AbstractThe effect of the inositol phospholipid-binding antibiotic neomycin was studied on high-affinity GTPase in human platelet membranes. At low concentrations (up to 1 mM), neomycin by itself stimulated a high-affinity GTPase. This GTPase stimulation was additive with that caused by the hormonal factors, prostaglandin E1 and epinephrine, but not with thrombin. At concentrations higher than 1 mM, neomycin reduced control GTPase activity and eliminated the stimulation caused by thrombin. The data suggest that neomycin by a presently unknown mechanism can regulate activity states of signal transducing GTP-binding proteins

Herrmann, Eva

Jakobs, Karl H.

Elsevier - Publisher Connector 

Volume 229, number 1, 49-53 FEB 05594 February 1988 
Stimulation and inhibition of human platelet membrane 
high-affinity GTPase by neomycin 
Eva Her rmann and Kar l  H. Jakobs  
Pharmakologisches In titut der Universitiit Heidelberg, Im Neuenheimer FeM 366, D-6900 Heidelberg, FRG 
Received 23 December 1987 
The effect of the inositol phospholipid-binding a tibiotic neomycin was studied on high-affinity GTPase in human platelet 
membranes. At low concentrations (up to 1 mM), neomycin by itself stimulated a high-affinity GTPas¢. This GTPase 
stimulation was additive with that caused by the hormonal factors, prostaglandin E 1and epinephrine, but not with 
thrombin. At concentrations higher than 1 raM, neomycin reduced control GTPas¢ activity and eliminated the stimula- 
tion caused by thrombin. The data suggest that neomycin by a presently unknown mechanism can regulate activity states 
of signal transducing GTP-binding proteins. 
Neomycin; GTP-binding protein; GTPase; Phospholipase C; (Human platelet) 
1. INTRODUCTION 
The aminoglycoside antibiotic neomycin binds 
specifically to polyphosphoinositides in com- 
parison to other phospholipids [1,2]. Therefore, 
this antibiotic has been used in many studies to in- 
hibit polyphosphoinositide-degrading phospho- 
lipase C and subsequent cellular processes [3-7]. 
Because of the polarity of the compound, neo- 
mycin is usually added to permeabilized cells or 
subcellular fractions in order to gain access of the 
antibiotic to the cytoplasmic site of the plasma 
membrane. 
In the last few years, ample functional evidence 
has been presented that receptor-mediated activa- 
tion of  phospholipase C by hormones and 
neurotransmitters involves the intermediate action 
of guanine nucleotide-binding proteins (G- 
proteins) [8-10], located at the inner surface of the 
plasma membrane. The nature of  the 
phospholipase C-coupling G-protein(s) has not 
been identified so far. In order to gain more insight 
Correspondence address: K.H. Jakobs, Pharmakologisches In- 
stitut der Universit~it Heidelberg, Im Neuenheimer Feld 366, 
D-6900 Heidelberg, FRG 
into the inhibitory action of neomycin on receptor- 
stimulated phospholipase C we studied whether, in 
addition to its well known binding to the substrates 
of phospholipase C, polyphosphoinositides, 
neomycin may also affect the activator of the 
phospholipase C, namely the G-proteins. The 
function of these proteins can be measured in 
crude membrane preparations as high-affinity 
GTPase activity [10,11]. In the course of these 
studies, we observed that in human platelet mem- 
branes neomycin by itself can markedly modify the 
activity of high-affinity GTPases. 
2. MATERIALS AND METHODS 
2.1. Materials 
Neomycin sulfate, thrombin (human), epinephrine, 
prostaglandin E1 (PGE1) and indomethacin were obtained from 
Sigma, Deisenhofen, FRG. GTP, ATP, creatine phosphate and 
crcatine kinase were purchased from Boehringer, Mannheim. 
[y-3~P]GTP was prepared as described in [12]. 
2.2. Preparation of human platelet membranes 
Crude membranes of human platelets were prepared as in 
[13] with 5 mM EDTA used throughout the membrane prepara- 
tion procedure. Small aliquots frozen at -70°C were thawed 
immediately before the GTPase assay and recentdfuged for 
10 min at 30000 x g in 10 mM triethanolamine-HCl, pH 7.4, 
Published by Elsevier Science Publishers B. V. (Biomedical Division) 
00145793/88/$3.50 © 1988 Federation of European Biochemical Societies 49 
Volume 229, number 1 FEBS LETTERS February 1988 
containing 5 mM EDTA, and the membranes were resuspended 
in 10 mM triethanolamine-HCl, pH 7.4. 
2.3. GTPase assay 
High-affinity GTPase activity was determined with a reaction 
mixture containing, if not otherwise indicated, 0.1-0.3/zM 
[9,-32p]GTP (0.1/zCi/tube), 0.1 mM ATP, 0.1 mM EGTA, 
2 mM MgC12, 1 mM dithiothreitol, 5 mM creatine phosphate, 
0.4 mg/ml creatine kinase, 2 mg/ml bovine serum albumin and 
the additions indicated in 50 mM triethanolamine-HC1, pH 7.4, 
in a total volume of 100/zl. Reactions were started by adding 
platelet membranes (5-10/~g protein/tube) to the pre- 
equilibrated reaction mixture and conducted for i0 min at 
25°C. Stopping of the reactions and isolation of 32pi formed 
were performed with the charcoal method as in [13]. High- 
affinity GTPase activity was calculated from the difference bet- 
ween total 32Pi formed and 32pi formed in the presence of 
50~M GTP, which amounted to 5-10070 of total 32pi for- 
mation. 
3. RESULTS 
When human platelet membrane GTPase activi- 
ty was studied with 0.1/zM GTP as substrate, 
neomycin added at concentrations from 0.1 to 
10 mM caused abiphasic response (fig. 1). At con- 
centrations up to about 1 mM, neomycin i creased 
GTP hydrolysis with a maximal increase of 
60-80070 observed at 0.8 mM neomycin. Half- 
maximal stimulation was observed at 0.1-0.2 mM 
neomycin. At above 1 mM, neomycin reduced 
GTPase activity, being similar to or slightly less 
than control activity at 10 mM neomycin. To 
determine whether the GTPase stimulation ob- 
served at the low concentrations of neomycin was 
due to a high-affinity GTPase, substrate saturation 
experiments were performed. As shown in fig.2, 
neomycin added at 0.8 mM increased GTPase ac- 
tivity for any low GTP concentration studied. The 
apparent Km values of the GTPase observed in the 
absence and presence of neomycin were very 
similar, 0.19 +_ 0.05 and 0.23 ± 0.03/zM (SE, n = 
3), respectively, while the Vm~ of the high-affinity 
GTPase was increased by neomycin by about 75 070. 
Since it has recently been described that 
neomycin's stimulatory effects on phospholipase C 
activity in permeabilized platelets are blocked by 
addition of EGTA or indomethacin [14], we per- 
formed similar experiments tothose shown in fig. 1 
in the absence of these agents and in the presence 
of either 1 mM EGTA or 10/~M indomethacin. As
illustrated in fig.3, neither the stimulatory nor the 
inhibitory phase of neomycin's actions on human 
, /I 
t- 
o 
E 
r" 
E 
o_ 
E 
/. 
04 ~ 0  \ 
Neomycin (raM) 
Fig.l. Influence of neomycin on platelet membrane GTPase 
activity. GTPase activity of human platelet membranes was 
studied with 0.1/zM GTP as substrate at the indicated 
concentrations of neomycin. 
platelet membrane GTPase was essentially altered 
by the presence of these agents. 
In human platelet membranes, a wide variety of 
hormone receptors after activation by their specific 
agonists interact with guanine nucleotide-binding 
proteins. This interaction can be monitored as an 
increase in the respective G-protein GTPase activi- 
ty [13,15-18]. Therefore, we were interested in 
whether the stimulation of the hormone receptor- 
dependent GTPases is modulated by neomycin be- 
ing present at either stimulatory or inhibitory con- 
centrations. As shown in fig.4, neomycin 
(0.8 raM) increased GTPase activity to an extent 
similar to that of PGE1 (10/zM) and epinephrine 
(30/zM), which activate the stimulatory (Gs) and 
inhibitory (Gi) G-protein of the adenylate cyclase 
system, respectively. When combined with 
neomycin at the low stimulatory concentration 
(0.8 raM), at least partial additivity between the ef- 
fects of the hormonal agents and that of neomycin 
was observed. The most effective GTPase ac- 
tivator in platelet membranes i  thrombin, which 
activates both (}i and an as yet unidentified G- 
protein coupling to phospholipase C [16-18]. In 
the presence of thrombin (0.1 U/ml), addition of 
neomycin (0.8 mM) had only a minimal effect. 
50 
Volume 229, number l FEBS LETTERS February 1988 
-~ 0.4 
~ 
" Co I 0 ° 
0.3 o o~.~I / , .  ~ 02 
~/  Neomycin 
o ~ ~b ~'s 
I/GTP (~M -1) 
Fig.2. Activation of a high-affinity GTPase by neomycin. 
GTPase activity was studied at various concentrations of GTP 
in the absence (o) and presence (o) of 0.8 mM neomycin. 
Double-reciprocal plots are shown. 
The effects of neomycin added at a complete 
concentration-response curve on basal and 
thrombin-stimulated GTPase are shown in fig.5. 
Up to 1 mM, neomycin had only a very small ef- 
• - f~l  , i , 
Indomet hac in /  ~@'@'~k 
• ~,_ %~.o  o. \ 
.<  . 
o 
E EGTA ~- 6 o4 
.-- Control ~ ~  
13.. 
a .  
4 
6 n dl ~ ib 
Neomycin (raM) 
Fig.3. Influence of EGTA and indomethacin on regulation of 
GTPase by neomycin. GTPas¢ activity was studied with 0.2/zM 
GTP as substrate in the absence (o) and presence of either 
1 mM EGTA (m) or 10/LM indomethacin (e) at the indicated 
concentrations of neomycin. 
t- 
O .4, 
O 
E 
"7 r- 
E 
&.  
O 
E t'~ 
12 
8 
0 
[ ]  : Control 
Basal PGE1 Epi Thrombin 
Fig.4. Influence of neomycin on hormone-stimulated GTPase 
activity. GTPase activity was studied with 0.1/zM GTP as 
substrate in the absence (open bars) and presence (hatched bars) 
of 0.8 mM neomycin without (basal) and with 10/zM PGEh 
30/zM epinephrine (Epi) or 0.1 U/ml thrombin as indicated. 
feet on thrombin-stimulated GTPase activity, 
while basal activity was increased by about 60°70. 
At higher concentrations, neomycin not only 
reduced control GTPase activity, but also even 
16 
"T 
E 
0 
,"% 12 
E 
E 
IE 
. -  8 
13_ 
0 
E 
Q.  
, I I  , , 
0.~ 0~0 
Thro!bin 
/o fO~O_ @~ 
o \ 
' o,\ 
o I \" 
Control O~I  
6" 0'.I ~ 1o 
Neomycin (raM) 
Fig.5. Influence of neomycin on thrombin-stimulated GTPase 
activity. GTPase activity was studied with 0.3/~M GTP as 
substrate in the absence (o). and presence (e) of 0.1 U/ml 
thrombin at the indicated concentrations of neomycin. 
51 
Volume 229, number 1 FEBS LETTERS February 1988 
more specifically reduced GTPase stimulation by 
thrombin, which was compleletely absent at 
10 mM neomycin. 
4. DISCUSSION 
The present data demonstrate that the 
aminoglycoside antibiotic neomycin at rather low 
concentrations can be a very effective stimulator of 
a high-affinity GTPase in platelet membranes. The 
extent of GTPase stimulation caused by neomycin 
was similar to that induced by the hormonal fac-. 
tors PGE1 and epinephrine, activating the 
stimulatory and inhibitory G-protein of the 
adenylate cyclase system, respectively. When com- 
bined with one of these agents, both at maximally 
effective concentrations, at least partial additivity 
in GTPase activation was observed. In contrast, in 
combination with thrombin, which activates Gi 
and the phospholipase C-coupling G-protein, at a 
just maximally effective concentration [ 16,17], no 
additivity was observed. At high concentrations 
(> 1 mM), neomycin reduced basal GTPase and 
even more specifically attenuated the GTPase 
stimulation caused by thrombin. 
These biphasic responses observed with 
neomycin on high-affinity GTPase are in excellent 
agreement with recently published data on 
neomycin's action on permeabilized human 
platelets [14,19]. At low concentrations, with a 
maximal response observed between 0.4 and 
1 mM, neomycin was shown to induce inositol 
phosphate and phosphatidic acid formation as well 
as arachidonic release, platelet aggregation and 
serotonin release. At higher concentrations, 
neomycin inhibited platelet aggregation and 
serotonin release. At 10 mM, the stimulatory ef- 
fects of neomycin were lost [14]. This bell-shaped 
pattern of concentration response curve was also 
found in studying neomycin's actions on high- 
affinity GTPase in platelet membranes. 
The intriguing question is how neomycin exerts 
its effects on high-affinity GTPase. From the func- 
tional studies, in which an activation of G-protein- 
regulated effector systems by neomycin has been 
observed [14,19], it appears to be unlikely that 
neomycin simply activates the GTP-hydrolyzing 
activity of a G-protein. Such increased hydrolysis 
would induce inhibition rather than stimulation of 
effector enzymes uch as the phospholipase C. It 
may be speculated that neomycin binds directly to 
a G-protein and, thereby, by itself mimics the in- 
teraction of an agonist-activated receptor with a G- 
protein. From the combined ata, it seems most 
likely that the G-protein activated by neomycin is 
of the same nature as that activated by thrombin. 
It is also feasible that the hydrophilic ompound 
neomycin somehow induces the coupling of an 
agonist-free receptor with the G-protein and, 
thereby, also mimics the action of an agonist- 
activated receptor. At high concentrations, 
neomycin may additionally inhibit the binding of 
GTP to the G-protein and, thereby, decrease con- 
trol activity and agonist (thrombin)-stimulated 
GTP hydrolysis. Last but not least, although not 
very likely from the functional studies, it cannot be 
excluded that neomycin by binding to 
polyphosphoinositides somehow regulates the ac- 
tivity state of membrane proteins such as G- 
proteins and phospholipase C. Recently, evidence 
has been presented that inositol-containing 
phospholipids can constitute an anchoring domain 
for certain plasma membrane proteins [20]. The 
final answer to this question may be obtained in 
studying neomycin's effects on the purified mem- 
brane proteins. 
Acknowledgements: Thi  work was supported by the Deutsche 
Forschungsgemeinsehaft and the Fonds der Chemischen In- 
dustrie. 
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Stimulation and inhibition of human platelet membrane high-affinity GTPase by neomycin 

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Stimulation and inhibition of human platelet membrane high-affinity GTPase by neomycin

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