G-proteins in mammalian gametes: an immunocytochemical study

1988 ◽  
Vol 91 (1) ◽  
pp. 21-31
Author(s):  
N.B. Garty ◽  
D. Galiani ◽  
A. Aharonheim ◽  
Y.K. Ho ◽  
D.M. Phillips ◽  
...  
Keyword(s):  
Monoclonal Antibody ◽  
G Protein ◽  
G Proteins ◽  
Cumulus Cells ◽  
Bovine Brain ◽  
Regulatory Proteins ◽  
Immunocytochemical Study ◽  
Cell Cytoplasm ◽  
Sperm Tail ◽  
Immunoreactive Material

The presence of transductory GTP(G)-regulatory proteins in mammalian gametes has been examined by indirect fluorescence immunocytochemistry. Using rabbit antisera to bovine rod beta gamma-transducin (RA beta gamma T), bovine rod holotransducin (AS-1), bovine rod alpha-transducin (RA alpha T), synthetic bovine rod alpha-transducin C-terminal decapeptide (AS-6), bovine brain alpha 39Go (RA alpha 39), and two mouse monoclonal antibodies raised against frog retinal transducin (4A), and rat brain beta-tubulin, we demonstrated the presence of corresponding immunoreactive material in both rat oocytes and bovine ejaculated sperm. Immunostaining in the oocyte was evenly distributed on the oolemma, excluding the cell cytoplasm and zona pellucida. Immunoreactive material was also present in the cumulus cells that encapsulate the oocyte. In contrast, the immunofluorescence corresponding to transductory G-proteins was confined in sperm to functionally defined regions in the head and tail, in a manner specific for each antibody. While RA beta gamma T, AS-1 and RA alpha 39 all stained the entire acrosome, AS-6 and RA alpha T stained only the acrosomal tip. Monoclonal antibody 4A stained the midpiece exclusively and anti-rat betaq-tubulin (a structural G-protein) stained the full length of the sperm tail. The existence of several G-protein types in mammalian gametes suggests their possible involvement in the regulation of various effector systems, in a manner reminiscent of somatic cells. The unique situation in sperm, where different G-proteins show distinct and specific patterns of distribution, further suggests their association with various effector systems in discrete functional domains.

Stimulation by G Protein βγ Subunits of Phospholipase C β Isoforms in Human Platelets

1998 ◽  
Vol 79 (05) ◽  
pp. 1008-1013 ◽  
Author(s):  
Yoshiko Banno ◽  
Tomiko Asano ◽  
Yoshinori Nozawa
Keyword(s):  
Phospholipase C ◽  
G Protein ◽  
G Proteins ◽  
Human Platelet ◽  
Minor Component ◽  
Similar Efficacy ◽  
Human Platelets ◽  
Bovine Brain ◽  
Γ Subunit ◽  
A Minor

SummaryDifferent phospholipase C (PLC) isoforms were located in human platelet cytosol and membranes. PLCγ2 and PLCβ3b were mainly located in the cytosol and PLCβ2 and PLCβ3a were in both cytosol and membranes by using specific antibodies against PLC isozymes (Banno Y, Nakashima S, Ohzawa M, Nozawa Y. J Biol Chem 1996; 271: 14989-94). Three PLC fractions activated by G protein βγ subunits were purified from human platelet cytosol and membrane fractions. Two PLC fractions from membranes were identified as PLCβ2 and PLCβ3a, and one from cytosol was PLCβ3b. These PLCβ isoforms were activated by the purified βγ subunits of brain G proteins in the order PLCβ3b > PLCβ3a > PLCβ2. Western blot analysis of γ subunits of the purified platelet G proteins with antibodies against various standard γ subunits revealed that the major component of the γ subunit of Gi2 and Gq was γ5, and that γ7 was a minor component. Studies using various subtypes of βγ subunits, βγ2, βγ3, and βγ7 purified from bovine brain, βγ5 from bovine lung, or βγ12 from bovine spleen, failed to show differences in their ability to stimulate the isolated platelet PLCβ isoforms. These results suggest that the βγ subunits of Gi2 and Gq have similar efficacy in regulation of effectors in human platelets.

scholarly journals G-protein inhibition of phospholipase C-β1 in membranes: role of G-protein βγ subunits

1996 ◽  
Vol 319 (1) ◽  
pp. 173-178 ◽  
Author(s):  
Irene LITOSCH
Keyword(s):  
Phospholipase C ◽  
G Protein ◽  
G Proteins ◽  
Plasma Membranes ◽  
Bovine Brain ◽  
Protein Inhibition ◽  
Low Concentrations ◽  
Dependent Inhibition ◽  
Cortical Membranes

Rat liver plasma membranes reconstituted with bovine brain phospholipase C β1 (PLC-β1) exhibit a dual regulation of PLC-β1 activity by G-proteins. Guanosine 5´-[γ-thio]triphosphate (GTP[S]; 0.1 nM) produced a 20-25% inhibition of PLC-β1 activity within 7 min of incubation. The addition of vasopressin resulted in near-basal levels of activity in the presence of 0.1 nM GTP[S]. Clonidine had little effect on the net inhibition due to GTP[S]. A similar antagonism between carbachol and GTP[S] occurred in cerebral cortical membranes containing endogenous PLC-β1 activity. αo/i-GDP (a mixture of GDP-liganded Goα and Giα) attenuated the GTP[S]-dependent inhibition of PLC-β1 whereas αo/i-GTP[S] had no effect, suggesting an involvement of G-protein βγ subunits in the inhibition of PLC-β1. Low concentrations of βγ subunits inhibited PLC-β1 activity. Inhibition was followed by reversal to basal activity and onset of stimulation as the βγ concentration was increased. Inhibition by βγ was dependent on the presence of membranes. These results indicate that G-protein βγ subunits constitute a mechanism by which G-proteins mediate a rapid and transient inhibition of PLC-β1.

scholarly journals Light-dependent GTP-binding proteins in squid photoreceptors

1990 ◽  
Vol 272 (1) ◽  
pp. 79-85 ◽  
Author(s):  
P R Robinson ◽  
S F Wood ◽  
E Z Szuts ◽  
A Fein ◽  
H E Hamm ◽  
...  
Keyword(s):  
Monoclonal Antibody ◽  
G Protein ◽  
G Proteins ◽  
Alpha Subunit ◽  
Protein G ◽  
Adp Ribosylation ◽  
Alpha Subunits ◽  
Gtp Binding ◽  
Molecular Masses ◽  
Lines Of Evidence

Previous biochemical and electrophysiological evidence suggests that in invertebrate photoreceptors, a GTP-binding protein (G-protein) mediates the actions of photoactivated rhodopsin in the initial stages of transduction. We find that squid photoreceptors contain more than one protein (molecular masses 38, 42 and 46 kDa) whose ADP-ribosylation by bacterial exotoxins is light-sensitive. Several lines of evidence suggest that these proteins represent distinct alpha subunits of G-proteins. (1) Pertussis toxin and cholera toxin react with distinct subsets of these polypeptides. (2) Only the 42 kDa protein immunoreacts with the monoclonal antibody 4A, raised against the alpha subunit of the G-protein of vertebrate rods [Hamm & Bownds (1984) J. Gen. Physiol. 84. 265-280]. (3) In terms of ADP-ribosylation, the 42 kDa protein is the least labile to freezing. (4) Of the 38 kDa and 42 kDa proteins, the former is preferentially extracted with hypo-osmotic solutions, as demonstrated by the solubility of its ADP-ribosylated state and by the solubility of the light-dependent binding of guanosine 5′-[gamma-thio]triphosphate. The specific target enzymes for the observed G-proteins have not been established.

Hormone signalling via G-protein: regulation of phosphatidylinositol 4,5-bisphosphate hydrolysis by G q

1992 ◽  
Vol 336 (1276) ◽  
pp. 35-42 ◽  
Keyword(s):  
Phospholipase C ◽  
G Protein ◽  
G Proteins ◽  
Regulatory Proteins ◽  
Affinity Reagents ◽  
Surface Receptors ◽  
New Class ◽  
Α Subunits ◽  
Hormone Signalling ◽  
G Protein Α Subunits

Heterotrimeric GTP-dependent regulatory proteins (G-proteins) mediate modulation by many cell surface receptors. Activation of the G-proteins promotes dissociation of their α and βγ subunits. The similarity of behaviour of βγ subunits derived from a variety of G-proteins has led to their use as affinity reagents for the analysis of the more unique a subunits. The evolution of these uses is presented. One of the more insightful results was the isolation of a new class of G -protein α subunits (the α q subfamily) which have been shown to regulate the activity of a phospholipase C (PLC) specific for phosphatidylinositols. The experimental evidence leading to this conclusion is discussed. The activation by α q increases the apparent V max of the β isoform of phosphatidylinositol-specific phospholipase C (PLCβ) and can be modulated by βγ subunits.

Regulation of Na+-K+-ATPase activity in kidney proximal tubules: involvement of GTP binding proteins

1989 ◽  
Vol 256 (1) ◽  
pp. F57-F62 ◽  
Author(s):  
A. Bertorello ◽  
A. Aperia
Keyword(s):  
Atpase Activity ◽  
G Protein ◽  
G Proteins ◽  
Pertussis Toxin ◽  
Rat Kidney ◽  
Inhibitory Effect ◽  
Proximal Tubules ◽  
Regulatory Proteins ◽  
Free Access ◽  
Gtp Binding

This study evaluates the involvement of GTP-dependent regulatory proteins (G-proteins) in the regulation of Na+-K+-ATPase activity in proximal convoluted tubule (PCT) segments. Single PCT segments were dissected from rat kidney and permeabilized to allow nucleotides and medium free access to the interior of the cell. A GDP analogue that blocks GTP-dependent activation of the G-protein, GDP beta S (400 microM) significantly inhibited PCT Na+-K+-ATPase activity when Na in the medium (Nam) was greater than or equal to 70 mM. The inhibition was attenuated when Nam was 55 and 35 mM and was no longer significant when Nam was 25 mM. GDP beta S had no inhibitory effect on the activity of purified Na+-K+-ATPase. A nonhydrolyzable GTP analogue, GppNHp (50 microM) significantly increased Na+-K+-ATPase activity when Nam was 25 and 35 mM, but not when Nam was 55-140 mM. Dopamine (DA) and DA1 plus DA2 agonists significantly inhibit Na+-K+-ATPase activity. DA inhibition was competitively abolished by GppNHp. In PCT segments from rats pretreated with pertussis toxin, DA and DA1 plus DA2 agonist inhibition of Na+-K+-ATPase activity was abolished. In PCT segments from rats pretreated with cholera toxin, basal Na+-K+-ATPase activity was increased, but DA significantly inhibited Na+-K+-ATPase activity. Na+-K+-ATPase activity in PCT segments is regulated via a G-protein that stimulates Na+-K+-ATPase activity and a DA-activated pertussis toxin-sensitive G-protein that inhibits Na+-K+-ATPase activity.(ABSTRACT TRUNCATED AT 250 WORDS)

scholarly journals A slowly ADP-ribosylated pertussis-toxin-sensitive GTP-binding regulatory protein is required for vasopressin-stimulated Ca2+ inflow in hepatocytes

1994 ◽  
Vol 299 (2) ◽  
pp. 399-407 ◽  
Author(s):  
L A Berven ◽  
B P Hughes ◽  
G J Barritt
Keyword(s):  
G Protein ◽  
G Proteins ◽  
Pertussis Toxin ◽  
Single Cells ◽  
Regulatory Protein ◽  
Regulatory Proteins ◽  
Inositol Polyphosphates ◽  
Gtp Binding ◽  
Inositol 1,4,5 Trisphosphate ◽  
Epidermal Growth

The roles of heterotrimeric GTP-binding regulatory proteins (G-proteins) and inositol polyphosphates in the mechanism by which vasopressin stimulates Ca2+ inflow in hepatocytes were investigated by using single cells loaded with fura2 by microinjection. Vasopressin-stimulated Ca2+ inflow was mimicked by microinjection of guanosine 5′-[gamma-thio]triphosphate (GTP[S]) or guanosine 5′-[beta gamma-imido]triphosphate to the cells, but not adenosine 5′-[gamma-thio]triphosphate (ATP[S]) or guanosine 5′-[beta-thio]diphosphate (GDP[S]). Extracellular Gd3+ (5 microM) inhibited both vasopressin- and GTP[S]-stimulated Ca2+ inflow. GDP[S], but not GMP, administered to hepatocytes by microinjection, completely inhibited vasopressin-stimulated Ca2+ inflow and partially inhibited vasopressin-induced release of Ca2+ from intracellular stores. The microinjection of pertussis toxin had no effect either on the release of Ca2+ from intracellular stores or on Ca2+ inflow induced by vasopressin, but completely inhibited changes in these processes induced by epidermal growth factor (EGF). Hepatocytes isolated from rats treated with pertussis toxin for 24 h exhibited no vasopressin- or GTP[S]-stimulated Ca2+ inflow, whereas the vasopressin-stimulated release of Ca2+ from intracellular stores was similar to that observed for control cells. Heparin or ATP[S] inhibited, or delayed the onset of, both vasopressin-induced release of Ca2+ from intracellular stores and vasopressin-stimulated Ca2+ inflow. Vasopressin-induced oscillations in intracellular [Ca2+] were observed in some heparin-treated cells. It is concluded that the stimulation by vasopressin of Ca2+ inflow to hepatocytes requires inositol 1,4,5-trisphosphate (InsP3) and, by implication, the pertussis-toxin-insensitive G-protein required for the activation of phospholipase C beta [Taylor, Chae, Rhee and Exton (1991) Nature (London) 350, 516-518], and another G-protein which is slowly ADP-ribosylated by pertussis toxin and acts between InsP3 and the putative plasma-membrane Ca2+ channel. EGF-stimulated Ca2+ inflow involves at least one G-protein which is rapidly ADP-ribosylated and is most likely required for InsP3 formation.

scholarly journals Calmodulin binding distinguishes between βγ subunits of activated G proteins and transducin

1992 ◽  
Vol 283 (3) ◽  
pp. 683-690 ◽  
Author(s):  
L A Mangels ◽  
R R Neubig ◽  
H E Hamm ◽  
M E Gnegy
Keyword(s):  
Adenylate Cyclase ◽  
G Protein ◽  
G Proteins ◽  
Bovine Brain ◽  
Cyclase Activity ◽  
Adenylate Cyclase Activity ◽  
Dependent Manner ◽  
Protein Activation ◽  
Calmodulin Binding ◽  
Gamma Subunits

The interactions between guanine nucleotide regulatory proteins and the Ca(2+)-binding protein calmodulin were studied using calmodulin-Sepharose affinity chromatography. Purified bovine brain beta gamma subunits bound to calmodulin-Sepharose in a Ca(2+)-dependent manner. On the contrary, beta gamma subunits produced in an activated Go/Gi preparation did not bind to calmodulin-Sepharose. The effect was independent of the type of bovine brain G protein (Go/Gi, Gs), method of activation and the presence of magnesium. To distinguish whether the binding of purified beta gamma subunits to calmodulin was unique to brain beta gamma or to the method of purification, similar experiments were performed using transducin. In contrast to bovine brain G proteins, both purified transducin beta gamma subunits and beta gamma released from rhodopsin-activated transducin bound to calmodulin-Sepharose in a Ca(2+)-dependent manner. To assess the functional significance of the binding of bovine brain beta gamma subunits to calmodulin, the ability of purified beta gamma and of beta gamma in unactivated and activated Go/Gi to inhibit partially purified calmodulin-sensitive adenylate cyclase was determined. Purified beta gamma was highly effective in inhibiting calmodulin-stimulated adenylate cyclase activity. However, unactivated Go/Gi and preactivated Go/Gi inhibited calmodulin-stimulated adenylate cyclase activity to the same extent. This Go/Gi-mediated inhibition also occurred in the presence of a 500-fold molar excess of calmodulin over added G protein. These results demonstrate: (1) that beta gamma subunits may not be completely released upon G protein activation, and (2) that inhibition of calmodulin-stimulated adenylate cyclase by beta gamma subunits does not appear to be mediated by a direct beta gamma-calmodulin interaction. Differences in the binding properties of activated bovine brain G proteins versus those of transducin could be explained by differences in the gamma subunit between the proteins, or by differences in affinities of the alpha and beta gamma subunits for each other and for calmodulin. The different functional properties of purified beta gamma subunits and beta gamma subunits produced in situ by activation of G proteins indicates that extrapolation from the effects of purified subunits to events occurring in membranes should be done with caution.

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