Regulation of Gq/11
by the Gonadotropin-Releasing Hormone Receptor
Dinesh Stanislaus,
Jo Ann Janovick,
Shaun Brothers and
P. Michael Conn
Department of Physiology and Pharmacology (D.S., P.M.C.),Oregon
Health Sciences University, Portland, Oregon 97201,
Oregon
Regional Primate Research Center,(D.S., J.A.J., S.B., P.M.C.),
Beaverton, Oregon 97006
 |
ABSTRACT
|
---|
Evidence from use of pertussis and cholera toxins
and from NaF suggested the involvement of G proteins in GnRH regulation
of gonadotrope function. We have used three different methods to assess
GnRH receptor regulation of Gq/11
subunits(Gq/11
). First, we used
GnRH-stimulated palmitoylation of Gq/11
to
identify their involvement in GnRH receptor-mediated signal
transduction. Dispersed rat pituitary cell cultures were labeled with
[9,10-3H(N)]-palmitic acid and
immunoprecipitated with rabbit polyclonal antiserum made against the
C-terminal sequence of Gq/11
. The
immunoprecipitates were resolved by 10% SDS-PAGE and quantified.
Treatment with GnRH resulted in time-dependent (0120 min) labeling of
Gq/11
. GnRH (10-12,
10-10, 10-8, or
10-6 g/ml) for 40 min resulted in
dose-dependent labeling of Gq/11
compared
with controls. Cholera toxin (5 µg/ml; activator of
Gs
), pertussis toxin (100 ng/ml; inhibitor
of Gi
actions) and Antide (50
nM; GnRH antagonist) did not stimulate
palmitoylation of Gq/11
above basal levels.
However, phorbol myristic acid (100 ng/ml; protein kinase C activator)
stimulated the palmitoylation of Gq/11
above
basal levels, but not to the same extent as
10-6 g/ml GnRH. Second, we used the ability of
the third intracellular loop (3i) of other
seven-transmembrane segment receptors that couple to specific G
proteins to antagonize GnRH receptor-stimulated signal transduction and
therefore act as an intracellular inhibitor. Because the third
intracellular loop of
1B-adrenergic receptor
(
1B3i) couples to
Gq/11
, it can inhibit
Gq/11
-mediated stimulation of inositol
phosphate (IP) turnover by interfering with receptor coupling to
Gq/11
. Transfection (efficiency 57%) with
1B3i cDNA, but not
the third intracellular loop of
M1-acetylcholine receptor (which also couples
toGq/11
), resulted in 1012% inhibition
of maximal GnRH-evoked IP turnover, as compared with
vector-transfected GnRH-stimulated IP turnover. The third
intracellular loop of
2A-adrenergic
receptor, M2-acetylcholine receptor (both
couple to Gi
), and
D1A-receptor (couples to
Gs
) did not inhibit IP turnover
significantly compared with control values. GnRH-stimulated LH release
was not affected by the expression of these peptides. Third, we
assessed GnRH receptor regulation of Gq/11
in a PRL-secreting adenoma cell line (GGH31')
expressing the GnRH receptor. Stimulation of
GGH31' cells with 0.1 µg/ml Buserelin (a
metabolically stable GnRH agonist) resulted in a 1520% decrease in
total Gq/11
at 24 h following agonist
treatment compared with control levels; this action of the agonist was
blocked by GnRH antagonist, Antide (10-6
g/ml). Neither Antide (10-6 g/ml, 24 h)
alone nor phorbol myristic acid (0.33100 ng/ml, 24 h) mimicked
the action of GnRH agonist on the loss of
Gq/11
immunoreactivity. The loss of
Gq/11
immunoreactivity was not due to an
effect of Buserelin on cell-doubling times. These studies provide the
first direct evidence for regulation of
Gq/11
by the GnRH receptor in primary
pituitary cultures and in GGH3 cells.
 |
INTRODUCTION
|
---|
The GnRH receptor, like other receptors in the 7-transmembrane
segment receptor superfamily, couples to multiple G proteins (1, 2). In
dispersed pituitary cell cultures, pertussis toxin (PTX) pretreatment
results in decreased inositol phosphate (IP) turnover compared with
medium-pretreated levels in response to GnRH (3), suggesting that a
PTX-sensitive G protein (such as Gi or Go)
couples the receptor to IP turnover. However, in GGH312'
cell cultures (GH3 cells stably transfected with the rat
GnRH-receptor cDNA), GnRH agonist-evoked IP turnover is insensitive to
PTX (4), indicating that a PTX-insensitive G protein may be involved in
signal transduction and the receptor may be coupled differently in
different cells.
Gq/11
immune-depletion studies show that in membranes
derived from
T31 cells, GnRH receptor is coupled to
Gq/11
(5). Furthermore, PRL synthesis in
GGH3 cell cultures in response to GnRH is mediated by cAMP,
implicating Gs
in this signal transduction pathway (6),
although cAMP does not mediate GnRH-stimulated hormone release from the
gonadotrope (7). It is evident from these studies that GnRH receptor is
able to couple to different G proteins in different cell lines.
Therefore, to investigate the G proteins that couple to the GnRH
receptor, it is important to undertake these studies in primary
pituitary cultures.
In the gonadotrope, the little that is known about G proteins
involved in GnRH receptor-mediated signal transduction has been
obtained from toxin studies and from second messenger studies (1, 2, 3).
PTX-sensitive G proteins have been implicated in the GnRH receptor/G
protein coupling. In addition, the observation that cholera toxin (CTX)
pretreatment enhances GnRH stimulated LH release (8) has implicated
Gs
in modulation of GnRH action. Such studies provide
only indirect evidence as to the identity of the G proteins involved in
GnRH receptor-mediated signal transduction. Complicating this further
is the observation that both protein kinase C (PKC) and protein kinase
A, regulated by different G proteins, are capable of regulating IP
turnover by phosphorylating phospholipase C (9). Therefore, the sole
use of IP turnover as a marker for a specific G protein activation
would lead to unclear results. Furthermore, cross-talk between
CTX-sensitive G protein and PKC (10) can further complicate the
identification of the G proteins that couple to the GnRH
receptor.
Palmitoylation (i.e. the addition of a 16-carbon fatty
acid to a cysteine residue through a thioester link) of G protein
-subunits is a dynamic process that is regulated after receptor
activation. Receptor-evoked palmitoylation of Gq/11
and
Gs
is a well characterized phenomenon (11, 12, 13) and
occurs in a time- and dose-dependent manner (11, 12). Furthermore, G
proteins that do not associate with a specific receptor do not
incorporate [3H]palmitic acid into their
-subunits
when that receptor is stimulated (11). In addition, mutationally
activated Gs
turns over [3H]palmitic acid
labeling more rapidly than wild type Gs
(14). These
studies suggest that activation of G protein is required for the
-subunit to undergo palmitoylation. In the present study, in order
to identify the moieties that are affected when cells are stimulated
with GnRH, we used the ability of G protein-coupled receptors to
stimulate the palmitoylation of G protein
-subunits they
activate.
Several reports (15, 16) have shown that the cellular expression of the
third intracellular loop of G protein-coupled receptors can inhibit
receptor- evoked second messenger production. This effect is greatest
when the peptide is derived from the same receptor, although expression
of heterologous third intracellular loops inhibits receptor-stimulated
second messenger production to a lesser extent as long as the
intracellular loop and the receptor couple to a common G protein. These
studies demonstrate that peptides derived from the third intracellular
loop of G protein-coupled receptors produce G protein-specific
inhibition of receptor-mediated signal transduction (15). We used the
ability of the third intracellular loop to act as an intracellular
inhibitor to corroborate our findings from the palmitoylation
studies.
To examine further the regulation of G proteins by the GnRH receptor,
we examined receptor-evoked down-regulation of G protein
-subunits.
Agonist-induced reduction in total cellular G protein
-subunits has
been observed for members of the G protein family (Gs
,
Gi
, Gq/11
; 17). This reduction is
observed for G proteins that interact with the activated receptor (17)
and can be used as a marker for receptor regulation of G proteins.
Therefore, we used an RIA developed in our laboratory to assess GnRH
agonist-evoked reduction of total cellular Gq/11
in a
rat pituitary adenoma cell line stably expressing the GnRH receptor. We
opted to use a homogeneous cell line instead of a dispersed rat
pituitary cell culture, because gonadotropes are only about 20% of
cells obtained from a rat pituitary cell dispersion (female weanlings),
and changes in total G protein content may be masked against a
relatively high background from nongonadotrope cells.
In this study, we assess evidence to indicate GnRH receptor regulation
of Gq/11
. Evidence for regulation of
Gq/11
by the receptor is present in rat pituitary cell
dispersions and also in a cell line stably expressing the GnRH
receptor.
 |
RESULTS
|
---|
The time course of GnRH-stimulated palmitoylation of
Gq/11
in pituitary cell cultures is shown in Table 1
and
Fig. 1
. Rat pituitary cell cultures were treated with
10-6 g/ml GnRH or cell culture medium in the presence of
[3H]palmitate for 0, 20, 40, 60, 90, and 120 min.
Immunoprecipitation of Gq/11
showed an increase in
[3H]palmitate incorporation with GnRH treatment. The
earliest detectable incorporation of the label is measurable at 20 min
after the addition of GnRH. GnRH-stimulated incorporation of the label
is detectable up to 120 min. The basal incorporation of
[3H]palmitate label increases with time, although in the
presence of GnRH there is an increased incorporation over basal
levels.

View larger version (34K):
[in this window]
[in a new window]
|
Figure 1. The Time Course for GnRH-Stimulated Incorporation
of [3H]Palmitic Acid into Gq/11
Dispersed rat pituitary cell cultures were incubated in the presence or
absence of 10-6 g/ml GnRH for the indicated times.
Incorporation of the label into Gq/11 was assayed by
immunoprecipitation then resolved by 10% SDS-PAGE, fluorography, and
densitometry. Data show band density in arbitrary optical density
units. The data are from one representative experiment. Three separate
experiments showed similar results.
|
|
The palmitate incorporation to Gq/11
was dose dependent
with GnRH (Fig. 2
). Pituitary cell cultures were treated
with medium and GnRH (10-12, 10-10,
10-8, and 10-6 g/ml) in the presence of
[3H]palmitate for 60 min. GnRH concentration of
approximately 10-9 g/ml produced a half-maximal
incorporation of palmitate label on Gq/11
.

View larger version (16K):
[in this window]
[in a new window]
|
Figure 2. The Dose-Response for GnRH-Stimulated Incorporation
of [3H]Palmitic Acid into Gq/11
Dispersed rat pituitary cell cultures were incubated for 60 min with
the indicated doses of GnRH. Incorporation of the label into
Gq/11 was assayed by immunoprecipitation then resolved
by 10% SDS-PAGE, fluorography, and densitometry. Data show band
density in arbitrary absorbance units. The data are from one
representative experiment. Three separate experiments showed similar
results.
|
|
To further assess the specificity of GnRH receptor-stimulated
palmitoylation of Gq/11
, we examined the ability of CTX
(5 µg/ml), PTX (100 ng/ml), phorbol myristic acid (PMA, a PKC
activator; 100 ng/ml), and Antide (GnRH antagonist; 50 nM)
to evoke palmitoylation of Gq/11
(Fig. 3
). Rat pituitary cell cultures were incubated in the
presence of the above agents for 40 min, and Gq/11
was
immunoprecipitated. Only PMA and GnRH increased the incorporation of
[3H]palmitate label on Gq/11
above basal
incorporation levels. The level of Gq/11
incorporation
of palmitate when treated with CTX, PTX, and Antide was not
significantly different from basal levels.

View larger version (24K):
[in this window]
[in a new window]
|
Figure 3. The Effect of the Indicated Agents on
Gq/11 Incorporation of [3H]Palmitic Acid
Dispersed rat pituitary cell cultures were incubated in the presence of
the indicated agents at the indicated concentrations for 40 min.
Incorporation of the label into Gq/11 was assayed by
immunoprecipitation then resolved by 10% SDS-PAGE, fluorography, and
densitometry. Data show band density in arbitrary absorbance units. The
data are from one representative experiment. Three separate experiments
showed similar results.
|
|
Gels treated with 1 M hydroxylamine before autoradiography
did not show any residual radioactivity (data not shown), indicating
that the radiolabel was alkali sensitive, as would be expected from a
thioester-linked palmitate label.
Transfection of primary cell cultures with the cDNA for the third
intracellular loops of the
1B-adrenergic receptor
(
1B3i),
2A-adrenergic
receptor (
2A3i), M1-muscarinic
receptor (M13i), M2-Muscarinic
receptor (M23i), and D1A-dopamine
receptor (D1A3i) did not inhibit
GnRH-stimulated LH release from vector-transfected levels (Fig. 4
). Primary cell cultures were transiently transfected
using lipofectamine, and GnRH-stimulated LH release was measured by
RIA. Transfection efficiency was measured to be 57%.

View larger version (25K):
[in this window]
[in a new window]
|
Figure 4. GnRH Stimulated LH Release in Transiently
Transfected Dispersed Rat Pituitary Cell Cultures
Cell cultures were transiently transfected, as described in
Materials and Methods, with the indicated third
intracellular loops of G protein-coupled receptors in pRK5 expression
vector. Twenty four hours after transfection, cells were stimulated for
2 h with the indicated concentrations of GnRH, and LH released
into the medium was assayed by RIA. The data are the mean of triplicate
transfections, and error bars show the SEM. Three separate
experiments showed similar results.
|
|
Although
1B3i did not inhibit
GnRH-stimulated LH release, transfection of this loop resulted in
approximately 10% inhibition of GnRH-stimulated IP turnover as
compared with vector-transfected values in rat pituitary cell cultures.
IP turnover was measured as described in Materials and
Methods. Transfection of cDNA for
2A3i,
M13i, M23i, and
D1A3i resulted in no significant inhibition of
GnRH-stimulated IP turnover (Fig. 5
).

View larger version (26K):
[in this window]
[in a new window]
|
Figure 5. GnRH Stimulated IP Turnover in Transiently
Transfected Dispersed Rat Pituitary Cell Cultures
Cell cultures were transiently transfected, as described in
Materials and Methods, with the indicated third
intracellular loops of G protein-coupled receptors in pRK5 expression
vector. Twenty four hours after transfection, the cellular inositol was
labeled with [3H]-myo-inositol for 18 h, after which
cells were stimulated for 2 h with the indicated concentrations of
GnRH, and total 3H-labeled IPs were determined by Dowex
anion exchange chromatography and liquid scintillation spectroscopy.
The data are the mean of triplicate transfections, and error bars show
the SEM. Three separate experiments showed similar
results.
|
|
Cellular expression of
2A3i was determined
by immunoblotting cell lysates with the peptide-specific antisera (Fig. 6
). Cell lysates were prepared from rat pituitary cell
cultures transiently transfected with the cDNA for
2A3i. A single band was seen at the apparent
molecular mass of approximately 21 kDa. This band was absent in cell
lysates obtained from pRK5-transfected rat pituitary cell cultures.

View larger version (45K):
[in this window]
[in a new window]
|
Figure 6. Protein Immunoblot Depicting Expression of the
Third Intracellular Loop of the 2A-Adrenergic Receptor
Rat pituitary cell cultures were transiently transfected with the third
intracellular loop of the 2A-adrenergic receptor or the
empty expression vector pRK5. Twenty-four hours after transfection,
cell cultures were lysed and expression of the peptide was detected
with immunoblotting using peptide-specific antisera as described in
Materials and Methods.
|
|
The time course of Buserelin-stimulated net loss of
Gq/11
immunoreactivity is shown in Fig. 7
. GGH31' cell cultures
(GH3-derived cell line stably expressing the GnRH receptor)
were treated for the indicated times with 0.1 µg/ml of Buserelin or
medium alone, and the total cellular Gq/11
was measured
by RIA as described in Materials and Methods. Consistent
with a cell-doubling time of approximately 1 day, the total amount of
Gq/11
increased with time both in control cells and in
cells treated with 0.1 µg/ml of Buserelin. However, in cells treated
with Buserelin, total levels of Gq/11
were consistently
1520% less than in control cells at 24 h. The reduction in
total cellular Gq/11
after Buserelin treatment was first
observed at 6 h.
The loss of Gq/11
immunoreactivity was dose-dependent
with Buserelin (Fig. 8
). GGH31' cells were
treated with the indicated doses of Buserelin for 24 h, and the
total cellular Gq/11
immunoreactivity was assayed by
RIA. Buserelin concentration of 10-9 g/ml produced a
half-maximal loss of Gq/11
immunoreactivity. The effect
of 10-9 g/ml Buserelin on assayable Gq/11
was blocked by 10-6 g/ml Antide (a GnRH antagonist;
340 ± 8 pg/60 µl), as compared with medium-treated levels
(339 ± 13 pg/60 µl). Furthermore, 10-6 g/ml Antide
alone (322 ± 8 pg/60 µl) did not produce a loss of
Gq/11
immunoreactivity compared with medium-treated
levels (339 ± 13 pg/60 µl).
PMA did not mimic this action of Buserelin treatment (Fig. 9
). GGH31' cells were treated with 0.33100
ng/ml of PMA for 24 h, and total cellular Gq/11
was
assayed by RIA. Treatment of GGH31' cells with PMA for
24 h did not result in any significant loss of immunoreactive
Gq/11
, as compared with medium-treated levels.
 |
DISCUSSION
|
---|
The data presented here demonstrate that Gq/11
is
palmitoylated in a time- and dose-dependent manner when the GnRH
receptor is occupied by an agonist. Antagonist occupancy of the
receptor does not lead to this effect. Stimulation of hormone release
from dispersed pituitary cell cultures with increasing concentrations
of GnRH resulted in the concurrent increase in incorporation of
[3H]palmitic acid label into Gq/11
. This
effect was time- and dose-dependent. Similarly, CTX and PTX did not
increase the incorporation of the label into Gq/11
.
Gq/11
is CTX- and PTX-insensitive, and, as expected,
these agents do not stimulate the incorporation of
[3H]palmitic acid. PMA, a PKC activator, stimulated
palmitate labeling of Gq/11
, although not to the same
extent as GnRH-stimulated levels. We also show that the transfection of
the
1B3i loop cDNA resulted in the partial
inhibition of GnRH receptor-mediated IP turnover in dispersed rat
pituitary cell cultures. The expression of this peptide, however, did
not significantly inhibit GnRH-stimulated LH release in these cultures.
Transfection of the cDNA sequences for
2A3i
loop, M13i loop, M23i
loop, and D1A3i loop did not significantly
inhibit GnRH-stimulated IP turnover or LH release. The expression of
one of the plasmids containing the cDNA sequence for the
2A3i loop was confirmed by Western analysis.
We were unable to perform Western analysis to confirm the expression of
other third intracellular loops due to the unavailability of antisera
against these peptides. Because the same expression vector (pRK5; 18 was used for all studies, it is reasonable to believe that these
would be expressed at similar levels as the
2A3i loop. The regulation of
Gq/11
by the GnRH receptor extends toward the
GGH31' cells. Buserelin treatment of these cells resulted
in the loss of total cellular Gq/11
as assessed by a
RIA. This effect of Buserelin was time- and dose-dependent and was
antagonized by GnRH antagonist, Antide. Neither Antide alone nor PMA
mimicked the actions of Buserelin.
This study was designed to investigate the G proteins that are
regulated by the GnRH receptor. G protein involvement in GnRH action in
the gonadotrope has been suggested by studies that show stimulation of
LH release by stable GTP analogs and by IP accumulation in
ATP-permeabilized cells (1, 21). Although ample evidence is available
to suggest G protein involvement in the gonadotrope, specific G
protein(s) are yet to be identified. In this study, we used G
protein-coupled receptor-evoked palmitoylation of G
to identify
specific moieties that are regulated by the GnRH receptor. This study
demonstrates that GnRH receptor regulates the palmitoylation of
Gq/11
. Because palmitoylation of Gq/11
is
dependent on this moiety being activated by a receptor, it suggests
that GnRH receptor is coupled to Gq/11
. PMA, a
PKC activator, stimulated Gq/11
incorporation
of [3H]palmitic acid, albeit less than GnRH-stimulated
levels. The effect of PMA on Gq/11
incorporation of
[3H]palmitic acid is puzzling, as PMA did not have any
significant effect on the Buserelin-evoked loss
ofGq/11
immunoreactivity in GGH3 cells.
The effect of PMA on Gq/11
palmitoylation may be the
result of activation of G protein palmitoyltransferase, the enzyme
responsible for addition and removal of palmitate from G proteins.
Alternatively, PMA may activate Gq/11
directly or
through PKC, thereby presenting it as an activated G protein for
palmitoyltransferase to palmitoylate, although there is no prior
evidence for this action of PMA. However, GnRH-evoked palmitoylation is
greater than PMA alone. This may indicate that GnRH receptor-evoked
palmitoylation reflects a direct activation of G proteins, while the
action of PMA (mediated through PKC) may be pharmacological. The
receptor-mediated component demonstrates that the GnRH
receptor is coupled to Gq/11
in the gonadotrope.
Inhibition of GnRH receptor-evoked IP turnover by transfecting the cDNA
for the
1B3i loop, whose cognate receptor
mediates IP turnover through a member of the Gq/11
family, demonstrated that the GnRH receptor is coupled to
Gq/11
and further confirmed the palmitoylation studies.
Luttrell et al. (15) showed that the
1B3i loop inhibited heterologous
receptor-mediated IP turnover, which is consistent with the data
presented in this study. Although M1Ach receptor mediates
IP turnover through a PTX-insensitive G protein, transfection of
M13i loop cDNA did not inhibit GnRH
receptor-mediated IP turnover. This may indicate that this loop is less
effective at inhibiting GnRH receptor-mediated IP turnover, because the
inhibition of heterologous receptor-mediated activity by third
intracellular loop peptides can vary (16). The lack of inhibition may
also be due to the fact that this loop is expressed to a lesser extent
than
1B3i loop, although this is unlikely as
both the
1B3i loop and the
M13i loop are translated by the same regulatory
elements, namely, the CMV promoter of the pRK5 vector. Transfection of
2A3i loop cDNA and
M23i loop cDNA did not inhibit GnRH
receptor-mediated signal transduction, although the cognate receptors
of these loops couple to Gi
(16), which has been
implicated in GnRH receptor-mediated PTX-sensitive IP turnover (3). The
lack of inhibition may be due to the same reasons that were mentioned
earlier, including the fact that GnRH receptor may not couple to these
G proteins. As would be expected, D1A3i loop,
which inhibits Gs
-mediated actions, did not inhibit
GnRH-evoked IP turnover or LH release because these events are not
thought to involve Gs
. The decrease in maximal effects
of GnRH-evoked IP turnover seen with
1B3i
loop transfection has also been observed by other investigators (16).
They have shown that the peptide- mediated inhibition of
receptor-evoked IP turnover is due to competition between the
hormone-receptor complex and the peptide for the common binding site on
G
-subunit. This competition can be overcome by increasing the
hormone-receptor coupled by increasing its transfected receptor
cDNA.
Previous work done in cell lines has shown that activation of
Gs
induces a conformational change that allows a loss of
membrane avidity and increases its degradation rate (19). Furthermore,
in
T31 cell lines GnRH agonist treatment results in an increased
degradation of Gq/11
(20). These observations support
our findings in the GGH3 cells; Buserelin treatment
resulted in the time- and dose-dependent decrease in cellular
Gq/11
. Using the increased degradation of activated G
as a marker, our results show that Gq/11
is activated by
the GnRH receptor. For technical reasons, these studies were done in an
immortalized cell line, because gonadotropes are only about 20% of the
cells in culture prepared from female weanling rat pituitaries, and
changes in G protein content can be masked by a relatively high
background from nongonadotrope cells. Although this study was done in a
cell culture, it corroborates well with our previous findings to show
that the GnRH receptor regulates Gq/11
. Furthermore, the
fact that PMA treatment did not mimic the actions of Buserelin on loss
of Gq/11
may indicate that PKC is not involved, and this
action of GnRH agonist is mediated through a direct
GnRH-receptor/Gq/11
interaction.
This study provides evidence for GnRH receptor regulation of
Gq/11
. We have shown that Gq/11
incorporates [3H]palmitic acid in a dose- and
time-dependent manner when treated with GnRH, and the third
intracellular loop of the
1B-adrenergic receptor, which
couples to Gq/11
, acts as an intracellular inhibitor of
GnRH receptor-mediated IP turnover. We have also shown that GnRH
agonist treatment results in a dose- and time-dependent loss of
Gq/11
immunoreactivity in GGH31' cells. The
observation made in this study suggest that GnRH receptor regulates the
activity of Gq/11
in rat gonadotropes and also in the
GGH31' cell line. The ability of the GnRH receptor to
regulate Gq/11
activity indicates that it is able to
couple to this G protein.
 |
MATERIALS AND METHODS
|
---|
Materials
Suppliers of materials used in this study were as follows: horse
serum and FCS, Hyclone Laboratories (Logan, UT); BSA, Irvine Scientific
(Santa Ana, CA); HEPES, United States Biochemical (Cleveland, OH);
collagenase, Worthington Biochemical (Freehold, NJ); formic acid,
Mallinkrodt (McGraw Park, IL); ammonium formate, sodium deoxycholate,
and EDTA, Fisher Scientific (Fairlawn, NJ); Nonidet P-40, Particle Data
Laboratory (Elm Hurst, IL); gentamicin sulfate, Gemini (Bio-products,
Calabasas, CA), hyaluronidase, DNAse I, and PMA, Sigma (St. Louis, MO);
and Antide (Ares-Serono, Geneva). Other reagents were obtained at the
highest grade available from commercial vendors as indicated. The G
protein-coupled receptor third intracellular loops expression plasmids
pRK
1B3i,
pRK
2A3i, pRKM13i,
pRKM23i, and pRKD1A3i
were a gift from Dr. R. J. Lefkowitz (Duke University, Durham, NC)
(3).
Preparation of Pituitary Cell Cultures
Pituitary cell cultures were prepared as previously described
(7). Briefly, pituitary glands were removed from 28-day-old female
Sprague-Dawley rats (B&K Universal, Inc, Kent, WA) and placed in medium
199 (Irvine Scientific) containing 0.3% (wt/vol) BSA and 10
mM HEPES, pH 7.4 (M199/BSA). The pituitaries were minced
and incubated in sterile M199/BSA containing 0.125% (wt/vol)
collagenase and 0.1% (wt/vol) hyaluronidase in a 37 C shaking water
bath for 15 min. The dissociated cells were filtered through organza
cloth, and the remaining tissue was incubated a second time with a
similar enzyme solution for another 15 min. The combined cells were
collected by centrifugation (10 min at 200 x g) and
resuspended in M199/BSA containing 10% (vol/vol) horse serum, 2.5%
(vol/vol) FCS, and 20 µg/ml gentamicin sulfate and filtered through
an organza cloth. For palmitoylation studies, cell suspensions were
plated at a cell density of 2.5 x 106 cells per well
in six-well culture plates (Costar, Cambridge, MA). Single cell
suspensions were obtained for transfection studies by resuspending the
cell pellet with M199/BSA/4 mM EDTA containing 0.2%
(wt/vol) collagenase and 0.2% (wt/vol) hyaluronidase and incubating
for 30 min at 37 C, and adding DNAse I (100 µg/ml) for the last 5 min
of the incubation. The dissociated cells were filtered through a
10-µm mesh cloth and incubated for another 15 min at 37 C with
M199/BSA/4 mM EDTA containing 0.2% collagenase and 0.2%
hyaluronidase. The cells were collected by centrifugation (15 min at
200 x g; 4 C) and resuspended in cold M199/BSA
containing 10% horse serum, 2.5% FCS, and 20 µg/ml gentamicin
sulfate. The cell suspensions were plated at cell density of 15 x
104 cells per well in 24-well culture plates (Costar,
Cambridge, MA). Cells were maintained for approximately 48 h at 37
C in a water-saturated atmosphere before experiments were begun.
Metabolic Labeling of G Proteins with
\[9,10-3H\]Palmitic Acid and
Immunoprecipitation
Pituitary cell cultures were washed twice with M199/BSA (pH
7.4), 2 h before labeling with \[9,10-3H\]palmitic
acid (specific activity 3060 Ci/mmol, 0.5 mCi/ml of M199/BSA; DuPont
NEN, Boston, MA) containing the indicated compounds for the indicated
times. Labeling was stopped at the appropriate times by aspirating the
labeling medium and washing once with cold Dulbeccos-PBS, and the
cells were lysed for 1 h on ice with 750 µl cold RIPA buffer
\[50 mM HEPES, pH 7.4, 150 mM NaCl, 1%
(vol/vol) Nonidet P-40, 0.5% (wt/vol) sodium deoxycholate, 1
mM EDTA and 2.5 mM MgCl2\]. The
insoluble material and nuclei were removed by centrifugation at
12,000 x g (Eppendorf microcentrifuge) for 3 min.
Nonspecific binding was removed by rocking the cell extract in 1.5-ml
Eppendorf tubes containing 75 µl Protein A-Sepharose 6MB (Pharmacia
Biotech, Piscataway, NJ), previously coupled to IgG from normal rabbit
serum, for 30 min at 4 C. After this step, the cell extract was
transferred to new 1.5-ml Eppendorf tubes containing 75 µl Protein
A-Sepharose coupled to our Q7 rabbit polyclonal antibody specific for
Gq/11
and immunoprecipitated overnight at 4 C. The cell
extract was centrifuged gently, the supernate was discarded, and the
beads were washed three times with 750 µl of cold RIPA buffer.
Finally the beads were resuspended in SDS-PAGE sample buffer (reducing
agents were omitted to prevent the hydrolysis of thioester-linked fatty
acids) and heated at 100 C for 2 min. The immunoprecipitates were
resolved by 10% SDS-PAGE, fixed, and prepared for fluorography with
Fluoro-Hance (RPI, Mt. Prospect, IL). The gels were exposed to Kodak
X-OMAT autoradiography film (Eastman Kodak, Rochester, NY) for
approximately 30 days at -70 C. In parallel experiments, gels were
treated with 1 M hydroxylamine (pH 7.0) after a 15-min
fixing period, before fluorography and exposure to autoradiography film
(22). Treatment with hydroxylamine cleaves the thioester bonds of
palmitic acids to G proteins, and not the amide bonds of myristic
acids, indicating palmitate labeling of G proteins as opposed to
myristate labeling (22).
Transfection of Primary Pituitary Cell Cultures
Transfection of primary cell cultures was done in 24-well plates
(Costar). Approximately 48 h after cell dispersion, cells were
washed with M199 (pH 7.4), and 0.4 µg DNA mixed with 2 µl
lipofectamine (GIBCO BRL, Gaithersburg, MD) in 0.25 ml of M199/BSA was
added to each well in triplicate. After 5 h at 37 C, 0.25 ml M199
containing 20% horse serum and 5% FCS was added to each well. After
24 h from start of transfection, medium was removed and plates
prepared for IP assays or LH RIA.
Measurement of IP Accumulation
After the transfection medium was removed, plates were washed
twice with a balanced salt solution (BSS) containing 0.3% BSA to
remove serum and unattached cells. Cellular inositol lipids were
labeled with [3H]myo-inositol (specific activity 3060
Ci/mmol, 4 µCi/ml; Dupont NEN) for 18 h. After inositol
labeling, cells were washed twice with BSS containing 5 mM
LiCl (BSS/LiCl) and stimulated for 2 h with the indicated GnRH
concentrations prepared in BSS/LiCl. The treatment solutions were
removed, and 1 ml of 0.1 M formic acid was added to each
well. The cells were freeze-thawed once to disrupt the cell membranes,
and the total 3H-labeled IPs were determined by Dowex anion
exchange chromatography and liquid scintillation spectroscopy (23).
Quantification of LH Release
After the transfection medium was removed, cells were washed
twice with M199/BSA and stimulated with the indicated GnRH
concentrations for 2 h. The medium was collected from the culture
wells. LH released was determined by RIA.
The RIA used a highly purified rat LH for iodination (24) and a
reference preparation (RP3) obtained from the NIDDK (Baltimore, MD). LH
antisera (C102) was prepared and characterized as previously described
(25). Bound and free hormone were determined with immobilized protein A
(26).
Western Blots
SDS-polyacrylamide gels (12% acrylamide) and Western transfers
to nitrocellulose paper (Hoefer Scientific Instruments, San Francisco,
CA) were performed as previously described (27). Polyclonal antisera
(Ref. 28; a gift from Dr. Hitoshi Kurose, University of Tokyo, Tokyo,
Japan) made against the third intracellular loop of the
2A-adrenergic receptor was used at 1:500 titer. Color
was developed on Western blots using 4-chloro-1-napthol (horseradish
peroxidase) color development reagent (Bio-Rad Laboratories, Richmond,
CA). Standards were color-stained proteins (rainbow markers, Amersham)
with the following molecular weights (including the dye): myosin
(200K), phosphorylase (92.5K), BSA (69K), ovalbumin (46K), carbonic
anhydrase (30K), trypsin inhibitor (21.5K), and lysozyme (14.3K).
Cell Culture and Transfection
GGH31' cells were derived from GH3 cells
stably transfected with the rat GnRH receptor cDNA as previously
reported (29). The GGH31' cells were maintained in an
atmosphere of 5% CO2 at 37 C in DMEM (GIBCO, Grand Island,
NY) containing 10% FCS and 20 µg/ml gentamicin. Cells were grown to
confluency in 162-cm2 T flasks (Costar), then scraped and
plated at a density of 350,000 cells per well in a 24-well culture
plate for 3640 h at 37 C in 5% CO2. Cells were washed
twice in DMEM, 0.1% BSA, and 20 µg/ml gentamicin and treated with
the indicated secretogogues for the indicated times.
Production of Polyclonal Gq/11
Antisera and Gq/11
RIA
Antisera were raised in rabbits using the C-terminal sequence
("CTS", QLNLKEYNLV) for the
-subunit of the Gq/11
family of guanyl nucleotide-linked proteins coupled to keyhole limpet
hemocyanin. This same CTS was radioiodinated to serve as the
immunoligand in the RIA. Unlabeled CTS was the standard; accordingly, a
molar correction factor of 0.03 should be used to adjust the values
obtained to account for the ratio of the mol wt of the standard (CTS)
to that of Gq/11
. The sensitivity and lower limit of
quantification values for this assay were <5 pg/tube at a final
antiserum titer of 1:100,000. The RIA was set up by disproportionation
for 12 h. The cellular Gq/11
proteins were measured
after solubilization of the cells in 0.1% Triton X-100. Bound and free
proteins were separated using the second antibody technique (26).
DNA Quantification
GGH31' cells were plated at 350,000 cells per well
in a 24-well plate and maintained at 37 C, 5% CO2, for
3640 h. Cells were treated with medium alone (control) or with the
GnRH agonist, Buserelin (Hoescht-Roussel Pharmaceuticals, Somerville,
NJ), 0.1 µg/ml for 0, 1, 2, 3, 4, 5, 6, and 24 h. The supernate
was removed and the cells were frozen. The previously frozen cells were
scraped, washed, and assayed in 0.44 N perchloric acid. The
DNA content per well for each treatment and time point were assessed
using the mini-diphenylamine assay (30).
 |
ACKNOWLEDGMENTS
|
---|
We thank Linda Wolf for preparing the manuscript and Dr.
Brian Hawes and Dr. Alfredo Ulloa-Aguirre for helpful
suggestions.
 |
FOOTNOTES
|
---|
Address requests for reprints to: Michael Conn, Oregon Regional Primate Research Center, 505 Northwest 185th Avenue, Beaverton, Oregon 97006.
This study was supported by NIH Grants HD-19899, HD-00163, and
HD-18185
Received for publication January 31, 1997.
Accepted for publication March 14, 1997.
 |
REFERENCES
|
---|
-
Hawes BE, Marzen JE, Waters SB, Conn PM 1992 Sodium
fluoride provokes gonadotrope desensitization to gonadotropin-releasing
hormone (GnRH) and gonadotrope sensitization to A23187: evidence for
multiple G protein in GnRH action. Endocrinology 130:24652475[Abstract]
-
Andrews WV, Conn PM 1986 Gonadotropin releasing hormone
stimulates mass changes in phosphoinositides and diacylglycerol
accumulation in purified gonadotrope cell cultures. Endocrinology 118:11481158[Abstract]
-
Hawes BE, Barnes S, Conn PM 1993 Cholera toxin and pertussis
toxin provoke differential effects on luteinizing hormone release,
inositol phosphate production, and gonadotropin releasing hormone
receptor binding in the gonadotrope: evidence for multiple guanyl
nucleotide binding proteins in GnRH action. Endocrinology 132:21242130[Abstract]
-
Janovick JA, Conn PM 1994 GnRH-receptor coupling to inositol
phosphate and prolactin production in GH3 cells stably
transfected with rat GnRH receptor cDNA. Endocrinology 135:22142219[Abstract]
-
Hsieh KP, Martin TFJ 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]
-
Kuphal D, Janovick JA, Kaiser UB, Chin WW, Conn PM 1994 Stable transfection of GH3 cells with rat
gonadotropin-releasing hormone receptor complementary deoxyribonucleic
acid results in expression of a receptor coupled to cyclic adenosine
3',5'-monophosphate-dependent prolactin release via a G-protein.
Endocrinology 135:315320[Abstract]
-
Conn PM, Morrell DV, Dufau ML, Catt KJ 1979 Gonadotropin
releasing hormone action in cultured pituicytes: independence of
luteinizing hormone release and adenosine 3',5'-monophosphate
production. Endocrinology 104:448453[Abstract]
-
Janovick JA, Conn PM 1993 A cholera toxin-sensitive guanyl
nucleotide binding protein mediates the movement of pituitary
luteinizing hormone into a releasable pool: loss of this event is
associated with the onset of homologous desensitization to
gonadotropin-releasing hormone. Endocrinology 132:21312135[Abstract]
-
Tsutsumi M, Zhou W, Millar RP, Mellon PL, Roberts JL,
Flanagan CA, Dong K, Gillo B, Sealfon SC 1992 Cloning and functional
expression of a mouse gonadotropin-releasing hormone receptor. Mol
Endocrinol 6:11631169[Abstract]
-
Barnes SJ, Conn PM 1993 Cholera toxin and dibutryl cyclic
adenosine 3',5'-monophosphate sensitize gonadotropin-releasing
hormone-stimulated inositol phosphate production to inhibition in
protein kinase-C (PKC)-depleted cells: evidence for cross-talk between
a cholera toxin-sensitive G-protein and PKC. Endocrinology 133:27562760[Abstract]
-
Degtyarev MY, Spiegel AM, Jones TLZ 1993 Increased
palmitoylation of Gs protein
subunit after activation
by the ß-adrenergic receptor or cholera toxin. J Biol Chem 268:2376923772[Abstract/Free Full Text]
-
Mumby SM, Kleuss C, Gilman AG 1994 Receptor regulation of
G-protein palmitoylation. Proc Natl Acad Sci USA 91:28002804[Abstract]
-
Wedegaertner PB, Chu DH, Wilson PT, Levis MJ, Bourne HR 1993 Palmitoylation is required for signalling functions and membrane
attachment of Gq
and Gs
. J Biol Chem 268:2500125008[Abstract/Free Full Text]
-
Wedegaertner PB, Bourne HB 1994 Activation and
depalmitoylation of Gs
. Cell 77:10631070[Medline]
-
Luttrell LM, Ostrowski J, Cotecchia S, Kendall H, Lefkowitz RJ 1993 Antagonism of catecholamine receptor signaling by expression of
cytoplasmic domains of the receptors. Science 259:14531457[Medline]
-
Hawes BE, Luttrell LM, Exum ST, Lefkowitz RJ 1994 Inhibition
of G-protein-coupled receptor signaling by expression of cytoplasmic
domains of the receptor. J Biol Chem 269:1577615785[Abstract/Free Full Text]
-
Milligan G 1993 Agonist regulation of cellular G protein
levels and distribution: mechanisms and functional implications. Trends
Pharmacol Sci 14:413418[CrossRef][Medline]
-
Eaton DL, Wood WI, Eaton D, Haas PE, Hollingshead P, Wion K,
Mather J, Lawn RM, Vehar GA, Gorman C 1986 Construction and
characterization of an active factor VIII variant lacking the central
one-third of the molecule. Biochemistry 25:83438347[Medline]
-
Levis MJ, Bourne HR 1992 Activation of the
subunit of
Gs in intact cells alters its abundance, rate of
degradation, and membrane avidity. J Cell Biol 119:12971307[Abstract]
-
Shah BH, MacEwan DJ, Milligan G 1995 Gonadotropin-releasing
hormone receptor agonist-mediated down-regulation of
Gq
/G11
(pertussis toxin-insensitive) G
proteins in
T31 gonadotroph cells reflects increased G protein
turnover but not alterations in mRNA levels. Proc Natl Acad Sci USA 92:18861890[Abstract]
-
Andrews WV, Staley DD, Huckle WR, Conn PM 1986 Stimulation of
luteinizing hormone (LH) release and phospholipid breakdown by
guanosine triphosphate in permeabilized pituitary gonadotropes:
antagonist action suggest association of a G protein and
gonadotropin-releasing hormone receptor. Endocrinology 119:25372546[Abstract]
-
Mumby SM, Buss JE 1990 Metabolic radiolabeling techniques for
identification of prenylated and fatty acylated proteins. Methods: A
Companion to Methods in Enzymology 1:216220
-
Huckle WR, Conn PM 1987 The relationship between
gonadotropin-releasing hormone-stimulated luteinizing hormone release
and inositol phosphate production: studies with calcium antagonists and
protein kinase C activators. Endocrinology 120:160169[Abstract]
-
Hunter WM, Greenwood FC 1962 Preparation of iodine-131 labeled
growth hormone of high specific activity. Nature 194:495496
-
Smith WA, Cooper RL, Conn PM 1982 Altered pituitary
responsiveness to gonadotropin-releasing hormone in middle-aged rats
with 4-day estrous cycles. Endocrinology 111:18431848[Abstract]
-
Gupta R, Morton DL 1979 Double antibody method and the
protein-A-bearing Staphylococcus aureus cells method
compared for separating bound and free antigen in radioimmunoassay.
Clin Chem 25:752756[Abstract/Free Full Text]
-
Conn PM, Janovick JA, Braden TD, Maurer RA, Jennes L 1992 SIIp: a unique secretogranin/chromogranin of the pituitary released in
response to GnRH. Endocrinology 130:30333040[Abstract]
-
Kurose H, Arriza JL, Lefkowitz RJ 1993 Characterization of
alpha 2-adrenergic receptor subtype-specific antibodies. Mol Pharmacol
43(3):44450
-
Kaiser UB, Katzenellenbogen R, Conn PM, Chin WW 1994 Evidence
that signalling pathways by which thyrotropin-releasing hormone and
gonadotropin-releasing hormone act are both common and distinct. Mol
Endocrinol 8:10381048[Abstract]
-
Burton K 1956 A study of the conditions and mechanisms of
diphenylamine reaction for the colorimetric estimation of
deoxyribonucleic acid. Biochem J 62:315323