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INTRODUCTION |
Heterotrimeric G proteins transduce signals generated by hormone
receptors with seven transmembrane domain
-helices to various effectors such as AC,1
phospholipase C, and ion channels (1-3). These G proteins are composed
of a G
subunit and a tight complex of G
and G
subunits. The
binding of agonist to receptors allows them to interact with G
proteins, which subsequently accelerates the rate of dissociation of
GDP and the binding of GTP to the G
subunits. Both GTP-liganded G
subunits and G
dimers then regulate downstream effector
activities. The intrinsic GTPase activity of G
subunits hydrolyzes
GTP to GDP to form G
-GDP, which then associates with G
dimers
to inactivate the complex.
The cyclic AMP-forming enzyme, adenylyl cyclase, is one of the
ubiquitous effectors that is regulated by G protein-coupled receptors.
The pathways of Gs-coupled receptor-induced stimulation of
AC have been well characterized (1, 4). However, the inhibitory
regulation of AC by Gi-coupled receptors is far less clear,
and the underlying mechanisms seem to be rather complex. For example,
in several cell systems, e.g. mouse Ltk
,
Rat-1, and NIH-3T3 fibroblast cells, inhibition of cAMP synthesis by
Gi-coupled receptors was only observed when AC was
activated by forskolin or Gs-coupled receptors (5-7). On
the other hand, in neuronal and endocrine cells, Gi-coupled
receptors inhibited both unstimulated and stimulated AC activity (2,
5, 8). In addition, some Gi-coupled receptors have even
been reported to stimulate AC, e.g.
2-adrenergic receptor in PC-12 pheocytochroma cells (7,
9) or 5-HT1A receptors in hippocampus (10, 11).
The specificity of distinct Gi proteins in receptor-AC
coupling remains incompletely understood. For example, using
anti-G
i subunit antibodies to block receptor coupling,
it has been reported that inhibition of AC by
2-adrenergic receptors in platelet membranes is mediated
by G
i2 (12) and that 5-HT1A receptor-induced inhibition of cAMP synthesis in HeLa cell membranes is preferentially mediated by
G
i3 (13). This approach, however, is limited to
cell-free preparations and depends on the specificity of the antibodies used. To assess the roles in receptor coupling of particular G proteins
in whole cells, transfection of PTX-insensitive G
i
mutant proteins has been used to rescue receptor-mediated signaling
following PTX pretreatment. For example, the dopamine-D2S receptor
appears to couple to G
i2 and G
i3 to
mediate inhibition of forskolin and Gs-stimulated AC,
respectively (14, 15). This approach depends on the specificity and
functionality of the G
i mutants. We have used expression
of antisense constructs to selectively deplete particular G proteins
and assessed their contribution to receptor coupling (16-18).
The aim of the present study was to evaluate the contribution of the
three known G
i subunits (19) in relaying inhibitory signals from 5-HT1A and muscarinic M4 receptors to AC in intact GH4C1
rat pituitary cells. We analyzed Gi protein subtype
specificity in receptor-effector coupling by stably introducing
distinct full-length rat G
i antisense constructs into
GH4ZD10 cells (GH4C1 cells transfected with the rat 5-HT1A receptor
(5)). This approach produced a specific block of the gene expression of
these proteins (18). Characterization of these different
G
i-deficient antisense clones indicates that
G
i proteins specifically link receptors to inhibition of
cAMP synthesis but not to closure of calcium channels and that the
combined presense of all three G
i subunits is essential
for receptor-mediated inhibition of unstimulated but not of VIP
receptor-stimulated cAMP synthesis. Strikingly, upon depletion of
distinct G
i subunits, the Gi-coupled 5-HT1A
and muscarinic M4 receptors switched to stimulate AC activity.
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EXPERIMENTAL PROCEDURES |
Materials--
5-HT, carbachol, VIP, PTX, and
isobutylmethylxanthine were purchased from Sigma. Hygromycin B was from
Calbiochem (La Jolla, CA). BayK-8644 was from Research Biochemicals
Inc. (Natick, MA). Fura 2-AM was from Molecular Probes (Eugene, OR).
PTX was from List Biological Laboratories (Campbell, CA). Rat G protein
G
i1, G
i2, and G
i3 subunits
and AC type II cDNAs were gifts of Dr. R. Reed. G protein
G
i subunit antibodies were kindly donated by Dr. D. Manning.
Cell Culture--
All cells were grown as monolayer in Ham's
F-10 medium with 8% fetal bovine serum at 37 °C in a humidified
atmosphere with 5% CO2. Media were changed 12-24 h
prior to experimentation.
Preparations of the Antisense Clones--
The 2.0-kb
EcoRI-EcoRI G
i1 cDNA fragment,
the 1.8-kb EcoRI-EcoRI G
i2
cDNA fragment, or the 3.1-kb EcoRI-EcoRI
G
i3 cDNA fragment (13) containing the full coding
sequences and 0.5 to 0.8 kb of 5' and 3' noncoding regions were excised
using EcoRI. The cDNA fragments were ligated into
pcDNA (Invitrogen) in the reverse orientation with respect to the
cytomegalovirus promoter, resulting in G
i1,
G
i2, or G
i3 antisense expression vectors. The constructs were confirmed by restriction enzyme analysis and by DNA
sequencing. A modified transfection procedure was used; 300-500 µg
of each G
i antisense construct was cotransfected
separately with 30 µg of pY-3 hygromycin B resistance plasmid into
G418-resistant GH4ZD10 cells by standard calcium phosphate
co-precipitation protocol (20). The selection was initiated after
24 h by adding 150 µg/ml hygromycin B into the culture medium to
select the clones with expression of the antisense RNAs and to allow
the clones to adapt the cytotoxicity of hygromycin B. After 2 weeks,
the concentration of hygromycin B was raised to 400 µg/ml to select
the clones that have the highest resistance to hygromycin B and that
most likely express the highest levels of the antisense RNAs. Isolated
clones were propagated, and total RNA was prepared from them and
screened by reverse transcription-PCR analysis using a pair of
oligonucleotides specific for each G
i cDNA
(G
i1, 5'-ACCAGACGAGTACTTATA-3' and 5'-TAGTCTGTGCAACGTTTA-3'; G
i2, 5'-CACTACCTGTGAGGAAGA-3'
and 5'-ACTCCTCCAGACATAGG-3'; G
i3,
5'-TGATATCAAATCTAGGGC-3' and 5'-TAGAACGCATTCCCAGAT-3') to identify positive clones. Total RNA from GH4ZD10 or the different G
i antisense clones was reverse-transcribed after
annealing the primer (sense or antisense) to RNA by heating the two
together at 90 °C and then chilling on ice. One-tenth of the
reverse-transcription mixture was used for PCR amplification. 50 µl
of PCR mixture contained 50 mM KCl, 25 mM
Tris-HCl, pH 8.3, 2.5 mM MgCl2, 1 mg/ml bovine serum albumin, 0.2 mM each dCTP, dATP, dGTP, and dTTP, 1 unit Hot-Tub DNA polymerase (Amersham Pharmacia Biotech) and 3 µM primer. The PCR cycle included denaturation for 1 min
at 94 °C, annealing for 1 min at 48 to 52 °C (varied with
different pairs of primers), and extension for 20 s at 72 °C.
For Western blotting, membranes (50 µg protein/lane) prepared from
the positive antisense clones were solubilized, electrophoresed,
transferred to nitrocullose sheets, and probed with specific antibodies
as described (16). Densitometric scanning of the blots was done on the
Scanmaster 3 densitometer (Howtek, Hudson, NH). The data were
digitized, quantitated using the Masterscan analysis program
(Scanalytric, Billerica, MA), and reconstructed as Masterscan images
presented in the figures. This analysis allows enhanced resolution of
weak signals. Western blots were quantitated from the Masterscan data. Optical densities of equal area samples from each band of interest were
subtracted from background for that lane and normalized (×100) to the
control samples to obtain the percentage of control.
Measurement of
[Ca2+]i--
Measurement of
[Ca2+]i was performed as described previously
(5). In brief, cells were harvested by incubation in calcium-free HBSS
containing 5 mM EDTA. The cells were washed once with HBSS
(118 mM NaCl, 4.6 mM KCl, 1 mM
CaCl2, 1 mM MgCl2, 10 mM D-glucose and 20 mM HEPES, pH
7.2) and then incubated for 30 min at 37 °C in the presence of 2 µM Fura 2-AM. The cells were then diluted to 10 ml,
centrifuged, washed twice with HBSS, resuspended in 2 ml HBSS, and
finally placed in a fluorescence cuvette. Change in fluorescence ratio
was recorded on a Perkin-Elmer (Buckinghamshire, UK) LS-50
spectrofluorometer and analyzed by computer, based on a
KD of 227 nM for the Fura 2-calcium
complex. Calibration of Rmax was performed by
addition of 0.1% Triton X-100 and 20 mM Tris base, and
calibration of Rmin was performed by addition of
10 mM EGTA. All experimental compounds were added directly to the cuvette from 200-fold concentrated test solutions.
cAMP Assay--
Measurement of cAMP was performed as described
previously (5). In brief, the cells were plated in six-well 35-mm
dishes. After removal of the medium, the cells were preincubated in 2 ml/well of HBSS for 5-10 min at 37 °C. The buffer was replaced by 1 ml of HBSS containing 100 µM isobutylmethylxanthine, a
cAMP phosphodiesterase inhibitor, and the incubation was continued for
another 5 min. Then the various test compounds were added to the wells,
and the cells were incubated at room temperature for 20 min. The buffer
was collected for cAMP assay using a specific radioimmunoassay (ICN) as
described before (5).
Determination of AC RNA Expression--
Cytosolic RNA was
extracted from rat brain tissue and GH4C1 cells using guanidium
thiocyanate extraction, and GH4C1 RNA was further purified on oligo(dT)
cellulose to obtain poly(A)+ RNA. The RNA was
reverse-transcribed using SuperScript II RNase H
reverse
transcriptase (Life Technologies Inc.) and random hexamer primers (50 ng). The cDNAs were subjected to PCR with the following primer
pairs (2 pmol/µl) designed using the Primer Select program (DNASTAR
Inc.) to amplify specific fragments of the Indicated sizes: AC I
(444 bp), 5'-CTGCGGGCGTGCGATGAGGAGTTC and 5'-GCGCACGGGCAGCAGGGCATAG; AC
II (425 bp), 5'-GCTGGCGTCATAGGGGCTCAAAA and
5'-GGCACGCGCAGACACCAAACAGTA; AC III (418 bp),
5'-GGACGCCCTTCACCCACAACCAA and 5'-AGACCACCGCGCACATCACTACCA; AC IV (454 bp), 5'-CACGGCCGGGATTGCGAGTAGC and 5'-TGCCGAGCCAGGACGAGGAGTGT; AC V
(412 bp), 5'-GAGCCCCAATGACCCCAGCCACTA and 5'-CGGGAGCGGCGCAATGATGAACT; and AC VI (362 bp), 5'-CCTGGCGGAAGCTGTGTCGGTTAC and
5'-GCGGTCAGTGGCCTTGGGGTTTG. The PCR reaction was performed with
different concentrations of cDNA (0.1, 0.5, and 1.0 µg/reaction)
and repeated at least twice. The amplified DNA fragment was subcloned
into pCR2.1 (Invitrogen) and sequenced by Sanger dideoxynucleotide
chain termination using modified T7 DNA polymerase (Amersham Pharmacia
Biotech). Northern blot analysis was performed on GH4C1
poly(A)+ RNA at high stringency as described (20), using
the 32P-labeled 1.203-kb EcoRI/EcoRV
fragment of rat AC II cDNA as probe (21).
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RESULTS |
Independent Depletion of Distinct G
i Subunits from
GH4ZD10 Cells--
Reverse transcription-PCR and Western blot analysis
indicated the presence of all three known G
i subunits in
GH4C1 cells (Fig. 1), although
G
i1 may be the least abundant based on the weakness of
the signal. The three G
i subunits are highly homologous in their coding regions but exhibit a clear variability in their 5'-
and 3'-noncoding sequences (19). To specifically block the protein
expression of distinct G
i subunits, the antisense
constructs were chosen to include the full coding sequences and, in
addition, 500-800 base pairs of the 5'- and 3'-untranslated sequences
(18). Selection for stably transfected clones exhibiting the highest levels of antisense RNA expression was accomplished by raising temporarily the concentration of hygromycin B to 400 µg/ml. The expression of antisense RNA was detected by reverse transcription-PCR analysis, using a pair of oligonucleotides specific for each
G
i subunit (not shown). The extent of depletion of
distinct G
i subunits was verified by Western blot
analysis, using antibodies specific for each G
i subunit
(22, 23). It was found that G
i1 and G
i2
subunits were virtually eliminated in clones Gi1ZD-3 and Gi1ZD-5 (Fig.
1A) and Gi2ZD-4 and Gi2ZD-5 (Fig. 1B),
respectively, whereas G
i3 subunits were largely depleted
in clones Gi3ZD-3 and Gi3ZD-4 (Fig. 1C). To examine the
extent of cross-hybridization of different G
i antisense
RNAs with sense RNAs of other G
i subunits, the membranes
prepared from different G
i antisense clones were also
probed with antibodies specific for other G
i and
G
o subunits. In G
i3-depleted cells, the
amount of G
i1 was similar to the control cells (Fig.
1D), whereas in G
i1- and
G
i3-depleted clones the amount of G
i2 or
G
o was unchanged (data not shown). Thus, stable
transfection of G
i antisense constructs can specifically eliminate their cognate G
i proteins without major
alterations in the amount of other G
i subunits.

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Fig. 1.
Western blot analysis of different
G i subunits in
G i-depleted antisense clone
membranes. Membranes of GH4ZD10 cells and various
G i antisense clones were probed with specific
antibodies, and the blots were analyzed by the Masterscan technique as
described under "Experimental Procedures." The percentage of
control (lane 1) optical density (background subtracted) for
each of the antisense clones is indicated in parentheses as the
means ± range of duplicate independent scans. A,
depletion of G i1 subunits in G i1
antisense clone membranes. Blot was probed with an antibody specific
for G i1 subunits. Lane 1, GH4ZD10; lane
2, Gi1ZD-3 (13 ± 2%); lane 3, Gi1ZD-5 (4 ± 1%). B, depletion of G i2 subunits in
G i2 antisense clone membranes. Blot was probed with an
antibody specific for G i2 subunits. Lane 1,
GH4ZD10; lane 2, Gi2ZD-4 (7 ± 1%); lane 3,
Gi2ZD-5 (12 ± 2%). C, depletion of G i3
subunits in G i3 antisense clone membranes. Blot was
probed with an antibody specific for G i3 subunits.
Lane 1, GH4ZD10; lane 2, Gi3ZD-3 (22 ± 3%); lane 3, Gi3ZD-4 (19 ± 6%). D,
Western blots of G i3 antisense clone membranes with an
antibody specific for G i1 subunits. Lane 1,
GH4ZD10; lane 2, Gi3ZD-3 (74 ± 4%); lane
3, Gi3ZD-4 (88 ± 7%).
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5-HT1A and Muscarinic M4 Receptor-mediated Inhibition of cAMP
Synthesis and Calcium Entry in GH4ZD10 Cells--
In previous studies
(5), it was found that agonist activation of the transfected 5-HT1A
receptor inhibits both cAMP accumulation and calcium entry in GH4ZD10
cells. Similarly, activation of endogenously expressed muscarinic M4
receptors (24) in GH4ZD10 cells also inhibited both cAMP accumulation
and calcium entry (Fig. 2, upper panel, and Table I). These receptor
actions were sensitive to PTX treatment (Fig. 2, lower
panel), suggesting the involvement of
Gi/Go proteins. Muscarinic M4 receptors
exhibited greater efficacies than 5-HT1A receptors (Fig. 2 and Table
I), although the number of endogenously expressed muscarinic M4
receptors in GH4ZD10 cells is 2-3-fold lower than that of the
transfected 5-HT1A receptors (5). Similarly distinct efficacies were
observed for agonist-stimulated GTPase activity by these two receptors
in membrane preparations (data not shown), suggesting that in this
cellular system, muscarinic M4 receptors couple more effectively to G
proteins than do 5-HT1A receptors.

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Fig. 2.
Receptor-mediated PTX-sensitive inhibition of
cAMP synthesis in GH4ZD10 cells. The cells plated on six-well
plates were incubated for 20 min at 25 °C in the presence of 100 µM isobutylmethylxanthine and the indicated compounds.
cAMP was determined as described under "Experimental Procedures."
For PTX pretreatment (lower panel), PTX (50 ng/ml) was added
to the medium 16 h prior to experimentation. Data averaged from
three independent experiments each done in triplicate are presented as
the means ± S.D. The concentrations of the agents used were: VIP,
0.2 µM; 5-HT, 1 µM; and carbachol
(Car), 10 µM.
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Table I
Effects of elimination of different G i subunits on
receptor-mediated inhibition of calcium entry
Data are presented as the means ± S.D. of three independent
experiments in which the actions of 5-HT (1 µM) and
carbachol (10 µM) on the BayK 8644 (1 µM)-induced increase in [Ca2+]i were
measured in GH4ZD10 and various G i antisense clones as
described under "Experimental Procedures." Values are expressed as
fold basal level of [Ca2+]i.
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Influence of Distinct G
i Subunit Knockout on
Receptor-mediated Inhibition of Calcium Entry--
To monitor the
roles of each G
i subunit in coupling of 5-HT1A and
muscarinic M4 receptors to inhibition of voltage-dependent calcium channels, the effects of the receptor agonists, 5-HT and carbachol, on calcium entry induced by the calcium channel opener, BayK
8644, in the different G
i-depleted clones were examined. In any of the G
i-depleted clones, both 5-HT1A and
muscarinic M4 receptors induced similar inhibition of the BayK
8644-induced increase in [Ca2+]i, as observed in
GH4ZD10 cells (Table I). These results indicate that no single class of
G
i subunits is essential for 5-HT1A or muscarinic M4
receptor-mediated closure of calcium channels.
Alteration of Receptor-induced Inhibition of AC upon Depletion of
Distinct G
i Subunits--
The consequence of depletion
of distinct G
i subunits on receptor-mediated inhibition
of cAMP accumulation was examined in unstimulated cells and in cells
stimulated by the Gs-coupled VIP receptor. In the control
cells, GH4ZD10, 5-HT, and carbachol inhibited both basal and
VIP-stimulated cAMP formation (Fig. 2). In contrast to calcium entry,
receptor-mediated inhibition of cAMP synthesis in the different
G
i-depleted clones was clearly and selectively altered.
Specifically, in G
i1-depleted clones, Gi1ZD-3, both 5-HT
and carbachol failed to inhibit basal cAMP production (Fig. 3A). Surprisingly, 5-HT
induced a 5-fold increase in basal cAMP accumulation in these cells, an
action almost as efficacious as that of VIP. The 5-HT-induced
stimulation of basal cAMP accumulation was blocked by PTX pretreatment,
suggesting mediation by Gi/Go proteins. This
novel stimulatory action of 5-HT on basal cAMP formation was also
observed in another G
i1 depleted clone, Gi1ZD-5 (Table
II). In the same
G
i1-depleted cells, however, 5-HT inhibited VIP-stimulated cAMP accumulation by some 45%, similar to the results obtained in GH4ZD10 cells. These data indicate that the presence of
G
i1 subunits is not essential for the 5-HT1A
receptor-mediated inhibition of Gs-stimulated cAMP
accumulation. In contrast to 5-HT1A receptor action, in both Gi1ZD-3
and Gi1ZD-5 clones, carbachol-induced inhibition of VIP-stimulated cAMP
accumulation was blocked (Fig. 3A).

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Fig. 3.
Effects of depletion of different
G i subunits on receptor-mediated
inhibition of cAMP accumulation. Measurements of cAMP were
performed as described in the legend to Fig. 2 in G i1
antisense clone Gi1ZD-3 (A), G i2 antisense
clone Gi2ZD-5 (B), and G i3 antisense clone
Gi3ZD-4 (C). For PTX pretreatment, PTX (50 ng/ml) was added
to the medium 16 h prior to experimentation. Data averaged from
three independent experiments each done in triplicate are presented as
the means ± S.D. The asterisk indicates significant
difference (p < 0.05) compared with VIP alone. The
concentrations of the agents used were: VIP, 0.2 µM;
5-HT, 1 µM; and carbachol (Car), 10 µM.
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Table II
Receptor-mediated inhibition of cAMP accumulation in
G i-depleted cells
The level of cAMP (pmol/dish) was determined as described in the legend
to Fig. 2 in parental GH4ZD10 cells (data from Fig. 2), and the
indicated G i1-, G i2-, and G i3
antisense cell lines. The cells were treated with the indicated
compounds: VIP (0.2 µM), 5-HT (1 µM), and
carbachol (10 µM) and PTX (50 ng/ml for 16 h). Data averaged
from three independent experiments each done in triplicate are
presented as the means ± S.D.
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As observed in G
i1-depleted clones, the ability of
5-HT1A and muscarinic M4 receptors to inhibit basal cAMP synthesis was also ablated in G
i2-depleted clones, Gi2ZD-4 (Fig.
3B). In addition, both receptors failed to inhibit
VIP-stimulated cAMP synthesis. Activation of 5-HT1A receptors
potentiated slightly VIP-stimulated cAMP accumulation by 35%, an
action sensitive to PTX treatment.
In G
i3-depleted clone Gi3ZD-3, activation of 5-HT1A or
muscarinic M4 receptors did not result in inhibition of basal cAMP accumulation (Fig. 3C). Curiously, carbachol induced a
3-fold increase in cAMP production in these antisense clones. This
stimulation was prevented by PTX pretreatment of the cells. In the same
G
i3-depleted clones, however, the inhibitory action of
muscarinic M4 receptors on VIP-stimulated cAMP synthesis remained
largely unaltered. In Gi3ZD-4 cells, another G
i3
depleted clone, carbachol-induced inhibition of VIP-stimulated cAMP
accumulation was also essentially unaltered (Table II). By contrast,
5-HT1A receptor-induced inhibition of VIP-stimulated cAMP accumulation
was completely blocked in G
i3-depleted cells.
Subtypes of AC Expressed in GH4C1 Cells--
The possible
mechanism of Gi-mediated stimulation of cAMP production
upon depletion of specific G
i subunits was addressed. Certain AC subtypes (types II, IV, and VII) have been demonstrated biochemically to be conditionally stimulated by G
subunits. In
addition AC II is known to mediate Gi-induced stimulation
of cAMP levels when co-transfected with specific Gi-coupled
receptors, such as the
2-adrenergic receptor (26). We
examined the RNA expression of the most extensively characterized
subtypes, AC types I-VI (4), using reverse transcription-PCR analysis
at different concentrations of cDNA (Table
III). In rat brain each subtype was
present, although type I was weakly expressed (25). The rank order of
expression of adenylyl cyclases in GH4C1 cells was II = VI > III
(I, IV, and V). Of particular interest was the predominant
expression in GH4C1 cells of AC type II, which was detected as a major
species of 6.7 kb by Northern blot analysis of poly(A)+ RNA
from GH4C1 cells (Fig. 4). By contrast,
AC type II RNA was undetectable in various fibroblast cell lines
(Ltk
and Balb/c-3T3), adrenocortical Y1, DDT1-MF2 smooth
muscle, or PC-12 pheocytochroma cells (data not shown and Refs. 15 and 25). Thus, AC II is abundantly expressed in pituitary GH4C1 cells and
brain tissue, permitting the possibility for receptor coupling to
Gi-dependent stimulation of cAMP accumulation
in these tissues.
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Table III
Expression of different adenylyl cyclase subtypes in GH4C1 cells
The PCR was performed for AC I-VI with 0.1, 0.5, and 1.0 µg of
cDNA synthesized from GH4C1 poly(A)+ RNA or rat brain
RNA as indicated. Data were obtained from at least two independent
experiments for each condition. Specific primers for each subtype of AC
amplified only a single product of the predicted size. Plus (+) and
minus ( ) signs indicate the presence and the absence of the specific
product on ethidium bromide stained gels, respectively, and ± represents a weakly detectable product.
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Fig. 4.
RNA expression of adenylyl cyclase type II in
GH4C1 cells. Poly(A)+ RNA (5 µg) was subjected to
Northern blot analysis under high stringency conditions using rat AC
type II cDNA as a probe (21). The migration of RNA molecular mass
markers (in kb) is indicated. A major RNA species of 6.7 kb in size was
detected with a minor species at 4.5 kb.
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DISCUSSION |
Using stable transfection of distinct G
i
full-length antisense constructs, we were able to specifically deplete
the protein expression of individual G
i subunits from
GH4C1 pituitary cells. It was found that knocking out of any of the
three G
i subunits specifically altered 5-HT1A and
muscarinic M4 receptor-mediated inhibition of AC but not the
receptor-induced inhibition of calcium entry. The latter was achieved
by specific ablation of
o subunits from GH4C1 cells
(17). These data confirm that G
i subunits mediate
inhibition of AC but not closure of calcium channels. Similar
specificities have been reported for the coupling of different receptors to voltage-dependent calcium channels by
oA or
oB proteins but not
G
i proteins in GH3 cells (27-29).
One of the important findings of the present study is that although the
three different G
i subunits all participate in
receptor-mediated inhibition of AC, each G
i subunit
apparently plays a distinct role in this signal transduction process.
First, the contemporaneous expression of all three G
i
subunits appeared to be essential for both 5-HT1A and muscarinic M4
receptor-mediated inhibition of unstimulated cAMP synthesis (Table
III). Depletion of any of the three G
i subunits led to
the inability of both receptors to inhibit basal cAMP synthesis in
GH4C1 cells. This is consistent with our previous observations in GH4C1
cells that inhibition of basal cAMP level by dopamine-D2S,
dopamine-D2L, and somatostatin receptors was blocked upon depletion of
G
i2 (17). In cell types in which two or fewer types of
G
i subunits are expressed, such as fibroblast Rat-1,
Chinese hamster ovary, or JEG-3 choriocarcinoma cells,
Gi-coupled receptors inhibit stimulated cAMP production but
do not inhibit basal cAMP accumulation (5-7, 30, 31). This supports
the hypothesis that Gi-coupled receptors require all three
G
i subunits for inhibition of basal cAMP accumulation. Second, different receptors link to different G
i
subunits to inhibit Gs-stimulated cAMP synthesis. For
example, 5-HT1A receptors couple to G
i2 and
G
i3 subunits to suppress cAMP accumulation stimulated by
the Gs-coupled VIP receptor, because depletion of either
subunit blocks this response. By contrast, muscarinic M4 receptors
apparently couple to G
i1 and G
i2 subunits
for inhibition of VIP-stimulated cAMP formation. Interestingly, each
receptor interacts with and activates more than one type of
Gi proteins to inhibit Gs-stimulated cAMP
synthesis. Using immunoprecipitation with specific G protein antibodies
and cholera toxin-catalyzed labeling of Gi proteins, it has
been shown that Gi-coupled receptors may simultaneously
activate more than one G protein and that different receptors
apparently exhibit different preferences to different G proteins (6, 7,
31, 32).
A third major conclusion to be drawn from the data presented is that a
receptor may dominantly link to different G
i subunits depending on whether Gs is engaged in stimulation of AC.
For example, depletion of G
i1 and G
i3
subunits led 5-HT1A and muscarinic M4 receptors, respectively, to
stimulate basal cAMP synthesis. These depletions had no effect on
inhibition of VIP-stimulated AC activity by the same receptors. These
results suggest that different G proteins regulate inhibition of basal
(primarily Gi1) and Gs-stimulated (primarily
Gi2) cAMP synthesis by the 5-HT1A receptor in GH4C1 cells.
Results from transfections of PTX-insensitive G proteins in
Ltk
cells indicate that the D2S receptor couples to
Gi2 to inhibit forskolin-induced cAMP accumulation but uses
Gi3 to inhibit Gs-stimulated action (15). Taken
together, these results are consistent with a
state-dependent inhibition of adenylyl cyclase by specific
G
i subunits.
A surprising finding of the present study was the efficient stimulation
of basal cAMP synthesis in G
i1- and
G
i3-depleted antisense clones by agonist-activated
5-HT1A and muscarinic M4 receptors, respectively. The weaker
stimulation by muscarinic compared with 5-HT1A receptors (3-fold
versus 5-fold basal cAMP) may reflect the less complete
depletion of G
i3 compared with G
i1 or
weaker efficacy of the muscarinic receptor for this signaling pathway.
This stimulation of cAMP synthesis by 5-HT or carbachol was sensitive
to PTX treatment, suggesting the involvement of the remaining
Gi/Go proteins to induce direct stimulation of
AC. Functional analysis of AC enzymes I-VI indicates that all of them are stimulated by G
s, and some of them (types I, V, and
VI) have been shown to be inhibited by G
i proteins (4,
33-36). Interestingly, specific G
dimer combinations potentiate
Gs stimulation of AC types II, IV, and VII while either
inhibiting (type I) or having no effect on the other isoenzymes
(36-38). In cotransfection experiments, Gi-coupled
receptors have been shown to potentiate Gs- or protein kinase C-stimulated cAMP synthesis by AC type II, apparently by release
of G
dimers (26, 39). Our observations indicate that in the
presence of multiple G
i subunits and AC subtypes, receptor-mediated inhibition is the dominant pathway. However, upon
depletion of particular G
i subunits, the remaining
PTX-sensitive G proteins stimulate cAMP levels. Because GH4C1 cells
appear to express AC type II, the enhancement of cAMP by these
receptors could be mediated by conditional activation of AC type II. We have identified Gi2 as the major G protein that mediates
coupling of the 5-HT1A receptor to AC type II upon cotransfection in
HEK-293 cells (40). These results suggest that upon depletion of
Gi1 in GH4C1 cells, G
subunits associated with
Gi2 mediate positive coupling to AC type II resulting in
enhanced cAMP production. Distinct G protein specificities of 5-HT1A or
muscarinic M4 receptors to enhance cAMP levels may reflect the
association of specific G protein complexes with each receptor. For
example coupling of somatostatin and muscarinic M4 receptors in
Go-mediated inhibition of calcium channels, a
G
-mediated response, is mediated by distinct combinations of
o and G
subunits (29). Similarly, specific
i and G
subunit combinations may mediate
receptor-specific activation of AC II to confer G protein-selective
enhancement of cAMP levels.
Another novel signal revealed by depletion of G
i
subunits was that 5-HT1A receptors potentiate VIP-stimulated cAMP
synthesis in G
i2-deficient antisense clones in a
PTX-sensitive manner, as observed for endogenously expressed
somatostatin receptors in G
i2-deficient antisense GH4C1
clones (17). Potentiation of VIP-stimulated adenylyl cyclase by 5-HT1A
receptors in G
i2-deficient antisense clones may be
caused by G
dimers released from Gi3, which weakly
couples this receptor to AC type II (40). It is interesting that
Gi2 is implicated in both 5-HT1A-mediated inhibition (rather than enhancement) of VIP-stimulated cAMP accumulation and in
stimulation of basal cAMP. This suggests that
G
i-mediated inhibition and G
-mediated activation
of AC subtypes may be triggered simultaneously and that the outcome of
receptor activation involves integration of both stimulatory and
inhibitory actions.
In summary, stable transfection of distinct G
i subunit
antisense constructs was used to eliminate their cognate proteins and
to study Gi protein subtype specificity of inhibitory
receptor coupling to AC. Characterization of the different
G
i antisense clones not only demonstrated Gi
protein specificity of receptor-mediated signal transduction pathways
but also revealed unexpected signaling mechanisms. The biological and
physiological significance of the novel stimulation of cAMP synthesis
by Gi-coupled receptors observed in distinct
G
i-deficient antisense clones is not yet clear. It has
been reported, however, that in hippocampal membranes activation of
5-HT1A receptors can stimulate basal adenylyl cyclase activity while
inhibiting forskolin-stimulated cAMP synthesis (10, 11, 41, 42).
Interestingly, AC type II is most abundant in regions of the brain
(e.g. hippocampus CA1 area) in which 5-HT1A receptor activation appears to stimulate cAMP levels (43). Finally, it is known
that the expression of G protein
subunits is regulated by a variety
of hormones and neurotransmitters (44-47). Thus, the present results
suggest that significant reduction of a G
subunit may not only
affect G protein-coupled receptor signaling quantitatively but may
qualitatively alter the receptor signaling phenotypes.