scholarly journals Differential effects of G-protein activators on 5-hydroxytryptamine and platelet-derived growth factor release from streptolysin-O-permeabilized human platelets

1996 ◽  
Vol 314 (1) ◽  
pp. 123-128 ◽  
Author(s):  
Philip J. PADFIELD ◽  
Ninder PANESAR ◽  
Patricia HENDERSON ◽  
Joseph J. BALDASSARE
Keyword(s):  
Growth Factor ◽  
G Protein ◽  
G Proteins ◽  
Protein S ◽  
Human Platelets ◽  
Dense Core ◽  
Platelet Derived Growth Factor ◽  
Permeabilized Cells ◽  
Streptolysin O ◽  
Dense Core Granules

In this paper we have used streptolysin O (SLO)-permeabilized human platelets to examine the G-protein(s) that control Ca2+-independent secretion from α and dense-core granules. As shown for electropermeabilized platelets, Ca2+ alone stimulated a concentration-dependent increase in 5-hydroxytryptamine (5-HT) (dense-core-granule marker) and platelet-derived growth factor (PDGF) (α-granule marker) release from the SLO-permeabilized cells. The EC50 values for Ca2+-dependent 5-HT and PDGF release were 5 μM and 10 μM respectively. Guanosine 5´-[γ-thio]triphosphate (GTP[S]) (100 μM) stimulated Ca2+-independent release from both α and dense-core granules. In contrast, AlF4- had no effect on Ca2+-independent release from either α or dense-core granules. Neither GTP[S] nor AlF4- appeared to have a significant effect on Ca2+-dependent release from α and dense-core granules. GTP[S] can activate both heterotrimeric and low-molecular-mass G-proteins, whereas AlF4- activates only heterotrimeric G-proteins. Our results, therefore suggest that secretion in the human platelet is regulated by a small G-protein. Both GTP[S]- and Ca2+-dependent secretion were effected by extending the time between permeabilization with SLO and stimulation of secretion. GTP[S]-stimulated secretion from α and dense-core granules decreased rapidly after permeabilization. In contrast, Ca2+-dependent 5-HT and PDGF release ran down at a much lower rate. These observations indicate that GTP[S] and Ca2+ act through parallel pathways to stimulate secretion from SLO-permeabilized platelets.

scholarly journals Exocytosis from permeabilized bovine adrenal chromaffin cells is differently modulated by guanosine 5′-[γ-thio]triphosphate and guanosine 5′-[β γ-imido]triphosphate. Evidence for the involvement of various guanine nucleotide-binding proteins

1992 ◽  
Vol 284 (2) ◽  
pp. 321-326 ◽  
Author(s):  
G Ahnert-Hilger ◽  
U Wegenhorst ◽  
B Stecher ◽  
K Spicher ◽  
W Rosenthal ◽  
...  
Keyword(s):  
G Protein ◽  
Chromaffin Cells ◽  
Inhibitory Effect ◽  
Adrenal Chromaffin Cells ◽  
Prolonged Incubation ◽  
Permeabilized Cells ◽  
Alpha Subunits ◽  
Cytoplasmic Material ◽  
Streptolysin O ◽  
Adrenal Chromaffin

1. In bovine adrenal chromaffin cells made permeable either to molecules less than or equal to 3 kDa with alphatoxin or to proteins less than or equal to 150 kDa with streptolysin O, the GTP analogues guanosine 5′-[beta gamma-imido]triphosphate (p[NH]ppG) and guanosine 5′-[gamma-thio]triphosphate (GTP[S]) differently modulated Ca(2+)-stimulated exocytosis. 2. In alphatoxin-permeabilized cells, p[NH]ppG up to 20 microM activated Ca(2+)-stimulated exocytosis. Higher concentrations had little or no effect. At a free Ca2+ concentration of 5 microM, 7 microM-p[NH]ppG stimulated exocytosis 6-fold. Increasing the free Ca2+ concentration reduced the effect of p[NH]ppG. Pretreatment of the cells with pertussis toxin prevented the activation of the Ca(2+)-stimulated exocytosis by p[NH]ppG. 3. In streptolysin O-permeabilized cells, p[NH]ppG did not activate, but rather inhibited Ca(2+)-dependent catecholamine release under all conditions studied. In the soluble cytoplasmic material that escaped during permeabilization with streptolysin O, different G-protein alpha-subunits were detected using an appropriate antibody. Around 15% of the cellular alpha-subunits were detected in the supernatant of permeabilized control cells. p[NH]ppG or GTP[S] stimulated the release of alpha-subunits 2-fold, causing a loss of about 30% of the cellular G-protein alpha-subunits under these conditions. Two of the alpha-subunits in the supernatant belonged to the G(o) type, as revealed by an antibody specific for G(o) alpha. 4. GTP[S], when present alone during stimulation with Ca2+, activated exocytosis in a similar manner to p[NH]ppG. Upon prolonged incubation, GTP[S], in contrast to p[NH]ppG, inhibited Ca(2+)-induced exocytosis from cells permeabilized by either of the pore-forming toxins. This effect was resistant to pertussin toxin. 5. The p[NH]ppG-induced activation of Ca(2+)-stimulated release from alphatoxin-permeabilized chromaffin cells may be attributed to one of the heterotrimeric G-proteins lost during permeabilization with streptolysin O. The inhibitory effect of GTP[S] on exocytosis is apparently not mediated by G-protein alpha-subunits, but by another GTP-dependent process still occurring after permeabilization with streptolysin O.

scholarly journals Differential mechanisms of inositol phosphate generation at the receptors for bombesin and platelet-derived growth factor

1989 ◽  
Vol 262 (2) ◽  
pp. 665-668 ◽  
Author(s):  
M G Cattaneo ◽  
L M Vicentini
Keyword(s):  
Growth Factor ◽  
Kinase Activity ◽  
Inositol Phosphate ◽  
Growth Factor Receptor ◽  
Platelet Derived Growth Factor ◽  
3T3 Cells ◽  
Tyrosine Kinase Activity ◽  
Independent Manner ◽  
Streptolysin O ◽  
Bombesin Receptor

We investigated the mechanism(s) whereby activation of a growth-factor receptor typically endowed with tyrosine kinase activity, such as the platelet-derived growth factor (PDGF) receptor, triggers phosphoinositide hydrolysis. In Swiss 3T3 cells permeabilized with streptolysin O, an analogue of GTP, guanosine 5′-[gamma-thio]triphosphate, was found to potentiate the coupling of the bombesin receptor to phospholipase C. In contrast, the activation of the enzyme by PDGF occurred in a GTP-independent manner. Moreover, the inactive analogue of GTP, guanosine 5′-[beta-thio]diphosphate, significantly inhibited the bombesin-induced InsP3 generation, whereas it did not decrease the same effect when stimulated by PDGF.

scholarly journals A major part of platelet-derived growth factor purified from human platelets is a heterodimer of one A and one B chain.

1988 ◽  
Vol 263 (31) ◽  
pp. 16493-16498 ◽  
Author(s):  
A Hammacher ◽  
U Hellman ◽  
A Johnsson ◽  
A Ostman ◽  
K Gunnarsson ◽  
...  
Keyword(s):  
Growth Factor ◽  
Major Part ◽  
Human Platelets ◽  
Platelet Derived Growth Factor

scholarly journals Tethering of the Platelet-derived Growth Factor β Receptor to G-protein-coupled Receptors

2001 ◽  
Vol 276 (30) ◽  
pp. 28578-28585 ◽  
Author(s):  
Forbes Alderton ◽  
Soma Rakhit ◽  
Kok Choi Kong ◽  
Timothy Palmer ◽  
Balwinder Sambi ◽  
...  
Keyword(s):  
Growth Factor ◽  
G Protein ◽  
G Protein Coupled Receptors ◽  
Platelet Derived Growth Factor ◽  
Β Receptor ◽  
G Protein Coupled

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 Characterization of the tyrosine phosphorylation of calpactin I (annexin II) induced by platelet-derived growth factor

1991 ◽  
Vol 278 (2) ◽  
pp. 447-452 ◽  
Author(s):  
R Brambilla ◽  
R Zippel ◽  
E Sturani ◽  
L Morello ◽  
A Peres ◽  
...  
Keyword(s):  
Growth Factor ◽  
Tyrosine Phosphorylation ◽  
Platelet Derived Growth Factor ◽  
Annexin Ii ◽  
Necessary Condition ◽  
Pdgf Receptor ◽  
Permeabilized Cells ◽  
Phosphorylated Proteins ◽  
3T3 Fibroblasts

Stimulation in vivo of Swiss 3T3 fibroblasts with platelet-derived growth factor (PDGF) in the presence of orthovanadate induces the tyrosine phosphorylation of a 39 kDa protein, identified as the phosphorylated slow-migrating form of calpactin I (annexin II) heavy chain, p36. In fact, in PDGF-stimulated cells, anti-(calpactin I) antibodies recognize a doublet of bands, p36 and p39, and the latter disappears upon treatment with phosphatase. In many regards phosphorylation of p39 differs from the rapid and transient phosphorylation of the PDGF receptor and of other substrates: (a) it has slower kinetics but is then stable for longer periods of time; (b) it occurs at 37 degrees C but not at 4 degrees C; and (c) whereas most of the tyrosine-phosphorylated proteins are associated with membrane-enriched preparations, membrane association of p39 only occurs in the presence of Ca2+. Moreover, calpactin I leaks out of permeabilized cells at 0.1 microM free Ca2+, whereas it remains associated with the cells at concentrations of Ca2+ greater than or equal to 1 microM. PDGF does not stimulate phosphoinositide turnover (and thus Ca2+ mobilization) at 4 degrees C; thus it can be suggested that the Ca(2+)-dependent translocation of the protein to membrane/cytoskeletal structures is a necessary condition for its phosphorylation. In addition, calpactin I may not be a direct substrate for the PDGF receptor kinase, but rather the substrate of another tyrosine kinase activated by the receptor.

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.

scholarly journals A predictive computational model reveals that GIV/girdin serves as a tunable valve for EGFR-stimulated cyclic AMP signals

2019 ◽  
Vol 30 (13) ◽  
pp. 1621-1633 ◽  
Author(s):  
Michael Getz ◽  
Lee Swanson ◽  
Debashish Sahoo ◽  
Pradipta Ghosh ◽  
Padmini Rangamani
Keyword(s):  
Growth Factor ◽  
G Protein ◽  
G Proteins ◽  
Cross Talk ◽  
Tyrosine Kinases ◽  
Control Valve ◽  
Camp Signaling ◽  
Nucleotide Exchange ◽  
Camp Concentration ◽  
Camp Levels

Cellular levels of the versatile second messenger cyclic (c)AMP are regulated by the antagonistic actions of the canonical G protein → adenylyl cyclase pathway that is initiated by G-protein–coupled receptors (GPCRs) and attenuated by phosphodiesterases (PDEs). Dysregulated cAMP signaling drives many diseases; for example, its low levels facilitate numerous sinister properties of cancer cells. Recently, an alternative paradigm for cAMP signaling has emerged in which growth factor–receptor tyrosine kinases (RTKs; e.g., EGFR) access and modulate G proteins via a cytosolic guanine-nucleotide exchange modulator (GEM), GIV/girdin; dysregulation of this pathway is frequently encountered in cancers. In this study, we present a network-based compartmental model for the paradigm of GEM-facilitated cross-talk between RTKs and G proteins and how that impacts cellular cAMP. Our model predicts that cross-talk between GIV, G αs, and G αi proteins dampens ligand-stimulated cAMP dynamics. This prediction was experimentally verified by measuring cAMP levels in cells under different conditions. We further predict that the direct proportionality of cAMP concentration as a function of receptor number and the inverse proportionality of cAMP concentration as a function of PDE concentration are both altered by GIV levels. Taking these results together, our model reveals that GIV acts as a tunable control valve that regulates cAMP flux after growth factor stimulation. For a given stimulus, when GIV levels are high, cAMP levels are low, and vice versa. In doing so, GIV modulates cAMP via mechanisms distinct from the two most often targeted classes of cAMP modulators, GPCRs and PDEs.

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