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and Pertussis Toxin-Catalyzed ADP-Ribosylation of Gi
Department of Cell and Molecular Biology (R.M.R.-G., M.H.-D)
Northwestern University Medical School Chicago, Illinois 60611
Department of Physiology and Biophysics and Psychiatry
(M.M.R.) University of Illinois College of Medicine Chicago,
Illinois 60680
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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proteins were associated
with the LH/CG receptor in ovarian follicular membranes. Since
hormone-dependent, CTX-catalyzed ADP ribosylation (AR) constitutes
evidence that a G protein is specifically associated with a
receptor, CTX-catalyzed AR of membrane proteins was examined both in
the presence and absence of guanine nucleotides to determine which G
proteins exhibit hCG-dependent labeling by
[32P]NAD. Results demonstrated the time- and
hCG-dependent AR of both a 45-kDa protein and a 48/50-kDa doublet as
well as a 40-kDa protein that was also sensitive to AR by PTX in a
time- and hCG-dependent manner. Using anti-G protein antisera to
specifically immunoprecipitate photoaffinity-labeled G proteins, we
were able to identify the 45- and 48/50 kDa proteins as the short and
long forms of Gs and the 40-kDa protein as
Gi. A monoclonal anti-hCG antibody
immunoprecipitated the activated LH/CG receptor along with the long and
short forms of Gs and
Gi. These results suggest that a portion of
Gi along with the long and short forms of
Gs are associated physically with the LH/CG
receptor in ovarian follicular membranes. |
INTRODUCTION |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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-, ß-, and 
-subunits. In
the inactive heterotrimeric conformation, GDP is bound to the
-subunit. Agonist stimulation of receptor promotes the rate-limiting
release of GDP from the -subunit and subsequent binding of GTP. The
GTP-bound -subunit, in its active conformation, activates the
appropriate effector. Then, through the intrinsic GTPase activity of
the -subunit, GTP is hydrolyzed to GDP resulting in the formation of
the inactive heterotrimeric G protein. This cycle continues as long as
agonist is available and able to activate receptor and as long as G
protein can stimulate effector activity.
Based upon their differential ability to covalently modify guanine
nucleotide-binding proteins, bacterial toxins have become useful as
biochemical tools in the study and identification of heterotrimeric G
proteins. The -subunit of G proteins contains a site that may be
covalently modified by the NAD-dependent ADP ribosylation catalyzed by
the bacterial toxins, cholera toxin (CTX) and/or pertussis toxin (PTX).
PTX catalyzes the transfer of ADP-ribose from NAD to a cysteine residue
four amino acids from the carboxyl terminus of Gi,
Go, and Gt (2, 3, 4, 5, 6). Because it is the
carboxyl terminus of the G protein that interacts with receptor, ADP
ribosylation prevents this interaction and results in uncoupling of the
G protein from receptor (7, 8).
CTX catalyzes the ADP ribosylation of an internal arginine residue (9)
of Gs, Golf, and Gt. The
modified arginine residue is located close to the GTP-binding domain of
the -subunit (10), and ADP ribosylation of this residue results in a
decreased rate of GTP hydrolysis (11, 12), leading to constitutive
activation of G proteins. Of the G proteins that are widely expressed,
Gs is the only well characterized G protein that
undergoes CTX-catalyzed ADP ribosylation (Arg 201 in the long form of
Gs, Arg 187 in the short form of Gs).
However, since all G proteins contain the substrate site for
CTX-catalyzed ADP-ribosylation (13), it has been predicted that G
proteins other than Gs might also be covalently modified
by CTX. Indeed, as detailed below, various laboratories have shown that
some PTX-sensitive G proteins (Gi2, Gi3,
Go) also exhibit hormone-dependent ADP ribosylation by
CTX under specific conditions. The site of CTX-catalyzed ADP
ribosylation on Gi has been identified as an arginine
residue that corresponds to Arg 201 of Gs (14, 15, 16, 17). When
membranes from Rat 1 fibroblasts transfected with the human
2-C10 adrenergic receptor were incubated with CTX and
[32P]NAD in the absence of added guanine nucleotides,
Milligan and co-workers (13) demonstrated the incorporation of an
ADP-ribose moiety into Gi2 and Gi3 in the
presence of agonist as well as the agonist-independent ADP ribosylation
of the long and short forms of Gs (13).
Although it has been well characterized that agonist stimulation of the
LH/CG receptor stimulates adenylyl cyclase activity (18), presumably
via Gs, and in some cells stimulates phospholipase C (PLC)
(19, 20, 21, 22, 23) via a PTX-sensitive G protein (24), there is no reported
evidence that indicates direct physical interaction between the
LH/CG receptor and any associated G protein(s). In this study,
CTX-catalyzed ADP ribosylation of porcine ovarian follicular membranes
was examined, both in the presence and absence of guanine nucleotides,
to determine which G proteins exhibit hCG-dependent ADP ribosylation.
Using anti-G protein antisera to specifically immunoprecipitate
radiolabeled G proteins as well as a monoclonal anti-hCG antibody,
which immunoprecipitates the activated LH/CG receptor (25) and
presumably any receptor-associated G proteins, we were able to identify
conclusively that both the long and short forms of Gs
interact with the LH/CG receptor. Our results also show that the LH/CG
receptor also couples to a portion of the Gi expressed
in ovarian follicular membranes.
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RESULTS |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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2, Gq/11,
Gi, G13, and ras are present in porcine
ovarian follicular membranes while Go and
Gz are absent (Table 1
). Antibodies used
in immunoblot analyses and immunoprecipitation studies are listed in
Table 2. In an effort to identify the G proteins in
porcine follicular membranes that are physically associated with the
LH/CG receptor, we used the ability of bacterial toxins to catalyze the
hormone-dependent ADP ribosylation of those G proteins that are
functionally coupled to LH/CG receptors.
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, lanes 5–8). This result was expected since the only
PTX-sensitive G protein in porcine follicular membranes is
Gi (Table 1
). Control studies indicated that the 40-kDa
band was specifically labeled by PTX since samples that were incubated
in the presence of excess cold NAD (400 µM; not shown) or
in the absence of PTX (Fig. 1B, lanes 5–8) did not exhibit the 40-kDa
radiolabeled band. On immunoprecipitation using
anti-Gi antibody (antiserum 117), the 40-kDa protein
ADP-ribosylated by PTX was immunoprecipitated (Fig. 2A, lane 2). These data lead us to conclude unambiguously that the
PTX-sensitive, radiolabeled 40-kDa band in porcine follicular membranes
is Gi.
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i a poorer substrate for ADP ribosylation by PTX and
resulting in decreased labeling of i with
[32P]NAD. Incubation of follicular membranes in the
presence of GTP resulted in an approximately 2-fold increase in the ADP
ribosylation of Gi when compared with incubations in the
absence of guanine nucleotides (Fig. 1A, lanes 5–8; Fig. 1B, lanes
1–4). However, PTX-catalyzed ADP ribosylation of Gi did
not appear to be affected, i.e., was not reduced, by LH/CG
receptor activation in this 30-min incubation (Fig. 1A, lanes 5–8;
Fig. 1B, lanes 1–4). A time course of PTX-sensitive ADP ribosylation
was also performed in the presence or absence of hCG. We observed that
ADP ribosylation of Gi was decreased in the presence of
agonist, especially at the earliest times of incubation, whether or not
exogenous GTP was included in the incubations (Fig. 3).
This result is consistent with LH/CG receptor activation of a portion
of the Gi in follicular membranes.
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, lanes 1 and
2). Equivalent results (i.e., hCG-independent CTX-catalyzed
ADP ribosylation of the 48/50 and 45-kDa bands) were obtained when
membranes were incubated with GDP (10 µM, not shown).
Immunoblotting of membrane proteins using anti-Gs (U-584
antiserum) showed immunoreactive bands that also migrated at 45 and 48
kDa (Fig. 4, lane 2). When incubations with CTX were
performed in the absence of added guanine nucleotides, we detected a
slight hormone-dependent increase in ADP ribosylation (
1.4-fold over
incubation performed in the absence of hCG or basal levels) of the
45-kDa band as well as a more distinct increase in labeling
(2.5-fold over basal levels) of the 48/50-kDa doublet in the
presence of hCG (Fig. 1A, lanes 3 and 4). A 40-kDa band was also
ADP-ribosylated by CTX in the absence, but not in the presence, of GTP
(Fig. 1A, lanes 3 and 4). CTX-catalyzed ADP ribosylation of the 40-kDa
protein was often slightly increased by hCG, consistent with coupling
of the activated LH/CG receptor to this protein.
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, compare
lanes 3 and 4 to lanes 5–8), which suggests that this CTX-labeled 40
kDa protein most likely represents Gi. To confirm the
identity of the 40-kDa band, membranes were incubated with CTX and
immunoprecipitated using anti-Gi antibody. As shown
in Fig. 5A, the 40-kDa CTX-catalyzed ADP-ribosylated
band was immunoprecipitated by anti-Gi antibody (as
indicated by the arrow in lane 3). This
anti-Gi antibody also immunoprecipitates a portion of
the CTX-labeled 45-kDa band (shown below to be the short form of
Gs3).
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(antiserum U-584) (Fig. 4), we determined
whether the CTX-catalyzed radiolabeled bands indeed represented the
long and short forms of Gs. Membranes were incubated
with [32P]NAD and CTX in the absence of GTP, since these
conditions appeared to be optimal for the ADP ribosylation of the 45-,
48/50-, and 40-kDa proteins in the same sample, and Gs
was then immunoprecipitated with anti-Gs antisera
(antiserum 1190). Both 45- and 48/50-kDa CTX-catalyzed ADP-ribosylated
bands, representing the short and long forms of Gs, were
immunoprecipitated using the anti-Gs antiserum (Fig. 5B, lane 4). Anti-Go antibody (antiserum 9072), as well as
preimmune sera, served as negative controls (Fig. 5B, lanes 2 and
3).
In view of the clear dependence on hCG of CTX-catalyzed ADP
ribosylation of the long form Gs in a 30-min reaction, a
time course of CTX-catalyzed ADP ribosylation was performed. When GTP
was added to the incubations, both the short and long forms of
Gs were increasingly ADP-ribosylated with increasing
time of incubation, exhibiting 2.3- and 4-fold increases at 30 min
relative to levels at 1 min of incubation, respectively, but in a
hormone-independent manner (Fig. 6A). When incubations
were conducted in the absence of GTP (Fig. 6B), a slight hCG-dependent
increase in the ADP ribosylation of the 45-kDa short form of
Gs was detectable as early as 1 min (1.5-fold over basal
levels) which persisted for at least 30 min (1.4-fold over basal
levels). However, more dramatic hCG-dependent labeling of the 48/50 kDa
long form of Gs was noted by 1 min (3.6-fold over basal
levels) and lasted at least 30 min (2.5-fold over basal levels). Like
labeling of the Gs forms, CTX-catalyzed ADP ribosylation
of Gi at 40 kDa increased with incubation time (Fig. 6B).
Human CG promoted a slight increase in ADP ribosylation of
Gi, especially at the early incubation times. When the same
study was performed using the hCG antagonist, deglycosylated hCG
(dhCG), instead of hCG, no change was observed in the level of ADP
ribosylation of the short or long forms of Gs or
Gi between membranes incubated in the absence of dhCG
compared with membranes incubated for the same amount of time with dhCG
(not shown). This result confirmed that ADP ribosylation of
Gs and Gi catalyzed by CTX in the absence of
GTP was specific for hCG-dependent receptor activation.
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in the absence of GTP was
dependent on the concentration of hCG in incubations. As shown in Fig. 7A (lanes 7–12), increased labeling of both short and
long forms of Gs was observed with increasing
concentrations of hCG while labeling of Gi was
hCG-independent in this 30-min incubation. Membranes incubated with CTX
in the presence of GTP (Fig. 7A, lanes 1–6) exhibited only
hormone-independent labeling of Gs as predicted from
results shown in Figs. 1 and 6A. The graphs in Fig. 7B quantify the
level of ADP ribosylation of the long and short forms of
Gs from three separate hCG dose-response studies
performed in the absence of GTP.
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protein at 42/43 kDa was detected in immunoprecipitates, we were unable
to detect any ADP ribosylation of this protein in incubations conducted
in the absence or presence of hCG and/or GTP (not shown). Thus, we
hypothesized that Gq/11 was not a good substrate for CTX
under the conditions used in our assay. To further investigate this,
membranes from SF9 cells that overexpress both Gq and
the muscarinic acetylcholine receptor (mAChR), which signals via
Gq/11, were incubated in the presence of CTX and in the
absence or presence of carbachol. Despite the presence of
Gq/11 protein in the membranes (Fig. 8, lane
1), no CTX-catalyzed ADP-ribosylated protein at 42/43 kDa was
immunoprecipitated by the anti-Gq/11 antibody from SF9 cell
membranes (Fig. 8, lanes 2 and 3). From these results we conclude that
Gq/11 is not an efficient substrate for CTX-catalyzed ADP
ribosylation under the conditions used in these studies.
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and
Gi with the LH/CG Receptor and a portion of the short form of both
Gs and Gi are functionally associated with
the LH/CG receptor, based on the hCG dependence of the CTX-catalyzed
ADP ribosylation of Gs and Gi and the
PTX-catalyzed ADP ribosylation of Gi, these results do not
provide any direct evidence that the LH/CG receptor physically
interacts with Gs or Gi. To obtain more direct
evidence for an association of the LH/CG receptor with G proteins, we
used an anti-hCG monoclonal antibody, B105, which has previously been
shown by our laboratory to immunoprecipitate hormone-activated
LH/CG receptor (25). B105 was used to immunoprecipitate the LH/CG
receptor from membranes with the idea that if receptor is physically
associated with one or more G protein(s), then B105 should be able to
immunoprecipitate the receptor/G protein(s) complex, as was shown for
the muscarinic acetylcholine receptor (28). Follicular membranes were
incubated with hCG in the absence of GTP and in the presence of CTX and
[32P]NAD, since these conditions were optimal for the ADP
ribosylation of both forms of Gs and of Gi, or
in the presence of PTX. Both the 45-kDa and 48/50-kDa Gs
forms were immunoprecipitated by B105 (Fig. 2A, lane 4). Based on the
amount of membrane protein that was labeled with CTX (see legend to
Fig. 2) and the ability of the anti-hCG antibody to immunoprecipitate
50% of activated LH/CG receptor (25), approximately 40% of the
CTX-labeled Gs is being immunoprecipitated with the
LH/CG receptor. ADP-ribosylated Gi from membranes incubated
with either CTX or PTX was also immunoprecipitated with the activated
LH/CG receptor (Fig. 2A, lanes 4 and 5). Based on the amount of
membrane protein that was labeled with CTX or PTX and the efficiency of
immunoprecipitation of the LH/CG receptor by the anti-hCG antibody
(25), approximately 7% of Gi is immunoprecipitated with
the activated LH/CG receptor.
Because the amount of Gi that was physically associated
with the LH/CG receptor was low (7%), a similar immunoprecipitation
experiment was performed using the anti-LH/CG receptor antibody, LHR38
(29), after membrane proteins were incubated with the
radiolabeled, nonhydrolyzable, photoaffinity GTP analog,
P3-(4-azidoanilido)-P1 5'-GTP
([32P]AAGTP) (Fig. 2B, lane 1). Indeed, anti-LH/CG
receptor antibody immunoprecipitated Gi (and
Gs) along with the LH/CG receptor. Normal mouse serum
was used as a negative control and was unable to immunoprecipitate any
[32P]AAGTP-bound proteins (Fig. 2B, lane 2).
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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Because the agonist-occupied LH/CG receptor has been shown to activate two distinct effectors, it is possible that this receptor is coupled to more than one G protein. Indeed, Herrlich et al. (24) have recently shown that the LH/CG receptor transfected into L cells is capable of coupling both to Gi to activate PLC and to inhibit adenylyl cyclase activities and to Gs to stimulate adenylyl cyclase activity. Receptor coupling to more than one G protein is not an unprecedented phenomenon since various investigators have demonstrated multiple G proteins coupled to a single receptor based upon immunoprecipitation of receptor-G protein complexes using G protein peptide-specific antisera (33), agonist-dependent bacterial toxin labeling of G proteins (34), and agonist-dependent stimulation of effector activity (24, 35). In light of the potential ability of the LH/CG receptor to couple to more than one G protein in a physiological cell model, CTX was used under specific conditions as a means of identifying LH/CG receptor-associated G proteins in porcine ovarian follicular membranes.
Many studies designed to detect receptor association to specific G proteins have used transfected cell systems or proteins reconstituted into phospholipid vesicles. Although these studies have been invaluable in identifying with which G proteins a receptor may couple, it is difficult to determine physiological interactions utilizing these methods because overexpressing receptors or G proteins into a cell system can skew the actual associations taking place between these signaling proteins. We performed our toxin studies using porcine ovarian follicular membranes, with no exogenous signal-transducing molecules introduced to the system, with the expectation of identifying physiological interactions between G proteins and the LH/CG receptor.
Studies were performed first to confirm LH/CG receptor association with
Gs. Our results provide evidence, for the first time, that
the LH/CG receptor is directly associated with both the long and short
forms of Gs in ovarian follicular membranes. Under
conditions used in our studies, ADP ribosylation of the long form of
Gs requires agonist-dependent activation of the LH/CG
receptor and is dependent on the concentration of agonist bound to the
receptor.
Next we wanted to determine whether the LH/CG receptor in porcine
follicular membranes was associated with any G proteins other than
Gs, possibly to activate an effector distinct from adenylyl
cyclase such as PLC. Early studies reported that hCG-dependent
activation of PLC appeared to be PTX-insensitive in L cells transfected
with the murine LH/CG receptor (19). As PLC can be activated by
Gq (36, 37, 38, 39), ß from Gi or potentially
Gs (40, 41, 42, 43), or by both Gq and ß from
Gi (44) and because G proteins that modulate PLC in
physiological cell models have not been elucidated, we determined
whether LH/CG receptor activation promoted CTX-catalyzed
ADP-ribosylation of Gq/11. However, we did not detect
CTX-catalyzed ADP-ribosylation of Gq/11 in porcine
follicular membranes under any of the labeling conditions that we used
(± GTP, ± hCG) or in SF9 cells (± carbachol) overexpressing mAChR
and Gq. This result does not eliminate the possibility that
Gq/11 proteins are activated by the LH/CG receptor but
rather indicates that Gq/11 proteins are not good
substrates for CTX-catalyzed ADP ribosylation under the conditions
used.
Recent studies have demonstrated that the murine LH/CG receptor
transfected into L cells couples both to PLC and to adenylyl cyclase
via Gi2 (24). In this model, PTX treatment augmented
hCG-stimulated cAMP production and greatly reduced PLC activity. Our
results provide evidence that the endogenous LH/CG receptor is also
coupled to Gi in follicular membranes, albeit a relatively
small percentage of the total Gi. This conclusion is based
on the following observations. First, some of the Gi is ADP
ribosylated in a hormone-dependent manner by CTX in the absence of
added GTP (see Figs. 1A and 6B). Second, we consistently observed a
distinct decrease of agonist-induced PTX-catalyzed ADP ribosylation
of Gi in follicular membranes consistent with
agonist-dependent activation of Gi (seen in Fig. 3). Third,
the anti-hCG antibody, B105, was able to immunoprecipitate both CTX-
and PTX-catalyzed ADP-ribosylated Gi from membranes, albeit
at very low levels relative to the amount of Gi in
membranes labeled with PTX or CTX and relative to the amount of
Gs in membranes that was immunoprecipitated with this
antibody. Fourth, anti-LH/CG receptor antibody, LHR38,
immunoprecipitated [32P]AAGTP-bound Gi from
membranes. Taken together, these data show that a portion of
Gi in ovarian follicular membranes is functionally and
physically associated with the LH/CG receptor. However, it is likely
that only a small percentage of the membrane Gi is coupled
to the LH/CG receptor, based on the following observations:
hCG-dependent CTX-catalyzed ADP ribosylation of Gi was not
observed in every experiment (compare Figs. 1 and 6B with Fig. 7A), and
when it was observed, it was not robust; the hCG-dependent reduction in
PTX-catalyzed ADP ribosylation of Gi was consistent but
weak; only a small percentage of membrane Gi (labeled with
PTX or CTX) immunoprecipitated with the LH/CG receptor.
The ability of the anti-hCG antibody (B105) to precipitate
LH/CG-activated receptor (25) and Gi ADP-ribosylated by PTX
appears contradictory because PTX-catalyzed ADP ribosylation
functionally uncouples Gi from the receptor. At this time
we are unable to explain the mechanism behind this observation;
however, it is possible that although most of the ADP-ribosylated
Gi is uncoupled from the LH/CG receptor, a small percentage
remains bound and is precipitated along with the activated LH/CG
receptor using B105. Alternatively, PTX-stimulated Gi
uncoupling from LH/CG receptor may be only functional and may not
abolish association of the G protein with receptor. That Gi
is a poorer substrate for PTX in the presence of hCG during the time
course of ADP-ribosylation (seen in Fig. 3) supports the observed
interaction between LH/CG receptor and Gi.
Several studies have been performed using CTX to ADP ribosylate
PTX-sensitive G proteins in the absence of GTP (13, 16, 45).
Investigators have observed that in order for -subunits other than
s to serve as optimal substrates for CTX-catalyzed ADP
ribosylation, the guanine nucleotide-binding pocket must be devoid of
nucleotide. Consequently, this technique can be used to demonstrate
specific receptor/G protein associations since GDP is released from the
-subunit upon receptor activation. When incubations are conducted in
the absence of exogenous guanine nucleotides, the guanine
nucleotide-binding domain then remains empty and the arginine substrate
site for ADP ribosylation in at least i proteins is
accessible to CTX. Gs also has been shown to be an optimal
substrate for CTX when coupled to an agonist-activated receptor in the
absence of GTP (45). In porcine ovarian follicular membranes,
hormone-dependent CTX-catalyzed ADP ribosylation of the long form of
Gs was observed in the absence but not in the presence of
either GTP or GDP. In the presence of guanine nucleotides, ADP
ribosylation of the long form of Gs appears to be
independent of hCG, suggesting that in the presence of GTP the
CTX-sensitive arginine is equally accessible for ADP ribosylation in
the absence or presence of hCG. In the absence of GTP, however, when
LH/CG receptor is activated by hCG, GDP is released from the G protein
and because GTP is not available to bind, the site is open and
accessible for CTX-catalyzed ADP ribosylation. It appears that guanine
nucleotides eliminate the hCG requirement for CTX-catalyzed ADP
ribosylation. In contrast to the long form of Gs, the
45-kDa short form of Gs appears to be a good substrate
for CTX-catalyzed ADP ribosylation whether or not the guanine
nucleotide-binding site is filled with GTP or GDP or is empty.
Gi also is a substrate for CTX-catalyzed ADP ribosylation
in the absence of GTP. It appears that like the long form of
Gs, the arginine substrate site of Gi is
optimally accessible to CTX only when the guanine nucleotide-binding
site is empty (seen in Fig. 1A, lanes 1–4).
It has long been recognized that the LH/CG receptor, unlike most other G protein-linked receptors, does not exhibit the typical guanine nucleotide dependence on agonist affinity (46, 47, 48). Thus, in the absence or presence of guanine nucleotides, the LH/CG receptor always exhibits high affinity for the agonist. Perhaps the basis for this observation lies in the sustained coupling, in the absence or presence of GTP and hormone, of the majority of the short form of Gs to the LH/CG receptor. Perhaps it is the long form of Gs that specifically couples to adenylyl cyclase. Further studies are required to elucidate potentially distinct roles for the long vs. the short forms of Gs in follicular membranes.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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(06–190; recognizes Gi, Go and
Gt) used in immunoblot analysis, UBI (Lake Placid, NY);
anti-Gq/11 (C-19, specific for Gq and
G11) used in immunoprecipitation studies, Santa Cruz
Biotechnology, Inc. (Santa Cruz, CA); LHR38, Transbio; coarse Sephadex
G25, Pharmacia Biotechnology Inc. (Piscataway, NJ); pertussis toxin,
List Biological Inc. (Campbell, CA) ; electrophoresis purity reagents,
Bio-Rad Laboratories (Richmond, CA); prestained mol wt markers,
Diversified Biotech; nytran, Schleicher & Schuell, Inc. (Keene, NH);
other reagents, Sigma (St. Louis, MO). Radiochemicals were used without
further purification.
Preparation of Ovarian Follicular Membranes
Walls from follicles that were 6–12 mm in diameter were
dissected from ovaries of nonpregnant pigs. A 10,000 x
g membrane fraction was then prepared as described
previously (49). Protein was determined by the method of Lowry et
al. (50).
ADP Ribosylation of Porcine Large Follicle Membranes by CTX or
PTX
Before addition to membrane preparations, CTX (2 mg/ml) was
activated by incubation at 37 C for 15 min with an equal volume of 40
mM dithiothreitol (DTT). The sample was then desalted on a
coarse Sephadex G25 column using 1 mg/ml BSA as the elution buffer. PTX
(50 µg) was dissolved in 250 µl distilled water (GIBCO, Grand
Island, NY) and activated by incubation with an equal volume of 50
mM DTT for 30 min at 30 C. Membranes (100 µg) were then
incubated for 30 min at 30 C for standard assays (varying incubation
times for time course studies) in a final volume of 100 µl containing
1 mM ATP, 15 mM thymidine, 5 mM
ADP-ribose, 20 mM L-Arg-HCl, 5 mM DTT, 25
mM Tris-HCl, pH 7. 5, 20 µM
[32P]NAD (1 Ci/mmol), 5 mM GTP, in the
absence or presence of 10 µg/ml hCG, and in the presence of either 50
µg/ml CTX or 2. 5 µg/ml PTX. Membranes were then washed with 1 ml
wash buffer (10 mM Tris-HCl, pH 7. 5, 1 mM
EDTA) and resuspended in 50 µl SDS sample buffer. The samples were
run on a 10.5% SDS-polyacrylamide gel (51). The gel was then either
stained, destained, dried, and exposed to Kodak X-Omat AR film (Eastman
Kodak, Rochester, NY) or transferred onto nytran overnight (4 C, 0.1
A).
Solubilization
Membranes (500 µg) were pelleted and resuspended to a final
concentration of 5 µg/µl (100–150 µl per Eppendorf tube) in
solubilization buffer (50 mM Tris-HCl,pH 7. 4, 1.0% Triton
X-100, 25% glycerol, 5 mM EDTA/5 mM EGTA, pH
7. 4, 1 mM phenylmethylsulfonylfluoride, 50 mM
benzamidine, 100 µM leupeptin, 5 µg/ml aprotinin, and
50 µg/ml soybean trypsin inhibitor). Membranes were allowed to stir
slowly in solubilization buffer at 4 C for 60 min at which time they
were diluted 10-fold in solubilization buffer in the absence of Triton
so that the Triton concentration in the final volume was 0.1%.
Nonsolubilized material was removed by centrifugation (100,000 x
g, 60 min, 4 C). Supernatant was either used in Western blot
analyses or immunoprecipitation studies.
Immunoprecipitation
Protein A-Sepharose (33%) (30 µl) was added to solubilized
membranes and rotated at 4 C for 2 h to bind any nonspecific
solubilized proteins. Sepharose was then pelleted and discarded, and
the immunoprecipitating antibody was added (1:50 dilution of anti-G
protein antibodies and preimmune sera, 75 µg B105) and allowed to
rotate at 4 C overnight. If B105 was the primary antibody used, 100
µl rabbit anti-mouse IgG were added the next morning and the mixture
was rotated for 2 h at 4 C. If a polyclonal antibody was used as
the immunoprecipitating antibody (G protein antibodies and preimmune
sera), no secondary antibody was added. Subsequently, 30 µl Protein
A-Sepharose (33%) were added, and the incubation was allowed to
continue for 2 h. Immunocomplexes were collected as Protein-A
Sepharose pellets by centrifugation (10,000 x g, 10
min, 4 C). These pellets were washed twice with 1 ml of a buffer
containing 50 mM Tris-HCl, 0. 1% Triton X-100, 0.1% BSA,
and 25% glycerol. The Sepharose pellet was then resuspended in 50–100
µl of 3-fold SDS sample buffer, vortexed briefly, and allowed to sit
at room temperature for at least 30 min. Samples were placed in a
boiling water bath (5 min) and centrifuged (10,000 x
g, 10 min), and supernatant was subjected to SDS-PAGE
(10.5% acrylamide, 50 mA).
Western Blot Analysis
To detect G proteins in porcine follicular membranes, 50–75
µg membranes (and up to 300 µg membranes to detect Gz)
in SDS sample buffer were routinely run on a 10.5% SDS-polyacrylamide
gel, after which the gel was transferred onto nytran (0.2-µm pore
size) overnight (4 C, 0.1 A). Nytran was blocked in blocking buffer (50
mM Tris-HCl, pH 8. 5, 150 mM NaCl, 3% BSA, 0.
1% NaN3) for 3 h and then incubated with primary
antibody in blocking buffer (overnight, 4 C, rotating). The next day,
nytran was washed sequentially in wash buffer (50 mM
Tris-HCl, pH 8. 5, 150 mM NaCl) for 10 min, detergent wash
buffer (wash buffer, 0. 5% BSA, 0.1% Triton X-100) twice for 10 min,
and wash buffer again for 10 min. Nytran was then incubated with
[125I]donkey anti-rabbit IgG for approximately 3 h,
rotating at room temperature. Then the wash sequence was repeated and
nytran was air dried and exposed to Kodak X-Omat AR film.
[32P]AAGTP-Labeling of Porcine Ovarian
Large Follicle Membrane Proteins
Porcine ovarian large follicle membranes were incubated in a
volume of 40 µl incubation medium containing 0.5 µM
[32P]AAGTP, 1.0 µg/ml hCG, 10 µM GDP,
31.25 mM 1,3-bis[tris(hydroxymethyl)-methylamino]propane,
pH 7.2, 6.25 mM MgCl2, 0.5/1.25 mM
EDTA/EGTA, 25 mM creatine phosphate, and 0.2 mg/ml creatine
phosphokinase. Incubation was allowed to proceed at 30 C for 20 min,
and the reaction was stopped by placing sample tubes on ice and adding
1 ml cold 10 mM Tris-HCl, pH 7.2 + 0.2 µM
ß-mercaptoethanol. Samples were then centrifuged (20,000 x
g, 5 min, 4 C), and membrane pellets were resuspended in 40
µl incubation medium that did not contain [32P]AAGTP or
hCG but did contain 1 µg/ml BSA. Samples were UV-irradiated for 3 min
at 4 C approximately 5 cm from the UV source. Samples were then
pelleted again and resuspended in solubilization buffer followed by
immunoprecipation with LHR38 or normal mouse sera. This method is
adapted from that of Rasenick et al. (61). Baculovirus
expression of Gq SF9 cells were infected with viruses
encoding mAChR (from Elliott Ross) and Gq (from James
Garrison). Membranes were prepared and photolabeled as described by
Popava et al. (62).
|
ACKNOWLEDGMENTS |
|---|
(antiserum 1190),
anti-Gi (antiserum 117), anti-Go
(antiserum 9072), and corresponding preimmune sera from Dr. David
Manning (Department of Pharmacology, University of Pennsylvania School
of Medicine, Philadelphia, PA); anti-Gs (antiserum
U-584) used for immunoblot analysis from Drs. Alfred Gilman and Susanne
Mumby (University of Texas Southwestern Medical Center, Dallas, TX);
anti-Gq/11 (antiserum B6T) used for immunoblot analysis
from Dr. Tom Martin (University of Wisconsin, Madison, WI);
anti-Gz (antiserum P-961) from Dr. Patrick Casey
(Department of Biochemistry, Duke University Medical Center, Durham,
NC); anti-ras (antiserum Y13–259) from Dr. Frank McCormick (Onyx
Pharmaceuticals, Richmond, CA); deglycosylated hCG was a gift from Dr.
Robert Ryan (Mayo Medical School, Rochester, MN). |
FOOTNOTES |
|---|
This research was supported by the US Department of Agriculture Grant NRICGP 9401432 (to M.H.D.) and USPHS Grant MH 39595 and The Council for Tobacco Research Grant 4089 (to M.M.R.).
1 Predoctoral appointee to the NIH Training Program in Reproductive
Biology (T32-HD 07068–17). 
2 The long form of Gs usually is
seen to resolve as a doublet upon CTX-catalyzed ADP-ribosylation but as
a single band in immunoblots.
3 This anti-Gi antibody also
immunoprecipitates [32P]-azidoanilido-GTP-labeled
Gs from porcine follicular membranes (R. M.
Rajagopalan-Gupta, M. M. Rasenick, and M. Hunzicker-Dunn, manuscript in
preparation).
Received for publication September 3, 1996. Revision received January 13, 1997. Accepted for publication February 18, 1997.
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