scholarly journals Membrane localization of the pertussis toxin-sensitive G-protein subunits alpha i-2 and alpha i-3 and expression of a metallothionein-alpha i-2 fusion gene in LLC-PK1 cells.

1990 ◽  
Vol 87 (12) ◽  
pp. 4635-4639 ◽  
Author(s):  
L. Ercolani ◽  
J. L. Stow ◽  
J. F. Boyle ◽  
E. J. Holtzman ◽  
H. Lin ◽  
...  
Keyword(s):  
G Protein ◽  
Pertussis Toxin ◽  
Fusion Gene ◽  
Membrane Localization ◽  
Protein Subunits

Characterization of heterotrimeric G-proteins in adult Acanthocheilonema viteae

1996 ◽  
Vol 320 (2) ◽  
pp. 459-466 ◽  
Author(s):  
GRANT Karen R. ◽  
Margaret M. HARNETT ◽  
Graeme MILLIGAN ◽  
William HARNETT
Keyword(s):  
G Protein ◽  
G Proteins ◽  
Pertussis Toxin ◽  
Eukaryotic Cells ◽  
Heterotrimeric G Proteins ◽  
Protein Subunits ◽  
Acanthocheilonema Viteae ◽  
Affinity Labelling ◽  
Α Subunits

Heterotrimeric G-proteins have been found in eukaryotic cells, from yeast to humans, but have received little attention, to date, with respect to parasitic organisms. We now present the first report of the characterization of heterotrimeric G-proteins expressed in a filarial nematode, Acanthocheilonema viteae. Using a combination of (i) affinity labelling with [α-32P]GTP; (ii) ADP-ribosylation with cholera toxin and pertussis toxin; (iii) Western blotting with a panel of anti-G-protein antibodies; and (iv) reverse transcriptase-PCR with degenerate G-protein oligonucleotide primers followed by hybridization analysis using oligonucleotides specific for individual G-protein subunits, we demonstrate that adult A. viteae expresses homologues of the β1-and/or β2-like subunits and α-subunits of the Gs, Gi, Gq and G12 subfamilies found in mammals. The role which these G-proteins may play in the biology of the organism is discussed.

Lidocaine Enhances GαiProtein Function

2003 ◽  
Vol 99 (5) ◽  
pp. 1093-1101 ◽  
Author(s):  
Claudia Benkwitz ◽  
James C. Garrison ◽  
Joel Linden ◽  
Marcel E. Durieux ◽  
Markus W. Hollmann
Keyword(s):  
Adenylate Cyclase ◽  
Signaling Pathway ◽  
G Protein ◽  
Pertussis Toxin ◽  
Chinese Hamster Ovary Cells ◽  
Phosphodiesterase Inhibitor ◽  
Cyclic Adenosine Monophosphate ◽  
Adenosine Monophosphate ◽  
Protein Subunit ◽  
Protein Subunits

Background Local anesthetics inhibit several G protein-coupled receptors by interaction with the Galphaq protein subunit. It is not known whether this effect on G protein function can be extrapolated to other classes of G proteins. The authors investigated interactions of lidocaine with the human adenosine 1 receptor (hA1R)-coupled signaling pathway. Activated A1Rs couple to adenylate cyclase via the pertussis toxin sensitive Galphai protein, thereby decreasing cyclic adenosine monophosphate formation. A1Rs are widely expressed and abundant in the spinal cord, brain, and heart. Interactions of LAs with the hA1R-coupled transduction cascade therefore might produce a broad range of clinically relevant effects. Methods The function of hA1Rs stably expressed in Chinese hamster ovary cells was determined with assays of cyclic adenosine monophosphate, receptor binding, and guanosine diphosphate/guanosine triphosphate gamma35S exchange by using reconstituted defined G protein subunits. Involvement of phosphodiesterase and Galphai was characterized by using the phosphodiesterase inhibitor rolipram and pertussis toxin, respectively. Results Lidocaine (10-9-10-1 M) had no significant effects on agonist or antagonist binding to the hA1R or on receptor-G protein interactions. However, cyclic adenosine monophosphate levels were reduced significantly to 50% by the LAs, even in the absence of an A1R agonist or presence of an A1R antagonist. This effect was unaffected by rolipram (10 mum), but abolished completely by pretreatment with pertussis toxin, which inactivates the Galphai protein. Therefore, the main target site for LAs in this pathway is located upstream from adenylate cyclase. Conclusions Lidocaine potentiates Galphai-coupled A1R signaling by reducing cyclic adenosine monophosphate production. The study suggests an interaction site for LAs in a Galphai-coupled signaling pathway, with the Galphai protein representing the prime candidate. Taken together with previous results showing inhibitory LA interactions on the Galphaq protein subunit, the data in the current study support the hypothesis that specific G protein subunits represent alternative sites of LA action.

scholarly journals G-protein from Medicago sativa: functional association to photoreceptors

1993 ◽  
Vol 291 (2) ◽  
pp. 383-388 ◽  
Author(s):  
J P Muschietti ◽  
H E Martinetto ◽  
O A Coso ◽  
M D Farber ◽  
H N Torres ◽  
...  
Keyword(s):  
Medicago Sativa ◽  
Cholera Toxin ◽  
G Protein ◽  
Pertussis Toxin ◽  
Polyclonal Antibodies ◽  
Red Light ◽  
Maximal Increase ◽  
Protein Subunits ◽  
Binding Rate ◽  
Polypeptide Band

G-protein subunits were characterized from Medicago sativa (alfalfa) seedlings. Crude membranes and GTP-Sepharose-purified fractions were electrophoresed on SDS/polyacrylamide gels and analysed by Western blotting with 9193 (anti-alpha common) and AS/7 (anti-alpha t, anti-alpha i1 and anti-alpha i2) polyclonal antibodies. These procedures led to the identification of a specific polypeptide band of about 43 kDa. Another polypeptide reacting with the SW/1 (anti-beta) antibody, of about 37 kDa, was also detected. The 43 kDa polypeptide bound specifically [alpha-32P]GTP by a photoaffinity reaction and was ADP-ribosylated by activated cholera toxin, but not by pertussis toxin. Irradiation of etiolated Medicago sativa protoplast preparations at 660 nm for 1 min produced a maximal increase in the guanosine 5′-[gamma-thio]triphosphate (GTP[35S])-binding rate. After this period of irradiation, the binding rate tended to decrease. The effect of a red-light (660 nm) pulse on the binding rate was reversed when it was immediately followed by a period of far-red (> 730 nm) illumination. These results may suggest that activation of GTP[S]-binding rate was a consequence of conversion of phytochrome Pr into the Ptr form.

scholarly journals Involvement of A pertussis Toxin Sensitive G-Protein in the Inhibition of Inwardly Rectifying K+Currents by Platelet-Activating Factor in Guinea-Pig Atrial Cardiomyocytes

1994 ◽  
Vol 3 (1) ◽  
pp. 45-51
Author(s):  
M. Gollasch ◽  
T. Kleppisch ◽  
D. Krautwurst ◽  
D. Lewinsohn ◽  
J. Hescheler
Keyword(s):  
Cyclic Amp ◽  
Guinea Pig ◽  
G Protein ◽  
Pertussis Toxin ◽  
Platelet Activating Factor ◽  
Resting Membrane Potential ◽  
Patch Clamp Technique ◽  
Guinea Pig Heart ◽  
Ventricular Cells ◽  
Inwardly Rectifying

Platelet-activating factor (PAF) inhibits single inwardly rectifying K+channels in guinea-pig ventricular cells. There is currently little information as to the mechanism by which these channels are modulated. The effect of PAF on quasi steady-state inwardly rectifying K+currents (presumably of the IK1type) of auricular, atrial and ventricular cardiomyocytes from guinea-pig were studied. Applying the patch-clamp technique in the whole-cell configuration, PAF (10 nM) reduced the K+currents in all three cell types. The inhibitory effect of PAF occurred within seconds and was reversible upon wash-out. It was almost completely abolished by the PAF receptor antagonist BN 50730. Intracellular infusion of atrial cells with guanine 5′-(β-thio)diphosphate (GDPS) or pretreatment of cells with pertussis toxin abolished the PAF dependent reduction of the currents. Neither extracellularly applied isoproterenol nor intracellularly applied adenosine 3′,5′-cyclic monophosphate (cyclic AMP) attenuated the PAF effect. In multicellular preparations of auricles, PAF (10 nM) induced arrhythmias. The arrhythmogenic activity was also reduced by BN 50730. The data indicate that activated PAF receptors inhibit inwardly rectifying K+currents via a pertussis toxin sensitive G-protein without involvement of a cyclic AMP-dependent step. Since IK1is a major component in stabilizing the resting membrane potential, the observed inhibition of this type of channel could play an important role in PAF dependent arrhythmogenesis in guinea-pig heart.

scholarly journals A Regulator of G Protein Signaling-containing Kinase Is Important for Chemotaxis and Multicellular Development inDictyostelium

2003 ◽  
Vol 14 (4) ◽  
pp. 1727-1743 ◽  
Author(s):  
Binggang Sun ◽  
Richard A. Firtel
Keyword(s):  
Protein Kinase ◽  
G Protein ◽  
Kinase Activity ◽  
Site Directed Mutagenesis ◽  
Membrane Localization ◽  
Pleckstrin Homology Domain ◽  
G Protein Signaling ◽  
Protein Signaling ◽  
Wild Type ◽  
Gene Encoding

We have identified a gene encoding RGS domain-containing protein kinase (RCK1), a novel regulator of G protein signaling domain-containing protein kinase. RCK1 mutant strains exhibit strong aggregation and chemotaxis defects. rck1 null cells chemotax ∼50% faster than wild-type cells, suggesting RCK1 plays a negative regulatory role in chemotaxis. Consistent with this finding, overexpression of wild-type RCK1 reduces chemotaxis speed by ∼40%. On cAMP stimulation, RCK1 transiently translocates to the membrane/cortex region with membrane localization peaking at ∼10 s, similar to the kinetics of membrane localization of the pleckstrin homology domain-containing proteins CRAC, Akt/PKB, and PhdA. RCK1 kinase activity also increases dramatically. The RCK1 kinase activity does not rapidly adapt, but decreases after the cAMP stimulus is removed. This is particularly novel considering that most other chemoattractant-activated kinases (e.g., Akt/PKB, ERK1, ERK2, and PAKa) rapidly adapt after activation. Using site-directed mutagenesis, we further show that both the RGS and kinase domains are required for RCK1 function and that RCK1 kinase activity is required for the delocalization of RCK1 from the plasma membrane. Genetic evidence suggests RCK1 function lies downstream from Gα2, the heterotrimeric G protein that couples to the cAMP chemoattractant receptors. We suggest that RCK1 might be part of an adaptation pathway that regulates aspects of chemotaxis in Dictyostelium.

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