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Endocrinology Vol. 140, No. 4 1612-1621
Copyright © 1999 by The Endocrine Society

ARTICLES

Roles of Gi and Gq/11 in Mediating Desensitization of the Luteinizing Hormone/Choriogonadotropin Receptor in Porcine Ovarian Follicular Membranes1

Rajsree M. Rajagopalan-Gupta2, Sutapa Mukherjee2, Xi Zhu, Yee-Kin Ho, Heidi Hamm, Mariel Birnbaumer, Lutz Birnbaumer and Mary Hunzicker-Dunn

Departments of Cell and Molecular Biology (R.M.R.-G., S.M.) and Molecular Pharmacology and Biological Chemistry (H.H.) and the Neuroscience Institute, Northwestern University Medical School, Chicago, Illinois 60611; Department of Pharmacology and the Neurobiotechnology Center, Ohio State University (X.Z.), Columbus, Ohio 43210; the Department of Anesthesiology, University of California School of Medicine (M.B., L.B.), Los Angeles, California 90095; and the Department of Biochemistry, University of Illinois (Y.-K.H.) Chicago, Illinois 60612

Address all correspondence and requests for reprints to: Dr. Mary Hunzicker-Dunn, Department of Cell and Molecular Biology, Northwestern University Medical School, 303 East Chicago Avenue, Chicago, Illinois 60611. E-mail: mhd{at}nwu.edu


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Although desensitization of most guanine nucleotide-binding (G) protein receptors is triggered by phosphorylation of the receptor, desensitization of the LH/CG receptor (-R) in porcine follicular ovarian membranes appears to be independent of LH/CG-R phosphorylation. We therefore evaluated whether desensitization of the LH/CG-R reflected a direct inhibition of adenylyl cyclase (AC) activity by either the {alpha}-subunit of Gi or ß{gamma}-subunits derived from any of the membrane G proteins activated in response to LH/CG-R activation or whether desensitization reflected a competition between Gs and a G protein that activated phospholipase C for binding sites on the LH/CG-R. The results showed that follicular membrane AC activity was not inhibited upon activation of the LH/CG-R despite evidence that the ACs in follicular membranes, when maximally activated by forskolin, could be inhibited when membrane G proteins were activated by guanyl-5'-yl imidodiphosphate, and that pertussis toxin pretreatment of membranes raised forskolin-stimulated AC activity, consistent with a tonic inhibition of follicular membrane AC activity. Similarly, agonist-stimulated desensitization of LH/CG-R-stimulated AC activity was not inhibited by pertussis toxin. Therefore, desensitization is not the result of inhibition of AC mediated by an inhibitory Gi subunit. Follicular membrane AC was also not inhibited by Gß{gamma} subunits freed with activation of Gs, Gq/11, or G13, based on the inabilities of exogenous Gß{gamma} to promote desensitization and of a protein that sequesters Gß{gamma} to inhibit desensitization. Desensitization was also not inhibited by a Gq/11 C-terminal peptide or antiserum directed toward the C-terminus of Gq/11, nor was it reversed with the addition of Gß{gamma} to membranes exhibiting desensitized LH/CG-R, suggesting that desensitization is independent of coupling of the LH/CG-R to Gq/11. These results indicate that agonist-dependent desensitization of LH/CG-R-stimulated AC activity is mediated by a unique mechanism.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
EXPOSURE of serpentine guanine nucleotide binding (G) protein-coupled receptors to saturating concentrations of agonist generally promotes an attenuation of receptor-specific effector activity, which is referred to as desensitization (1). For most of these receptors, it is phosphorylation of the receptor itself that triggers agonist-induced desensitization (2, 3). For the well studied ß-adrenergic receptor, desensitization in response to fractional receptor occupancy is triggered by receptor phosphorylation catalyzed by cAMP-dependent [protein kinase A (PKA)] and lipid-dependent (protein kinase C) protein kinases (1, 4), whereas desensitization to saturating agonist additionally involves phosphorylation of the receptor by a G protein-coupled receptor kinase (GRK) (1, 2). Mutagenesis of these phosphorylation sites eliminates desensitization (2). Activation at least of GRK2 and -3 is regulated in part by their interaction with heterotrimeric G protein ß{gamma}-subunits, which promote translocation of the cytoplasmic GRKs to the plasma membrane (2). After ß-adrenergic receptor phosphorylation, the protein ß-arrestin binds to the phosphorylated receptor and quenches coupling of the receptor to Gs (5). A growing list of receptors has been shown to be phosphorylated by one or more GRKs, leading to desensitization (6).

The LH/CG receptor (-R) is a member of the superfamily of seven transmembrane-spanning receptors that demonstrates agonist-induced activation and subsequently desensitization of adenylyl cyclase (AC) activity. However, the mechanism(s) that mediates desensitization of the LH/CG-R is incompletely understood. While the LH/CG-R in follicular membranes can be (substoichiometrically) phosphorylated by PKA, PKA-mediated LH/CG-R phosphorylation is not responsible for desensitization of agonist-stimulated AC activity (7). Moreover, although the LH/CG-R transfected into HEK 293 or L cells is readily phosphorylated in an agonist-dependent manner (8, 9, 10), truncation of the cytoplasmic tail of this receptor to remove phosphorylated serine and threonine residues (8, 10, 11, 12) or mutation of phosphorylated serine and threonine residues in the C-terminal tail to alanines (13) only reduces the extent of desensitization from about 80% in cells containing wild-type LH/CG-R to about 65% in cells with mutated LH/CG-R (60 min after agonist addition) (8, 13). This result suggests that about 65% of the desensitization response of the LH/CG-R is independent of receptor phosphorylation. Consistent with this conclusion, we have shown that desensitization of the LH/CG-R in porcine follicular membranes, like that of the serotonin type II, cholecystokinin, and secretin receptors (14, 15, 16), is not dependent on receptor phosphorylation, based on the absence of detectable phosphate incorporation into immunoprecipitated LH/CG-R (7), the inability of protein kinase inhibitors to block desensitization (17), and the occurrence of desensitization in the presence of the ATP antagonist adenyl-5'-yl imidodiphosphate (AMP-PNP) (7, 18, 19).

Desensitization, however, is dependent on GTP. Desensitization of follicular membrane LH/CG-R requires nanomolar concentrations of GTP (Km = 68 nM) (18, 19, 20, 21) and is reversed by guanosine-5'-O-(2-thiodiphosphate) (GDPßS) (22). We have also shown that hyperdesensitization (up to 100%) of LH/CG-R-stimulated AC activity is achieved by incubating membranes with 8–10% ethanol3 (23), and that this hyperdesensitization retains its GTP dependence (23) and is reversed by GDPßS (Hunzicker-Dunn, M., personal observation). These results suggest that a G protein, small or large, is required for desensitization of the LH/CG-R. We have recently demonstrated that activation of the LH/CG-R promotes activation of Gs{alpha}, Gi{alpha}, Gq/11{alpha}, and G13{alpha}, based upon the ability of hCG to stimulate binding of a GTP photoaffinity analog to these G{alpha} proteins (24). We therefore considered the possibility that desensitization reflected a direct inhibition of AC activity by either the {alpha}-subunit of Gi or ß{gamma}-subunits derived from any of the membrane G proteins activated by hCG. Alternatively, we considered the possibility that desensitization of the LH/CG-R reflected a competition between Gs and a G protein that activated phospholipase C (PLC) for binding sites on the LH/CG-R. These two hypothesis are tested below. The results reveal that desensitization of the LH/CG-R is not mediated by direct inhibition of the AC enzyme, nor is it the result of competition with Gq/11 protein subunits.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Materials
Purified hCG (batch CR-125) was provided by the Center for Population Research, NICHHD; anti-Gq/11{alpha} (B6T, C-terminal Gq/11{alpha} peptide antibody) was provided by Dr. T. Martin (25). Synthetic G protein peptides were synthesized and purified as previously described (26). Bovine rod transducin ß{gamma} was purified as previously described (27). Glutathione-S-transferase (GST)-ß-adrenergic receptor kinase (ßARK) (C-terminal) fusion protein was purified by affinity chromatography on glutathione-Sepharose 4B by standard protocols; purity was assessed by Coomassie blue staining of SDS-PAGE-purified material. Materials were purchased from the following sources: anti-Gq/11{alpha} (C-19, C-terminal peptide antibody specific for Gq{alpha} and G11{alpha}), Santa Cruz Biotechnology, Inc. (Santa Cruz, CA); creatine phosphokinase, Calbiochem (La Jolla, CA); pertussis toxin (PTX), List Biological Laboratories, Inc. (Campbell, CA); [2,8-3H]cAMP (41 Ci/mmol), Amersham (Arlington Heights, IL); [{alpha}-32P]ATP (10–50 Ci/mmol), [32P]NAD (30 Ci/mmol), and myo-[2-3H]inositol (21 Ci/mmol), New England Nuclear Research Products (Boston, MA); U-73122, BIOMOL Research Laboratories, Inc. (Plymouth Meeting, PA); DMEM, FBS, G418, HBSS, Life Technologies (Gaithersburg, MD); nucleotides and most other reagents, Sigma Chemical Co. (St. Louis, MO).

Preparation of ovarian follicular membranes
Pig ovaries were obtained from a local slaughterhouse and immediately transported to the laboratory on ice. The walls from follicles larger than 6 mm in diameter were dissected, and a membrane fraction enriched in AC activity was prepared (19). All membrane preparations were stored at -70 C at a protein concentration of 3–5 mg/ml in 10 mM Tris-HCl, pH 7.2. Protein concentrations were determined using BSA as standard (28).

Desensitization reaction and AC assay
The AC assay was conducted in a 50-µl reaction volume at 30 C for 5 or 10 min, as indicated, in an incubation medium/ATP regenerating system (IMRS) consisting of 25 mM 1,3-bis[Tris(hydroxymethyl)-methylamino]propane-HCl (pH 7.2), 0.4 mM EDTA, 1 mM EGTA, 20 mM phosphocreatine, 0.2 mg/ml creatine phosphokinase (163 U/mg), 5 mM MgCl2, 1 mM AMP-PNP, and 1 mM [3H]cAMP (~10,000 cpm). The following was added to this IMRS: 100 µM GTP, [{alpha}-32P]ATP (~5 µCi; 100–200 cpm/pmol), 10 µg/ml BSA or hCG, unless otherwise indicated, and follicular membrane protein (~30 µg). The reaction was stopped by the addition of a 100-µl volume of 40 mM ATP, 10 mM cAMP, and 1% SDS followed by boiling for 3 min in a water bath. [32P]cAMP was purified and quantitated (29). All determinations were run in triplicate or quadruplicate. Membranes were also subjected to various types of preincubation reactions. For the standard desensitization incubation, a two-stage reaction was conducted. In the stage 1 desensitization reaction, membranes were incubated at 30 C for 40 min in a 40-µl volume of IMRS, as indicated above, plus 10 µM GTP and 1 µg BSA or hCG. Immediately after the stage 1 preincubation, an assay for AC activity (stage 2) was preformed as described above with the addition of a 10-µl volume containing the following components: 100 µM GTP, [{alpha}-32P]ATP and 10 µg/ml BSA or hCG. The presence of BSA in stages 1 and 2 indicated basal AC activity; BSA in stage 1 and hCG in stage 2 indicated full hCG-stimulated AC activity; hCG in stages 1 and 2 indicated hCG-induced desensitization of AC activity. The percent reduction of full hCG-stimulated AC activity above basal AC activity, expressed as the percent desensitization, was used as a measure of the extent of LH/CG-R desensitization. In some instances, membranes were subjected to a preincubation reaction before the stage 1 desensitization reaction. When the preincubation was with an antibody or peptide, preincubated membranes were added to the stage 1 desensitization reaction without washing away antibody or peptide, as described in the text. When the preincubation was with cholera toxin (CTX) or PTX, membrane batches (~1 mg) were preincubated 30 min at 30 C in the absence (vehicle) or presence of activated CTX or PTX (30) in 1 ml buffer containing 25 mM Tris-HCl (pH 7.2), 5 mM ADP ribose, 20 mM L-arginine, 0.5 mM GTP, 1 mM ATP, 15 mM thymidine, 20 µM NAD, 5 mM dithiothreitol, and activated toxin at the indicated concentrations. The reaction was stopped by dilution with cold 10 mM Tris-HCl (pH 7.2), followed by centrifugation at 10,000 x g for 5 min at 4 C. The washing procedure was repeated, and membranes were resuspended in 10 mM Tris-HCl, pH 7.2. Vehicle- or toxin-treated membranes (~30 µg membrane protein) were then subjected to stage 1 desensitization reaction followed by stage 2 AC assay. When indicated, membranes were subjected to a three-stage reaction (22). Stage 1 consisted of the desensitization reaction; membranes were then diluted 10-fold with 10 mM Tris-HCl (pH 7.2), followed by centrifugation at 10,000 x g for 5 min at 4 C. Membrane pellets were resuspended in 10 mM Tris-HCl, pH 7.2, and incubated for 30 min at 30 C for stage 2 in a 40-µl volume of IMRS, substituting ATP for AMP-PNP, and other additions, as indicated; membranes were diluted, centrifuged, and resuspended in 10 mM Tris-HCl, as described above, and subjected to the standard AC (stage 3) assay. Unless otherwise stated, the concentrations of reagents in a final 50-µl reaction volume are given.

LH/CG-R mutations and generation of stable cell lines expressing LH/CG-R
Site-directed mutagenesis of the murine LH/CG-R was performed by a PCR-based method (31). The resulting constructs were sequenced by the dideoxy chain termination method (32). For stable expression in HEK 293 cells, the complementary DNAs were subcloned into the expression vector pcDNA3 (Invitrogen, Carlsbad, CA) and transfected (31). G418-resistant transfectants were expanded and analyzed for AC activities in response to stimulation by hCG in an in situ assay (33). Primary clones were subjected to clonal selection by limiting dilution to isolate clonal cell lines. Experiments were performed on three wild-type clones, three clones with the D564G LH/CG-R mutation, and three clones with the A568I mutation. Cells were grown on 100-mm plates in DMEM containing 5% FBS, 1% penicillin/streptomycin, and 400 µg/ml G418. To prepare membranes for AC assay, cells were briefly trypsinized, washed in HBSS, then homogenized in 27% (wt/wt) sucrose, 1 mM EDTA, and 10 mM Tris-HCl, pH 7.2, with 15 strokes of a tight pestle Dounce homogenizer (Kontes Co., Vineland, NJ) (34). The homogenate was centrifuged at 800 x g for 5 min at 4 C to remove cell debris and nuclei, and the supernatant was centrifuged at 10,000 x g for 10 min at 4 C to yield a membrane pellet. This pellet was resuspended in 10 mM Tris-HCl, pH 7.2, and frozen at -70 C until used for AC assays.

PLC assay
Cell PLC activity was measured as described by Lomasney (35). Briefly, inositol pools in cells were labeled by incubating cells overnight with 4 µCi/ml myo-[3H]inositol, cells were then incubated for 30 min with 20 mM LiCl and for 30 min with vehicle or 10 µg/ml hCG. Incubations were terminated by the addition of perchloric acid, and total inositol phosphates were collected by elution over AG 1-X8 resin (Bio-Rad Laboratories, Inc., Richmond, CA).

Statistics
Results (mean ± SEM) were analyzed using Student’s t test (P <= 0.05) (36).


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Role of Gi in desensitization of LH/CG-R-stimulated AC activity
We have previously shown that porcine ovarian follicular membranes express Gi, based on immunoblot analysis (24), and that hCG promotes rapid activation of Gi, based on the ability of hCG to stimulate both the binding of a photoaffinity GTP analog to Gi{alpha} (24) and CTX-catalyzed ADP ribosylation of Gi{alpha} (30). We therefore were interested to determine whether activation of follicular membrane Gi proteins led to inhibition of AC activity, whether hCG activated Gi to inhibit AC, and whether desensitization was the result of inhibition of AC by a Gi protein.

To determine whether the AC(s) in follicular membranes could be inhibited by an endogenous Gi, membrane G proteins were activated by the poorly hydrolyzable GTP analog guanyl-5'-yl imidodiphosphate (GMP-PNP). The results shown in Fig. 1Go revealed the classic evidence for Gi-mediated inhibition of AC (37). Although basal AC activity was activated 10-fold by GMP-PNP with an ED50 of approximately 5 µM, in the presence of forskolin GMP-PNP exerted a biphasic effect on AC activity, i.e. inhibition with lower concentrations of GMP-PNP (ED50, ~0.1 µM GMP-PNP) followed by activation with higher concentrations of GMP-PNP (ED50, ~50 µM GMP-PNP). These results are consistent with activation of Gi to inhibit AC activity at lower concentrations of GMP-PNP, followed by activation of Gs with higher concentrations of GMP-PNP (38).



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  		Figure 1. Porcine follicular membranes exhibit guanine nucleotide-dependent inhibition of AC activity. AC activity was measured in a 5-min assay in the presence of 1.25 mM ATP and the indicated additions of GMP-PNP in the absence ({circ}) and presence (•) of 100 µM forskolin, as described in Materials and Methods. Results are the mean ± SEM of triplicate determinations of a single assay and are representative of two separate experiments. The shaded line is an extension of forskolin-stimulated AC activity ± SEM in the absence of GMP-PNP.

 
We next determined whether hCG promoted activation of Gi to inhibit AC activity in follicular membranes. The standard procedures to show that a hormone activates Gi are 1) to pretreat membranes with CTX (to cause constitutive activation of Gs{alpha}), then to demonstrate hormone-dependent inhibition of forskolin-stimulated AC activity; and 2) to pretreat membranes with PTX (which uncouples receptor from Gi), then to show increased hormone-stimulated AC activity in PTX-treated membranes compared with those not pretreated with PTX (39). Using CTX and PTX at concentrations that promote maximal ADP ribosylation of Gs{alpha} and Gi{alpha} (30), we evaluated whether LH/CG-R activation led to inhibition of AC activity. The results show that activation of the LH/CG-R does not activate Gi to inhibit AC in follicular membranes. hCG at concentrations of 0.001–10 µg/ml did not inhibit forskolin-stimulated AC activity in CTX-pretreated membranes; rather, hCG promoted a dose-dependent increase in forskolin-stimulated AC activity in CTX-pretreated membranes (Fig. 2AGo). Similarly, no inhibition of forskolin-stimulated AC activity was seen when Gs was activated with the addition of CTX, FSH, and hCG (Fig. 2BGo, compare bar 6 in CTX- vs. vehicle-treated cells). In membranes pretreated with PTX vs. those treated with the vehicle control, rather than a selective rise in hCG-stimulated AC activity, basal, hormone-stimulated, and forskolin-stimulated AC activities were all raised 30–50%, and AC activity measured in the presence of hCG, FSH, and forskolin was raised 70% (Fig. 2BGo, compare bar 6 in vehicle- and CTX-treated membranes with that in PTX-treated membranes). This result suggests that the LH/CG-R is not functionally coupled to Gi to inhibit AC activity, but, rather, that PTX relieves inhibition by an unidentified ligand. However, none of the classical activators of Gi, including met-enkephalin, somatostatin, angiotensin II, acetylcholine, or epinephrine, inhibited forskolin-stimulated AC activity in follicular membranes (not shown).



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  		Figure 2. Effect of pretreatment of porcine follicular membranes with CTX or PTX on hCG- and forskolin-stimulated AC activities. A, Follicular membranes were preincubated with 50 µg/ml activated CTX for 30 min at 30 C, and membranes were washed and subjected to a 5-min AC assay in the presence of BSA, 10 µM forskolin, or 10 µM forskolin plus the indicated concentrations of hCG. Results are the mean ± SEM of triplicate determinations of a single assay. B, Follicular membranes were preincubated with vehicle, 50 µg/ml activated CTX, or 2.5 µg/ml activated PTX for 30 min at 30 C, and membranes were washed and subjected to a 10-min AC assay in the presence of the indicated additions of 10 µg/ml hCG, 10 µg/ml FSH, and/or 10 µM forskolin. Results are the mean ± SEM of triplicate determinations of a single assay. This result is representative of three separate experiments.

 
Although our results indicate that LH/CG-R does not activate Gi to inhibit AC in a 10-min assay, we were interested to determine whether a more prolonged incubation time consistent with desensitization (7, 23, 29, 40) was required to activate Gi. If desensitization is mediated by LH/CG-R activation of Gi, then pretreatment of membranes with PTX would block the development of desensitization. The results in Fig. 3Go show that PTX treatment of follicular membranes, at concentrations of PTX previously shown to ADP ribosylate Gi{alpha} (30), did not affect desensitization of LH/CG-R-stimulated AC activity, i.e. did not increase hCG-stimulated AC activity in membranes incubated with hCG in stages 1 and 2 (hCG/hCG). Taken together, these results show that the LH/CG-R does not activate Gi to inhibit AC activity in follicular membranes, and that desensitization of LH/CG-R-stimulated AC activity is not mediated by Gi.



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  		Figure 3. Effect of pretreatment of porcine follicular membranes with PTX on hCG-stimulated desensitization of AC activity. Follicular membranes were pretreated with the indicated concentrations of PTX, and membranes were washed and then subjected to a two-stage desensitization incubation, as described in Materials and Methods. Stage 1 contained 1.25 mM ATP. BSA or hCG additions to stage 1/stage 2 are indicated. The percent desensitization was calculated from AC activities as described in Materials and Methods and represents the reduction in hCG-stimulated AC activity when incubations contained hCG in stage 1 compared with activity measured with BSA in stage 1 over basal (BSA/BSA) AC activities. Results are the mean ± SEM of triplicate determinations for a single assay and are representative of two separate assays.

 
Role of Gß{gamma} subunits in desensitization of LH/CG-R-stimulated AC activity
We considered the possibility that desensitization reflected an inhibition of AC type I by Gß{gamma}-subunits derived, potentially, from any of the G proteins activated by the LH/CG-R (except for Gi, based on the results presented above). We do not know which of the nine identified forms of AC (41) are expressed in follicular membranes. However, our preliminary studies suggest that neither type V nor type VI, which are inhibited by low micromolar Ca2+ concentrations (42), is strongly expressed in porcine follicular membranes, based on the insensitivity of the follicular AC to Ca2+ inhibition (Rajagopalan-Gupta, R. M., and M. Hunzicker-Dunn, personal observation). We therefore wished to determine whether exogenous or endogenous Gß{gamma} promoted inhibition of follicular AC. We used various approaches to modulate membrane levels of Gß{gamma}. In the first approach, which was based on the ability of AlF4- to nonspecifically activate all heterotrimeric G proteins, we determined whether an inhibition of AC activity occurred when membrane G proteins were activated with AlF4-. A two-stage desensitization reaction was conducted for this experiment; stage 1 contained BSA or hCG and 8% ethanol, and either did or did not contain AlF4-, as indicated in Fig. 4AGo. Stage 1 was also conducted in the absence of added GTP or AMP-PNP, conditions in which desensitization does not occur (18, 19), or the presence of GTP and AMP-PNP, as indicated. The results in Fig. 4AGo show that desensitization of hCG-stimulated AC activity (hCG in stages 1 and 2) required GTP and AMP-PNP in stage 1 (compare bars in third group with those in first group), and that AlF4--stimulated G protein activation did not result in a reduction of AC activity. Indeed, the AC activity achieved when membranes were incubated with AlF4- in stage 1 and with hCG in stage 2 (solid bar in last group of bars, Fig. 4AGo) was equivalent to that achieved when AC was fully activated by forskolin and hCG (Fig. 4BGo). However, as the results with AlF4- are complicated by the ability of AlF4- not only to free Gß{gamma}-subunits but also to activate Gs{alpha}, we used two additional, more direct approaches to determine whether Gß{gamma} inhibited follicular membrane AC activity. In the second approach, we determined whether the direct addition of exogenous ß{gamma} to stage 1 of the two-stage reaction promoted inhibition (desensitization) of AC activity. The results presented in Fig. 5Go show that 1 µM transducin ß{gamma} did not inhibit AC activity and did not promote desensitization. In the third approach to evaluate whether desensitization reflected inhibition by Gß{gamma}, we determined whether the addition of a reagent that sequestered endogenous ß{gamma} prevented the development of desensitization. Addition to stage 1 of the desensitization incubation of the C-terminal 222 amino acids of ßARK ligated to GST as a GST-ßARK peptide fusion protein, which has been shown to be an effective ß{gamma} scavenger (43), did not prevent the development of hyperdesensitization (i.e. did not raise hCG-stimulated AC activity; Fig. 6Go). Taken together, these results show that desensitization of LH/CG-R-stimulated AC activity is not mediated by LH/CG-R-stimulated ß{gamma}-dependent inhibition of AC.



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  		Figure 4. A, Effect of addition of AlF4- to stage 1 of desensitization incubation on AC activities in porcine follicular membranes. Membranes were incubated in a two-stage desensitization incubation. Stage 1 contained indicated additions of no nucleotides (H2O) or 1 mM AMP-PNP, 10 µM GTP, 8% ethanol, BSA or hCG, and H2O or 10 mM NaF and 20 µM AlCl2 (AlF4-). Stage 2 AC assay additions contained BSA or hCG, as indicated; all samples for AC assay received 100 µM GTP, and 1 mM AMP-PNP was added to tubes that contained no nucleotide additions in stage 1, so that all AC assay tubes contained 1 mM AMP-PNP and 100 µM GTP. Results are the mean ± SEM of triplicate determinations of a single assay. B, Activation of follicular membrane AC activity by AlF4- or hCG and forskolin. Membranes were incubated in a two-stage desensitization incubation. Stage 1 contained 1 mM AMP-PNP, 10 µM GTP, 9% ethanol, and the indicated additions of 10 µM forskolin or AlF4-. Stage 2 AC assay additions of 10 µg/ml hCG, AlF4- and/or forskolin are as indicated. Results are the mean ± SEM of triplicate determinations and are representative of two separate experiments.

 


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  		Figure 5. Effect of addition of transducin Gß{gamma} on hCG-stimulated desensitization of porcine follicular membrane AC activity. Membranes were subjected to a two-stage desensitization incubation. Stage 1 contained 1 mM AMP-PNP and 1 µM transducin Gß{gamma} or vehicle [10 mM 3-[N-morpholino]propanesulfonic acid (pH 7.5), 250 mM NaCl, 1 mM dithiothreitol, 2 mM MgCl2, and 40% glycerol], as indicated. Stage 2 was the 5-min AC assay. Results are the mean ± SEM of triplicate determinations of a single experiment. Equivalent results were obtained in separate experiments using 200, 400, and 600 nM transducin Gß{gamma} (with the vehicle indicated above) and using 400 nM rat liver Gß{gamma} (and detergent-containing vehicle).

 


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  		Figure 6. Effect of addition of GST-C-terminal ßARK peptide fusion protein on hCG-stimulated desensitization of porcine follicular membrane AC activity. Membranes were subjected to a two-stage desensitization incubation. Stage 1 contained BSA or hCG as indicated, 10% ethanol, and 14 µM GST-C-terminal ßARK peptide fusion protein or vehicle control (water). Stage 2 was the 5-min AC assay. Results are the mean ± SEM of quadruplicate determinations of a single experiment and are representative of two separate experiments.

 
Role of Gq/11 in desensitization of LH/CG-R-stimulated AC activity
As LH/CG-R activation of PLC requires saturating concentrations of agonist and develops more slowly than activation of AC (44, 45, 46, 47), we considered the possibility that desensitization of LH/CG-R-stimulated AC activity reflected a competition between Gs and the G protein that activated PLC for binding sites on the LH/CG-R. Although the G protein that activates PLC in follicular cells has not been identified, PLCs in other cellular models are activated by the {alpha}-subunit from Gq/11 and/or by ß{gamma}-subunits from Gq/11 or Gi (48). PLC activation in the 11/6 fibroblast cell line on agonist activation of transfected LH/CG-R is PTX sensitive (49), consistent with an intermediary role for Gi. However, as we have shown that desensitization in follicular membranes is independent of Gi, we determined whether agents that prevented coupling of the LH/CG-R to Gq/11 and PLC blocked the development of LH/CG-R-stimulated desensitization of AC. We used various approaches to assess the roles of Gq/11 and PLC in LH/CG-R-stimulated AC desensitization in follicular membranes.

If desensitization is mediated by activation of Gq/11 via Gq/11{alpha}, then addition of excess ß{gamma} to membranes containing desensitized LH/CG-R should promote reassociation of the Gq/11{alpha}- and ß{gamma}-subunits to the heterotrimeric state, inactivation of the G protein-coupled, and reversal of desensitization. Using this same rationale and experimental design, we have previously shown that addition of GDPßS (to inactivate G proteins) to membranes containing desensitized LH/CG-R promotes reversal of desensitization (22). However, as shown in Fig. 7Go, the addition of 200 nM transducin ß{gamma}-subunits to desensitized membranes (i.e. addition of Gß{gamma} to stage 2 of a three-stage incubation) did not reverse desensitization. Desensitization of hCG-stimulated AC activity [membranes incubated with hCG in stage 1 and in AC assay (hCG/hCG)] in membranes incubated with Gß{gamma} was maintained at a level equivalent to that in the vehicle control.



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  		Figure 7. Effect of addition of 200 nM Gß{gamma} to stage 2 of a three-stage AC incubation. Follicular membranes were subjected to the stage 1 desensitization incubation (without ethanol; see Materials and Methods) in the presence of 1 mM AMP-PNP, 10 µM GTP, and BSA or hCG additions, as indicated. Membranes were then washed, and incubated for 30 min (stage 2) in the presence of 1 mM ATP, 5 mM MgCl2, and the indicated additions of 0.1 mM GTP, 200 nM transducin Gß{gamma}, or vehicle (see Fig. 5Go), as indicated. Membranes were again washed, then subjected to a 5-min (stage 3) AC assay in the presence of 1 mM ATP, 100 µM GTP, and the indicated additions of BSA or hCG. Results are the mean ± SEM of triplicate determinations of a single experiment.

 
We also determined whether the addition of a synthetic peptide corresponding to the C-terminal portion of Gq/11{alpha} and peptide antibodies directed to the C-terminal portion of Gq/11{alpha} blocked the development of desensitization. Synthetic C-terminal G{alpha} peptides have been shown in various cellular models to compete specifically with the G{alpha} protein for binding to a receptor and therefore to inhibit downstream responses (26, 48, 50, 51, 52, 53, 54). The use of G{alpha} C-terminal peptide-directed antisera is also an established technique to inhibit receptor coupling to specific G{alpha} proteins (25, 52, 55, 56, 57, 58, 59, 60, 61, 62, 63). A synthetic C-terminal Gi{alpha} peptide served as a negative control, as we have shown that Gi does not participate in LH/CG-R desensitization. Consistent with this conclusion, the Gi{alpha} peptide (which should block i{alpha} signaling to AC) did not increase hCG-stimulated AC activities (Fig. 8Go). Rather, the C-terminal Gi{alpha} peptide prompted an apparent nonspecific reduction in hCG-stimulated AC activity. The Gs{alpha}354–372 peptide (which should block s{alpha} signaling to AC) served as a positive control and inhibited full hCG-stimulated AC activity by nearly 50%,4 consistent with a previous report for this peptide (26), and did not inhibit desensitization. A second Gs{alpha}384–395 peptide did not modulate AC activities, consistent with a previous report (26). The C-terminal peptide of Gq/11{alpha} (which should block q/11{alpha} signaling to PLC) did not promote an increase in desensitized hCG-stimulated AC activity (hCG/hCG in stages 1/2), suggesting that Gq/11{alpha} does not participate in LH/CG-R desensitization. Rather, this synthetic peptide, like that for Gi{alpha}, promoted an equivalent reduction in both full (BSA/hCG in stages 1/2) and desensitized (hCG/hCG in stages 1/2) hCG-stimulated AC activities, and therefore did not inhibit desensitization. Consistent with results in Fig. 8Go, results in Fig. 9Go showed that desensitization was also not prevented by peptide antisera directed to the C-terminal region of Gq/11{alpha}.



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  		Figure 8. Effect of addition of C-terminal peptides for Gs{alpha}, Gq/11{alpha}, or Gi{alpha} on hCG-stimulated desensitization of follicular membrane AC activity. Follicular membranes (~30 µg in 10 µl) were preincubated at 4 C for 1 h with the indicated C-terminal peptides at a concentration of 250 µM (final concentration in AC assay of 100 µM). Membranes were then subjected to a two-stage desensitization incubation at 30 C (without ethanol). Results are the mean ± SEM of triplicate determinations of a single experiment and are representative of two separate experiments.

 


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  		Figure 9. Effect of addition of Gq/11{alpha} C-terminal peptide-directed antibody on hCG-stimulated desensitization of follicular membrane AC activity. Follicular membranes (~30 µg in 10 µl) were preincubated with 10 µl anti-Gq/11{alpha} peptide antibody (Santa Cruz Biotechnology, Inc.) or nonimmune serum (NIS) for 10 min at room temperature, then for 1 h at 4 C. The membranes were then subjected to a two-stage desensitization reaction (without washing) with the indicated additions of BSA or hCG in the presence of 8% ethanol to stage 1; stage 2 was the 5-min AC assay. Equivalent results were obtained in three additional experiments (with ethanol) using the Santa Cruz Biotechnology antibody and in two separate experiments (without ethanol) using a Gq/11{alpha} C-terminal peptide antibody (22 µg; provided by Dr. Tom Martin) that has been shown to inhibit the coupling of receptors to Gq/11{alpha} (25 ).

 
We evaluated whether PLC activation was necessary for LH/CG-R-stimulated AC desensitization. Addition of 0.15–15 µM of the PLC inhibitor U-73122 (64) to stage 1 of the desensitization reaction (in the presence of 9% ethanol) did not modulate any parameter of AC activity measured (BSA/BSA, BSA/hCG, hCG/hCG, BSA/forskolin in stages 1/2; not shown).

Finally, based on evidence for the rat TSH-R that an alanine in the C-terminal portion of the third intracellular loop (Ala623) is necessary for coupling to PLC (65), and as this alanine is conserved in rat, mouse, pig, and human LH/CG-Rs, we mutated the equivalent alanine in the C-terminal region of the third intracellular loop of the murine LH/CG-R to an isoleucine (A564I) and stably transfected HEK 293 cells with the receptor expressing this mutation. We also constructed one additional group of stable cell lines expressing a mutation of the murine LH/CG-R D564G that has been found in the human LH/CG-R to lead to moderate constitutive AC activity (66). If desensitization of LH/CG-R-stimulated AC activity reflected a competition between Gs and Gq/11 for binding sites on the LH/CG-R, then the LH/CG-R that does not couple to PLC should not exhibit desensitization of AC activity, and receptors that constitutively activate AC (and couple to Gs) should show reduced activation of PLC and reduced desensitization. Cells expressing wild-type or mutated LH/CG-R were then evaluated for hCG-stimulated PLC activity, and the membranes from these cells were evaluated for their ability to show cell-free desensitization of AC activity (in a two-state desensitization reaction). The results in Fig. 10Go show that cells expressing wild-type LH/CG-R exhibited hCG-stimulated PLC activity, and membranes from these cells exhibited 40% desensitization to LH/CG-R activation. The A568I mutation, which in the TSH-R abolished coupling to PLC (65), did not reduce coupling of the LH/CG-R to PLC and did not reduce the extent of desensitization of AC activity. Cells expressing the D564G mutation exhibited PLC and AC activities and LH/CG-R desensitization very much like wild-type cells.



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  		Figure 10. hCG-stimulated PLC activity and cell-free hCG-stimulated desensitization of AC activity in HEK 293 cells stably transfected with wild-type or mutated murine LH/CG-R. For AC assays, membranes were prepared from clonal cells expressing wild-type murine LH/CG-R, LH/CG-R mutation D564G, or LH/CG-R mutation A568I and subjected to a 2-stage desensitization incubation. Stage 1 contained 1 mM AMP-PNP, 10 µM GTP, and BSA or hCG, as indicated; stage 2 was the 5-min AC assay. Results are expressed as a percentage over basal AC activities. For all results, the means of triplicate determinations for 2 or 3 different clones were averaged, and the SEM is the variation from these means. For wild-type receptor, basal AC activity was 78 ± 9 pmol cAMP formed/min·mg protein (n = 3 different clonal wild-type cell lines); for mutation D564G, basal AC activity was 160 ± 30 (n = 4 for 2 different clonal cell lines); for mutation A568I, basal AC activity was 196 ± 13 (n = 10 for 3 different clonal cell lines). hCG-stimulated PLC activity was determined as described in Materials and Methods. Results are counts per min of inositol phosphates formed in cells treated for 30 min with hCG divided by counts per min of product in cells treated for 30 min with vehicle and are the mean ± SEM of the indicated numbers of separate determinations: wild-type, n = 21; D564G mutation, n = 10; A568I mutation, n = 8. The percent desensitization of hCG-stimulated AC activities is shownabove each set of values. Results are the mean ± SEM of 3 different cell clones for wild-type receptor, 5 determinations for 2 different clonal cell lines containing D564G mutation, and 10 determinations for 3 different clonal cell lines containing the A568I mutation.

 
Taken together, these results indicate that LH/CG-R-stimulated desensitization of AC is independent of coupling to Gq/11 and PLC, and that the LH/CG-R can functionally and simultaneously couple to Gs to activate AC and to one or more G proteins to activate PLC.


   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
Because desensitization of LH/CG-R-stimulated AC activity in ovarian follicular membranes is not dependent upon LH/CG-R phosphorylation yet requires GTP and is reversed by GDPßS, as reviewed in the introduction, we tested alternate mechanisms that could account for agonist-dependent desensitization in follicular membranes. We considered the possibility that desensitization reflected a direct inhibition of AC activity either by the {alpha}-subunit of Gi or by ß{gamma}-subunits derived from Gi, Gq/11, Gs, or G13, the G proteins activated in follicular membranes by the LH/CG-R (24, 30). Our results demonstrated that desensitization is not mediated by Gi{alpha}- or Giß{gamma}-subunits, based on the inability of PTX to reduce the extent of desensitization. Furthermore, and in contrast to results found for the murine LH/CG-R transfected into 11/6 cells (49), agonist-dependent activation of the porcine LH/CG-R in follicular membranes does not activate Gi to inhibit AC activity. The basis for the inability of the LH/CG-R to activate Gi to inhibit AC in follicular membranes, in contrast to results observed in 11/6 cells, is not known. As we have shown that the porcine LH/CG-R in follicular membranes can rapidly activate Gi, albeit rather minimally compared with other G proteins (24, 30), either follicular membranes do not contain sufficient levels of Gß{gamma}- inhibitable type I AC (67) or of i{alpha}-inhibitable ACs (68, 69, 70), or LH/CG-R activation of Gi is too minimal to result in detectable inhibition of membrane AC activity. We have shown that follicular membrane AC is tonically inhibited through an unknown receptor-Gi protein, based on the ability of PTX to enhance Gs-stimulated AC activity (activated by hCG and/or FSH) by 50–70%, showing that Gi (via i{alpha} or ß{gamma}) functionally inhibits one or more ACs in follicular membranes. Perhaps this tonic inhibition is mediated by Gß{gamma}-subunits, based on the ability of the Gß{gamma} scavenger GST-ßARK to raise forskolin-stimulated AC activity (~35%; see Fig. 6Go). However, generation of Gß{gamma} in the membranes by AlF4--dependent G protein activation did not result in a corresponding inhibition of forskolin-stimulated AC activity (see Fig. 4Go), perhaps because the Gß{gamma}-binding sites on AC type I are already saturated. Taken together, these results suggest that the inability of the LH/CG-R to activate Gi to promote detectable inhibition of AC activity in follicular membranes is most likely the result of the minimal activation of Gi by the LH/CG-R and is not due to the absence of the appropriate AC enzyme.

Our results also suggest that agonist-dependent desensitization of hCG-stimulated AC activity is not the result of inhibition of AC activity mediated by Gß{gamma}s derived from Gs, Gq/11, or G13. Additionally, our results suggest that agonist-dependent desensitization of hCG-stimulated AC activity does not reflect competition between Gs and Gq/11 for binding sites on the LH/CG-R. We showed that the conserved alanine in the C-terminal portion of the third intracellular loop of the LH/CG-R is not necessary for LH/CG-R activation of PLC, in contrast to results reported for this conserved amino acid in the TSH-R (65), and we were therefore unable to test the hypothesis that a LH/CG-R that does not couple to PLC does not exhibit desensitization of AC activity. However, our results demonstrate that C-terminal Gq/11{alpha} peptides and antisera, both of which are well established reagents that reduce signaling in a number of cellular models to effectors such as PLC (25, 26, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 71), do not prevent agonist-dependent desensitization of hCG-stimulated AC activity in follicular membranes. Our results suggest that the LH/CG-R can functionally and simultaneously couple to Gs to activate AC and to one more G proteins to activate PLC. This latter conclusion is reinforced by the recent finding of a mutation of the human LH/CG-R, D578Y, that yields apparently maximal constitutive activation of both AC and PLC (72).

In summary, these results rule out direct inhibition of AC by Gi{alpha} as the basis for agonist-dependent desensitization of hCG-stimulated AC activity. Our results further suggest that LH/CG-R desensitization is not mediated by Gß{gamma}-subunits or by competition of Gs and Gq/11 for LH/CG-R-binding sites. These results also reduce the likelihood that activation of a heterotrimeric G protein is the basis for the GTP requirement for desensitization of LH/CG-R-stimulated AC activity, based on the inability of exogenous Gß{gamma} added to desensitized membranes to reverse desensitization of agonist-dependent hCG-stimulated AC activity. Perhaps the GTP requirement for agonist-dependent LH/CG-R desensitization is fulfilled by a monomeric small mol wt G protein, activated directly or indirectly by LH/CG-R activation. As LH/CG-R desensitization in follicular membranes does not appear to depend on LH/CG-R phosphorylation, on direct inhibition of AC by G protein subunits, or on competition among Gs and Gq/11 subunits for binding to the LH/CG-R, desensitization in this model must be mediated by a unique mechanism. Future studies are directed toward elucidation of this unique mechanism of agonist-dependent desensitization and whether a small mol wt G protein fulfills the GTP requirement for agonist-dependent LH/CG-R desensiti-zation.


   Acknowledgments
 
We thank Dr. M. Marlene Hosey for the generous gift of Escherichia coli-overexpressing GST fused to the C-terminal 222 amino acids of ßARK, Dr. Jon Lomasney for assistance with the PLC assay, and Dr. T. Martin for the generous gift of anti-{alpha}q/11 antibody B6T.


   Footnotes
 
1 This work was supported by USDA Grant NRICGP 9401432 (to M.H.D.) and NIH Grants EY-06062 (to H.H.), DK-41244 (to M.B.), and DK-19318 (to L.B.). Back

2 Predoctoral appointee to the NIH Training Program in Reproductive Biology (Grant T32-HD-07068). Back

3 Ethanol at 8–10% in stage 1 of desensitization incubation selectively reduces AC activity when hCG is present in stages 1 and 2, but does not affect basal (BSA in stages 1 and 2), full hCG-stimulated (BSA in stage 1, hCG in stage 2), or fluoride-stimulated (hCG and fluoride in stage 1, hCG in stage 2) AC activities (23 ). At higher ethanol concentrations, ethanol inhibits all AC parameters. Back

4 Calculation is based on subtracting basal AC activity (BSA in stages 1 and 2) from full hCG-stimulated AC activity (BSA in stage 1, hCG in stage 2). Back

Received September 4, 1998.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

  1. Hausdorff WP, Caron MG, Lefkowitz RJ 1990 Turning off the signal: desensitization of ß-adrenergic receptor function. FASEB J 4:2881–2889[Abstract]
  2. Lefkowitz RJ 1993 G Protein-coupled receptor kinases. Cell 74:409–412[CrossRef][Medline]
  3. Sibley DR, Benovic JL, Caron MG, Lefkowitz RJ 1987 Regulation of transmembrane signaling by receptor phosphorylation. Cell 48:913–922[CrossRef][Medline]
  4. Dohlman HG, Thorner J, Caron MG, Lefkowitz RJ 1991 Model systems for the study of seven-transmembrane segment receptors. Annu Rev Biochem 60:653–688[CrossRef][Medline]
  5. Lohse MJ,, Andexinger S, Pitcher J, Trukawinski S, Codina J, Faure J-P, Caron MG, Lefkowitz RJ 1992 Receptor-specific desensitization with purified proteins. J Biol Chem 267:8558–8564[Abstract/Free Full Text]
  6. Ferguson SSG, Barak LS, Zhang J,Caron MG 1996 G-Protein-coupled receptor regulation: role of G-protein-coupled receptor kinases and arrestins. Can J Physiol Pharmacol 74:1094–1110
  7. Lamm MLG, Hunzicker-Dunn M 1994 Phosphorylation-independent desensitization of the luteinizing hormone/chorionic gonadotropin receptor in porcine follicular membranes. Mol Endocrinol 8:1537–1546[Abstract]
  8. Wang Z, Hipkin RW,Ascoli M 1996 Progressive cytoplasmic tail truncations of the lutropin-choriogonadotropin receptor prevent agonist- or phorbol ester-induced phosphorylation, impair agonist- or phorbol ester-induced desensitization, and enhance agonist-induced receptor down-regulation. Mol Endocrinol 10:748–759[Abstract]
  9. Hipkin RW, Sanchez-Yague J, Ascoli M 1993 Agonist-induced phosphorylation of the luteinizing hormone/chorionic gonadotropin receptor expressed in a stably transfected cell line. Mol Endocrinol 7:823–832[Abstract]
  10. Hipkin RW, Wang Z, Ascoli M 1995 Human chorionic gonadotropin (CG)- and phorbol ester-stimulated phosphorylation of the luteinizing hormone/CG receptor maps to serines 635, 639,649, and 652 in the C-terminal cytoplasmic tail. Mol Endocrinol 9:151–158[Abstract]
  11. Zhu X, Gudermann T, Birnbaumer M, Birnbaumer L 1993 A luteinizing hormone receptor with a severely truncated cytoplasmic tail (LHR-ct628) desensitizes to the same degree as the full-length receptor. J Biol Chem 268:1723–1728[Abstract/Free Full Text]
  12. Sanchez-Yague J, Rodriguez MC, Segaloff DL, Ascoli M 1992 Truncation of the cytoplasmic tail of the lutropin/choriogoandotropin receptor prevents agonist-induced uncoupling. J Biol Chem 267:7217–7220[Abstract/Free Full Text]
  13. Wang Z, Liu X, Ascoli M 1997 Phosphorylation of the lutropin/choriogonadotropin receptor facilitates uncoupling of the receptor from adenylyl cyclase and endocytosis of the bound hormone. Mol Endocrinol 11:183–192[Abstract/Free Full Text]
  14. Vouret-Craviari V, Auberger P, Pouyssegur J, Van Obberghen-Schilling E 1995 Distinct mechanisms regulate 5-HT2 and thrombin receptor desensitization. J Biol Chem 270:4813–4821[Abstract/Free Full Text]
  15. Roettger BF, Rentsch RU, Hadac EM, Hellen EH, Burghardt TP, Miller LJ 1995 Insulation of a G protein-coupled receptor on the plasmalemma surface of the pancreatic acinar cell. J Cell Biol 130:579–590[Abstract/Free Full Text]
  16. Holtmann MH, Roettger BF, Pinon DI, Miller LJ 1996 Role of receptor phosphorylation in desensitization and internalization of the secretin receptor. J Biol Chem 271:23566–23571[Abstract/Free Full Text]
  17. Lamm MLG, Ekstrom RC, Maizels ET, Rajagopalan RM, Hunzicker-Dunn M 1994 The effect of protein kinases on desensitization of the porcine follicular membrane luteinizing hormone/chorionic gonadotropin-sensitive adenylyl cyclase. Endocrinology 134:1745–1754[Abstract]
  18. Ekstrom RC, Hunzicker-Dunn M 1989 Guanosine triphosphate fulfills a complete and specific nucleotide requirement for luteinizing hormone-induced desensitization of pig ovarian adenylyl cyclase. Endocrinology 125:2470–2474[Abstract]
  19. Ekstrom RC, Hunzicker-Dunn M 1989 Homologous desensitization of ovarian luteinizing hormone/human chorionic gonadotropin-responsive adenylyl cyclase is dependent upon GTP. Endocrinology 124:956–963[Abstract]
  20. Ezra E, Salomon Y 1980 Mechanism of desensitization of adenylate cyclase by lutropin. J Biol Chem 255:653–658[Abstract/Free Full Text]
  21. Ezra E, Salomon Y 1981 Mechanism of desensitization of adenylate cyclase by lutropin. J Biol Chem 256:5377–5382[Free Full Text]
  22. Ekstrom RC, Carney EM, Lamm MLG, Hunzicker-Dunn M 1992 Reversal of the desensitized state of pig ovarian follicular human choriogonadotropin-sensitive adenylylcyclase by guanosine 5'-O-(2-thiodiphosphate). J Biol Chem 267:22183–22189[Abstract/Free Full Text]
  23. Ekstrom RC, Hunzicker-Dunn M 1990 Opposing effects of ethanol on pig ovarian adenylyl cyclase desensitized by human choriogonadotropin or isoproterenol. Endocrinology 127:2578–2586[Abstract]
  24. Rajagopalan-Gupta RM, Lamm ML, Mukherjee S, Rasenick MM, Hunzicker-Dunn M 1998 Luteinizing hormone/choriogonadotropin receptor-mediated activation of heterotrimeric guanine nucleotide binding proteins in ovarian follicular membranes. Endocrinology 139:4547–4555[Abstract/Free Full Text]
  25. Hsieh KP, Martin TF 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 6:1673–1681[Abstract]
  26. Rasenick MM, Watanabe M, Lazarevic MB, Hatta S, Hamm HE 1994 Synthetic peptides as probes for G protein function. J Biol Chem 269:21519–21525[Abstract/Free Full Text]
  27. Ho YK,Fung BK 1984 Characterization of transducin from bovine retinal rod outer segments. The role of sulfhydryl groups. J Biol Chem 259:6694–6699[Abstract/Free Full Text]
  28. Lowry OW, Rosebrough NJ, Farr AL, Randall RJ 1951 Protein measurement with the Folin phenol reagent. J Biol Chem 193:265–275[Free Full Text]
  29. Bockaert J, Hunzicker-Dunn M, Birnbaumer L 1976 Hormone-stimulated desensitization of hormone-dependent adenylyl cyclase. J Biol Chem 251:2653–2663[Abstract/Free Full Text]
  30. Rajagopalan-Gupta RM, Rasenick MM, Hunzicker-Dunn M 1997 LH/choriogonadotropin-dependent, cholera toxin-catalyzed adeonsine 5'-diphosphate (ADP)-ribosylation of the long and short forms of Gsalpha and pertussis toxin-catalyzed ADP-ribosylation of Gi{alpha}. Mol Endocrinol 11:538–549[Abstract/Free Full Text]
  31. Innamorati G, Sadeghi H, Eberle AN, Birnbaumer M 1997 Phosphorylation of the V2 vasopressin receptor. J Biol Chem 272:2486–2492[Abstract/Free Full Text]
  32. Sanger F, Nicklen S, Coulson AR 1977 DNA sequencing with chain-terminating inhibitors. Proc Natl Acad Sci USA 74:5463–5467[Abstract/Free Full Text]
  33. Themmen AP, Hinrichs V, Birnbaumer M 1993 In situ assay of hormone-stimulated adenylyl cyclase in 96-well microtitration plates: an aide to rapid identification of transformed cell clones. J Rec Res 13:69–78
  34. Ekstrom RC, Hunzicker-Dunn M 1990 Comparison of the luteinizing hormone-sensitive adenylyl cyclase of the pig ovarian follicle and corpus luteum and its susceptibility to in vitro hormone-dependent desensitization. Endocrinology 126:1191–1198[Abstract]
  35. Martin TF 1983 Thyrotropin-releasing hormone rapidly activates the phosphodiester hydrolysis of phosphoinositides in GH3 pituitary phospholipase C in hormone action. J Biol Chem 258:14816–14822[Abstract/Free Full Text]
  36. Bender FE, Douglass LW, Kramer A 1982 Statistical methods for Food and Agriculture. AVI, Westport, pp 87–107
  37. Katada T, Northup JK, Bokoch GM, Ui M, Gilman AG 1984 The inhibitory guanine nucleotide-binding regulatory component of adenylate cyclase. J Biol Chem 259:3578–3585[Abstract/Free Full Text]
  38. Hunt TW, Carroll RC, Peralta EG 1994 Heterotrimeric G proteins containing G{alpha}i3 regulate multiple effector enzymes in the same cell. J Biol Chem 47:29565–29570
  39. Eason MG,Liggett SB 1995 Identification of a Gs coupling domain in the amino terminus of the third intracellular loop of the {alpha}2A-adrenergic receptor. J Biol Chem 270:24753–24760[Abstract/Free Full Text]
  40. Hunzicker-Dunn M 1981 Rabbit follicular adenylyl cyclase activity. II. Gonadotropin-induced desensitization in granulosa cells and follicle shells. Biol Reprod 24:279–286[Abstract]
  41. Taussig R, Gilman AG 1995 Mammalian membrane bound adenylyl cyclases. J Biol Chem 270:1–4[Free Full Text]
  42. Cooper DMF, Mons N, Karpen JW 1995 Adenylyl cyclases and the interaction between calcium and cAMP signalling. Nature 374:421–424[CrossRef][Medline]
  43. Premont RT, Macrae AD, Stoffel RH, Chung N, Pitcher JA, Ambrose C, Inglese J, MacDonald ME, Lefkowitz RJ 1996 Characterization of the G protein-coupled receptor kinase GRK4. J Biol Chem 271:6403–6410[Abstract/Free Full Text]
  44. Guderman T, Birnbaumer M, Birnbaumer L 1992 Evidence for dual coupling of the murine luteinizing hormone receptor to adenylyl cyclase and phosphoinositide breakdown and Ca2+ mobilization. J Biol Chem 267:4479–4488[Abstract/Free Full Text]
  45. Zhu X, Gilbert S, Birnbaumer M, Birnbaumer L 1994 Dual signaling potential is common among Gs-coupled receptors and dependent on receptor density. Mol Pharmacol 46:460–469[Abstract]
  46. Davis JS, West LA, Farese RV 1984 Effects of luteinizing hormone on phosphoinositide metabolism in rat granulosa cells. J Biol Chem 259:15028–15034[Abstract/Free Full Text]
  47. Davis JS, Weakland LL, Farese RV, West LA 1987 Luteinizing hormone increases inositol triphosphate and cytosolic free Ca2+ in isolated bovine luteal cells. J Biol Chem 262:8515–8521[Abstract/Free Full Text]
  48. Birnbaumer L 1992 Receptor-to-effector signaling through G proteins: roles for ß{gamma} dimers as well as {alpha} subunits. Cell 71:1069–1072[CrossRef][Medline]
  49. Herrlich A, Kuhn B, Grosse R, Schmid A, Schultz G, Guderman T 1996 Involvement of Gs and Gi proteins in dual coupling of the luteinizing hormone receptor to adenylyl cyclase and phospholipase C. J Biol Chem 271:16764–16772[Abstract/Free Full Text]
  50. 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 269:15766–15785
  51. Luttrell LM, Ostrowski J, Cotecchia S, Kendall H, Lefkowitz RJ 1993 Antagonism of catecholamine receptor signalling by expression of cytoplasmic domains of receptors. Science 259:1453–1457[Abstract/Free Full Text]
  52. Shenker A, Goldsmith P, Unson CG, Spiegel AM 1991 The G protein coupled to the thromboxane A2 receptor in human platelets is a member of the novel Gq family. J Biol Chem 266:9309–9313[Abstract/Free Full Text]
  53. Martin EL, Rens-Domiano S, Schatz PJ, Hamm HE 1996 Potent peptide analogues of a G protein receptor-binding region obtained with a combinatorial library. J Biol Chem 271:361–366[Abstract/Free Full Text]
  54. Mumby SM, Heukeroth RO, Gordon JI, Gilman AG 1990 G-protein {alpha}-subunit expression, myristoylation, and membrane association in COS cells. Proc Natl Acad Sci USA 87:728–732[Abstract/Free Full Text]
  55. James SR, Vaziri C, Walker TR, Milligan G, Downes CP 1994 The turkey erythrocyte ß-adrenergic receptor couples to both adenylate cyclase and phospholipase C via distinct G-protein {alpha} subunits. Biochem J 304:359–364
  56. Jouneaux C, Mallat A, Serradeil-Le Gal C, Goldsmith P, Hanoune J, Lotersztajn S 1994 Coupling of endothelin B receptors to the calcium pump and phospholipase C via Gs and Gz in rat liver. J Biol Chem 269:1845–1851[Abstract/Free Full Text]
  57. Courney AJ, Tjian R 1988 Analysis of Sp1 in vivo reveals multiple transcription domains, including a novel glutamine-rich activation motif. Cell 55:887–898[CrossRef][Medline]
  58. Zhang J, Pratt RE 1996 The AT2 receptor selectively associates with Gi{alpha}2 and Gi{alpha}3 in the rat fetus. J Biol Chem 271:15026–15033[Abstract/Free Full Text]
  59. Murphy KS, Coy DH, Makhlouf GM 1996 Somatostatin receptor-mediated signaling in smooth muscle. J Biol Chem 271:23458–23463[Abstract/Free Full Text]
  60. Murray-Whelan R, Schlegel W 1992 Brain somatostatin receptor-G protein interaction. J Biol Chem 267:2960–2965[Abstract/Free Full Text]
  61. Eason MG, Kurose H, Holt BD, Raymond JR, Liggett SB 1992 Simultaneous coupling of alpha2-adrenergic receptors to two G-proteins with opposing effects. J Biol Chem 267:15795–15801[Abstract/Free Full Text]
  62. Eason MG, Kurose H, Holt BD, Raymond JR, Liggett SB 1992 Simultaneous coupling of the {alpha}2-adrenertic receptors to two G-proteins iwth opposing effects. Subtype-selective coupling of {alpha}2C10, {alpha}2C4, and {alpha}2C2 adrenergic receptors to Gi and Gs. J Biol Chem 267:15795–15801
  63. Wu D, Jiang H, Simon MI 1995 Different {alpha}1-adrenergic receptor sequences required for activating different G{alpha} subunits of Gz class of G proteins. J Biol Chem 270:9828–9832[Abstract/Free Full Text]
  64. Babich M, Alford GE, Nissenson RA 1994 A novel phospholipase C inhibitor and phorbol esters reveal selective regulation of thrombin- and parathyroid hormone-stimulated signaling pathways in rat osteosarcoma cells. J Pharmacol Exp Ther 269:172&#