Role Of GTP-binding Proteins Research Articles

Role of GTP-binding protein in histamine release from mast cells

It is my great pleasure and honor to participate in this special symposium in honor of Dr. Elmer Becker. On this memorable occasion I would like to present our recent studies on the possible role of guanine nucleotide (GTP)-binding protein in mediator release from mast cells. Mast cells and basophils are target cells for IgE and the reaction of cell-bound IgE antibodies with multivalent antigen results in the release of a variety of inflammatory mediators (1). The same mediators are also released by various IgEindependent stimuli (2). Analysis of biochemical events involved in the mediator release revealed that the activation of various membrane-associated enzymes appears to be involved in the transduction of triggering signals (3-7). The results also suggest that enzymes involved in the mediator release are different depending on stimuli (8). Recent studies on hormonal regulation of adenylate cyclase resulted in the discovery of a pair of GTP-binding regulatory proteins. Receptors for various hormones link to one of the two GTP-binding proteins Gs or Gi, which mediates either the activation (Gs) or inhibition (Gi) of adenylate cyclase (9). It was demonstrated that pertussis toxin (PT) induces the ADP ribosylation of the cx subunit of Gi (Gia ) (10, 1 l), while cholera toxin ADP ribosylates Gsa (12, 13). Accumulated evidence also suggests that GTP-binding regulatory proteins are involved in the signal transduction in various types of cells (14-17). In view of these observations, we studied the possible participation of GTPbinding proteins in the mediator release from mast cells and basophils.

Relationship of GTP-binding proteins, phospholipase C, and PGE2 synthesis in rat glomerular mesangial cells

We evaluated the role of GTP-binding proteins in the activation of phospholipase C, release of arachidonic acid, and synthesis of prostaglandin (PG) E2 in response to platelet-activating factor (PAF) and angiotensin II (ANG II) in cultured rat mesangial cells. Pretreatment with pertussis toxin (PT) decreased PGE2 formation and arachidonic acid release in response to PAF and ANG II but not that to A 23187. PT pretreatment also inhibited formation of inositol trisphosphate (IP3) in response to ANG II or PAF but did not significantly alter the rise in intracellular calcium detected by fura-2. PT catalyzed ADP ribosylation of two proteins of molecular mass approximately 40 and 41 kDa. Further evidence for involvement of GTP-binding protein in phospholipase C activation was that GTP-gamma S stimulated IP3 generation. Immunoblots with antibodies directed against different inhibitory alpha subunits of GTP-binding proteins showed that the major 40-kDa PT substrate reacted with an antibody directed against a decapeptide of the G protein subunit alpha i2 that is also found in leukocytes. This was further confirmed by Northern blot that showed the existence of mRNA in mesangial cells that hybridized with a cDNA probe for G alpha i2. In addition lesser amounts of mRNA hybridized with a restriction fragment cDNA probe for G alpha i3, which corresponds to the 41-kDa substrate for PT ribosylation. These results show that phospholipase C activation by PAF and ANG II in mesangial cells involves a specific G protein, most likely G alpha i2.(ABSTRACT TRUNCATED AT 250 WORDS)

O-227 - A role of GTP-binding proteins in growth factor-induced proliferation of rat hepatocytes in primary culture

O-227 - A role of GTP-binding proteins in growth factor-induced proliferation of rat hepatocytes in primary culture

Mechanism of activation of lymphocyte Na+/H+ exchange by concanavalin A. A calcium- and protein kinase C-independent pathway.

Treatment of thymic lymphocytes with the mitogenic lectin concanavalin A (ConA) increases the intracellular free Ca2+ concentration and stimulates phosphoinositide turnover. ConA also induced a rapid, amiloride-sensitive, Na+-dependent increase in cytosolic pH of 0.13 +/- 0.01, indicative of stimulation of the Na+/H+ antiport. To investigate the mechanism underlying activation of Na+/H+ exchange by ConA, the intracellular free Ca2+ concentration changes induced by this lectin were precluded by loading the cells with Ca2+-buffering agents and suspension in Ca2+-free media. Under these conditions, the ConA-induced cytoplasmic alkalinization proceeded normally. Two approaches were used to assess the role of protein kinase C. First, this enzyme was inhibited by the addition of 1-(5-isoquinolinysulfonyl)-2-methylpiperazine. In the presence of this potent antagonist, stimulation of the antiport by 12-O-tetradecanoylphorbol-13-acetate was greatly inhibited. In contrast, stimulation by ConA was unaffected. Second, protein kinase C was depleted by overnight incubation with phorbol esters. Following this treatment, Na+/H+ exchange was no longer activated by 12-O-tetradecanoyl-13-acetate, but was still stimulated by ConA. These data suggest that a Ca2+- and protein kinase C-independent mechanisms mediates the activation of Na+/H+ exchange by ConA. The possible role of GTP-binding proteins in the activation was also studied. The antiport was not stimulated by either fluoroaluminate or vanadate. Moreover, pretreatment with pertussis toxin failed to inhibit the ConA-induced cytoplasmic alkalinization. In contrast, preincubation with cholera toxin partially inhibited activation. Under these conditions, cholera toxin significantly elevated intracellular cAMP levels. Inhibition was also observed in cells treated with forskolin at concentrations that increased [cAMP]. The data suggest that a novel cAMP-sensitive signaling mechanism not involving Ca2+ and protein kinase C is involved in the stimulation of Na+/H+ exchange by mitogens in T lymphocytes.

Short-term desensitization of muscarinic K+ channel current in isolated atrial myocytes and possible role of GTP-binding proteins

The short-term desensitization of the acetylcholine (ACh)-induced K+ channel current was examined in single atrial cells of guinea-pig heart. The tight-seal whole cell voltage clamp technique was used. The solution in the pipettes contained GTP or guanosine-5'-O-(3-thiotriphosphate) (GTP-gamma S, a non-hydrolyzable GTP analogue). In GTP-loaded cells, ACh evoked a specific K+ channel current via GTP-binding proteins (G) in a dose-dependent manner. The K+ current showed agonist-dependent desensitization similar to those reported in other cardiac tissues (Nilius 1983; Carmeliet and Mubagwa 1986). The cellular response to ACh was also desensitized by activation of P1-purinergic receptors with adenosine (Ado). In GTP-gamma S-loaded cells, the K+ current was gradually induced even in the absence of agonists, probably due to direct activation of G proteins by GTP-gamma S. In the early phase of the spontaneous current increase, ACh evoked a large current transiently. As the GTP-gamma S-induced activation of the current progressed, the magnitude of the ACh-evoked current transient became smaller and finally negligible. Similar results were obtained when Ado was used as an agonist instead of ACh to induce the K+ current. Therefore, it is indicated that the agonist-receptor interaction may not be essential for the desensitization of ACh-induced K+ current in atrial myocytes.

S9-4 - Role of GTP-binding proteins in muscarinic and adenosine receptor-mediaton inhibitory regulation of cardiac function

S9-4 - Role of GTP-binding proteins in muscarinic and adenosine receptor-mediaton inhibitory regulation of cardiac function

Light activation of phospholipase A2 in rod outer segments of bovine retina and its modulation by GTP-binding proteins.

Light stimulates phospholipase A2 activity in rod outer segments (ROS) of bovine retina as measured by the liberation of arachidonate from phosphatidylcholine, in in vitro assays of dark-adapted ROS. A role for GTP-binding proteins (G or N proteins) in the light activation of phospholipase A2 is suggested by the capacity for guanosine 5'-O-(thiotriphosphate) (GTP gamma S) to activate phospholipase A2 in dark-adapted ROS. In contrast, addition of GTP gamma S coincident with light exposure inhibited the light activation of phospholipase A2, suggesting that phospholipase A2 activity in the ROS is under dual regulation by G proteins. Transducin, the major G protein of the ROS, mediates the activation of cGMP phosphodiesterase by light and is a substrate for both cholera and pertussis toxin. Treatment of dark-adapted ROS with either toxin inhibits both basal and light-activated phospholipase A2, mimicking the action of the toxins on the light-induced cGMP phosphodiesterase activity of ROS. There is a loss of light-sensitive phospholipase A2 activity coincident with extraction of transducin from ROS membranes. In addition, the light-sensitive phospholipase A2 activity can be partially restored by the addition of purified transducin to the extracted ROS membranes. Light activation of phospholipase A2 in ROS membranes thus occurs by a transducin-dependent mechanism.

The role of GTP-binding proteins in receptor activation of phospholipase C.

Recent studies indicate that phospholipase C-induced degradation of the inositol phospholipids produces the formation of two molecules that have second messenger properties. The two molecules are 1, 2-diacylglycerol, which stimulates protein kinase C, and 1, 4, 5-inositol trisphosphate, which can mobilize Ca2+ from the endoplasmic reticulum to the cytosol. Inositol phospholipid degradation occurs in a wide variety of cells responding to specific stimuli. In platelets, it has been shown that agonists such as thrombin, collagen, platelet-activating factor, vasopressin, serotonin, prostaglandin-endoperoxides and thromboxane A2 stimulate the metabolism of the inositol phospholipids (Lapetina, 1986a). This receptor-coupled reaction seems to be an early step in the transduction mechanism of those agonists. Recent advances indicate that regulation of inositol phospholipid degradation might be modulated by GTP-binding proteins (Lapetina, 1986a,b; Cockcroft and Gomperts, 1985; Haslam and Davidson, 1984; Lapetina et al., 1986; Litosch et al., 1985; Wallace and Fain, 1985; Ui, 1984), much like the regulation of adenylate cyclase (Katada et al., 1984; Hanski and Gilman, 1982). In this case, two guanine nucleotide-binding regulatory proteins, Gs(Ns) and Gi (Ni), modulate the stimulation and inhibition of adenylate cyclase, respectively. These proteins contain three subunits recognized as α, β and γ Cholera toxin catalyzes ADP-ribosylation of the αs-subunit of Gs and stimulates adenylate cyclase. Pertussis toxin, on the other hand, ADP-ribosylates the αi-subunit of Gi.

Receptor-stimulated phosphoinositide metabolism: a role for GTP-binding proteins?

Receptor-stimulated phosphoinositide metabolism: a role for GTP-binding proteins?

Beta-adrenergic stimulation of adenylate cyclase and alpha-adrenergic inhibition of adenylate cyclase: GTP-binding proteins as macromolecular messengers.

This review, which is a summary of an oral presentation made at the International Study Group for Research in Cardiac Metabolism, is a brief synopsis of work in our laboratory concerning the mechanism of hormonal regulation of adenylate cyclase. By its very nature, it refers almost entirely to our own work. However, since a number of laboratories have contributed to the field in ways at least as significant as our own, I recommend reading the reviews cited as references 4, 7 and 11 for a more comprehensive summary of the literature. The emphasis of this review is the role of GTP-binding proteins as important communicators between hormone-occupied receptors and the catalytic subunit of adenylate cyclase. The experimental work summarized emphasizes the extent to which one can determine the “intactness” receptor-effector coupling by focusing on receptor-agonist interactions, and modulation of these interactions by guanine nucleotides.

Activation and attenuation of adenylate cyclase. The role of GTP-binding proteins as macromolecular messengers in receptor--cyclase coupling.

Research Article| April 01 1981 Activation and attenuation of adenylate cyclase. The role of GTP-binding proteins as macromolecular messengers in receptor–cyclase coupling L E Limbird L E Limbird Search for other works by this author on: This Site PubMed Google Scholar Biochem J (1981) 195 (1): 1–13. https://doi.org/10.1042/bj1950001 Views Icon Views Article contents Figures & tables Video Audio Supplementary Data Peer Review Share Icon Share Twitter LinkedIn Cite Icon Cite Get Permissions Citation L E Limbird; Activation and attenuation of adenylate cyclase. The role of GTP-binding proteins as macromolecular messengers in receptor–cyclase coupling. Biochem J 1 April 1981; 195 (1): 1–13. doi: https://doi.org/10.1042/bj1950001 Download citation file: Ris (Zotero) Reference Manager EasyBib Bookends Mendeley Papers EndNote RefWorks BibTex toolbar search Search Dropdown Menu toolbar search search input Search input auto suggest filter your search All ContentAll JournalsBiochemical Journal Search Advanced Search This content is only available as a PDF. © 1981 London: The Biochemical Society1981 Article PDF first page preview Close Modal You do not currently have access to this content.