Stimulation of cAMP Synthesis by Gi-coupled Receptors upon Ablation of Distinct Galpha i Protein Expression
Gi SUBTYPE SPECIFICITY OF THE 5-HT1A RECEPTOR*

Ya Fang LiuDagger §, Mohammad H. GhahremaniDagger , Mark M. Rasenick, Karl H. Jakobsparallel , and Paul R. Albert**Dagger Dagger

From the Dagger  Department of Pharmacology and Therapeutics, McGill University, Montreal, Quebec H3G 1Y6, Canada, the  Departments of Physiology and Biophysics and Psychiatry, University of Illinois at Chicago, Chicago, Illinois 60612-7342, the parallel  Institut für Pharmakologie, Universität GH Essen, Hufelandstrasse 55, D-45122 Essen, Germany, and the ** Neuroscience Research Institute, University of Ottawa, Ottawa, Ontario K1H 8M5, Canada

    ABSTRACT
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

The three Galpha i subunits were independently depleted from rat pituitary GH4C1 cells by stable transfection of each Galpha i antisense rat cDNA construct. Depletion of any Galpha i subunit eliminated receptor-induced inhibition of basal cAMP production, indicating that all Galpha i subunits are required for this response. By contrast, receptor-mediated inhibition of vasoactive intestinal peptide (VIP)-stimulated cAMP production was blocked by selective depletions for responses induced by the transfected serotonin 1A (5-HT1A) (Galpha i2 or Galpha i3) or endogenous muscarinic-M4 (Galpha i1 or Galpha i2) receptors. Strikingly, receptor activation in Galpha i1-depleted clones (for the 5-HT1A receptor) or Galpha i3-depleted clones (for the muscarinic receptor) induced a pertussis toxin-sensitive increase in basal cAMP production, whereas the inhibitory action on VIP-stimulated cAMP synthesis remained. Finally, in Galpha i2-depleted clones, activation of 5-HT1A receptors increased VIP-stimulated cAMP synthesis. Thus, 5-HT1A and muscarinic M4 receptor may couple dominantly to Galpha i1 and Galpha i3, respectively, to inhibit cAMP production. Upon removal of these Galpha i subunits to reduce inhibitory coupling, stimulatory receptor coupling is revealed that may involve Gbeta gamma -induced activation of adenylyl cyclase II, a Gi-stimulated cyclase that is predominantly expressed in GH4C1 cells. Thus Gi-coupled receptor activation involves integration of both inhibitory and stimulatory outputs that can be modulated by specific changes in alpha i subunit expression level.

    INTRODUCTION
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Heterotrimeric G proteins transduce signals generated by hormone receptors with seven transmembrane domain alpha -helices to various effectors such as AC,1 phospholipase C, and ion channels (1-3). These G proteins are composed of a Galpha subunit and a tight complex of Gbeta and Ggamma 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 Galpha subunits. Both GTP-liganded Galpha subunits and Gbeta gamma dimers then regulate downstream effector activities. The intrinsic GTPase activity of Galpha subunits hydrolyzes GTP to GDP to form Galpha -GDP, which then associates with Gbeta gamma 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. alpha 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-Galpha i subunit antibodies to block receptor coupling, it has been reported that inhibition of AC by alpha 2-adrenergic receptors in platelet membranes is mediated by Galpha i2 (12) and that 5-HT1A receptor-induced inhibition of cAMP synthesis in HeLa cell membranes is preferentially mediated by Galpha 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 Galpha i mutant proteins has been used to rescue receptor-mediated signaling following PTX pretreatment. For example, the dopamine-D2S receptor appears to couple to Galpha i2 and Galpha i3 to mediate inhibition of forskolin and Gs-stimulated AC, respectively (14, 15). This approach depends on the specificity and functionality of the Galpha 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 Galpha 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 Galpha 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 Galpha i-deficient antisense clones indicates that Galpha 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 Galpha i subunits is essential for receptor-mediated inhibition of unstimulated but not of VIP receptor-stimulated cAMP synthesis. Strikingly, upon depletion of distinct Galpha i subunits, the Gi-coupled 5-HT1A and muscarinic M4 receptors switched to stimulate AC activity.

    EXPERIMENTAL PROCEDURES
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
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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 Galpha i1, Galpha i2, and Galpha i3 subunits and AC type II cDNAs were gifts of Dr. R. Reed. G protein Galpha 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 Galpha i1 cDNA fragment, the 1.8-kb EcoRI-EcoRI Galpha i2 cDNA fragment, or the 3.1-kb EcoRI-EcoRI Galpha 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 Galpha i1, Galpha i2, or Galpha 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 Galpha 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 Galpha i cDNA (Galpha i1, 5'-ACCAGACGAGTACTTATA-3' and 5'-TAGTCTGTGCAACGTTTA-3'; Galpha i2, 5'-CACTACCTGTGAGGAAGA-3' and 5'-ACTCCTCCAGACATAGG-3'; Galpha i3, 5'-TGATATCAAATCTAGGGC-3' and 5'-TAGAACGCATTCCCAGAT-3') to identify positive clones. Total RNA from GH4ZD10 or the different Galpha 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).

    RESULTS
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Independent Depletion of Distinct Galpha i Subunits from GH4ZD10 Cells-- Reverse transcription-PCR and Western blot analysis indicated the presence of all three known Galpha i subunits in GH4C1 cells (Fig. 1), although Galpha i1 may be the least abundant based on the weakness of the signal. The three Galpha 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 Galpha 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 Galpha i subunit (not shown). The extent of depletion of distinct Galpha i subunits was verified by Western blot analysis, using antibodies specific for each Galpha i subunit (22, 23). It was found that Galpha i1 and Galpha i2 subunits were virtually eliminated in clones Gi1ZD-3 and Gi1ZD-5 (Fig. 1A) and Gi2ZD-4 and Gi2ZD-5 (Fig. 1B), respectively, whereas Galpha i3 subunits were largely depleted in clones Gi3ZD-3 and Gi3ZD-4 (Fig. 1C). To examine the extent of cross-hybridization of different Galpha i antisense RNAs with sense RNAs of other Galpha i subunits, the membranes prepared from different Galpha i antisense clones were also probed with antibodies specific for other Galpha i and Galpha o subunits. In Galpha i3-depleted cells, the amount of Galpha i1 was similar to the control cells (Fig. 1D), whereas in Galpha i1- and Galpha i3-depleted clones the amount of Galpha i2 or Galpha o was unchanged (data not shown). Thus, stable transfection of Galpha i antisense constructs can specifically eliminate their cognate Galpha i proteins without major alterations in the amount of other Galpha i subunits.


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Fig. 1.   Western blot analysis of different Galpha i subunits in Galpha i-depleted antisense clone membranes. Membranes of GH4ZD10 cells and various Galpha 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 Galpha i1 subunits in Galpha i1 antisense clone membranes. Blot was probed with an antibody specific for Galpha i1 subunits. Lane 1, GH4ZD10; lane 2, Gi1ZD-3 (13 ± 2%); lane 3, Gi1ZD-5 (4 ± 1%). B, depletion of Galpha i2 subunits in Galpha i2 antisense clone membranes. Blot was probed with an antibody specific for Galpha i2 subunits. Lane 1, GH4ZD10; lane 2, Gi2ZD-4 (7 ± 1%); lane 3, Gi2ZD-5 (12 ± 2%). C, depletion of Galpha i3 subunits in Galpha i3 antisense clone membranes. Blot was probed with an antibody specific for Galpha i3 subunits. Lane 1, GH4ZD10; lane 2, Gi3ZD-3 (22 ± 3%); lane 3, Gi3ZD-4 (19 ± 6%). D, Western blots of Galpha i3 antisense clone membranes with an antibody specific for Galpha i1 subunits. Lane 1, GH4ZD10; lane 2, Gi3ZD-3 (74 ± 4%); lane 3, Gi3ZD-4 (88 ± 7%).

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 Galpha 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 Galpha i antisense clones as described under "Experimental Procedures." Values are expressed as fold basal level of [Ca2+]i.

Influence of Distinct Galpha i Subunit Knockout on Receptor-mediated Inhibition of Calcium Entry-- To monitor the roles of each Galpha 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 Galpha i-depleted clones were examined. In any of the Galpha 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 Galpha 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 Galpha i Subunits-- The consequence of depletion of distinct Galpha 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 Galpha i-depleted clones was clearly and selectively altered. Specifically, in Galpha 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 Galpha i1 depleted clone, Gi1ZD-5 (Table II). In the same Galpha 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 Galpha 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 Galpha i subunits on receptor-mediated inhibition of cAMP accumulation. Measurements of cAMP were performed as described in the legend to Fig. 2 in Galpha i1 antisense clone Gi1ZD-3 (A), Galpha i2 antisense clone Gi2ZD-5 (B), and Galpha 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 Galpha 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 Galpha i1-, Galpha i2-, and Galpha 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.

As observed in Galpha i1-depleted clones, the ability of 5-HT1A and muscarinic M4 receptors to inhibit basal cAMP synthesis was also ablated in Galpha 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 Galpha 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 Galpha i3-depleted clones, however, the inhibitory action of muscarinic M4 receptors on VIP-stimulated cAMP synthesis remained largely unaltered. In Gi3ZD-4 cells, another Galpha 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 Galpha i3-depleted cells.

Subtypes of AC Expressed in GH4C1 Cells-- The possible mechanism of Gi-mediated stimulation of cAMP production upon depletion of specific Galpha i subunits was addressed. Certain AC subtypes (types II, IV, and VII) have been demonstrated biochemically to be conditionally stimulated by Gbeta gamma 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 alpha 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.


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Using stable transfection of distinct Galpha i full-length antisense constructs, we were able to specifically deplete the protein expression of individual Galpha i subunits from GH4C1 pituitary cells. It was found that knocking out of any of the three Galpha 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 alpha o subunits from GH4C1 cells (17). These data confirm that Galpha 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 alpha oA or alpha oB proteins but not Galpha i proteins in GH3 cells (27-29).

One of the important findings of the present study is that although the three different Galpha i subunits all participate in receptor-mediated inhibition of AC, each Galpha i subunit apparently plays a distinct role in this signal transduction process. First, the contemporaneous expression of all three Galpha 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 Galpha 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 Galpha i2 (17). In cell types in which two or fewer types of Galpha 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 Galpha i subunits for inhibition of basal cAMP accumulation. Second, different receptors link to different Galpha i subunits to inhibit Gs-stimulated cAMP synthesis. For example, 5-HT1A receptors couple to Galpha i2 and Galpha 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 Galpha i1 and Galpha 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 Galpha i subunits depending on whether Gs is engaged in stimulation of AC. For example, depletion of Galpha i1 and Galpha 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 Galpha i subunits.

A surprising finding of the present study was the efficient stimulation of basal cAMP synthesis in Galpha i1- and Galpha 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 Galpha i3 compared with Galpha 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 Galpha s, and some of them (types I, V, and VI) have been shown to be inhibited by Galpha i proteins (4, 33-36). Interestingly, specific Gbeta gamma 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 Gbeta gamma dimers (26, 39). Our observations indicate that in the presence of multiple Galpha i subunits and AC subtypes, receptor-mediated inhibition is the dominant pathway. However, upon depletion of particular Galpha 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, Gbeta gamma 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 Gbeta gamma -mediated response, is mediated by distinct combinations of alpha o and Gbeta gamma subunits (29). Similarly, specific alpha i and Gbeta gamma 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 Galpha i subunits was that 5-HT1A receptors potentiate VIP-stimulated cAMP synthesis in Galpha i2-deficient antisense clones in a PTX-sensitive manner, as observed for endogenously expressed somatostatin receptors in Galpha i2-deficient antisense GH4C1 clones (17). Potentiation of VIP-stimulated adenylyl cyclase by 5-HT1A receptors in Galpha i2-deficient antisense clones may be caused by Gbeta gamma 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 Galpha i-mediated inhibition and Gbeta gamma -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 Galpha 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 Galpha 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 Galpha 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 alpha  subunits is regulated by a variety of hormones and neurotransmitters (44-47). Thus, the present results suggest that significant reduction of a Galpha subunit may not only affect G protein-coupled receptor signaling quantitatively but may qualitatively alter the receptor signaling phenotypes.

    ACKNOWLEDGEMENTS

We thank Dr. R. Reed for providing rat Galpha i subunit and AC type II cDNA clones, Dr. D. Manning for Galpha subunit antibodies, and Dr. G. Almazan for pY-3 plasmid. We gratefully acknowledge F. Belga and P. Cheng for assistance in experimentation.

    FOOTNOTES

* This work was supported by grants from the National Institutes of Health (to M. M. R.), the Deutsche Forschungsgemeinschaft (to K. H. J.), the National Cancer Institute, and Medical Research Council of Canada (to P. R. A.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§ Present address: Dept. of Pharmacology, Brown University, 75 Waterman St., Providence, RI 02912.

Dagger Dagger Recipient of the Novartis/Medical Research Council Michael Smith Chair in Neurosciences. To whom correspondence should be addressed: Neuroscience Research Inst., University of Ottawa, 451 Smyth Rd., Ottawa, ON K1H-8M5, Canada. Tel.: 613-562-5800 (ext. 8307); Fax: 613-562-5403; E-mail: palbert{at}uottawa.ca.

    ABBREVIATIONS

The abbreviations used are: AC, adenylyl cyclase; 5-HT, serotonin; PTX, pertussis toxin; VIP, vasoactive intestinal peptide; kb, kilobase pair(s); PCR, polymerase chain reaction; HBSS, Hanks' balanced salt solution; bp, base pairs.

    REFERENCES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
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