From the Departments of Pharmacology and Toxicology
and § Medicinal Chemistry, The University of Kansas,
Lawrence, Kansas 66045-7582 and ¶ Department of Chemistry and
Physics, Southeastern Louisiana University, Hammond, Louisiana
70402-0878
Received for publication, October 3, 2002, and in revised form, December 13, 2002
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ABSTRACT |
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Xanthine nucleotide-selective small GTP-binding
proteins with an Asp/Asn mutation are valuable for the analysis of
individual GTP-binding proteins in complex systems. Similar
applications can be devised for heterotrimeric G-proteins. However,
Asp/Asn mutants of G G-proteins are heterotrimeric ( G Our study aim was to generate xanthine nucleotide-selective mutants of
G Materials--
Baculovirus encoding G NTP Construction of cDNAs for G Sf9 Cell Culture and Membrane Preparation--
Sf9
cells were cultured in 250-ml disposable Erlenmeyer flasks at 28 °C
under shaking at 125 rpm in SF 900 II medium (Invitrogen). Recombinant
baculoviruses were generated in Sf9 cells using the BaculoGOLD
transfection kit (Pharmingen) as described previously (23). For protein
expression, Sf9 cells were infected with 1:100 dilutions of high
titer baculovirus stocks and cultured for 48 h (17). Sf9
membranes were prepared as described previously (17). Membranes were
suspended in binding buffer (12.5 mM MgCl2, 1 mM EDTA, and 75 mM Tris/HCl, pH 7.4) at a
concentration of ~1-2 mg protein/ml and stored at AC Activity--
The determination of AC activity in Sf9
membranes was performed as described previously (23). Briefly, tubes
(30 µl) contained membranes (20-40 µg protein/tube), 5 mM MgCl2, 0.4 mM EDTA, 30 mM Tris/HCl, pH 7.4, and nucleotides at various
concentrations. Some experiments were conducted in the presence of ISO
or ICI 118,551. Tubes were incubated for 3 min at 37 °C before the
addition of 20 µl of reaction mixture containing (final)
[ NTPase Activity--
High-affinity GTPase activity in Sf9
membranes expressing Immunoblot Analysis--
Membrane proteins were separated on
SDS-polyacrylamide gels containing 10% (w/v) acrylamide. Proteins were
transferred onto Immobilon-P transfer membranes (Millipore, Bedford,
MA). Membranes were reacted with anti-G Miscellaneous--
Protein concentrations were determined with
the Bio-Rad DC protein assay kit (Bio-Rad). Data shown in Figs. 2-4
were analyzed by nonlinear regression using the Prism 3.02 software
(GraphPad, Prism, San Diego, CA).
Expression of G Regulation of AC Activity by NTPs, NTP
G
In membranes expressing G
In agreement with the NTP Regulation of AC Activity by NTPs, NTP
Similar to the observations for the
G Regulation of AC Activity in Sf9 Membranes Expressing
G Role of GDP Affinity in the Function of Xanthine
Nucleotide-selective G
G
The requirement for the additional Gln/Leu mutation in
G Perturbation of the Catalytic Site Alters Nucleotide Selectivity of
G
The Gln/Leu mutation perturbs the structure of the catalytic site as
well (6). In G Conclusions--
Go, G
11, and
G
16 were inactive. An additional Gln/Leu mutation in the
catalytic site, reducing GTPase activity and increasing GDP
affinity, was required to generate xanthine nucleotide-selective unspecified G-protein
-subunit (G
). Our study aim was to
generate xanthine nucleotide-selective mutants of G
s,
the stimulatory G-protein of adenylyl cyclase. The short splice variant
of G
s (G
sS) possesses higher GDP affinity
than the long splice variant (G
sL). Nucleoside
5'-[
-thio]triphosphates (NTP
Ss) and nucleoside 5'-[
,
-imido]triphosphates effectively activated a
G
sS mutant with a D280N exchange
(G
sS-N280), whereas nucleotides activated a
G
sL mutant with a D295N exchange
(G
sL-N295) only weakly. The Gln/Leu mutation enhanced
G
sL-N295 activity. NTP
Ss activated G
sS-N280 and a G
sL mutant with a Q227L
and D295N exchange (G
sL-L227/N295) with similar
potencies, whereas xanthosine 5'-triphosphate and xanthosine
5'-[
,
-imido]triphosphate were more potent than GTP and
guanosine 5'-[
,
-imido]triphosphate, respectively.
G
sS-N280 interacted with the
2-adrenoreceptor and exhibited high-affinity XTPase
activity. Collectively, (i) G
sS-N280 is the first
functional xanthine nucleotide-selective G
with the Asp/Asn mutation
alone; (ii) sufficiently high GDP affinity is crucial for G
Asp/Asn mutant function; (iii) with nucleoside 5'-triphosphates and nucleoside 5'-[
,
-imido]triphosphates, G
s-N280 and
G
sL-L227/N295 exhibit xanthine nucleotide
selectivity, whereas NTP
Ss sterically perturb the catalytic site of
G
and annihilate xanthine selectivity.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-structure) and mediate
transmembrane signal transfer between receptors and effectors (1-3). Activated receptor promotes GDP dissociation from
G
.1 GDP dissociation is
the rate-limiting step of the G-protein cycle. Subsequently, receptor
catalyzes GTP binding to G
. GTP binding to G
induces the active
conformation of the G-protein, leading to the dissociation of the
heterotrimer into G
-GTP and the
-complex. Both G
-GTP and
regulate the activity of effector systems. G
possesses GTPase
activity. The GTPase hydrolyzes GTP into GDP and Pi and
thereby deactivates the G-protein. G
-GDP and
reassociate, completing the G-protein cycle. The GTP analogs GTP
S and GppNHp are
GTPase-resistant and persistently activate G-proteins (1, 4). The
-thiophosphate of GTP
S is bulkier than the
-phosphate of
GppNHp/GTP (4, 5). As a result of these chemical differences, GTP
S,
unlike GppNHp, sterically perturbs the structure of the catalytic site
of G
(6). These crystallographic data imply that G
-GppNHp
resembles G
-GTP more closely than G
-GTP
S.
consists of the ras-like domain that is structurally
similar to small GTP-binding proteins and the
-helical domain that is unique in G
. The two domains embed the nucleotide-binding pocket
(3, 7). Nucleotide binding to G
involves several hydrogen and ionic
bonds. Of particular importance for guanine selectivity is an aspartate
that is conserved among small GTP-binding proteins and G
(3, 7-11).
Exchange of this aspartate against asparagine (Asp/Asn mutation) in
small GTP-binding proteins switches base selectivity from guanine to
xanthine. Such mutants are valuable to study a specific small
GTP-binding protein in complex systems containing multiple GTP-binding
proteins (8-11). Similar applications can be devised for G-proteins.
Unexpectedly, Asp/Asn mutants of G
o, G
11,
and G
16 were inactive (5, 12, 13). However, the
additional exchange of a conserved glutamine against leucine (Gln/Leu
mutation) in the catalytic site of G
(3, 7) resulted in active G
with the expected xanthine nucleotide selectivity (5, 12, 13). The
Gln/Leu mutation reduces GTPase activity and increases GDP-affinity of
G
(14, 15).
s. G
s mediates coupling of the
2AR to AC (1, 2). G
s exists as two splice
variants, G
sS and G
sL, with G
sS possessing ~2-3-fold higher GDP-affinity than
G
sL (16). We generated G
sS-N280,
G
sS-L212, G
sS-L212/N280,
G
sL-N295, G
sL-L227, and
G
sL-L227/N295 and analyzed these G
s
mutants with guanine-, hypoxanthine-, and xanthine-substituted NTPs,
NTP
Ss, and NppNHps. We used Sf9 cells as an expression system
because this system is suitable for G
s analysis, and
there is no interference of nucleoside diphosphokinase-mediated
trans(thio)phosphorylation reactions with the effects of NTPs/NTP
Ss
on G
s (17-19).
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
sS was
kindly donated by Dr. A. G. Gilman (Department of Pharmacology,
University of Southwestern Medical Center, Dallas, TX). Baculovirus
encoding G
sL was kindly donated by Dr. H. Bourne
(Department of Pharmacology, University of California, San Francisco,
CA). [
-32P]GTP (6000 Ci/mmol),
[
-32P]ATP (3000 Ci/mmol),
[32P]Pi (8500-9000 Ci/mmol), and
[3H]dihydroalprenolol (85-90 Ci/mmol) were from
PerkinElmer Life Sciences. Unlabeled ATP (special quality, <0.01%
(w/w) GTP as assessed by high pressure liquid chromatography, # 519 979), GDP, GTP, GTP
S, GppNHp, adenosine
5'-[
,
-imido]triphosphate, and adenosine
5'-[
-thio]triphosphate were of the highest quality available and
were obtained from Roche Molecular Biochemicals. [
-32P]XTP (~6000 Ci/mmol) was synthesized as
described previously (20). ISO, XDP, IDP, bovine liver
nucleoside diphosphokinase, and the M1 monoclonal antibody (anti-FLAG
Ig) were from Sigma. IppNHp and XppNHp were obtained from
JenaBioscience (Jena, Germany). ICI 118,551 was from RBI (Natick, MA).
Pfu DNA polymerase was from Stratagene (La Jolla, CA). All
restriction enzymes and DNA-modifying enzymes were from New England
Biolabs (Beverly, MA). The anti-G
s Ig (C-terminal) was
from Calbiochem (La Jolla, CA).
S Synthesis--
ITP
S and XTP
S were synthesized by
nucleoside diphosphokinase-catalyzed transthiophosphorylation as
described previously (21), with modifications. Briefly, reaction
mixtures contained 10 mM IDP or XDP and 5 mM
adenosine 5'-[
-thio]triphosphate in 30 mM Tris/HCl, pH
8.0, supplemented with 5.0 mM dithiothreitol and 5.0 mM MgCl2 in a total volume of 1.0 ml. The
reaction was initiated by the addition of 78 IU nucleoside
diphosphokinase and conducted at 37 °C for 24-48 h until the
reaction equilibrium resulted in maximum NTP
S product. The product
NTP
S was then purified by fast protein liquid chromatography mono Q
ion-exchange chromatography using either a 30 min linear gradient from
0.1-2 M ammonium acetate, pH >8.0, or by isocratic
elution in the same buffer (the gradient method allowed the recovery of
starting IDP/XDP). The collected product peak was lyophilized and, if
necessary, repurified using a shallow gradient. The final product was
analyzed for purity by both isocratic and gradient elution from the
same chromatography systems used for the purification. The pH > 8.0 ammonium acetate purification buffer produced synthetic NTP
S of
>98% purity based on fast protein liquid chromatography elution. At
lower pH, the product was a mixture of NTP
S and IDP/XDP at a ratio
of >88:12. Purified products contained 0.75-1.2 µmol of NTP
S as
assessed by UV absorption at
max relative to IDP/XDP standard.
s
Mutants--
cDNAs for G
s mutants were generated by
overlap extension PCR (22, 23) using
pGEM-3Z-
2AR-G
sL as template. Sense and antisense primers encoding for the Q227L mutation (L227) created a
diagnostic BanII site; sense and antisense primers encoding for the D295N mutation (N295) resulted in the loss of a
BglII site. For generation of G
sL-L227/N295
cDNA, pGEM-
2AR-G
sL-L227 served as
template, using the primers for generation of the D295N mutation.
Recombinant pGEM-3Z-
2AR-G
sL plasmids were
digested with EcoRI and XbaI and cloned into
pVL1392-
2AR-G
sL digested with
EcoRI and XbaI. To generate the corresponding
G
sS cDNAs, the NheI/EcoRI
fragment of pGEM-3Z-
2AR-G
sS replaced the
NheI/EcoRI fragment of
pGEM-3Z-
2AR-G
sL mutants. For generation
of pVL1392-G
s plasmids devoid of
2AR
cDNA, pVL1392-
2AR-G
s plasmids were
digested with SacI and PflMI to eliminate
2AR cDNA. The noncoherent DNA ends were filled with
Klenow fragment, and the pVL1392-G
s plasmids were
religated. Extensive restriction enzyme diagnostics and enzymatic sequencing confirmed all mutations.
80 °C until
use. Expression levels of
2AR-G
s fusion
proteins were determined by [3H]dihydroalprenolol
saturation binding (17). Immediately before AC and NTPase experiments,
membrane aliquots were thawed and centrifuged for 15 min at 4 °C and
15,000 × g to remove, as far as possible, endogenous
nucleotides (23).
-32P]ATP (0.5-1.5 µCi/tube) plus 40 µM ATP, 2.7 mM mono(cyclohexyl)ammonium phosphoenolpyruvate, 0.125 IU pyruvate kinase, 1.0 IU myokinase, and
0.1 mM cAMP. Reactions were conducted for 20 min. Stopping of reactions and separation of [
-32P]ATP from
[32P]cAMP were performed as described previously (23). In
some experiments, the incubation temperature was varied from
16-37 °C.
2AR-G
s fusion
proteins was determined as described previously (23). Briefly, tubes
(80 µl) contained membranes (10 µg protein/tube), 1.0 mM MgCl2, 0.1 mM EDTA, 0.1 mM ATP, 1 mM adenosine
5'-[
,
-imido]triphosphate, 5 mM creatine phosphate,
40 µg creatine kinase, 30 nM to 10 µM unlabeled GTP, 10 µM ISO, and 0.05% (w/v) bovine serum
albumin in 50 mM Tris/HCl, pH 7.4. Reaction mixtures were
incubated for 3 min at 25 °C before the addition of 20 µl of
[
-32P]GTP (2.0 µCi/tube). Nonenzymatic
[
-32P]GTP hydrolysis was determined in the presence of
a large excess of unlabeled GTP (1 mM) and amounted to
<1% of total [
-32P]GTP hydrolysis. Reactions were
conducted for 20 min. Stopping of reactions and recovery of
[32P]Pi were performed as described
previously (23). XTPase activity was determined as GTPase activity,
except that XTP and [
-32P]XTP were used.
s Ig or anti-FLAG
Ig (1:1000 each). Immunoreactive bands were visualized by donkey
anti-rabbit IgG (anti-G
s Ig) or sheep anti-mouse IgG
(anti-FLAG Ig) coupled to peroxidase at 1:1000 dilutions, using
o-dianisidine and H2O2 as
substrates. Immunoblots were scanned using a Molecular Imager FX and
evaluated with the Quantity One 4.3 software (Bio-Rad, Hercules, CA),
using
2AR-G
s fusion proteins (25-75
µg/lane) expressed at defined levels
([3H]dihydroalprenolol saturation binding) as standard.
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
s- and
2AR-G
s Proteins in Sf9
Membranes--
In mammalian cells, G
sS and
G
sL possess apparent molecular masses of 45 and 52 kDa,
respectively (24-26). The same was true for G
sS and
G
sL expressed in Sf9 membranes (Fig.
1A). In agreement with a
recent report (27), G
sL expressed in Sf9
membranes also showed a ~40-kDa proteolytic fragment. In contrast,
G
sS did not exhibit major proteolytic fragments.
However, in membranes expressing G
sS-L212,
G
sS-N280, and G
sS-L212/N280, a prominent
~39-kDa proteolytic fragment became apparent (Fig. 1B). In
membranes expressing the corresponding G
sL mutants, the
~40-kDa fragment was present, and an additional ~46-kDa proteolytic
fragment emerged. These data indicate that G
s and
G
s mutants possess different conformations, i.e. proteases access G
s mutants more readily
than G
s. G
s and G
s mutants
were expressed at similar levels (9-15 pmol/mg). We also expressed
2AR-G
s fusion proteins. Fusion proteins
allow for the sensitive analysis of
2AR/G
s coupling, particularly with
respect to NTP hydrolysis (17, 18).
2AR-G
sS and
2AR-G
sL proteins exhibited the expected
molecular masses of ~100 and 106 kDa, respectively (Fig.
1C) (22). The expression levels of
2AR-G
s proteins ranged between 4 and 7 pmol/mg. Collectively, G
s and G
s mutants
in the nonfused and fused state were expressed at similar levels in
Sf9 membranes. For brevity, we only show the AC data with
nonfused G
s. Studies with
2AR-G
s proteins yielded essentially the
same results.
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Fig. 1.
Analysis of the expression of
G s- and
2AR-G
s
proteins in Sf9 membranes. Sf9 cells were infected
with the baculoviruses indicated below A-C and incubated
for 48 h before membrane preparation. Sf9 cell membranes
(60 µg protein/lane) were separated on SDS gels containing 10% (w/v)
acrylamide as described under "Experimental Procedures." Proteins
were transferred onto Immobilon-P transfer membranes and probed
with anti-G
s Ig (C-terminal) (A and
B) or anti-FLAG Ig (C). The expression levels of
nonfused G
s proteins were estimated using
2AR-Gs
L (7.0 pmol/mg as assessed by
[3H]dihydroalprenolol saturation binding) as standard.
Numbers on the left indicate molecular masses (in
kDa) of marker proteins.
Ss, and NppNHps in
Sf9 Membranes Expressing G
sL and
G
sL Mutants--
GTP, ITP, and XTP were essentially
devoid of stimulatory effects on AC in membranes expressing
G
sL, G
sL-N295, and
G
sL-L227 (Fig. 2,
A, B, and D). A striking difference
among those three constructs was the extremely high basal AC activity
in the absence of GTP, ITP, or XTP in membranes expressing
G
sL-L227. This finding could be explained by the GTPase
deficiency brought about by the Gln/Leu mutation (14, 15). However, the
Gln/Leu mutation also increases the GDP affinity of G
sL
(14, 15), and G
s-GDP activates AC as efficiently as
G
s-GTP
S, provided that the concentration of
G
s-GDP is sufficiently high (28). As will be shown at
the end of "Results," the
2AR agonist ISO
efficiently reduces AC activity in the absence of GTP in
membranes co-expressing the
2AR plus
G
sL-L227/N295 through GDP dissociation and generation of
nucleotide-free G
s. Nucleotide-free G
s is
less efficient at activating AC than G
s-GDP (22, 23).
These data indicate that the increase in GDP affinity induced by the
Gln/Leu mutation critically contributes to the high basal AC activity
in membranes expressing such mutants.
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Fig. 2.
Effects of NTPs,
NTP Ss, and NppNHps on AC activity in
Sf9 membranes expressing
G
sL proteins. AC activity in
Sf9 membranes was determined as described under "Experimental
Procedures." Reaction mixtures contained Sf9 membranes (20-40
µg protein/tube) expressing G
sL (A,
E, and I), G
sL-N295 (B,
F, and J), G
sL-L227/N295
(C, G, and K), or
G
sL-L227 (D, H, and L)
in the presence of NTPs (A-D), NTP
Ss (E-H),
or NppNHps (I-L) at the concentrations indicated on the
abscissa. Log
10 designates the absence of added guanine,
hypoxanthine, or xanthine nucleotide. Note that because of the high AC
activities with membranes expressing G
sL-L227, the scale
of the y axis in D, H, and
L is different from that in the other panels. Data were
analyzed by nonlinear regression and best fitted to sigmoid
concentration-response curves. Data shown are the means ± S.D. of
three to six experiments performed in duplicates.
sL-N295 and G
sL-L227/N295 were expected
to possess decreased GDP affinity (5, 12, 13) and basal AC activity
relative to G
sL and G
sL-L227,
respectively. The experimental data were in agreement with these
assumptions (Fig. 2, A-D). In contrast to membranes
expressing G
sL and G
sL-N295, NTPs
significantly increased AC activity in membranes expressing
G
sL-L227/N295, compatible with a reduction of NTPase
activity of G
sL-L227/N295 (14, 15). In fact, ISO had
virtually no stimulatory effect on GTP and XTP hydrolysis in membranes
expressing
2AR-G
sL-L227/N295 (data not
shown). In agreement with data for the rab5-N136 mutant (29), the order of potency of NTPs at G
sL-L227/N295 was
XTP > GTP > ITP.
sL, NTP
Ss stimulated AC
activity in the expected order of potency, GTP
S > ITP
S > XTP
S (Fig. 2E) (18, 29). In membranes expressing
G
sL-N295, the order of potency was XTP
S > ITP
S > GTP
S, but the maximum AC activities in those
membranes amounted to only ~20% of the AC activities in membranes
expressing G
sL. These data indicate that
G
sL-N295, despite its efficient expression in Sf9
membranes (Fig. 1B), exhibited only low functional activity.
Routinely, we conducted AC assays at 37 °C. Intriguingly, the
G
sL-A366S mutant, which, like G
sL-N295, possesses lower GDP affinity than G
sL, denatures at
37 °C and requires temperatures of <33 °C to be active (30).
Therefore, we varied the incubation temperature in the AC assay from
16 °C to 37 °C, but these maneuvers did not increase AC
activation by G
sL-N295 (data not shown). These data
indicate that G
sL-N295 was largely expressed as a
functionally inactive protein, although the incubation temperature of
Sf9 cells was rather low (28 °C). The poor activity of
G
sL-N295 is reminiscent of the properties of the
analogous Asp/Asn mutants of G
o, G
11, and
G
16 (5, 12, 13). In G
o,
G
11, and G
16 Asp/Asn mutants, the
additional Gln/Leu mutation resulted in XTP
S-selective G
mutants
(5, 12, 13). However, the switch in base selectivity in
G
sL-L227/N295 was incomplete, i.e. GTP
S,
ITP
S, and XTP
S were similarly potent (Fig. 2G). In
membranes expressing G
sL-L227, NTP
Ss were similarly potent at moderately reducing AC activity (Fig.
2H).
S data, the order of potency of NppNHps at
activating AC in membranes expressing G
sL was
GppNHp > IppNHp > XppNHp, but the potencies of NppNHps were
lower than the potencies of the corresponding NTP
Ss (Fig. 2,
E and I). In membranes expressing
G
sL-N295, NppNHps stimulated AC in the order of potency XppNHp > GppNHp > IppNHp, but again, maximum
AC activities were very low (Fig. 2J). XppNHp increased AC
activity in membranes expressing G
sL-L227/N295 with
~10-fold higher potency than GppNHp (Fig. 2K). These data
fit to the data obtained with XTP/GTP (~20-fold potency difference)
(Fig. 2C). IppNHp was virtually inactive in membranes
expressing G
sL-L227/N295 (Fig. 2K). In
contrast to NTP
Ss, NppNHps had only minimal inhibitory effects on AC
activity in membranes expressing G
sL-L227 (Fig. 2,
H and L).
Ss, and NppNHps in
Sf9 Membranes Expressing G
sS and
G
sS Mutants--
There were no notable differences in
the effects of NTPs, NTP
Ss, and NppNHps at G
sS and
G
sS-L212 relative to the corresponding G
sL constructs, except that AC inhibition by
NTP
Ss in membranes expressing G
sS-L212 was only
marginal (Figs. 2H and
3H). Compared with membranes
expressing G
sS, basal AC activity in membranes expressing G
sS-N280 was lower, indicative for the
reduction in GDP affinity of the G
sS mutant (Fig. 3,
E and F). In contrast to the observations made
for the G
sL/G
sL-N295 couple (Fig. 2, E, F, I, and J),
G
sS-N280 was as efficacious as G
sS in
activating AC in the presence of NTP
Ss or NppNHps at saturating
concentrations (Fig. 3, E, F, I, and
J). As was true for G
sL-L227/N295 (Fig. 2G), G
sS-N280 did not discriminate between
GTP
S, ITP
S, and XTP
S (Fig. 3F). In contrast,
G
sS-N280 exhibited ~20-fold higher potency for XppNHp
than for GppNHp/IppNHp (Fig. 3J). Relative to
G
sS, the potency of XppNHp at G
sS-N280
was increased ~240-fold, whereas the potency of GppNHp was reduced
~20-fold (Fig. 3, I and J).
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Fig. 3.
Effects of NTPs,
NTP Ss, and NppNHps on AC activity in
Sf9 membranes expressing
G
sS proteins. AC activity in
Sf9 membranes was determined as described under "Experimental
Procedures." Reaction mixtures contained Sf9 membranes (20-40
µg protein/tube) expressing G
sS (A,
E, and I), G
sS-N280 (B,
F, and J), G
sS-L212/N280
(C, G, and K), or
G
sS-L212 (D, H, and L)
in the presence of NTPs (A-D), NTP
Ss (E-H),
or NppNHps (I-L) at the concentrations indicated on the
abscissa. Log
10 designates the absence of added guanine,
hypoxanthine, or xanthine nucleotide. Note that because of the high AC
activities with membranes expressing G
sS-L212, the scale
of the y axis in D, H, and
L is different than from in the other panels. Data were
analyzed by nonlinear regression and best fitted to sigmoid
concentration-response curves. Data shown are the means ± S.D. of
four to six experiments performed in duplicates.
sL-L227/G
sL-L227/N295 couple (Fig. 2,
C and D), introduction of the N280 mutation into G
sS-L212 decreased basal AC activity (Fig. 3,
C and D), indicative for a decrease in GDP
affinity. The introduction of the Leu212 mutation into
G
sS-N280 had considerable impact on the potencies of
NTP
S and NppNHps. Specifically, XTP
S activated
G
sS-L212/N280 with ~10-fold higher potency than
GTP
S and ITP
S, whereas G
sS-N280 did not
discriminate between NTP
Ss (Fig. 3, F and G).
By analogy, the G
o, G
11, and
G
16 Gln/Leu- Asp/Asn double mutants exhibited XTP
S
selectivity (5, 12, 13). Moreover, at G
sS-L212/N280, IppNHp was only 2-fold less potent than XppNHp, whereas at
G
sS-N280, IppNHp was ~20-fold less potent than XppNHp
(Fig. 3, J and K). We also noted similar NTP
potencies at G
sS-L212/N280 (Fig. 3C).
2AR plus G
s Proteins--
We wished to
determine whether G
s mutants interact with the
2AR. The
2AR is constitutively active,
i.e. even in the absence of agonist,
2AR
promotes nucleotide exchange at G
s, resulting in
stimulatory effects of NTPs on basal AC activity (17, 18). The inverse
agonist ICI 118,551 inhibits the effects of agonist-free
2AR. In membranes co-expressing
2AR and
G
sS-N280, XTP increased basal AC activity with
~50-fold higher potency than GTP (Fig. 4, A and C). This
potency difference fits with the observations made for XppNHp/GppNHp
(Fig. 3J) and contrasts with the lack of XTP
S selectivity
(Fig. 3F). ICI 118,551 abrogated the stimulatory effects of
GTP and XTP on basal AC activity in membranes co-expressing
2AR and G
sS-N280, indicating that the
agonist-free
2AR stimulated nucleotide exchange at
G
sS-N280. Additionally, the
2AR agonist ISO further increased AC activities in the presence of GTP and XTP
(Fig. 4, A and C), supporting the view that
G
sS-N280 couples to the
2AR. Based on the
high potency of XTP at G
sS-N280 in terms of AC
activation, we expected G
sS-N280 to exhibit
high-affinity XTPase activity. In fact, in membranes expressing
2AR-G
sS-N280, ISO stimulated steady-state
XTP hydrolysis with a Km of 240 ± 80 nM and a Vmax of 0.27 ± 0.02 min
1 (means ± S.D., n = 3). The
Km value for GTP hydrolysis was reduced to >4
µM.
View larger version (27K):
[in a new window]
Fig. 4.
Analysis of AC activity in Sf9
membranes expressing 2AR plus
G
s proteins. AC activity in
Sf9 membranes was determined as described under "Experimental
Procedures." Reaction mixtures contained Sf9 membranes (20-30
µg protein/tube) expressing the
2AR plus
G
sS-N280 (A and C) or the
2AR plus G
sL-L227/N295 (B and
D). Reaction mixtures contained GTP (A and
B) or XTP (C and D) at the
concentrations indicated on the abscissa in the presence of
solvent (basal), 10 µM ISO, or 1 µM ICI
118,551 (ICI). Log
10 designates the absence of added GTP
or ITP. Data were analyzed by nonlinear regression and best fitted to
sigmoid concentration-response curves. Data shown are the means ± S.D. of four to six experiments performed in duplicates.
sL-N295 failed to reconstitute
2AR-stimulated AC activity with GTP or XTP (data not
shown), corroborating the weak functional activity of this mutant.
Similar to the data obtained with membranes expressing
G
sL-L227/N295 alone (Fig. 2C), XTP was
severalfold more potent than GTP at activating AC in membranes
co-expressing
2AR plus G
sL-L227/N295
(Fig. 4, B and D). The inverse agonist ICI
118,551 did not inhibit AC stimulation by NTPs, supporting the
conclusion that the effects of NTPs were due to their hydrolysis resistance at G
sL-L227/N295. ISO strongly
reduced basal AC activity in membranes co-expressing the
2AR and G
sL-L227/N295. A model in which
ISO stimulates GDP dissociation from G
sL-L227/N295 and increases the abundance of nucleotide-free G
sL-L227/N295
explains these data. Nucleotide-free G
s is less
efficient than G
s-GDP at activating AC (22, 23). ISO
increased the diminished basal AC activity in the presence of GTP and
XTP, indicating that the
2AR also stimulated GTP/XTP
binding to G
sL-L227/N295 (Fig. 4, B and
D). However, the AC activities in the presence of ISO always remained well below the activities in the absence of ISO, indicating that nucleotide dissociation dominated nucleotide binding. Finally,
2AR-G
sL-L227/N295 did not exhibit
ISO-stimulated GTPase or XTPase activity (data not shown). Thus,
receptor regulation was intact in G
sS-N280 but defective
in G
sL-L227/N295 and G
sL-N295.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
Proteins--
In small GTP-binding proteins,
the Asp/Asn mutation switches the base selectivity from guanine to
xanthine and is valuable for the analysis of a specific GTP-binding
protein in complex systems (8-11). Similar applications could be
devised for xanthine nucleotide-selective G
proteins. Because of the
conserved guanine nucleotide binding in small GTP-binding proteins and
G
(3, 7), the achievement of this goal was thought to be
straightforward. However, G
o, G
11,
G
16, and G
sL Asp/Asn mutants were
inactive or exhibited weak activity at best (Fig. 2, B,
F, and J) (5, 12, 13). An explanation for the
poor activity of these proteins could be their low GDP affinity, a
consequence of the Asp/Asn mutation. Because cellular GDP
concentrations are much higher than XDP concentrations (5), G
Asp/Asn mutants are likely to exist predominantly in the
nucleotide-free state, particularly if GDP affinity of the parent G
is low anyway. In fact, G
o and G
sL
possess low GDP affinities (16, 31). However, in the nucleotide-free
state, G
is instable and denatures (30). Thus, because of their low
GDP affinity, G
o, G
11, and
G
sL Asp/Asn mutants may already denature during
expression. Denaturation apparently happens at temperatures as low as
28 °C, i.e. the incubation temperature for Sf9
cells. G
o, G
11, and G
16
Asp/Asn mutants were expressed at 37 °C (5, 12, 13), further
increasing the probability of denaturation.
sS possesses ~2-3-fold higher GDP affinity than
G
sL (16). This difference in GDP affinity is too small
to result in different basal AC activities in membranes expressing
G
sS and G
sL (Figs. 2A and
3A). Only in the presence of agonist-free
2AR, which preferentially stimulates GDP dissociation
from G
sL, do different basal AC activities with
G
sS and G
sL become evident (22).
Apparently, the moderately higher GDP affinity of G
sS
relative to G
sL is sufficient to prevent denaturation of
G
sS-N280 during expression because
G
sS-N280 was as effective as G
sS at
activating AC (Fig. 3, E, F, I, and J). Moreover, G
sS-N280 possesses the expected
XTP selectivity in terms of AC activation and NTP hydrolysis and is
receptor-regulated (Fig. 4, A and C). Compared
with G
sS, the Asp/Asn mutation increased the potency of
XppNHp by ~250-fold (Fig. 3, I and J). This
increase in potency equals or even exceeds the increases in xanthine
nucleotide potency at Asp/Asn mutants of small GTP-binding proteins
(8-11). Accordingly, XppNHp at 1 µM almost maximally
activates G
sS-N280 without a stimulatory effect on
G
sS or G
sL (Figs. 2I and 3, I and J). These potency differences render XppNHp
a highly selective G
sS-N280 activator. Thus, to the best
of our knowledge, G
sS-N280 represents the first fully
functional xanthine nucleotide-selective G
with the Asp/Asn mutation
alone. Accordingly, G
sS-N280 provides an excellent model
to clarify the still poorly understood role of G
sS
relative to the role of G
sL in signal transduction (16, 22, 24-26, 32).
o, G
11, G
16, and
G
sL Asp/Asn mutants for functionally active xanthine nucleotide-selective G
proteins can be explained by the
mutation-induced increase in GDP affinity, preventing G
denaturation
during expression (Fig. 2, C, G, and
K) (5, 12-15). The increase in GDP affinity in
G
sL-L227/N295 relative to G
sL-N295 is
reflected by the strong increase in basal AC activity (Fig. 2,
B and C) and ISO-induced AC inhibition
in the absence of GTP or XTP (Fig. 4, B and D). However, the introduction of the Gln/Leu mutation in
G
sL-N295 increases the GDP affinity and basal AC
activity so strongly that the signal-to-noise ratio of this
G
s mutant is poor, i.e. the stimulatory
effects of NTP
Ss and NppNHps are much smaller than those with
G
sS-N280 (Fig. 2, G and K and Fig.
3, F and J). In addition, the high GDP affinity
and reduction of NTPase activity in G
sL-L227/N295
compromises receptor regulation of this G
mutant (Fig.
4, B and D). However, by systematically mutating Gln227 in G
sL against other amino acids than
leucine or targeting other conserved amino acids that regulate GDP
affinity such as Ala366 in G
sL (30), it
could be possible to adjust the GDP affinity of any G
to a level
that ensures both functional expression and excellent signal-to-noise
ratio. Fortuitously, G
sS possesses the optimal GDP
affinity to fulfill both prerequisites for a xanthine nucleotide-selective G
without the need for mutagenesis.
Proteins--
GTP
S, but not GppNHp, sterically perturbs the
structure of the catalytic site of G
(6). Accordingly, G
-GppNHp
resembles G
-GTP more closely than G
-GTP
S. Our studies with
G
mutants support the conclusions of the crystallographic studies.
Most strikingly, G
sS-N280 and
G
sL-L227/N295 did not discriminate between NTP
Ss, and
NTP
Ss exhibited ~5-10-fold reduced potencies compared with the
potencies of GTP
S at G
sS and G
sL
(Figs. 2G and 3F). In contrast to the NTP
S
data, G
sS-N280 and G
sL-L227/N295 exhibited selectivity for XTP and XppNHp relative to GTP and GppNHp, respectively (Fig. 2, C and K, Fig.
3J, and Fig. 4, A-D). Thus, steric perturbation
of the catalytic site of G
s Asp/Asn mutants by NTP
Ss
annihilates xanthine nucleotide selectivity. Apparently, perturbation
of the catalytic site propagates a conformational change in
G
s, reorienting Asn280/Asn295,
so that these amino acids do not participate in hydrogen bonding of
NTP
Ss anymore. As consequences of the functional neutralization of
Asn280/Asn295, base selectivity is lost, and
NTP
S affinity of G
s mutants is reduced. Thus, to take
advantage of the xanthine nucleotide selectivity of
G
sS-N280 and G
sL-L227/N295, it is crucial
to use XppNHp or XTP but not XTP
S. Based on all these findings, we
predict that the crystal structures of the catalytic site of G
-Asp/Asn-XppNHp/XTP resemble the corresponding structures of G
-GppNHp/GTP. Moreover, the crystal structures of the catalytic site
of G
-Asp/Asn-XTP
S and G
-GTP
S should be similar.
sL, the Gln/Leu mutation converted NTP
Ss from activators into inhibitors and
abolished base selectivity (Fig. 2H). The Gln/Leu mutation
also profoundly changed the interactions of G
sS-N280
with nucleotides. Most notably, at G
sS-L212/N280, XTP
S was ~10-fold more potent than GTP
S, whereas at
G
sS-N280, GTP
S and XTP
S were similarly potent
(Fig. 3, F and G). Thus, a combination of two
perturbing factors in the catalytic site (NTP
S and Gln/Leu mutation)
revealed xanthine nucleotide selectivity of
G
sS-L212/N280. One can envisage that the conformational
change induced by the Gln/Leu mutation is functionally compensated by the NTP
S-induced conformational change, leaving the orientation of
Asn280 intact and preserving XTP
S selectivity of
G
sS-L212/N280. Such a process also apparently took place
in the Gln/Leu-Asp/Asn double mutants of G
o,
G
11, and G
q (5, 12, 13). In contrast to
NTP
Ss, NppNHps and NTPs should not neutralize the conformational change induced by the Gln/Leu mutation in G
sS-N280. In
fact, xanthine nucleotide selectivity for NTPs and NppNHps in
G
sS-L212/N280 is substantially reduced relative to
G
sS-N280 (Fig. 3C, J, and K and Fig. 4, A and C).
sS-N280 is a fully functional
xanthine nucleotide-selective G
and provides an excellent model to
study the specific roles of G
sS in signal transduction.
A sufficiently high GDP affinity of G
Asp/Asn mutants is crucial for
expression of functionally active proteins. The order state of the
catalytic site of G
critically determines nucleotide-G
interactions. Specifically, NTP
Ss and the Gln/Leu mutation perturb
the catalytic site, but in the case of G
sS-L212/N280,
the combination of two perturbing factors actually results in xanthine
nucleotide selectivity. Moreover, our data show that XTP
S and XppNHp
are not functionally equivalent activators of G
Asp/Asn mutants, and
caution must be exerted when making the decision which nucleotide to
use. Furthermore, our data explain some as yet poorly understood
properties of xanthine nucleotide-specific G
mutants (5, 12, 13). We
anticipate that functionally active xanthine nucleotide-selective G
mutants with high signal-to-noise ratio can be generated for any given
G
, but an individualized mutagenesis approach for each G
has to
be taken. The availability of an array of xanthine nucleotide-selective
G
mutants will complement gene knockout approaches to study the
functions of individual G
proteins in intact cell systems.
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ACKNOWLEDGEMENTS |
---|
We thank Dr. H.-Y. Liu for conducting some preliminary studies for this project and Dr. S. R. Sprang (Howard Hughes Medical Institute, The University of Texas Southwestern Medical Center, Dallas, TX) for stimulating discussions.
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FOOTNOTES |
---|
* This work was supported by Grant 0051404Z of the Heartland Affiliate of The American Heart Association (to R. S.), the J. R. & Inez Jay Biomedical Research Award of The University of Kansas (to R. S.), and a predoctoral fellowship of the Studienstiftung des Deutschen Volkes (to A. G.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
This paper is in remembrance of the 12th anniversary of the reunification of Germany on October 3rd, 2002, without which this project would not have been conducted.
To whom correspondence should be addressed: Dept. of
Pharmacology and Toxicology, The University of Kansas, Malott Hall, Rm. 5064, 1251 Wescoe Hall Dr., Lawrence, KS 66045-7582. Tel.:
785-864-3525; Fax: 785-864-5219; E-mail: rseifert@ku.edu.
Published, JBC Papers in Press, December 23, 2002, DOI 10.1074/jbc.M210162200
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ABBREVIATIONS |
---|
The abbreviations used are:
G, unspecified
G-protein
-subunit;
AC, adenylyl cyclase;
2AR,
2-adrenoceptor;
2AR-G
s, fusion protein containing the
2AR and a
G
s protein;
G
sL, long splice variant of
G
s;
G
sS, short splice variant of
G
S;
G
sL-L227, G
sL mutant
with a Q227L exchange;
G
sL-N295, G
sL
mutant with a D295N exchange;
G
sL-L227/N295, G
sL mutant with a Q227L- and D295N exchange;
G
sS-L212, G
sS mutant with a Q212L
exchange;
G
sS-N280, G
sS mutant with a
D280N exchange;
G
sS-L212/N280, G
sS mutant
with a Q212L- and D280N exchange;
GppNHp, guanosine
5'-[
,
-imido]- triphosphate;
GTP
S, guanosine
5'-[
-thio]triphosphate;
IppNHp, inosine
5'-[
,
-imido]triphosphate;
ISO, (-)-isoproterenol;
ITP
S, inosine 5'-[
-thio]triphosphate;
NppNHp, nucleoside
5'-[
,
-imido]triphosphate;
NTP, nucleoside 5'-triphosphate;
NTP
S, nucleoside 5'-[
-thio]triphosphate;
XppNHp, xanthosine
5'-[
,
-imido]triphosphate;
XTP, xanthosine 5'-triphosphate;
XTP
S, xanthosine 5'-[
-thio]triphosphate;
IDP, inosine
5'-diphosphate;
XDP, xanthosine 5'-diphosphate.
![]() |
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