(Received for publication, September 20, 1996, and in revised form, November 21, 1996)
From the Department of Pharmacology, University of Texas Southwestern Medical Center, Dallas, Texas 75235-9041
A GTPase-activating protein (GAP) specific for
Gz was identified in brain, spleen, retina, platelet, C6
glioma cells, and several other tissues and cells. Gz GAP
from bovine brain is a membrane protein that is refractory to
solubilization with most detergents but was solubilized with warm
Triton X-100 and purified up to 50,000-fold. Activity is associated
with at least two separate proteins of Mr
~22,000 and 28,000, both of which have similar specific activities.
In an assay that measures the rate of hydrolysis of GTP pre-bound to
detergent-soluble G
z, the GAP accelerates hydrolysis
over 200-fold, from 0.014 to 3 min
1 at 15 °C, or to
20 min
1 at 30 °C. It does not alter rates of
nucleotide association or dissociation. When co-reconstituted into
phospholipid vesicles with trimeric Gz and m2 muscarinic
receptor, Gz GAP accelerates agonist-stimulated
steady-state GTP hydrolysis as predicted by its effect on the
hydrolytic reaction. In the single turnover assay, the
Km of the GAP for G
z-GTP is 2 nM. Its activity is inhibited by
G
z-guanosine 5
-O-thiotriphosphate
(G
z-GTP
S) or by
G
z-GDP/AlF4 with Ki
~1.5 nM for both species; G
z-GDP does not
inhibit. G protein
subunits inhibit Gz GAP activity,
apparently by forming a GTP-G
z
complex that is a poor GAP substrate. Gz GAP displays little GAP activity
toward G
i1 or G
o, but its activity with
G
z is competitively inhibited by both G
i1
and G
o at nanomolar concentrations when they are bound
to GTP
S but not to GDP. Neither phospholipase C-
1 (a
Gq GAP) nor several adenylyl cyclase isoforms display
Gz GAP activity.
G proteins mediate numerous cellular processes by traversing a cycle of GTP binding and hydrolysis. Bound GTP activates a G protein such that it can stimulate a downstream effector protein. Activation is terminated when the bound GTP is hydrolyzed to GDP, which does not activate. Each step of the cycle is controlled, such that both steady-state GTPase activity and the concentrations of the active and inactive forms are highly regulated.
Activation of heterotrimeric G proteins is promoted by seven-span cell-surface receptors that facilitate GDP release and GTP binding. Small monomeric G proteins (Ras, Rac, Rho, Arf, etc.) are activated by cytosolic proteins that similarly facilitate GTP binding.
In many cases, hydrolysis of bound GTP, the deactivation step, is
accelerated by GTPase-activating proteins, or
GAPs1 (1-4). GAPs appear to fulfill at
least one of four definable roles. Some GAPs for monomeric signaling G
proteins, such as Ras GAP, appear to attenuate G protein signal
amplitude in response to inputs from inhibitory signaling pathways (1, 2, for review). GAPs for the monomeric G proteins involved in
cytoplasmic vesicle trafficking are thought to act by terminating a G
protein-dependent assembly or transit step. Some effector
proteins that are regulated by heterotrimeric G proteins also act as
GAPs for their G protein regulators. The GAP activities of these
effectors, such as phospholipase C- (5, 6) and the cyclic GMP
phosphodiesterase
subunit (7, 8), may allow effector-specific
modulation of response times or may enhance the selectivity of
receptor-G protein signaling (3). A fourth class of GAPs, also for the
trimeric G proteins, includes members of the recently identified RGS
protein family (4, 9-13). Little is known of the physiology of RGS
proteins, but they can contribute to desensitization toward a prolonged signal (Sst2p in yeast; Refs. 10, 14, 15) or act as long term
attenuators of signal amplitude (Egl-10 protein in Caenorhabditis elegans; Ref. 9).
G protein GAP activity can potentially be used to identify and purify regulators of G protein function or to point to novel inputs to G protein signaling pathways. For GAPs that are also effectors, their identification can indicate what downstream signals the G protein mediates.
We began a search for new GAPs for heterotrimeric G proteins by looking for a GAP for Gz, a pertussis toxin-insensitive member of the Gi family that is abundant in brain, adrenal medulla, and platelets (16-19). There were three reasons for this choice. Although Gz can mediate inhibition of adenylyl cyclase (20-22) and respond to receptors that regulate other Gi family members (21, 23), the signaling pathway(s) mediated by Gz remains unknown, and a Gz GAP might be an effector. Second, Gz hydrolyzes bound GTP very slowly (16). Its activation lifetime is about 7 min, which seems incompatible with normal signaling functions unless a GAP accelerates deactivation. Last, the slow hydrolytic rate simplifies design of an assay for a GAP. We describe here the detection of Gz GAP activity in brain and other tissues, substantial purification of a Gz GAP from bovine brain, and several aspects of its mechanism of action and regulation.
Procedures for purification of Gz
(20), G
i1 (24, 25), G
o (24, 26),
G
s (24), G
q (6), G
(6), m2
muscarinic cholinergic receptor (23), and phospholipase C-
1 (6) have been described. Sf9 membranes that contain recombinant adenylyl cyclase
isoforms (27) and purified G
12 (20) were gifts from Drs.
Carmen Dessauer and Tohru Kozasa (this department).
G
z-agarose was prepared according to Mumby et
al. (28) and phenyl-Sepharose was purchased from Pharmacia Biotech
Inc. Cholic acid was purified as described (29), and other detergents
were purchased from various suppliers. [
-32P]GTP was
either purchased or synthesized (30) and purified as described (6).
[-32P]GTP was bound
to G
z by incubating 10-50 pmol of G
z for
20 min at 30 °C in 200 µl of 25 mM NaHepes (pH 7.5), 3 mM DTT, 0.1% Triton X-100, 1 mM EDTA, 2.5 µM [
-32P]GTP (20-80 cpm/fmol), and
sufficient MgCl2 to provide 1 µM free Mg2+. After incubation, [
-32P]GTP and
[32P]orthophosphate were removed by chromatography on a
2-ml column of Sephadex G25, which was run in the same buffer but
without GTP or MgCl2. G
z-bound
[
-32P]GTP was determined by nitrocellulose binding
assay (31) and was usually 25% of the total G
z, with
the remainder bound to GDP.
Hydrolysis of Gz-bound [
-32P]GTP in
routine GAP assays was measured by incubation of the substrate (usually
~1-2 nM) in the buffer used for its preparation but
including 1 mM free Mg2+, 10 µg/ml bovine
albumin, and 5 mM nonradioactive GTP. Unlabeled GTP was
added to inhibit nucleoside triphosphatase activity present in crude
GAP fractions in the event that any [
-32P]GTP
dissociated from G
z. Assays were carried out at 15 °C
for times that varied from 30 s to 60 min. Hydrolysis of bound
[
-32P]GTP was measured as release of
[32P]orthophosphate (31). Hydrolysis followed a single
exponential time course. Hydrolysis is expressed either as the amount
of bound GTP hydrolyzed at early times (quasilinear time course) or as a first-order rate constant.2
GAP-independent hydrolysis is subtracted from all data except in Figs.
1, 6, and 8B.
Other GTPase Assays
Steady-state GTPase assays were
performed as described (31) at 30 °C in buffer that contained 20 mM NaHepes (pH 8.0), 25 mM NaCl, 0.1 mM EDTA, 1.1 mM MgCl2, 20 µg/ml
bovine albumin, 50 nM GDP, 100 nM
[-32P]GTP, and either 100 µM carbachol
or 5 µM atropine. The rate of hydrolysis of
[
-32P]GTP bound to G
o and
G
i1 was measured at 15 °C essentially as described
previously (26), except that the buffer for the initial
[
-32P]GTP binding reaction contained 5 mM
EDTA, and hydrolysis was initiated by adding 6.0 mM
MgCl2 plus 0.1 mM unlabeled GTP. The concentration of G
-bound [
-32P]GTP at initiation of
the hydrolysis reaction is given as the total amount of
[32P]orthophosphate released during the reaction.
Gz GAP was
purified from bovine cerebral cortical membranes prepared according to
Sternweis and Pang (32). All procedures were performed at 0-4 °C
except where explicitly noted. In practice, we pool active fractions
from multiple runs of the Q-Sepharose column before gel filtration and
pool several gel filtration peaks for Gz affinity
chromatography. Combining preparations in this way improves both the
yield and purification. However, Table II is an example of an early
preparation that was completed exactly as described below.
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Membranes were washed once in 50 mM Tris-Cl (pH 7.5), 1 mM DTT, 1 mM EDTA, 0.5 M NaCl, and
0.1 mM PMSF and resuspended to 2.5 mg/ml in 20 mM NaHepes (pH 7.5), 0.1 mM EDTA, 0.3 mM PMSF, and 2% Triton X-100. The suspension was sloshed
for 25 min at 30 °C and centrifuged for 40 min at 150,000 × g. The supernatant was loaded on a DEAE-Sephacel column (700 ml) that was equilibrated with Buffer 1 (20 mM NaHepes (pH
7.5), 0.1 mM EDTA, 1% Triton X-100, 0.1 mM
PMSF). The column was washed with Buffer 1 and eluted with a gradient
of 0-300 mM NaCl in Buffer 1. Both Gz GAP
activity and protein were eluted in parallel as a broad, asymmetric
peak. Active fractions were pooled and diluted 3-fold with Buffer 2 (20 mM NaHepes (pH 7.5), 0.1 mM EDTA, 1 mM DTT, 1% cholate, 0.1 mM PMSF). DTT was
added to a final concentration of 20 mM, and the solution
was applied to a column of Q-Sepharose that had been equilibrated with
Buffer 2. The column was washed sequentially with Buffer 2, Buffer 2 plus 0.1 M NaCl, and Buffer 2 plus 0.25 M NaCl,
and then eluted with a gradient of 0.25-0.55 M NaCl in Buffer 2. Protein and GAP activity again eluted as a broad peak. Active
fractions were concentrated on an Amicon PM30 membrane and
chromatographed on a column of Ultrogel AcA-34 equilibrated with Buffer
2 plus 0.1 M NaCl. A typical elution profile is shown in
Fig. 2. The second peak of GAP activity was pooled and concentrated by
adsorption to Mono Q and elution with a gradient of 0.1-0.55 M NaCl in Buffer 2.
For affinity chromatography, pooled Mono Q fractions were diluted
20-fold with Buffer 3 (25 mM NaHepes (pH 7.5), 1 mM EDTA, 1 mM DTT, 0.1% Triton X-100, 0.25%
cholate, 0.1 mM PMSF, 10 µM GDP, 30 µM AlCl3, 5 mM MgCl2,
10 mM NaF) and applied to a column of
Gz-agarose that was equilibrated with Buffer 3. The
column was washed with Buffer 3 plus 25 mM NaCl, and GAP
activity was eluted with a gradient of 25-500 mM NaCl in
Buffer 4 (Buffer 3 but containing 0.5% cholate and without
AlCl3, MgCl2, or NaF). Peak fractions were
pooled, concentrated by adsorption and elution from Q Sepharose as
described above, and diluted 8-fold with Buffer 5 (20 mM
NaHepes (pH 7.5), 1 mM DTT, 0.1 mM EDTA, 1 M NaCl, 0.1 mM PMSF). The pool was applied to a
column of phenyl-Sepharose equilibrated with Buffer 5 plus 0.2%
cholate and washed with 5 volumes of the same buffer. GAP activity was
eluted with a discontinuous gradient of 0.2-1.0% cholate (0.2%
steps) in Buffer 5 but without NaCl (see Fig. 3).
Miscellaneous Methods
Gz GAP was
co-reconstituted with Gz
and m2 muscarinic
cholinergic receptor into unilamellar phospholipid vesicles
(phosphatidylserine:phosphatidylethanolamine:cholesteryl hemisuccinate,
5:8:1) according to the method of Parker et al. (23).
Trimeric Gz was prepared by mixing GDP-bound
G
z with G
1
2 (
:
= 0.4) before reconstitution (26). G
z was routinely quantitated according to the binding of 10 µM
[35S]GTP
S for 1.5 h at 30 °C (31). Binding of
[
-32P] and [
-32P]GTP to
Gz was also measured by the nitrocellulose binding assay (31). Protein was measured by amido black binding (33).
Standard procedures were used for SDS-gel electrophoresis (34) and staining with Coomassie Blue or silver (35). For samples in which GAP activity was to be measured after electrophoresis, samples were denatured in sample buffer (34) that contained 1% SDS and 10 mM DTT. Gz GAP activity was extracted from slices of SDS-polyacrylamide gels and renatured by homogenizing the gel in 5-10 volumes of renaturation buffer (20 mM NaHepes (pH 7.5), 1 mM DTT, 0.1 mM EDTA, 1% Triton X-100) and shaking overnight at 0 °C.
The concentration of free Mg2+ in assay buffers that contained significant concentrations of both EDTA and GTP was calibrated as described by Huskens and Sherry (36).
We used the
Gz-[
-32P]GTP complex as substrate to
test for the presence of proteins that can increase the rate of
hydrolysis of G
z-bound GTP. Purified G
z
hydrolyzes bound GTP very slowly (16), such that
G
z-[
-32P]GTP can be prepared and
purified with good yield. About 25% of total G
z is
bound to [
-32P]GTP after gel filtration, with the rest
bound to unlabeled GDP. Under standard assay conditions at 15 °C,
GTP bound to G
z is hydrolyzed with a rate constant,
khydrol, of about 0.014 min
1,
which corresponds to a t1/2 of about 50 min (Fig.
1).
Addition of a crude membrane fraction from bovine brain increased the
rate of hydrolysis of Gz-bound GTP dramatically (Fig. 1A). Hydrolysis was a single component, first-order reaction
over a 10-fold range of rates, and rate constants increased linearly with increasing amounts of membrane protein (Fig. 1B). These
data indicate the existence of a GTPase-accelerating activity in bovine brain, i.e. a Gz GAP. Based on the linearity of
GTP hydrolysis with added membrane protein, we defined a unit of
Gz GAP activity as an increment in the hydrolytic rate
constant of 1.0 min
1. Both the basal rate of GTP
hydrolysis by G
z and the GAP activities of several
tissues were quite reproducible in this assay. The hydrolysis-accelerating activity is evidently that of a protein. In
membranes, activity was destroyed by incubation with 6.7 µg/ml trypsin, 20 µg/ml chymotrypsin plus detergent or 0.5 mM
N-ethylmaleimide. Added detergent markedly sensitized the
GAP to proteolysis.
Release of [32P]orthophosphate in GAP assays such as
shown in Fig. 1 reflects only hydrolysis of [-32P]GTP
bound to G
z rather than dissociation followed by
hydrolysis catalyzed by other nucleoside triphosphatases in the
membranes. Dissociation of GTP from G
z was unmeasurably
slow, as estimated either by comparing the loss of bound
[
-32P]GTP with the appearance of
[32P]orthophosphate or by monitoring the dissociation of
[
-32P]GTP. The rate of formation of
[32P]orthophosphate was equal both to the rate of loss of
G
z-bound [
-32P]GTP and to the rate of
conversion of bound [
-32P]GTP to bound
[
-32P]GDP (not shown), either with or without added
membrane protein. Furthermore, the reaction is carried out in the
presence of 5 mM free GTP to block any other nucleoside
triphosphatase activity. In control experiments, no hydrolysis was
observed when free [
-32P]GTP was substituted for
G
z-[
-32P]GTP (not shown).
Homogenates of several mammalian tissues and cultured cells were
screened for Gz GAP activity (Table I), and
its distribution was found to be similar to that of Gz
itself (16-19; confirmed qualitatively by anti-
z
immunoblot durning this study). Activity was highest in cerebral
cortex, although there was considerable activity in membranes of spleen
and retina. Peripheral fat, lung, platelets, and testis also displayed
readily measurable activity. Several other tissues displayed low
activity, which may represent contamination by adipose, neuronal, or
vascular tissue or the action of other GAPs with low activity toward
Gz. We do not know how many proteins in these tissues
display Gz GAP activity. Among the cultured cells tested,
C6 glioma cells displayed activity similar to that of brain, 20-50
units/mg depending upon the source of the cells and the culture
conditions. Several other cell lines displayed more modest activities.
Gz GAP activity was low in S49 murine lymphoma cells and
was barely detected in Sf9 cells. Because activity was highest in brain
and bovine brain is readily obtainable, we concentrated on this source
and have not attempted to determine the multiplicity of Gz
GAPs in other tissues.
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There is no soluble Gz GAP activity detectable in brain homogenates (<2%). Gz GAP behaves as an integral membrane protein and is difficult to solubilize. It is not extracted by washing at high or low ionic strength and is not solubilized by many detergents. At 0 °C, neither cholate, deoxycholate, Lubrol PX, Triton X-100, CHAPS, digitonin, nor several other detergents solubilized any Gz GAP activity. Small and irreproducible amounts of activity were solubilized by dodecyl maltoside and lauroyl sucrose. Fortunately, incubation of membranes with 2% Triton X-100 at 30 °C released considerable Gz GAP activity into a 200,000 × g supernatant. This apparently soluble GAP was still badly aggregated, however, and substantial increases in specific activity were not obtained by chromatography in multiple systems. Two sequential rounds of anion exchange chromatography, which included transfer from Triton X-100 to cholate, provided little purification and substantial loss of activity (Table II) but did allow subsequent purification. Although about half of the Gz GAP activity remained aggregated, the other half behaved as an apparently monodisperse species of reasonable molecular size and was thus purified about 20-fold by gel filtration in cholate (Fig. 2). This soluble material was used for further purification.
After gel filtration, Gz GAP activity was appreciably
purified by affinity chromatography on Gz-agarose. GAP
activity bound to the column when the covalently coupled
G
z was activated with either GTP
S or with
GDP/AlF4 but not when the G
z was in the nonactivated, GDP-bound form (not shown). For purification, peak fractions from gel filtration were applied to
Al3+/F4
-activated
G
z-agarose, and after extensive washing, GAP activity was eluted by removal of Al3+, F
, and
Mg2+ and increasing the concentration of detergent and
salt.
Further purification of Gz GAP was achieved by
phenyl-Sepharose chromatography (Fig. 3). Gz
GAP activity was consistently eluted from phenyl-Sepharose in two
peaks. This was true for multiple elution protocols that included
increasing the concentration of detergent and/or decreasing the
concentration of salt, although it was possible to alter the peak
shapes and the total and specific activities in each peak. Typically,
both peaks were purified about 5000-12,000-fold relative to the Triton
extract (Table II shows data for pools). Neither peak contained
homogeneous protein, as shown by SDS-gel electrophoresis (Fig.
3B). The peak fractions contained a large but reproducible
group of proteins, the only identifiable one of which was the G protein
subunit. G
has no GAP activity (see below), and GAP activity
was not co-immunoprecipitated by anti-G
antibodies (not shown). Its
appearance is apparently an artifact of G
z affinity
chromatography. No single polypeptide obviously co-fractionated exactly
with Gz GAP activity. The phenyl-Sepharose pools contained
no GTP
S binding activity (<0.015 mol/mol GAP).
We attempted to identify the polypeptide(s) that accounts for
Gz GAP activity by further fractionating phenyl-Sepharose
peak fractions on SDS-polyacrylamide gels and then renaturing the GAP protein eluted from individual gel slices (see "Experimental
Procedures"). About 50-75% of Gz GAP activity was
recovered from the gel. As shown in Fig. 4, activity was
broadly distributed on SDS gels between 20 and 30 kDa, with two peaks
of activity reproducibly appearing at about 22 and 28 kDa. Smearing
during electrophoresis is an unlikely cause of this behavior because
both activity and discrete protein bands eluted from individual gel
slices retain their distinct electrophoretic mobilities upon extraction
and a second round of gel electrophoresis (Fig. 4B). The
22-kDa peak of activity corresponds to a relatively blurry band, but we
have not assigned the 28-kDa activity to a specific stained band. In addition to the two peaks of activity, Gz GAP activity is
readily detected throughout the region between 22 and 28 kDa,
indicating considerable heterogeneity. It is unclear whether the 22-kDa
band and intermediate forms are proteolytic products of the 28-kDa form
or whether they are unique Gz GAP species. SDS gel analysis of GAP activity in membranes and earlier fractions during the purification have not clarified this question, although they indicate the presence of GAP activity with molecular size up to 40 kDa. Thus,
the data of Fig. 4 indicate that Gz GAP activity results from monomeric proteins in the size range 22-28 kDa, but the number of
species remains uncertain.
In addition to indicating which molecular weight species contribute to Gz GAP activity, SDS-gel electrophoresis and renaturation provide substantial purification of GAP activity. The specific activity of Gz GAP extracted from SDS gels is increased more than 50,000-fold relative to the Triton extract. It is likely that these fractions are essentially pure. Such extensive purification indicates that Gz GAP is of low abundance even in brain.
Mechanism of Action of Gz GAPA single GAP
molecule can turn over multiple molecules of Gz-GTP
(Fig. 5). Its behavior is most readily analyzed when it is considered as an enzyme that acts upon the substrate
G
z-GTP and converts it to the products
G
z-GDP plus orthophosphate. Its Km
for G
z-GTP is about 2 nM, which represents
sufficiently high affinity binding to be physiologically reasonable.
The maximum GAP-stimulated hydrolysis rate can be estimated two ways.
Because velocity increases linearly with the amount of GAP at low GAP concentrations (Figs. 1 and 5B), the maximum GAP-stimulated
hydrolytic rate constant (kgap2) can
be calculated by dividing Vmax by the molar
concentration of GAP, which is calculated according to its estimated
purity and approximate molecular weight. This calculation yields a
kgap of 3 min
1 at 15 °C. The
other estimate of kgap derives from a titration of GAP when the concentration of G
z-GTP is maintained at
or above the Km (Fig. 5B). The maximum in
such an experiment, 1.8 min
1, can be corrected for the
subsaturating concentration of G
z-GTP to yield a true
maximum kgap of 3.1 min
1. Thus,
both determinations of kgap are about 3 min
1, more than a 200-fold stimulation over
khydrol for G
z-GTP. We estimate
that kgap at 30 °C is over 20 min
1, which corresponds to an average lifetime for
activated Gz of <2 s. Thus, because
kgap is fast and because Gz GAP has
a high affinity for its G
z-GTP substrate
(Km ~2 nM), its action is sufficient
to allow Gz-mediated signal transduction with
physiologically appropriate rates.
Gz can hydrolyze bound GTP at very low concentrations of
free Mg2+, and its intrinsic khydrol
is independent of the concentration of Mg2+ up to 10 mM (Fig. 6). In contrast, Gz GAP
activity displays a marked Mg2+ optimum at ~1
mM. Although the GAP is active over a wide range of
Mg2+ concentrations, stimulation is ~4-fold higher at the
optimum. Neither Ca2+ nor Mn2+ exerted a unique
regulatory effect on Gz GAP, although either can replace
Mg2+ over approximately the same range of concentrations
(not shown).
Gz GAP binds tightly to Gz, but only in its
GTP-activated form. Both G
z-GTP
S and
G
z-GDP/AlF4 thus inhibited Gz
GAP activity, with a Ki of ~1.5 nM for
either nucleotide (Fig. 7). This value of
Ki is similar to the Km for the substrate G
z-GTP, which suggests that
G
z-GTP, G
z-GTP
S, and G
z-GDP/AlF4 all bind the GAP at a common
site and with similar affinities. G
z-GDP did not inhibit
at concentrations up to 20 nM. Note that in the experiment
of Fig. 7, G
z-GDP/AlF4 was formed in the
reaction mixture by the addition of Al3+ plus
F
, which appear to inhibit GAP activity by themselves.
This inhibition results from the presence of G
z-GDP in
the preparation of G
z-GTP substrate, such that about 6 nM G
z-GDP/AlF4 is present in the assay even when no excess G
z-GDP was added. Selectivity
of the GAP for the active conformation of G
z is
confirmed by the selective binding of the GAP to
G
z-agarose when it is activated by either GTP
S or
GDP/AlF4.
The following small molecules had no effect upon Gz GAP
activity: inositol trisphosphate, cyclic AMP, cyclic GMP, GTP, GTPS, and ATP (not shown).
The selectivity of brain
Gz GAP among different G subunits was tested initially
by comparing their abilities to compete with G
z-GTP in
the standard assay (Fig. 7, Table III). Both
myristoylated, recombinant G
i1 and bovine brain
G
o inhibited competitively, G
o-GTP
S
with a Ki of ~5 nM (Fig. 7) and
G
i1-GTP
S with a Ki of about 20 nM. Their GDP-bound forms did not inhibit (not shown).
Inhibition by G
o or G
i required that they be myristoylated; nonmyristoylated G
subunits inhibited weakly or
not at all. Based on these data, we measured the ability of the
Gz GAP to accelerate hydrolysis of G
o-GTP
and G
i1-GTP, using the single turnover assay of
Higashijima et al. (26) to accommodate the faster basal
hydrolytic rates of these G
subunits. Although both
G
o and G
i1 hydrolyze bound GTP much
faster than does G
z, the relative effect of
Gz GAP on both G
subunits was minimal when compared with
G
z (Table IV): 30% and 7% compared with
more than a 30-fold effect on G
z. The GTP
S-bound
forms of G
s, G
q, and G
12
did not compete significantly in the Gz GAP assay (Table III), and their activities as GAP substrates were not tested.
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Although G protein subunits had little if any
effect on the rate of hydrolysis of G
z-bound GTP,
G
inhibited Gz GAP activity up to 80% (Fig.
8). Inhibition was most marked at low concentrations of
G
z and is caused by an increase in Km
of at least 5-fold (Fig. 8A). No effect of G
on
Vmax was detected, but we were unable to achieve
saturation with G
z-GTP at high G
concentrations, and we may have failed to observe a slight decrease in
Vmax. Gz GAP was inhibited
approximately equally by G
1
2,
G
2
2, and
G
2
3 (not shown). The increase in
Km caused by G
apparently reflects formation
of the GTP-bound G
z
heterotrimer. The
IC50 for G
1
2 (~400
nM, Fig. 8B) agrees well with its affinity for GTP-bound G
z (37), and we have found no evidence for
G
binding directly to the GAP (which would yield classical
competitive inhibition). G
z
-GTP may be a low
affinity (high Km) GAP substrate or it may simply
block GAP binding to G
z-GTP. These alternatives are
potentially distinguishable according to the dependence of the apparent
Km on the concentration of G
, but we have been
unable to determine Km accurately over a high enough range of G
concentrations to answer this question.
In addition to inhibiting the GAP, G decreased the rate of
dissociation of GDP, but not GTP, from G
z, as is true
for other G
subunits (38). We observed no other effects of G
in this system.
To study the effect of Gz GAP on the
receptor-stimulated steady-state GTPase cycle, we co-reconstituted
Gz GAP with m2 muscarinic cholinergic receptor and
heterotrimeric Gz into unilamellar phospholipid vesicles.
When the muscarinic agonist carbachol was added to promote receptor-catalyzed exchange (23), Gz GAP increased the
steady-state GTPase rate by about 2.5-fold (Fig.
9A). This effect seems small in comparison to
the 200-fold maximum effect of the GAP on hydrolysis of preformed
Gz-GTP, but the steady-state concentration of
G
z-GTP in the vesicles is in significant molar excess
over that of the GAP. The effect of the GAP on the steady-state GTPase
rate is consistent with its observed activity in the single turnover
assay. The effect of GAP on steady-state GTPase rates is evidently
exerted only at the hydrolytic step. In the absence of agonist, where steady-state GTPase activity is limited by the GDP/GTP exchange rate,
GAP had no effect, and GAP also had no effect on the rates of
nucleotide binding (Fig. 9B) or release (not shown).
Consistent with its effect on hydrolysis of bound GTP, Gz
GAP decreased the steady-state concentration of the active
Gz-GTP complex during stimulation by agonist. When assayed
at either 5 or 15 min in the system shown in Fig. 9A, the
addition of Gz GAP decreased the concentration of
Gz-GTP by about 30%. Both of these sample times are after
steady-state was reached, as indicated by the constant concentration of
bound GTP in this interval and by the completion of agonist-stimulated
GTPS binding in about 2 min (Fig. 9B). Simple kinetic
models predict that the relative effect of GAP on the accumulation of
Gz-GTP would be greater in the absence of agonist, but we
were unable to measure accurately the small amount of binding of
[
-32P]GTP binding that occurred without agonist.
The results of the experiments shown in Fig. 9 indicate that purified Gz GAP can reassociate with membrane lipids and regulate Gz appropriately in (or on the surface of) a phospholipid bilayer. The addition of detergent-soluble Gz GAP to preformed vesicles had no effect on the steady-state GTPase rate (not shown), which is consistent with the idea that the GAP is an integral membrane protein.
Gz hydrolyzes bound GTP (deactivates) extremely slowly. Although it is activated at a normal rate in response to receptors (23), its active state decays with an average lifetime of about 7 min at physiological temperature (16). With these kinetics, it would be hard to understand how Gz can mediate signaling responses in a reasonable way, although it clearly does.
The data presented here describe the identification of a GAP for
Gz in brain membranes, its purification, and mechanistic behavior. Similar activity was identified in membranes of several other
tissues and cultured cells. By accelerating GTP hydrolysis, a
Gz GAP reconciles aberrant deactivation kinetics with
normal signaling functions. However, its precise role in signaling
physiology remains unclear. Gz GAP may be a
Gz-regulated effector protein, in analogy with
phospholipase C- and cyclic GMP phosphodiesterase. These effectors
are both regulated by G proteins and have GAP activity specific for
their G protein regulators, Gq and Gt (5, 7,
8). The low Km of the Gz GAP, about 2 nM, is in the same range of affinities as that displayed by
G
q, G
s, or G
t for their
effectors (5, 6, 39, 40), and its selectivity for the activated form of
G
z is also consistent with this role. Alternatively, the
GAP may be a negative regulatory component of the Gz
pathway, involved either in desensitization or in mediating negative
input from another signaling pathway. The model for such regulation
could be either the GAPs for p21RAS and related small,
monomeric G proteins (2) or the RGS proteins, a large family of related
proteins that inhibit signaling (4, 9, 10) and whose prototypes are
GAPs (12). Whether the cerebral Gz GAP is an effector or a
modulator of inhibition will probably be elucidated when its cDNA
can be used to manipulate its expression in cells.
Gz GAP was initially purified about
12,000-fold according to the specific activity of phenyl-Sepharose
fractions, and the specific activity of the purest fractions from SDS
gels is about 4-fold higher (Fig. 4). Gz GAP is thus a rare
protein in brain, its richest source, and is perhaps expressed in only
a few cell types. By rough comparison with published data (16, 41) and with immunoblots performed during this study (not shown),
Gz GAP is about 5-10-fold less abundant than is
Gz. This is not surprising, however, whether the GAP is
an effector or purely a negative regulator. G proteins are generally in
molar excess over their effector proteins. Alternatively, because
Gz GAP acts catalytically, it could readily function as an
efficient inhibitor of Gz signaling.
Despite extensive purification, preparations of Gz GAP remain heterogeneous. GAP activity in peak fractions from phenyl-Sepharose is distributed bimodally between 22 and 28 kDa. We do not know if the 22-kDa GAP is distinct from the 28-kDa GAP or if it is a proteolytic product, although we have been unable to proteolyze the larger form to the smaller form. There is also obvious GAP activity and protein between the two major peaks. Because the ratio of GAP activity to silver-stained protein is low between the peaks, we suspect that major silver-stained bands in this region are contaminants and that the activity represents distribution of active proteolytic fragments of the 28-kDa GAP. We approached the question of proteolysis during purification by analyzing unfractionated brain membranes by SDS-gel electrophoresis. The principal peak of activity was at about 28 kDa, with tailing to about 20 kDa, but we could also detect small peaks of activity higher in the gel. These larger forms are not observed in purified preparation; peptides of Mr > 30,000 in the phenyl-Sepharose fractions have no GAP activity.
Mechanism, Selectivity, and Regulation of Gz GAPPurified cerebral Gz GAP is highly specific in
its action on Gz. It displayed only slight activity with
either G
i1-GTP or G
o-GTP as substrates
under conditions where hydrolysis of G
z-GTP was
accelerated over 30-fold (Table IV). According to competitive inhibition, however, the affinity of the GAP for G
z is
only about 3-fold greater than that for G
o and about
10-fold higher than for G
i1 (Table III). Evidently,
Gz GAP can bind these other G proteins with high affinity
but cannot efficiently promote their deactivation. This unusual pattern
of selectivity suggests that other members of the Gi family
may inhibit Gz GAP in cells, where they are much more
abundant than is Gz. Given the selectivity of the purified
GAP for G
z, it was initially surprising that there is
significant activity in cells and tissues that express little if any
G
z (Table I). It is likely that this activity is that of
GAPs for other Gi family members but which act on
Gz with low efficiency (47, 48).
Cerebral Gz GAP behaves generally as an integral membrane protein, although it was unusually refractory to solubilization by nondenaturing detergents. This behavior is reminiscent of caveolar proteins (42-44). However, caveolae are reported to be solubilized by octyl glucoside (Gz GAP was not) and, in one experiment, Gz GAP activity did not co-fractionate with caveolin in lysates of MA104 cells. We suspect that Gz GAP is a markedly hydrophobic protein because of its resistance to solubilization and its tendency to aggregate. This conclusion is supported by its functional co-reconstitution with m2 muscarinic receptors and trimeric Gz into phospholipid vesicles (Fig. 9), in contrast to its inactivity when added to preformed receptor-Gz vesicles. We have been unable to perform the standard tests for monomeric solubility of purified Gz GAP because removing detergent by dilution or chromatography before assay led to loss of GAP activity. This was true even though the assay was performed in the presence of Triton X-100. Some of this behavior is similar to difficulties encountered in solubilizing adenylyl cyclase, a G protein-regulated effector that is a much larger, multi-span membrane protein. There is inadequate information to compare this aspect of Gz GAP with RGS proteins, although RGS4 and GAIP are both water-soluble (11, 12) and Sst2p binding to membranes is sensitive to ionic strength (15).
The enzymologic mechanism of Gz GAP action is apparently
straightforward.2 Gz-GTP is essentially
stable over the usual assay interval. The GAP binds the GTP-bound form
of G
z with nanomolar affinity (Figs. 5 and 7), and the
GAP-G
z-GTP complex then hydrolyzes GTP fairly quickly
(t1/2 ~15 s at 15 °C; t1/2
<2 s at 30 °C). The GAP binds G
z-GDP weakly if at
all, such that the complex rapidly dissociates after hydrolysis. This
mechanism allows Gz GAP to act catalytically;
i.e. one GAP molecule can cycle among multiple molecules of
G
z-GTP. A corollary to this behavior is that the rate-limiting step in the GAP-mediated GTPase reaction is hydrolysis of
the GAP-G
z-GTP complex. This conclusion is supported by
the finding that the maximum reaction rate at saturating and
super-stoichiometric concentrations of GAP (Fig. 5B) is the
same as the maximum specific activity of the GAP at saturating
G
z-GTP (Fig. 5A). In apparent contrast, the
effect of RGS4 on Go and Gi seems to be limited by substrate binding (12).
The activity of Gz GAP during receptor-stimulated
steady-state GTP hydrolysis is evident when it is co-reconstituted in
phospholipid vesicles with Gz and m2 muscarinic receptor
(Fig. 9). The relative effect of the GAP activity was limited by its
concentration and/or by the ratio of its concentration to that of
Gz. Gz was in molar excess over GAP in the
vesicles, the probable physiologic condition, and stimulation of
steady-state hydrolysis increased if either more GAP or less
Gz was used. We have not yet pursued this relationship quantitatively because the availability of GAP and its concentration in
stock solutions were both limiting. The need for G in the vesicles to permit receptor-G
z coupling probably
diminished the effect of the GAP (Fig. 8), and we also suspect that
there was less than one molecule of GAP per vesicle. It is important to note that GAP does not alter the rates of dissociation of either GDP or
GTP from G
z. It will not, therefore, influence
activation rates and will only accelerate the deactivation limb of the
GTPase cycle.
Gz GAP binds the activated form of Gz with
about the same affinity when it is bound to GTP (according to
Km), to GTP
S, or to GDP/AlF4
(according to Ki). In contrast, RGS4, a GAP for the
Gi family and Gq, is inhibited much more
potently by an GDP/AlF4-bound G
than by the same G
bound to GTP
S (47, 48). Because GDP/AlF4 binds to
G
i1 and G
t as a transition state analog
(45, 46), these authors suggested that RGS4 acts as a GAP by favoring
the transition state structure of a G
over its GTP-bound form. This
mechanism would presumably differentiate the GAP activity of RGS
proteins from that of effectors, which are activated both by GTP
S-
and GDP/AlF4-liganded G proteins, and would thus suggest
that the cerebral Gz GAP is an effector. This distinction
may not be generally valid, however, or may perhaps not extend to
Gz. In preliminary experiments, we found that RGS4 is
potently inhibited by the GTP
S-bound form of G
z, a
behavior similar to that of the cerebral Gz GAP. The active
site and enzymatic properties of Gz differ markedly from
those of other Gi family members (16-18), and it is
possible that GDP/AlF4 is not a transition state analog at
the active site of G
z. If true, however, this argument
would favor an effector function for the Gz GAP.
Regardless of any yet unknown cellular roles of the Gz GAP,
its presence and regulation will influence Gz-modulated
signaling. The first mode of regulation so far observed is inhibition
of the GAP by G subunits. Inhibition of GAP activity by G
over a reasonable range of concentrations allows modulation of
Gz signaling by other G protein pathways, where activation
will release G
in large excess over G
z. Other
controls of GAP activity are also likely, and their understanding
should help us understand the cellular pathways uniquely regulated by
Gz.
![]() |
(Eq. 1) |
We thank Juriaan Huskens and A. Dean Sherry
(University of Texas, Dallas) for generously providing the reagents and
NMR facilities for measuring free Mg2+ and for help with
the measurements themselves. We also thank Gloria Biddlecome for
extensive and insightful discussion of the manuscript, Karen Chapman
for help performing Sf9 cell culture, Carmen Dessauer (UT-Southwestern)
for samples of adenylyl cyclase isoforms, Tohru Kozasa
(UT-Southwestern) for a sample of G12 and for advice on
the expression and purification of G
z, and other members
of this department for various cultured cells. Pilot measurements of
Km for Gz-GTP were performed by
Yoon-Hang Kim.