Luteinizing Hormone/Choriogonadotropin-Dependent, Cholera Toxin-Catalyzed Adenosine 5'-Diphosphate (ADP)-Ribosylation of the Long and Short Forms of Gs
and Pertussis Toxin-Catalyzed ADP-Ribosylation of Gi
Rajsree M. Rajagopalan-Gupta1,
Mark M. Rasenick and
Mary Hunzicker-Dunn
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
 |
ABSTRACT
|
---|
Although it is well established that activated
LH/human (h) CG receptor stimulates adenylyl cyclase activity (via the
heterotrimeric stimulatory guanine nucleotide-binding protein,
Gs) and in some cells stimulates phospholipase
C activity, there is no evidence for a direct physical interaction
between the LH/CG receptor and Gs or any other
G protein(s). We conducted studies using cholera toxin (CTX) and
pertussis toxin (PTX) to determine which G
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
|
---|
Members of the seven transmembrane-spanning domain receptor
family, including the LH/CG receptor, transduce information from
extracellular signals, such as hormones, neurotransmitters, and sensory
stimuli, to cellular effector enzymes or ion channels via
membrane-associated heterotrimeric GTP-binding proteins (G proteins)
(1). These G proteins are composed of
-, 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.
 |
RESULTS
|
---|
Hormone-Dependent ADP Ribosylation of G Proteins
Immunoblot analysis showed that
Gs
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.
Incubation of follicular membranes under ADP ribosylation conditions
using [32P]NAD and PTX in the absence and presence of hCG
and in the absence or presence of GTP resulted in the ADP ribosylation
of a single protein that migrated on SDS-PAGE at 40 kDa, consistent
with the molecular mass of Gi (Fig. 1A
, lanes 58). 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 58) 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
.

View larger version (57K):
[in this window]
[in a new window]
|
Figure 1. CTX- and PTX-Catalyzed ADP Ribosylation of
Follicular Membrane Proteins
A, SDS-PAGE of membranes incubated with[32P]NAD in
the presence of either CTX or PTX. Membranes (100 µg protein) were
incubated at 30 C for 30 min in the presence of either CTX (lanes 14)
or PTX (lanes 58), in the presence (lanes 1, 2, 5, 6) or absence
(lanes 3, 4, 7, 8) of 5 mM GTP, and in the absence (lanes
1, 3, 5, 7) or presence (lanes 2, 4, 6, 8) of 10 µg/ml hCG in a
reaction mix as described in Materials and Methods. The
gels were exposed to x-ray film with an intensifying screen. Molecular
masses of labeled proteins (kDa) are indicated at the
left and were calculated based on the migration of
protein standards (shown at right). Coommassie staining
of the gel showed that equal levels of proteins were loaded in each
lane. Equivalent results were obtained in three separate experiments.
B, SDS-PAGE of membranes indicating specificity of PTX-catalyzed ADP
ribosylation of membrane proteins. Membranes (100 µg protein) were
incubated in the presence (lanes 14) or absence (lanes 58) of PTX
(2. 5 µg/ml) at 30 C for 30 min in the presence of 10 µg/ml BSA
(lanes 2, 4, 6, 8) or hCG (lanes 1, 3, 5, 7) and in the presence (lanes
1, 2, 5, 6) or absence (lanes 3, 4, 7, 8) of 5 mM GTP in a
reaction mix prepared as described in Materials and
Methods. For the remainder of details, see legend in panel A.
Equivalent results were obtained in two separate experiments.
|
|

View larger version (50K):
[in this window]
[in a new window]
|
Figure 2. Immunoprecipitation of Bacterial Toxin-Catalyzed
ADP-Ribosylated Membrane Proteins
Membranes (100 µg in lanes 1 and 3; 250 µg in lanes 2, 4, and
5) were incubated at 30 C for 30 min in the presence of either PTX (2.
5 µg/ml, lanes 1, 2, and 5) or CTX (50 µg/ml CTX, lanes 3 and 4)
and in the presence of 10 µg/ml hCG in a reaction mix prepared as
described in Materials and Methods. Membranes were then
either pelleted immediately (lanes 1 and 3) or incubated with
anti-Gi (antiserum 117) (lane 2) or anti-hCG (B105) (lanes
4 and 5) for subsequent immunoprecipitation as described in
Materials and Methods. Samples were subjected to 10.5%
SDS-PAGE, and this figure represents an autoradiogram of the dried gel.
For the remainder of details see legend to Fig. 1 . Equivalent results
were obtained in three separate experiments. B,
[32P]AAGTP-labeled membrane proteins immunoprecipitated
with anti-LH/CG receptor monoclonal antibody. Membranes (200 µg) were
incubated with 1.0 µg/ml hCG for 20 min in a reaction mix as
described in Materials and Methods. Membrane proteins
were immunoprecipitated using either anti-LH/CG receptor antibody, LHR
38 (lane 1), or normal mouse serum (lane 2) as described in
Materials and Methods. Proteins were separated by
SDS-PAGE, and the gel was exposed to x-ray film with an intensifying
screen. Molecular masses of labeled proteins are indicated at the
left and were calculated based on the migration of
protein standards, which are shown at the right. This
result is representative of two experiments.
|
|
It has been established that in order for Gi to serve as an
optimal substrate for ADP ribosylation by PTX, it should be in its
heterotrimeric conformation (26, 27). Upon receptor activation,
receptor-coupled Gi subunits uncouple, rendering
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 58; Fig. 1B
, lanes
14). 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 58;
Fig. 1B
, lanes 14). 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.

View larger version (42K):
[in this window]
[in a new window]
|
Figure 3. Time Course of PTX-Catalyzed ADP Ribosylation of
Membrane Proteins
Membranes (100 µg protein) were incubated at 30 C for varying time
periods (130 min) in the presence of PTX (2. 5 µg/ml) and 5
mM GTP, in the presence or absence of 10 µg/ml hCG (as
indicated), in a reaction mix as described in Materials and
Methods. The dried gel was exposed to x-ray film with an
intensifying screen for 2 days. Equivalent results were obtained in two
separate experiments.
|
|
When membranes were incubated with CTX and [32P]NAD in
the presence of GTP, we observed the hCG-independent ADP ribosylation
of a 45-kDa band as well as a 48/50-kDa doublet (Fig. 1A
, 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.

View larger version (31K):
[in this window]
[in a new window]
|
Figure 4. Migration of CTX-Catalyzed ADP-Ribosylated Membrane
Proteins Compared with Migration of Immunoreactive Gs ,
Gq/11 , and Gi in Follicular Membranes
For lane 1, membranes (100 µg) were incubated at 30 C for 30 min in
the presence of 10 µg/ml hCG and 50 µg/ml CTX, as described in
Materials and Methods, pelleted, and boiled in SDS
sample buffer. For lanes 24, the same set of membranes was boiled in
SDS sample buffer. Sample proteins were resolved on a 10.5%
SDS-polyacrylamide gel and transferred onto nytran. Lanes 24 were
then subjected to immunoblot analysis using either
anti-Gs (antiserum U-584), anti-Gq/11
(antiserum B6T), or anti-Gi (UBI) as the primary
antibodies. Antiserum U-584 is consistently immunoreactive with the
45-kDa short form and 48/50 kDa-long form of Gs ,
antiserum B6T immunoreacts with the 42/43 kDa Gq/11
proteins, and UBIs anti-Gi is immunoreactive to the 40
kDa Gi band. Note that the 48/50-kDa doublet and the 40-kDa
band are faint in lane 1; however, these bands are usually distinctly
radiolabeled (see Fig. 1A , lane 4, and Fig. 6B , lanes 4, 6, 8, 10, 12).
Molecular masses of the proteins are indicated at the
left and were calculated from migration of protein
standards. Equivalent results were obtained in two separate
experiments.
|
|
This smaller CTX-catalyzed ADP-ribosylated band migrated at the
same molecular mass as PTX-sensitive Gi (Fig. 1A
, compare
lanes 3 and 4 to lanes 58), 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
Gs
3).

View larger version (47K):
[in this window]
[in a new window]
|
Figure 5. Immunoprecipitation of CTX-Catalyzed
ADP-Ribosylated Membrane Proteins Using G Protein-Specific Antisera
A, Immunoprecipitation of CTX-catalyzed ADP-ribosylated membrane
proteins using anti-Gi . Membranes (100 µg in lane 1;
250 µg in lanes 2 and 3) were incubated at 30 C for 30 min in the
presence of CTX (50 µg/ml CTX) and 10 µg/ml hCG using
[32P]NAD as described in Materials and
Methods. Membranes were then either pelleted immediately (lane
1) or immunoprecipitated using anti-Gi (antiserum 117)
or preimmune (PI) sera (lanes 2 and 3). Samples were boiled and
subjected to 10.5% SDS-PAGE, and this figure represents an
autoradiogram of the dried gel. Molecular masses of labeled proteins
are indicated at the left and were calculated based on
the migration of protein standards shown at right.
Equivalent results were obtained in two separate experiments. B,
Immunoprecipitation of CTX-catalyzed ADP-ribosylated membrane proteins
using anti-Gs . Membranes (100 µg in lane 1; 250 µg
in lanes 24) were incubated at 30 C for 30 min in the presence of CTX
(50 µg/ml CTX) and 10 µg/ml hCG using [32P]NAD.
Membranes were then either pelleted immediately (lane 1) or
immunoprecipitated using anti-Go (antiserum 9072),
preimmune (PI) sera, or anti-Gs (antiserum 1190) (lanes
24). For the remainder of details, see legend in panel A. Equivalent
results were obtained in three separate experiments.
|
|
Because the CTX-catalyzed ADP-ribosylated 45- and 48/50 kDa proteins
migrated at the same molecular mass as bands that were immunoreactive
with anti-Gs
(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.

View larger version (77K):
[in this window]
[in a new window]
|
Figure 6. Time Course of CTX-Catalyzed ADP Ribosylation of
Membrane Proteins
Membranes (100 µg protein) were incubated at 30 C for varying time
periods (130 min) in the presence of CTX (50 µg/ml), in the
presence (A) or absence (B) of 5 mM GTP, and in the
presence or absence of 10 µg/ml hCG, as indicated and as described in
Materials and Methods. Molecular masses of labeled
proteins are indicated at the left and were calculated
based on the migration of protein standards (shown at
right). Equivalent results were obtained in three
separate experiments.
|
|
We next determined whether CTX-catalyzed ADP ribosylation of the long
and short forms of Gs
in the absence of GTP was
dependent on the concentration of hCG in incubations. As shown in Fig. 7A
(lanes 712), 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 16) 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.

View larger version (30K):
[in this window]
[in a new window]
|
Figure 7. Human CG Dose-Dependence of CTX-Catalyzed
ADP-Ribosylated Follicular Membrane Proteins
A, hCG dose-dependence of CTX-catalyzed ADP-ribosylated membrane
proteins. Membranes (100 µg protein) were incubated at 30 C for 30
min in the presence of either 10 µg/ml BSA (lanes 1 and 7) or
indicated concentration of hCG in the presence or absence of 10
µM GTP, as indicated, in a reaction mix containing CTX
(50 µg/ml) as described in Materials and Methods.
Molecular masses of labeled proteins are indicated at the
left and were calculated based on the migration of
protein standards. Equivalent results were obtained in three separate
experiments. B, Quantification of levels of ADP-ribosylated
Gs(long) and Gs(short) upon incubation with
increasing levels of hCG. Levels of radiolabel incorporated into the
long and short forms of Gs from autoradiograph in panel A
were quantified by PhosphoImager (Molecular Dynamics, Sunnyvale, CA)
and graphed as means ± SEM.
|
|
As it is not known whether activated LH/CG receptor stimulates PLC in
physiologically relevant cellular models via a PTX-sensitive G protein,
as is the case with LH/CG receptor transfected into L cells (24), or
via a PTX-insensitive G protein (19), which is likely Gq/11
(see Discussion), we also performed an immunoprecipitation
study using an anti-Gq/11 antibody to determine whether
Gq/11 is ADP-ribosylated by CTX in an hCG-dependent manner
in porcine ovarian follicular membranes. Although Gq/11
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.

View larger version (29K):
[in this window]
[in a new window]
|
Figure 8. Immunoprecipitation of CTX-Catalyzed
ADP-Ribosylated SF9 Cell Lysate Proteins Using
anti-Gq/11
Lysate (250 µg in lanes 2 and 3) was incubated at 30 C for 30 min in
the presence of CTX (50 µg/ml) and in the presence or absence of 1
mM carbachol using [32P]NAD in a reaction mix
prepared as described in Materials and Methods. Proteins
were immunoprecipitated using anti-Gq/11 (C-19; Santa Cruz
Biotechnology) as described in Materials and Methods.
For lane 1, cell lysate (50 µg) was boiled in SDS sample buffer and
run with samples in lanes 2 and 3 on the same 10.5% SDS-polyacrylamide
gel. Proteins were transferred onto nytran, and immunoblot analysis was
performed on lane 1 as described in Materials and
Methods using anti-Gq/11. Nytran containing lanes 2
and 3 was dried and exposed to film. Molecular mass of the labeled
protein is indicated between lane 1 and lanes 2 and 3.
|
|
Coimmunoprecipitation of Gs
and
Gi
with the LH/CG Receptor
Although our results suggest that the majority of the long form of
Gs
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).
 |
DISCUSSION
|
---|
Although it is well established that stimulation of the LH/CG
receptor by agonist results in the subsequent activation of its
effector enzyme, adenylyl cyclase (18), several groups have recently
shown that agonist-stimulated LH/CG receptor can also activate PLC in a
number of cellular models (20, 21, 22, 23, 30). Davis and co-workers (20, 21, 22, 23)
reported that hCG can mobilize phosphoinositides and increase
[Ca2+]i in luteal and granulosa cells (22, 31). Recent studies using the murine LH/CG receptor transfected into L
cells have shown that activated LH/CG receptor stimulates both adenylyl
cyclase (leading to increases in cAMP) and PLC (leading to the
formation of inositol phosphates and elevations in
[Ca2+]i) (19, 24). The cloned dog TSH
receptor has also been shown to stimulate the generation of both cAMP
and inositol phosphates, so the ability of the LH/CG receptor to
stimulate both adenylyl cyclase and PLC is not unique (32).
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 14).
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.
 |
MATERIALS AND METHODS
|
---|
Materials
Purified hCG (CR-127) was provided by the Center for Population
Research, NICHHD. Materials were purchased from the following sources:
[32P]NAD (30 Ci/mmol), Dupont-New England Nuclear
(Boston, MA); [125I]donkey anti-rabbit IgG (100
µCi/ml), Amersham (Arlington Heights, IL); anti-Gi
(06190; 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 612 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 (100150 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 50100
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, 5075
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
|
---|
We would like to thank the following investigators for their
generous gifts of antibodies, which were crucial to the studies
performed in this manuscript: anti-hCG (monoclonal B105) from Dr. John
OConnor (Irving Center for Clinical Research, Department of Medicine,
College of Physicians and Surgeons of Columbia University, New York,
NY); anti-LH/CG receptor (LHR38) from Dr. Edwin Milgrom (Hormones et
Reproduction, Hopital de Bicetre, Inserm Unite 135,
Kremlin-Bicetre-France); anti-Gs
(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 Y13259) from Dr. Frank McCormick (Onyx
Pharmaceuticals, Richmond, CA); deglycosylated hCG was a gift from Dr.
Robert Ryan (Mayo Medical School, Rochester, MN).
 |
FOOTNOTES
|
---|
Address requests for reprints to: Dr. Mary Hunzicker-Dunn, Department of Cell and Molecular Biology, Northwestern University Medical School, 303 East Chicago Avenue, Chicago, Illinois 60611.
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 0706817). 
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.
 |
REFERENCES
|
---|
-
Hepler JR, Gilman AG 1992 G proteins. Trends Biochem Sci 17:383387[CrossRef][Medline]
-
West RE, Moss J, Vaughan M, Liu T 1985 Pertussis
toxin-catalyzed ADP-ribosylation of transducin. J Biol Chem 260:1442814430[Abstract/Free Full Text]
-
Hoshino S, Kikkawa S, Takahashi K, Itoh H, Kaziro Y, Kawasaki
H, Suziki K, Katada T 1990 Identification of sites for alkylation by
N-ethylmaleimide and pertussis toxin-catalyzed ADP-ribosylation on
GTP-binding proteins. FEBS Lett 276:227231[CrossRef][Medline]
-
Katada T, Ui M 1982 ADP-ribosylation of the specific membrane
protein of C6 cells by islet-activating protein associated with
modification of adenylate cyclase activity. J Biol Chem 257:72107216[Abstract/Free Full Text]
-
Codina J, Hildebrandt J, Iyengar R, Birnbaumer L, Sekura RD,
Manclark CR 1983 Pertussis toxin substrate, the putative Ni component
of adenylyl cyclases, is an
ß heterodimer regulated by guanine
nucleotide and magnesium. Proc Natl Acad Sci USA 80:42764280[Abstract]
-
Bokoch GM, Katada T, Northup JK, Hewlett EL, Gilman AG 1983 Identification of the predominant substrate for ADP-ribosylation by
islet activating protein. J Biol Chem 258:20722075[Abstract/Free Full Text]
-
Sullivan KA, Miller RT, Masters SB, Beiderman B, Heideman
W, Bourne HR 1987 Identification of receptor contact site involved in
receptor-G protein coupling. Nature 330:758760[CrossRef][Medline]
-
Masters SB, Sullivan KA, Miller RT, et al 1988 Carboxyl
terminal domain of Gs
specifies coupling of receptors to stimulation
of adenylyl cyclase. Science 241:448451[Medline]
-
Van Dop C, Tsubokawa M, Bourne HR, Ramachandran J 1984 Amino
acid sequence of retinal transducin at the site ADP-ribosylated by
cholera toxin. J Biol Chem 259:696698[Abstract/Free Full Text]
-
Tong L, de Vos AM, Milburn MV, et al 1989 Structural
differences between a ras oncogene protein and the normal protein.
Nature 337:9093[CrossRef][Medline]
-
Freissmuth M, Gilman AG 1989 Mutations of Gs
designed to
alter the reactivity of the protein with bacterial toxins. J Biol
Chem 264:2190721914[Abstract/Free Full Text]
-
Masters SB, Miller RT, Chi M, et al 1989 Mutations in the
GTP-binding site of Gs
alter stimulation of adenylyl cyclase. J
Biol Chem 264:1546715474[Abstract/Free Full Text]
-
Milligan G, Carr C, Gould GW, Mullaney I, Lavan BE 1991 Agonist-dependent, cholera toxin-catalyzed ADP-ribosylation of
pertussis toxin-sensitive G proteins following transfection of the
human
2-C10 adrenergic receptor into rat 1 fibroblasts. J Biol
Chem 266:64476455[Abstract/Free Full Text]
-
Gierschik P, Jakobs KH 1987 Receptor-mediated ADP-ribosylation
of a phospholipase C-stimulating G protein. FEBS Lett 224:219223[CrossRef][Medline]
-
Milligan G, McKenzie FR 1988 Opioid peptides promote
cholera-toxin-catalyzed ADP-ribosylation of the inhibitory
guanine-nucleotide-binding protein in membranes of neuroblastoma -
glioma hybrid cells. Biochem J 252:369373[Medline]
-
Iiri T, Tohkin M, Morishima N, Ohoka Y, Ui M, Katada T 1989 Chemotactic peptide receptor-supported ADP-ribosylation of a
pertussis toxin substrate GTP-binding protein by cholera toxin in
neutrophil-type HL-60 cells. J Biol Chem 264:2139421400[Abstract/Free Full Text]
-
Gierschik P, Sidiropoulos D, Jakobs KH 1989 Two distinct
Gi-proteins mediate formyl peptide receptor signal transduction in
human leukemia cells. J Biol Chem 264:2147021473[Abstract/Free Full Text]
-
Marsh JM 1970 The stimulatory effect of luteinizing hormone on
adenylyl cyclase in the bovine corpus luteum. J Biol Chem 245:15961603[Abstract/Free Full Text]
-
Gudermann T, Birnbaumer M, Birnbaumer L 1992 Evidence for dual
coupling of the murine luteinizing hormone receptor to adenylyl cyclase
and phosphoinositide breakdown and calcium mobilization. J Biol
Chem 267:44794488[Abstract/Free Full Text]
-
Davis JS, Weakland LL, Coffey RG, West LA 1989 Acute effects
of phorbol esters on receptor-mediated IP3, cAMP, and progesterone
levels in rat granulosa cells. Am J Physiol 256:E368E374
-
Davis JS, Weakland LL, West LA, Farese RV 1986 Luteinizing
hormone stimulates the formation of inositol triphosphate and cyclic
AMP in rat granulosa cells. Biochem J 238:597604[Medline]
-
Davis JS, Weakland LL, Farese RV, West LA 1987 Luteinizing
hormone increases inositol triphosphate and cytosolic free Ca2+ in
isolated bovine luteal cells. J Biol Chem 262:85158521[Abstract/Free Full Text]
-
Davis JS 1992 Modulation of leutinizing hormone-stimulated
inositol phosphate accumulation by phorbol esters in bovine luteal
cells. Endocrinology 131:749757[Abstract]
-
Herrlich A, Kuhn B, Grosse R, Schmid A, Schultz G, Guderman T 1996 Involvement of Gs and Gi proteins in dual
coupling of the luteinizing hormone receptor to adenylyl cyclase
and phospholipase C. J Biol Chem 271:1676416772[Abstract/Free Full Text]
-
Lamm MLG, Hunzicker-Dunn M 1994 Phosphorylation-independent
desensitization of the luteinizing hormone/chorionic gonadotropin
receptor in porcine follicular membranes. Mol Endocrinol 8:15371546[Abstract]
-
Milligan G 1987 Guanine nucleotide regulation of the pertussis
and cholera toxin substrates of rat glioma C6 BU1 cells. Biochem
Biophys Acta 929:197202[CrossRef][Medline]
-
Neer EJ, Lok JM, Wolf LG 1984 Purification and properties of
the inhibitory guanine nucleotide regulatory unit of brain adenylate
cyclase. J Biol Chem 259:1422214229[Abstract/Free Full Text]
-
Matesic DF, Manning DR, Luthin GR 1991 Tissue-dependent
association of muscarinic acetylcholine receptors with guanine
nucleotide-binding regulatory proteins. Mol Pharmacol 40:347353[Abstract]
-
Vuhai-Luuthi MT, Jolivet A, Jallal B, et al 1990 Monoclonal
antibodies against luteinizing hormone receptor. Immunochemical
characterization of the receptor. Endocrinology 127:20902098[Abstract]
-
Dimino MJ, Snitzer J, Brown KM 1987 Inositol phosphates
accumulation in ovarian granulosa after stimulation by luteinizing
hormone. Biol Reprod 37:11291134[Abstract]
-
Davis JS, West LA, Farese RV 1984 Effects of luteinizing
hormone on phosphoinositide metabolism in rat granulosa cells. J
Biol Chem 259:1502815034[Abstract/Free Full Text]
-
Van Sande J, Raspe E, Perret J, et al 1990 Thyrotropin
activates both the cyclic AMP and the PIP2 cascades in CHO cells
expressing the human cDNA of TSH receptor. Mol Cell Endocrinol 74:16[CrossRef][Medline]
-
Law SF, Yasuda K, Bell GI, Reisine T 1993 Gi
3 and Go
selectively associate with the cloned somatostatin receptor subtype
SSTR2. J Biol Chem 268:1072110727[Abstract/Free Full Text]
-
DellAcqua ML, Carroll RC, Peralta EG 1993 Transfected m2
muscarinic acetylcholine receptors couple to G
i2 and G
i3 in
Chinese hamster ovary cells. J Biol Chem 268:56765685[Abstract/Free Full Text]
-
Chabre O, Conklin BR, Brandon S, Bourne HR, Limbird LE 1994 Coupling of the
2A-adrenergic receptor to multiple G proteins.
J Biol Chem 269:57305734[Abstract/Free Full Text]
-
Smrcka AV, Hepler JR, Brown KO, Sternweis PC 1991 Regulation
of polyphosphoinositide-specific phospholipase C activity by purified
Gq. Science 251:804807[Medline]
-
Taylor SJ, Chae HZ, Rhee SG, Exton JH 1991 Activation of the
beta 1 isozyme of phospholipase C by alpha subunits of the Gq class of
G proteins. Nature 350:516518[CrossRef][Medline]
-
Wu D, Lee CH, Rhee SG, Simon MI 1992 Activation of
Phospholipase C by the alpha subunits of the Gq and G11 proteins in
transfected Cos-7 cells. J Biol Chem 267:18111817[Abstract/Free Full Text]
-
Lee CH, Park D, Wu D, Rhee SG, Simon MI 1992 Members of the
Gq
subunit gene family activate phospholipase C beta isozymes.
J Biol Chem 267:1604416047[Abstract/Free Full Text]
-
Waldo GL, Boyer JL, Morris AJ, Harden TK 1991 Purification of
an Aluminum Fluoride and G-protein ß/
-subunit-regulated
phospholipase C-activating protein. J Biol Chem 266:1421714225[Abstract/Free Full Text]
-
Carozzi A, Camps M, Gierschik P, Parker PJ 1993 Activation of
phosphatidylinositol lipid-specific phospholipase C-ß3 by G-protein
ß/
subunits. FEBS Lett 315:340342[CrossRef][Medline]
-
Katz A, Wu D, Simon MI 1992 Subunits ß/
of heterotrimeric
G protein activate ß 2 isoform of phospholipase C. Nature 360:686689[CrossRef][Medline]
-
Camps M, Carozzi A, Schnabel P, Scheer A, Parker PJ, Gierschik
P 1992 Isozyme-selective stimulation of phospholipase C-ß2 by G
protein ß/
-subunits. Nature 360:684686[CrossRef][Medline]
-
Lee SB, Shin SH, Hepler JR, Gilman AG, Rhee SG 1993 Activation
of phospholipase C-ß2 mutants by G protein
q and ß
subunits.
J Biol Chem 268:2595225957[Abstract/Free Full Text]
-
Bornancin F, Audigier Y, Chabre M 1993 ADP-ribosylation of Gs
by cholera toxin is potentiated by agonist activation of ß-adrenergic
receptors in the absence of GTP. J Biol Chem 268:1702617029[Abstract/Free Full Text]
-
Amir-Zaltsman Y, Salomon Y 1980 Studies on the receptor for
luteinizing hormone in a purified plasma membrane preparation from rat
ovary. Endocrinology 106:11661172[Medline]
-
LaBarbera AR, Richert ND, Ryan RJ 1980 Nucleotides do not
modulate rat luteocyte human chorionic gonadotropin responsiveness by
inhibiting human chorionic gonadotropin binding. Arch Biochem Biophys 200:177185[Medline]
-
Abramowitz J, Iyengar R, Birnbaumer L 1982 Guanine nucleotide
and magnesium ion regulation of the interaction of gonadotropic and
ß-adrenergic receptors with their hormones: a comparative study using
a single membrane system. Endocrinology 110:336346[Medline]
-
Ekstrom RC, Hunzicker-Dunn M 1989 Homologous desensitization
of ovarian luteinizing hormone/human chorionic gonadotropin-responsive
adenylyl cyclase is dependent upon GTP. Endocrinology 124:956963[Abstract]
-
Lowry OW, Rosebrough NJ, Farr AL, Randall RJ 1951 Protein
measurement with the Folin phenol reagent. J Biol Chem 193:265275[Free Full Text]
-
Laemmli UK 1970 Cleavage of structural proteins during the
assembly of the head of bacteriophage T4. Nature 227:680685[Medline]
-
Law SF, Manning D, Reisine T 1991 Identification of the
subunits of GTP-binding proteins coupled to somatostatin receptors.
J Biol Chem 266:1788517897[Abstract/Free Full Text]
-
Williams AG, Woolkalis MJ, Poncz M, Manning DR, Gerwirtz
AM, Brass LF 1990 Identification of the pertussis toxin-sensitive G
proteins in platelets, megakaryocytes, and human erythroleukemia cells.
Blood 76:721730[Abstract]
-
Bucci C, Parton RG, Mather IH, et al 1992 The small GTPase
rab5 functions as a regulatory factor in the early endocytic pathway.
Cell 70:715728[Medline]
-
Mumby SM, Kahn RA, Manning DR, Gilman AG 1986 Antisera of
designed specificity for subunits of guanine nucleotide-binding
regulatory proteins. Proc Natl Acad Sci USA 83:265269[Abstract]
-
Singer WD, Miller WT, Sternweis PC 1994 Purification and
characterization of the alpha subunit of G13. J Biol Chem 269:1979619802[Abstract/Free Full Text]
-
Okuma Y Reisine T 1992 Immunoprecipitation of
2a-adrenergic
receptor-GTP-binding protein complexes using Gtp-binding protein
selective antisera. J Biol Chem 267:1482614831[Abstract/Free Full Text]
-
Law SF, Reisine T 1992 Agonist binding to rat brain
somatostatin receptors alters the interaction of the receptors with
guanine nucleotide-binding regulatory proteins. Mol Pharmacol 42:398402[Abstract]
-
Hsieh KP 1992 Thyrotropin-releasing hormone and
gonadotropin-releasing hormone receptors activate phospholipase C
by coupling to the guanosine triphosphate-binding proteins Gq and G11.
Mol Endocrinol 6:16731681[Abstract]
-
Casey PJ, Fong HKW, Simon MI, Gilman AG 1990 Gz, a guanine
nucleotide-binding protein with unique biochemical properties. J
Biol Chem 265:23832390[Abstract/Free Full Text]
-
Rasenick MM, Talluri M, Dunn III WJ 1994 Photoaffinity
guanosine 5'-triphosphate analogs as a tool for the study of
GTP-binding proteins. Methods Enzymol 237:100110[Medline]
-
Popova JS, Garrison JC, Rhee SG, Rasenick MM 1997 Tubulin,
Gq, and phosphatidylinositol 4,5-bisphosphate interact to
regulate phospholipase cß1 signaling. J Biol Chem 272:67606765[Abstract/Free Full Text]