Pertussis Toxin-sensitive GTP-binding Proteins in Neuronal Tissues: Recent Insights into Expression and Function

1990 ◽  
pp. 77-107 ◽  
Author(s):  
Graeme Milligan
Keyword(s):  
Pertussis Toxin ◽  
Binding Proteins ◽  
Gtp Binding Proteins ◽  
Gtp Binding ◽  
Neuronal Tissues ◽  
And Function

Purification and identification of two pertussis-toxin-sensitive GTP-binding proteins of bovine spleen

1989 ◽  
Vol 161 (3) ◽  
pp. 1280-1285 ◽  
Author(s):  
Rika Morishita ◽  
Tomiko Asano ◽  
Kanefusa Kato ◽  
Hiroshi Itoh ◽  
Yoshito Kaziro
Keyword(s):  
Pertussis Toxin ◽  
Binding Proteins ◽  
Gtp Binding Proteins ◽  
Gtp Binding

A survey of GTP-binding proteins and other potential key regulators of exocytotic secretion in eosinophils. Apparent absence of rab3 and vesicle fusion protein homologues

1995 ◽  
Vol 108 (11) ◽  
pp. 3547-3556
Author(s):  
P. Lacy ◽  
N. Thompson ◽  
M. Tian ◽  
R. Solari ◽  
I. Hide ◽  
...  
Keyword(s):  
Western Blotting ◽  
Binding Proteins ◽  
Peak Plasma ◽  
Gradient Centrifugation ◽  
Vesicle Fusion ◽  
Beta Subunits ◽  
Marker Enzyme ◽  
Gtp Binding Proteins ◽  
Gtp Binding ◽  
Neuronal Tissues

We set out to identify potential key regulators of exocytotic fusion in the eosinophil, in the knowledge that granule exocytosis can be stimulated in these cells by intracellular application of nonhydrolyzable analogues of guanosine triphosphate, with Ca2+ acting as a modulator of guanine nucleotide-dependent secretion. To screen for GTP-binding proteins, guinea pig eosinophils were purified from peritoneal washings and subjected to western blotting analysis using specific immune sera raised against recombinant proteins or consensus peptide sequences within proteins of interest. We found a number of heterotrimeric G proteins (G alpha i3, G alpha o, G alpha q11, G alpha s and G beta subunits) and members of the small GTP-binding proteins expressed in eosinophils. Two subtypes of G-protein alpha subunits (G alpha i1 and G alpha z) could not be detected. Separation of subcellular organelles from homogenized eosinophils by density gradient centrifugation revealed that all of the detected GTP-binding proteins were mainly expressed in fractions containing peak plasma membrane and Golgi marker enzyme activities, while G beta subunits were also detected in secretory granule fractions. However, isoforms of Rab3, a putative GTP-binding regulator of exocytotic fusion, were undetectable in eosinophils. Neither, with the exception of syntaxin-3, could we detect any of the proteins belonging to the proposed synaptic vesicle fusion complex (SNAP-25; synaptobrevin (VAMP) and its non-neuronal homologue, cellubrevin; synaptophysin; synaptotagmin). The results from this study, based on western blotting, suggest that eosinophils express a different class of exocytotic fusion complex proteins from those found in neuronal tissues, although a number of potential candidates fulfilling the role of GE were identified in this important inflammatory cell.

THE ROLE OF INOSITIDES, PHOSPHOLIPASE C AND G-PROTEINS IN RECEPTOR TRANSDUCTION

1987 ◽  
Author(s):  
Eduardo G Lapetina
Keyword(s):  
Phospholipase C ◽  
Pertussis Toxin ◽  
Binding Proteins ◽  
Binding Protein ◽  
Gtp Binding Protein ◽  
Gtp Binding Proteins ◽  
Cytosolic Phospholipase ◽  
Gtp Binding ◽  
Membrane Bound ◽  
Stimulation Of

It is now widely recognized that the activation of phospholipase C by specific agonists leads to the formation of two second messengers: (1) inositol trisphosphate, which releases Ca2+ from the endoplasmic reticulum to the cytosol and (2) 1,2- diacylglycerol, which stimulates protein kinase C. In the past few years, GTP-binding proteins have been associated with the regulation of phospholipase C. However, the identity of the GTP-binding protein involved and the type of association with phospholipase C is not yet known. It is now recognized that there are two types of phospholipase C enzymes: (a) a soluble enzyme that has been characterized in several tissues and does not preferentially hydrolyze polyphospholinositides and (b) membrane-bound enzymes that are coupled to the receptors, specifically hydrolyzing polyphosphoinositides and activated by membrane guanine nucleotide-binding proteins. Recent reports have tried to assess the involvement of GTP-binding proteins in the agonist-induced stimulation of phospholipase C, and various related aspects have been reported. These are concerned with: (a) detection of various GTP-binding proteins in platelets, (b) the effects of known inhibitors of GTP-binding proteins such as GDPgS or pertussis toxin on the agonist-induced stimulation of phospholipase C, (c) the direct effects of stimulators of GTP-binding proteins such as GTP, GTP-analogs and fluoride on phospholipase C activity, (d) the possible association of GTP-binding proteins to cytosolic phospholipase C that would then lead to degradation of the membrane-bound inositides and (e) cytosolic phospholipase C response to the activation of cell surface receptors. The emerging information has had contradictory conclusions. (1) Pretreatment of saponin-permeabilized platelets with pertussis toxin has been shown to enhance and to inhibit the thrombin-induced activation of phospholipase C. Therefore, it is not clear if a G protein that is affected by pertussis toxin in a manner similar to Gi or Go plays a central role in activation of phospholipase C. (2) Studies on the effect of GDPβ;S are also conflicting indicating that there may be GTP-independent and/or -dependent pathways for the activation of phosphoinositide hydrolysis. (3) A cytosolic phospholipase C is activated by GTP, and it has been advanced that this activity might trigger the hydrolysis of membrane-bound inositides. A cytosolic GTP-binding protein might be involved in this action, and it is speculated that an α-subunit might be released to the cytoplasm by a receptor-coupled mechanism to activate phospholipase C. However, no direct evidence exists to support this conclusion. Moreover, the exact contribution of phospholipase C from the membranes or the cytosol to inositide hydrolysis in response to cellular agonists and the relationship of those activites to membrane-bound or soluble GTP-binding proteins are unknown. Our results indicate that the stimulation of phospholipase C in platelets by GDPβS and thrombin are affected differently by GDPβS. GDPgSinhibits the formation of inositol phosphates produced by GTPγS but not that induced by thrombin. Thrombin, therefore, can directly stimulate phospholipase C without the involvement of a “stimulatory” GTP-binding protein, such as Gs, for the agonist stimulation of adenylate cyclase. However, an “inhibitory” GTP-binding protein might have some influence on thrombin-stimulated phospholipase C, since in the presence of GDPγS thrombin produces a more profound stimulation of phospholipase C.This “inhibitory” GTP-binding protein might be ADP-ribosylated by pertussis toxin because pertussis toxin can also enhance thrombin action on phospholipase C activity. Therefore, phospholipase C that responds to thrombin could be different from the one that responds to GTPγS. Cytosolic phospholipase C can be activated by GTP or GTP analogs, and the one that responds to thrombin should be coupled to the receptors present in the plasma membrane. The initial action of thrombin is to directly activate the plasma membrane-bound phospholipase C and the mechanism of this activation is probably related to the proteolytic action of thrombin or the activation of platelet proteases by thrombin. In agreement with this, trypsin can also directly activate platelet phospholipase C and, subsequently, GTPyS produces further activation of phospholipase C. If these two mechanisms are operative in platelets, the inhibition of cytosolic phospholipase C by GDPβS would allow a larger fraction of inositides for degradation of the thrombin-stimulated phospholipase C, as our results show.

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