) mimicked the GTP gamma S-induced, persistent ICa. GppNHp sometimes induced a persistent ICa, but only if GppNHp was present at high concentration before Iso exposure. Inhibitors of protein kinase A inhibited both the GTP gamma S- and ATP gamma S-induced, persistent ICa. We conclude that: (a) GTP gamma S is less effective than GppNHp in inhibiting adenylyl cyclase (AC) via the inhibitory G protein, Gi; and (b) the persistent ICa results from a long-lived Gs-GTP gamma S complex that can activate AC in the absence of Iso. These results suggest that different hydrolysis-resistant nucleotide analogues may behave differently in activating G proteins and imply that the efficacy of G protein-effector molecule interactions can depend on the GTP analogue with which the G protein is activated.

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 Regulation of chemoattractant receptor interaction with transducing proteins by organizational control in the plasma membrane of human neutrophils.

1989 ◽  
Vol 109 (6) ◽  
pp. 2783-2790 ◽  
Author(s):  
A J Jesaitis ◽  
J O Tolley ◽  
G M Bokoch ◽  
R A Allen
Keyword(s):  
Plasma Membrane ◽  
G Protein ◽  
G Proteins ◽  
Membrane Fraction ◽  
Human Neutrophils ◽  
Membrane Domain ◽  
Plasma Membrane Fraction ◽  
A Minor ◽  
Plasma Membrane Domain ◽  
Gamma S

Isolated purified plasma membrane domains from unstimulated human neutrophils were photoaffinity labeled with F-Met-Leu-Phe-N epsilon-(2-(p-azido-[125I]salicylamido)ethyl- 1,3'-dithiopropionyl)-Lys also referred to as FMLPL-SASD[125I]. Most of the photoaffinity-labeled N-formyl peptide receptors were found in light plasma membrane fraction (PM-L) which has been previously shown to be enriched in guanyl nucleotide binding proteins and the plasma membrane marker alkaline phosphatase (Jesaitis, A. J., G. M. Bokoch, J. O. Tolley, and R. A. Allen. 1988. J. Cell Biol. 107:921-928). Furthermore, the heavy plasma membrane fraction (PM-H), which is enriched in actin and fodrin, was depleted in receptors. Solubilization of PM-L and PM-H in divalent cation-free buffer containing octylglucoside and subsequent sedimentation at 180,000 g in detergent-containing sucrose gradients revealed two receptor forms. The major population, found in PM-L sedimented as a globular protein with an apparent sedimentation coefficient of 6-7S, while a minor fraction found in the PM-H fraction sedimented as a 4S particle. In addition, the 6-7S form could be converted to the 4S form by inclusion of guanosine 5'-O-(3-thiotriphosphate) (GTP gamma S) in the extraction buffer (ED50 = 10-30 nM). ATP was not effective at doses of up to 10 microM. In contrast, isolation and solubilization of receptors from desensitized cells (photoaffinity labeled after a 15 degrees C incubation with FMLPL-SASD[125I]) revealed that the majority of receptors (greater than 60-90%), which are found in PM-H, sedimented as 4S particles. A minor fraction of receptors found in the PM-L sedimented as 6-7S species. The receptors in the PM-H fraction, however, were still capable of interacting with G-proteins, since addition of unlabeled PM-L membrane fraction as a G-protein source reconstituted a more rapidly sedimenting form showing sensitivity to GTP gamma S. These results suggest that receptors in unstimulated human neutrophils have a higher probability of interacting with G-proteins because they are in the light plasma membrane domain. The results also suggest that receptors that have been translocated to the heavy plasma membrane domain during the process of desensitization or response termination have a lower probability of interacting with G-protein. Since the latter receptors are still capable of forming G protein associations, then their lateral segregation would represent a mechanism of controlling of receptor G-protein interactions. This reorganization of the plasma membrane, therefore, may form the molecular basis for response termination or homologous desensitization in human neutrophils.

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.

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.

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