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Endocrinology Vol. 139, No. 11 4547-4555
Copyright © 1998 by The Endocrine Society

ARTICLES

Luteinizing Hormone/Choriogonadotropin Receptor-Mediated Activation of Heterotrimeric Guanine Nucleotide Binding Proteins in Ovarian Follicular Membranes1

Rajsree M. Rajagopalan-Gupta2, Marilyn L. G. Lamm3, Sutapa Mukherjee, Mark M. Rasenick and Mary Hunzicker-Dunn

Department of Cell and Molecular Biology (R.M.R.-G., M.L.G.L., S.M., M.H.-D.), Northwestern University Medical School, Chicago Illinois 60611; and Departments of Physiology and Biophysics and Psychiatry (M.M.R.), University of Illinois College of Medicine, Chicago, Illinois 60608

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
 
The LH/CG receptor signals to adenylyl cyclase via the stimulatory heterotrimeric GTP binding regulatory protein, Gs, and to phospholipase C and potentially to other effectors, such as ion channels, via a G protein or proteins that have not been identified in gonadal cells. To identify G proteins activated in a physiological membrane environment upon LH/CG receptor activation, we used the ability of activated G proteins to bind GTP and incubated ovarian follicular membranes with the photoaffinity GTP analog, P3-(4-azidoanilido)-P1-5'-GTP ([32P]AAGTP). Results showed that human CG (hCG) stimulated the binding of [32P]AAGTP to a 45-kDa protein(s) in follicular membranes that comigrated with immunoreactive G{alpha}s, G{alpha}q/11, and G{alpha}13. When G{alpha} proteins were immunoprecipitated from Triton X-100 solubilized membrane extracts after photoaffinity labeling with [32P]AAGTP, a time-dependent increase in hCG-dependent [32P]AAGTP binding to G{alpha}s, G{alpha}q/11, and G{alpha}i was detected. hCG-dependent [32P]AAGTP binding to G{alpha}13 was also detected. These results demonstrate that agonist-dependent LH/CG receptor activation promotes the activation of Gs, Gi, Gq/11, and G13 in porcine ovarian follicular membranes. These results further suggest that G{alpha}s remains coupled to the agonist-bound LH/CG receptor during at least the initial 10 min after agonist-dependent receptor activation.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
LH, LIKE MANY hormones, neurotransmitters, and sensory stimuli, initiates its biological actions by binding to a heptaspanning cell surface membrane receptor (1, 2). Agonist binding to these receptors consequently activates heterotrimeric guanine nucleotide binding proteins (G proteins) to stimulate enzyme or ion channel effector(s) (3, 4, 5). G protein activation is characterized by the rate-limiting release of GDP from the {alpha}-subunit, followed by the subsequent binding of GTP and consequent dissociation of G protein G{alpha}GTP and Gß{gamma}-subunits (6). The activated state persists until GTP is hydrolyzed to GDP, resulting in formation of {alpha}GDP and reassociation with ß{gamma}. Most G protein-coupled receptors share the ability to be desensitized, a process whereby they become refractory to further stimulation after an initial response, despite the continued presence of a stimulus of constant intensity. Desensitization of most G protein-coupled receptors results from phosphorylation of the receptor by G protein-coupled receptor kinases (7), subsequent binding of the clathrin adaptor protein arrestin (8) [which leads to the functional uncoupling of the receptor from its associated G protein (9)], receptor sequestration, and later, to receptor internalization (10).

LH/CG receptor activation in follicular target cells leads to activation of adenylyl cyclase (11) and phospholipase C (PLC) (12, 13). The continued presence of LH or human CG (hCG) results in desensitization of adenylyl cyclase (14). Each of these effects, including desensitization (15, 16), requires activation of one or more G proteins. To begin to understand LH/CG receptor-stimulated signaling pathways in ovarian follicles, we sought to identify, in a physiological membrane environment, G proteins activated upon LH/CG receptor activation. Taking advantage of the ability of activated G proteins to preferentially bind guanine nucleotides, we incubated porcine ovarian follicular membranes with the radiolabeled, photoaffinity, poorly hydrolyzable GTP analog, P3-(4-azidoanilido)-P1- 5'-GTP ([32P]AAGTP) (17) to identify G proteins activated in response to LH/CG receptor activation. Our results show that LH/CG receptor agonist promotes the time-dependent activation of G{alpha}s, G{alpha}q/11, G{alpha}13, and G{alpha}i.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Materials
Purified hCG (CR-127) was provided by the Center for Population Research, NICHHD. Materials were purchased from the following sources: [32P]nicotinamide adenine dinucleotide (NAD) (30 Ci/mmol), Dupont-New England Nuclear (Boston, MA); anti-G{alpha}i (06–190; G{alpha}i-3 peptide antibody, which recognizes G{alpha}i, G{alpha}o, and G{alpha}t), UBI; anti-G{alpha}i [117; C-terminal peptide antibody, kindly provided by Dr. D. Manning, which recognizes G{alpha}i1 = G{alpha}i1 > G{alpha}i3; (18, 19)]; anti-G{alpha}13 (B860), C-terminal peptide antibody, which is specific for G{alpha}13 (20), kindly provided by Dr. P. Sternweis; anti-G{alpha}q/11 (C-19, specific for G{alpha}q and G{alpha}11), anti-G{alpha}i3 (C-10; C-terminal peptide antibody to G{alpha}i3, which reacts with all G{alpha}i’s), anti-G{alpha}s/olf (C-18, C-terminal peptide antibody to G{alpha}s), and Protein A/G PLUS-Agarose, Santa Cruz Biotechnology, Inc. (Santa Cruz, CA); anti-G{alpha}s [1190, C-terminal G{alpha}s antibody, kindly provided by Dr. D. Manning (21)]; anti-G{alpha}q/11 [B6T, C-terminal G{alpha}q/11 peptide antibody, kindly provided by Dr. T. Martin (22)]; anti-LH/CG receptor antibody, kindly provided by Dr. E. Milgrom (23); coarse Sephadex G25, Pharmacia Biotechnology Inc.; creatine phosphokinase, Calbiochem; pertussis toxin (PTX), List Biological Inc.; electrophoresis purity reagents, Bio-Rad Laboratories, Inc. (Richmond, CA); enhanced chemiluminescence detection reagents, Amersham (Arlington Heights, IL); prestained molecular weight markers, Diversified Biotech (Newton Center, MA); nytran, Schleicher & Schuell, Inc. (Keene, NH); Hybond, Amersham; other reagents, Sigma Chemical Co., (St. Louis, MO). Radiochemicals were used without further purification.

Preparation of ovarian follicular membranes
Pig ovaries were collected at a local slaughterhouse (AMPAC) and were transported to the laboratory on ice. Walls from follicles, which were 6–12 mm in diameter, were dissected from ovaries of nonpregnant pigs, and a partially purified membrane fraction was isolated after sucrose density gradient centrifugation and stored at -70 C until use, as previously described (16, 24). Protein was determined by the method of Lowry (25).

[32P]ADP ribosylation of follicular membrane proteins
[32P]ADP-ribosylation of G{alpha}s and G{alpha}i proteins in follicular membranes, in the presence of activated cholera toxin (CTX) or PTX and [32P]NAD, was as previously described (24).

[32P]AAGTP-photoaffinity labeling of follicular membrane proteins
Follicular membranes (50–200 µg membrane protein, as indicated) were incubated in a vol of 40 µl, containing either 1 µg/ml BSA or indicated concentrations of hCG, in an incubation medium (IM) consisting of 10 µM GDP, 31.25 mM Bis-Tris-Propane (pH 7.2), 6.25 mM MgCl2, 0.5 mM EDTA, 1.25 mM EGTA, 25 mM creatine phosphate, and 0.2 mg/ml creatine phosphokinase. Reactions were initiated with the addition of 0.5 µM [32P]AAGTP in the dark and were conducted at 30 C for indicated lengths of time. The reaction was stopped by placing sample tubes on ice and adding 1 ml cold 10 mM Tris-HCl (pH 7.2), 0.2 µM ßME. Samples were centrifuged (20,000 x g, 5 min, 4 C), and membrane pellets were resuspended in 40 µl IM, which did not contain [32P]AAGTP or hCG but did contain 1 µg/ml BSA. Samples were UV-irradiated for 3 min at 4 C (approximately 5 cm from the UV source) to covalently bind [32P]AAGTP to membrane G proteins. This protocol was adapted from Rasenick et al. (26). Membranes were then either pelletted (to be resuspended in solubilization buffer for immunoprecipitation studies, as described below) or 20 µl of 3X SDS-sample buffer was added, samples were boiled 5 min, and membrane proteins were separated by SDS-PAGE containing 8 or 10.5% acrylamide (24). The gel was then either stained, destained, dried, and exposed to Kodak X-Omat AR film Eastman Kodak (Rochester, NY) or transferred onto nitrocellulose membrane overnight (4 C, 0.1 A).

Solubilization
After [32P]ADP-ribosylation (using 500 µg membrane protein) or [32P]AAGTP-labeling (using 200 µg membrane protein), pelletted membranes were resuspended to a final concentration of 5 µg/µl (100–150 µl per ependorf tube) in solubilization buffer [buffer B: 50 mM Tris-HCl (pH 7.4), 1.0% Triton X-100, 25% glycerol, 5 mM EDTA, 5 mM EGTA (pH 7.4), 1 mM phenylmethlysulfonylflouride, 50 mM benzamidine, 100 µM leupeptin, 5 µg/ml aprotinin, and 50 µg/ml soybean trypsin inhibitor], as previously described (24, 27), stirred at 4 C for 60 min, and diluted 10-fold in buffer B minus the Triton, so that the Triton concentration in the final volume was 0.1%. Triton-insoluble (TI) material was removed by centrifugation (100,000 x g, 60 min, 4 C). Triton-soluble (TS) supernatant was either used in Western blot analyses, immunoprecipitation studies, or LH/CG receptor binding assays. When indicated, the TI-pellet was either resuspended by sonication on ice (1 min) in a volume equal to that of the final TS fraction (buffer B minus Triton) or solubilized by boiling (5 min) in 3x SDS-sample buffer.

Immunoprecipitation
Thirty microliters of protein A-agarose (33%) was added to TS membrane extract and rotated at 4 C for 2 h to bind any nonspecific solubilized proteins. Agarose was then pelletted and discarded, and the immunoprecipitating antibody was added (1:20 dilution of anti-G protein antibodies and nonimmune or preimmune sera), and samples were allowed to rotate at 4 C overnight. Subsequently, 30 µl protein A/G-agarose was added, and the incubation was allowed to continue for 2 h. Immunocomplexes were collected as agarose pellets by centrifugation. These pellets were washed twice with 1 ml of a buffer containing 50 mM Tris-HCl, 0.1% Triton X-100, 0.1% BSA, and 25% glycerol. Pellets were then resuspended in 50–100 µl 3x SDS-sample buffer, vortexed briefly, and allowed to sit at room temperature for 30 min. Samples were placed in a boiling water bath (5 min), centrifuged (10,000 x g, 10 min), and supernatant was subjected to SDS-PAGE.

Western blot analysis
Protocols were as previously described (24), only antigen-antibody reactions were detected primarily by enhanced chemiluminescence per manufacturers instructions, incubating blots 1 h each at room temperature with primary and then with horseradish peroxidase-linked secondary antibody.

hCG-dependent redistribution of membrane proteins
To determine whether hCG-dependent receptor activation promoted the redistribution of LH/CG receptor or G{alpha} proteins between TS and TI membrane fractions, follicular membranes were incubated under optimal conditions to promote hCG-dependent adenylyl cyclase desensitization (28). To this end, membranes (100 µg membrane protein) were incubated in IM (only without [32P]AAGTP) in which 10 µM GDP was replaced with 10 µM GTP and 1 µM ATP in the presence of 1 µg/ml BSA or hCG, and 8% ethanol, for 40 min at 30 C. The reaction was then placed on ice, diluted with the addition of 3 ml ice-cold 10 mM Tris-HCl (pH 7.2), and membranes were pelletted. Pelletted membranes were then subjected to solubilization in 0.4 ml buffer B containing 1% Triton X-100, as described above, resulting in TS supernatant and TI pellet fraction. For Western blot analyses, 0.5 vol SDS-sample buffer was added to TS fraction, and 75 µl SDS-sample buffer was added to TI pellet, and extracts were placed in a boiling water bath for 5 min. For SDS-PAGE, 0.2 ml (33%) of TS extract and all of TI extract was loaded. For LH/CG receptor binding assay, both TS and TI fractions from BSA- and hCG-treated membranes were brought to 0.4 ml in buffer B and were then briefly acidified to remove hCG from receptor by incubating 2 min with HBSS (0.4 ml, pH 2.75) followed immediately by neutralization with 2 µl of 2 M Tris base. Preliminary results showed that without acid treatment, [125I]hCG binding was undetectable in membranes incubated 40 min with hCG, consistent with earlier reports (29, 30). With acid treatment, approximately 95% of [125I]hCG binding activity was recovered. [125I]hCG binding to BSA-treated membranes was not altered by the acid treatment.

LH/CG receptor binding activity
The ligand binding assay was conducted as previously described (27), except that [125I]hCG was obtained from INCSTAR Corp. (Stillwater, MN).


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
hCG-dependent [32P]AAGTP labeling of membrane G proteins
Our purpose was to identify G proteins activated in follicular membranes upon LH/CG receptor activation. Whereas guanine nucleotides are slowly exchanged on G{alpha} proteins in the absence of receptor activation, guanine nucleotides including [32P]AAGTP readily bind to the empty guanine nucleotide binding site of receptor-activated G{alpha} proteins (31, 32). Hormone-dependent binding of [32P]AAGTP to G{alpha} proteins, therefore, provides a technique to identify G proteins activated by a specific receptor.

We initially established conditions to detect optimal hCG-dependent binding of [32P]AAGTP to follicular membrane G proteins. Incubation of follicular membranes with [32P]AAGTP for 2 min resulted in the photoaffinity labeling of a number of membrane proteins, none of which showed increased binding of [32P]AAGTP upon LH/CG receptor activation by 1 µg/ml hCG (Fig. 1AGo, lanes 1 and 2). When 3 µM GDP was included in the incubation, binding of [32P]AAGTP to most of these membrane proteins was attenuated, and a distinct hCG-dependent increase (2.1 ± 0.2, n = 7) in binding of [32P]AAGTP to a 45-kDa protein(s) was readily detected (Fig. 1BGo, lanes 1 and 2). Equivalent reduced nonspecific labeling and increased detectability of hormone-dependent [32P]AAGTP binding to G proteins in the presence of GDP was observed for opioid receptor activation of Go and Gi2 in membranes of neuroblastoma x glioma hybrid cells (33). Incubation of follicular membranes with [32P]AAGTP for 10 min resulted in increased [32P]AAGTP incorporation into the 45-kDa protein(s) and also into a 40-kDa protein(s), both in the presence and absence of hCG; [32P]AAGTP binding to the 45-kDa protein(s) was still slightly enhanced (1.4 ± 0.2, n = 11) in membranes incubated with 1 µg/ml hCG, compared with BSA controls (Fig. 1BGo, lanes 3 and 4). The corresponding Coomassie-stained gels show protein loaded in each lane. [32P]AAGTP radiolabeling of the 40- and 45-kDa proteins was specific based on the decreased ability of each to incorporate [32P]AAGTP when excess (125 µM) unlabeled GTP (but not ATP, not shown) was added to the assay (Fig. 1CGo, lanes 1 and 2). Specificity of [32P]AAGTP radiolabeling of the 40- and 45-kDa bands is also argued by the hCG-insensitivity of labeling of the approximately 30-kDa bands (Fig. 1BGo, lanes 1–4). hCG-stimulated binding of [32P]AAGTP to membrane proteins in the Mr range of dynamin (100 kDa) or of small G proteins (20–25 kDa) was never detected (see Fig. 1BGo). The following experiments were designed to identify these G{alpha} protein(s) at 45 kDa, activated in follicular membranes by LH/CG receptor activation.



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  		Figure 1. hCG-dependent binding of [32P]AAGTP to membrane G proteins is enhanced by incubation with GDP and is reduced on incubation with excess GTP. A, Follicular membranes (50 µg protein) were incubated at 30 C for 2 min, in the absence of added GDP and in the absence or presence of 1 µg/ml hCG in a reaction mix, as described under Materials and Methods. Proteins were resolved by SDS-PAGE, and gel was exposed to x-ray film with an intensifying screen (lanes 1 and 2). Lanes 3 and 4 are the Coomassie blue-stained proteins in the first two lanes and show that equal protein was loaded per lane. Molecular masses of protein standards (kDa) are indicated at the right. Results are representative of three experiments. B, Follicular membranes (50 µg protein) were incubated at 30 C for 2 or 10 min, in the presence of 3 µM GDP and in the absence (-) or presence (+) of 1 µg/ml hCG in a reaction mix, as described under Materials and Methods. Proteins were resolved by SDS-PAGE, and the gel was exposed to x-ray film (lanes 1–4). Lanes 5–8 are corresponding Coomassie blue-stained proteins and show that equal protein was loaded per lane. Molecular masses of protein standards (kDa) are indicated at the left. The fold-increase over BSA control is for the 45-kDa protein(s) in this experiment. Results are representative of 7 experiments. C, Membranes (50 µg membrane protein) were incubated at 30 C for 10 min, in the absence or presence of 125 µM unlabeled GTP in a reaction mix, as described under Materials and Methods. Proteins were resolved by SDS-PAGE, and the gel was exposed to x-ray film with an intensifying screen (lanes 1 and 2). Coomassie staining of the gel is shown in lanes 3 and 4. Results are representative of three experiments.

 
Migration positions on SDS-PAGE of G{alpha} proteins in follicular membranes
We have previously determined that porcine ovarian follicular membranes contain G{alpha}i (40 kDa), G{alpha}q/11 (42/43 kDa), G{alpha}13 (42 kDa), the long (48/50 kDa) and short (45 kDa) forms of G{alpha}s, ras (21 kDa), and dynamin (100 kDa)4, but G{alpha}o (39 kDa) and G{alpha}z (41 kDa) are absent (24). To identify the G{alpha} proteins in follicular membranes that bound [32P]AAGTP in an hCG-dependent manner, we initially compared the migration positions of [32P]AAGTP photoaffinity-labeled G{alpha} proteins with those of membrane proteins immunoreactive with G{alpha} protein-selective antisera and with those of G{alpha} proteins ADP-ribosylated with CTX and PTX. Results demonstrated that the PTX-labeled G{alpha}i band (Fig. 2BGo, lane 1) comigrated with the 40-kDa [32P]AAGTP-labeled protein band (Fig. 2BGo, lane 3), suggesting that this band corresponded to a G{alpha}i. However, anti-G{alpha}s, anti-G{alpha}q/11, and anti-G{alpha}13 immunoreactive bands (Fig. 2AGo, lanes 2, 3, and 5) and the CTX-labeled G{alpha}sS band (Fig. 2BGo, lane 4) comigrated with the 45-kDa [32P]AAGTP photoaffinity-labeled band (Fig. 2AGo, lane 1; Fig. 2BGo, lane 3). Consistent with this result, hCG could be stimulating [32P]AAGTP binding at 45 kDa to G{alpha}s and/or G{alpha}q/11 and/or G{alpha}13. Identification of the G{alpha} protein(s) activated by LH/CG receptor thus required immunoprecipitation of G{alpha} proteins.



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  		Figure 2. Identification of [32P]AAGTP-labeled follicular membrane proteins. A, Alignment of [32P]AAGTP-labeled proteins with G{alpha}s-, G{alpha}q/11-, G{alpha}i-, and G{alpha}13-reactive proteins in follicular membranes. Follicular membranes (100 µg protein) were incubated at 30 C for 30 min, in the presence of 1 µg/ml hCG in a reaction mix, as described under Materials and Methods. After SDS-PAGE, proteins were transferred to nitrocellulose membrane (Hybond), and membrane strips were either exposed to x-ray film with an intensifying screen (lane 1) or were subjected to Western blot analysis using anti-G{alpha}s (lane 2), anti-G{alpha}q/11 (lane 3), or no antibody (lane 4). In a separate experiment, follicular membranes (100 µg) were subjected to Western blot analysis using anti-G{alpha}13 (lane 5). Molecular masses of labeled proteins are indicated at the left and were calculated based on the migration of protein standards. Results are representative of two separate experiments. B, Anti-G{alpha}i antibody preferentially immunoprecipitates G{alpha}i. For lanes 1–4, follicular membranes were incubated 30 min with 1 µg/ml hCG, [32P]NAD and PTX (100 µg membrane protein in lane 1), 1 µg/ml hCG and [32P]AAGTP (1 mg membrane protein in lane 2 and 200 µg protein in lane 3), or 1 µg/ml hCG, [32P]NAD and CTX (100 µg membrane protein in lane 4). For lanes 5 and 6, in a separate experiment, follicular membranes were incubated 20 min with 1 µg/ml hCG and [32P]AAGTP (600 µg membrane protein). Follicular membranes were then either solubilized in SDS sample buffer (lanes 1, 3, and 4) or (lanes 2, 5, and 6) were solubilized in 1% Triton X-100 for immunoprecipitation (IP) from the TS fraction with anti-G{alpha}i sera (lanes 2 and 5) or with preimmune sera (lane 6). G{alpha}i antibody immunoprecipitates predominately G{alpha}i and a small amount of G{alpha}sL (indicated by open arrow). Results are representative of two separate experiments. C, Anti-G{alpha}q/11 antibody immunoprecipitates both G{alpha}q and G{alpha}11. Follicular membranes were not incubated (lanes 1 and 2; 175 µg membrane protein) or were incubated 20 min with [32P]AAGTP and 10 µg/ml hCG (200 µg membrane protein), membranes were solubilized with 1% Triton X-100 and subjected to immunoprecipitation with anti-G{alpha}q/11 or nonimmune sera. After separation of immunoprecipitated proteins by SDS-PAGE, Hybond membrane was probed with anti-G{alpha}q/11 antisera (lanes 1 and 2) or subjected to autoradiography (lane 3). Upper band (lanes 1 and 2) reflects IgG bands. Results are representative of three experiments. D, Anti-G{alpha}s/olf antibody immunoprecipitates both the long and short forms of G{alpha}s. Follicular membranes (100 µg in lanes 1, 3, and 4; 300 µg in lane 2) were incubated 30 min at 30 C with [32P]NAD, 10 µg/ml hCG, and either CTX (lanes 1 and 2) or PTX (lane 4), or were not incubated (lane 3). Membrane proteins were either solubilized in SDS sample buffer (lanes 1, 3, and 4) or (lane 2) solubilized in 1% Triton X-100 and subjected to immunoprecipitation with anti-G{alpha}s/olf antibody, as described in Materials and Methods. Recovery of CTX-labeled G{alpha}s in immunoprecipitates is 76%, based on scanned densitometry of G{alpha}sS and G{alpha}sL in lanes 1 and 2, relative to total starting protein.

 
Distribution of LH/CG receptor, G{alpha}s, and G{alpha}q/11 in TS vs. TI follicular membrane fractions
Because immunoprecipitation of G{alpha} proteins requires that the membrane proteins be solubilized in detergent, we evaluated the solubility of G{alpha}s and G{alpha}q/11 and the LH/CG receptor in Triton X-100. Triton X-100 was the detergent of choice, based on the reported solubility of the LH/CG receptor in this detergent (30). Results in Fig. 3Go and Table 1Go showed that the majority of G{alpha}s, G{alpha}q/11, and LH/CG receptor was soluble in Triton X-100 and that incubation of membranes [under conditions that lead to LH/CG receptor activation and, subsequently, to desensitization (28)] did not result in the movement of any of these protein complexes into the TI membrane fractions. Indeed, LH/CG receptor activation, under conditions which lead to maximal desensitization (28), resulted in relocation of all of the LH/CG receptor into the TS fraction, whereas minimal amounts G{alpha}s and G{alpha}q/11 moved out of the TI fraction. Because the majority of G{alpha}s and G{alpha}q/11 was localized to the TS fractions under control and receptor activated conditions, we elected to immunoprecipitate G{alpha} proteins from the TS fraction after incubation of membranes with hCG or BSA and [32P]AAGTP.



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  		Figure 3. Distribution of G{alpha}s and G{alpha}q/11 in Triton X-100-soluble and -insoluble follicular membrane fractions. Follicular membranes (100 µg) were incubated in IM in the presence of 1 µg/ml BSA or hCG and 8% ethanol for 40 min at 30 C, pelletted, and solubilized in buffer B containing 1% Triton X-100, as described in Materials and Methods. TS-soluble and -insoluble fractions were separated by centrifugation, fractions were boiled 5 min in SDS-sample buffer, proteins were separated by SDS-PAGE, and Western blots were performed using G{alpha}s- and G{alpha}q/11-specific antisera, as described in the legend to Fig. 2Go. One-hundred percent of the TI fraction was loaded onto the SDS-PAGE gel; 33% of the TS fraction was loaded onto the SDS-PAGE gel, as described in Materials and Methods. To calculate total G{alpha}, densitometry of TS fraction was divided by 0.33 then summed with densitometry of TI fraction. Results are representative of two experiments for G{alpha}q/11 and four experiments for G{alpha}s.

 

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  		Table 1. Distribution of LH/CG receptor, G{alpha}s, and G{alpha}q/11 in follicular membrane fractions: effect of hCG

 
Specificity of G protein antisera
To identify whether hCG-dependent [32P]AAGTP binding at 45 kDa was to G{alpha}s, to G{alpha}13, and/or to G{alpha}q, G{alpha} protein-selective antisera were used to immunoprecipitate [32P]AAGTP photoaffinity labeled G{alpha} proteins from the Triton X-100-solubilized fraction of follicular membranes. However, before immunoprecipitation experiments with [32P]AAGTP-labeled membrane proteins, control experiments were performed to confirm that the antibodies used successfully immunoprecipitated the correct proteins and that immunoprecipitations were selective and specific. Results showed that the antibody used to immunoprecipitate G{alpha}i selectively immunoprecipitated [32P]AAGTP-labeled G{alpha}i, compared with immunoprecipitates with preimmune serum (Fig. 2BGo, lanes 2, 5, and 6). The G{alpha}i antisera also immunoprecipitated a small amount of [32P]AAGTP-labeled G{alpha}s (Fig. 2BGo, lanes 2 and 5, open arrow), consistent with our earlier report on the ability of this antibody to immunoprecipitate a small portion of CTX-catalyzed [32P]ADP-ribosylated G{alpha}s (24). The antibody used to immunoprecipitate G{alpha}q/11 selectively immunoprecipitated [32P]AAGTP-labeled G{alpha}q/11, compared with immunoprecipitates with nonimmune serum (Fig. 2CGo). The antibody used to immunoprecipitate G{alpha}s immunoprecipitated both long and short forms of G{alpha}s (Fig. 2DGo, lane 2), recovering approximately 75% of CTX-labeled G{alpha}s in immunoprecipitates from Triton-solubilized membrane extracts, as well as G{alpha}i ADP-ribosylated by CTX (seen more clearly on longer exposures). The G{alpha}13 antibody is specific for G{alpha}13 and does not react with other G{alpha} proteins (20).

[32P]AAGTP-labeling of G{alpha}s, G{alpha}q/11, and G{alpha}i
After incubation of membranes with [32P]AAGTP, G{alpha} proteins were immunoprecipitated from TS follicular membrane extracts. Immunoprecipitation results with anti-G{alpha}s antibody showed that G{alpha}sS exhibited an increased ability to bind [32P]AAGTP (in a 10-min incubation) that was dependent on the concentration of hCG (Fig. 4AGo). Time course studies showed hCG-dependent binding of [32P]AAGTP to G{alpha}sS at 5 and 10 min of incubation (Fig. 5AGo). By 20 min, photoaffinity labeling of G{alpha}sS in the absence of hCG was markedly increased, but increased binding with hCG was still detected, as shown graphically (Fig. 5AGo, inset). These results show that, during at least the initial 10 min after LH/CG receptor activation, hCG continues to stimulate [32P]AAGTP binding to G{alpha}sS. Results in Fig. 5AGo also show that hCG stimulated [32P]AAGTP binding to a 40-kDa band that aligns with G{alpha}i in total membrane extracts (Fig. 5AGo, lane 9).



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  		Figure 4. hCG dose-dependent binding of [32P]AAGTP to G{alpha}s and G{alpha}q/11. A, Membranes (200 µg protein) were incubated with indicated concentrations of hCG for 10 min in a reaction mix, as described under Materials and Methods. G{alpha}s was immunoprecipitated from TS membrane fraction using anti-G{alpha}s/olf antibody (SC, C-18), as described in Materials and Methods. Proteins were separated by SDS-PAGE, and the gel was exposed to x-ray film with an intensifying screen. B, Membranes (200 µg protein) were incubated as described above, in the absence (lane 1) or presence (lane 2) of 10 µg/ml hCG. G{alpha}q/11 was immunoprecipitated using anti-G{alpha}q/11 antibody (SC, C-19) from TS membrane fraction. Proteins were separated and visualized as described above. This result is representative of four experiments.

 


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  		Figure 5. Time course of [32P]AAGTP binding to G{alpha}s, G{alpha}q/11, and G{alpha}i. Membranes (200 µg protein) were incubated with indicated concentrations of hCG for indicated times, membrane proteins were solubilized in 1% Triton X-100, and TS extracts were subjected to immunoprecipitation with anti-G{alpha}s/olf antibody (SC, C-18; panel A), anti-G{alpha}q/11 antibody (SC, C-19; panel B), or anti-G{alpha}i antibody (117; panel C). Proteins were separated by SDS-PAGE, and the gel was exposed to x-ray film with an intensifying screen. The lower band in panel B migrates at 40 kDa. Graphed data in panel A show results from two separate experiments; data from the corresponding autoradiogram are the open circles. Graphed data in panels B and C reflect the corresponding autoradiograph. Values were obtained with the Bio Image program.

 
An equivalent protocol was followed using anti-G{alpha}q/11 to determine whether G{alpha}q/11 was activated upon LH/CG receptor activation. Immunoprecipitation results from TS membrane extracts showed that G{alpha}q/11 exhibited an increased ability to bind [32P]AAGTP when membranes were incubated with hCG (Fig. 4BGo). A 2-fold increase in [32P]AAGTP binding to Gq/11 was observed in the presence of hormone, compared with membranes incubated in the absence of hormone. hCG-dependent [32P]AAGTP incorporation into G{alpha}q/11 increased with time of incubation, exhibiting a 2.7-fold increase in [32P]AAGTP binding in the presence of hCG at 20 min of incubation, compared with BSA controls (Fig. 5BGo).

Based on evidence in Fig. 5AGo, that hCG may also promote activation of G{alpha}i, G{alpha}i was immunoprecipitated from the TS extract of follicular membranes incubated for indicated times, with or without hCG. In agreement with results in Fig. 5AGo, immunoprecipitation results using G{alpha}i-selective antisera showed that hCG indeed stimulated a time-dependent increase in [32P]AAGTP binding to G{alpha}i (Fig. 5CGo). Immunoprecipitation of G{alpha}13 from the TS extract of follicular membranes with anti-G{alpha}13-specific antibody showed that hCG also promoted increased [32P]AAGTP binding to G{alpha}13 over BSA (1.7 ± 0.2, n = 2) in a 20-min incubation (Fig. 6Go).



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  		Figure 6. hCG-dependent binding of [32P]AAGTP to G{alpha}13. Membranes (150 µg protein) were incubated with 1 µg/ml hCG or BSA for 20 min in a reaction mix, as described under Materials and Methods. G{alpha}13 was immunoprecipitated from TS membrane fraction using anti-G{alpha}13 antibody (B860), as described in Materials and Methods. Control immunoprecipitation with preimmune (PI) serum is also shown. Results are representative of two independent experiments.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
This study was initiated to identify G proteins activated in ovarian follicular membranes in response to LH/CG receptor activation. Using [32P]AAGTP to bind specifically to LH/CG receptor-activated G proteins, LH/CG receptors were shown to associate with G{alpha}s, G{alpha}q/11, G{alpha}13, and G{alpha}i in porcine ovarian follicular membranes. It is not unprecedented that a single receptor may stimulate multiple effectors through the activation of several different G proteins (33, 34, 35, 36, 37, 38, 39, 40), and there is evidence that the LH/CG receptor is coupled, directly or indirectly, to at least two effectors.

First, activated LH/CG receptor signals to adenylyl cyclase via G{alpha}s, leading to increased levels of intracellular cAMP (11). In agreement with this result, our laboratory has demonstrated the functional and direct physical association of LH/CG receptor to both the long and short forms of Gs, based on the ability of each form to undergo CTX-catalyzed ADP-ribosylation in the presence of hCG (24). Second, LH/CG receptor activation in rat granulosa and bovine luteal cells (41, 42, 43) and in L cells transfected with cDNA of the LH/CG receptor (12) leads to stimulation of PLC activity, and constitutively active LH/CG receptor transfected into Cos-7 cells strongly activates both adenylyl cyclase and PLC (13). Generally, receptor-induced stimulation of PLC has been shown to occur via PTX-insensitive G{alpha}q/11 or ß{gamma} from activated PTX-sensitive G{alpha}i (44, 45, 46). Recent results indicate that LH/CG receptor activation of PLC in L cells transfected with the LH/CG receptor is partially PTX-sensitive and most likely mediated by ß{gamma} from both G{alpha}i and G{alpha}s (47). Our laboratory has demonstrated functional coupling and physical association of the LH/CG receptor with G{alpha}i in porcine follicular membranes. However, this may be a select pool of the total Gi in follicular membranes, because less than 10% of the total PTX-labeled membrane G{alpha}i immunoprecipitates with the LH/CG receptor (24).

Until now, there has been no direct evidence that the LH/CG receptor also associates with Gq/11 or with G13. Data in the present investigation reveal that G{alpha}q/11 and G{alpha}13 show increased binding of [32P]AAGTP in the presence of hCG. [32P]AAGTP binding to G{alpha}q/11 is detectable only at later time points of incubation. This may be linked to activation of PLC. Herrlich’s group (47) demonstrated the involvement of Gi, and possibly Gs (ß{gamma}-subunits) in PLC activation in L cells, transfected with the LH/CG receptor but were unable to demonstrate increased activation of Gq/11 or G13 using [32P]AAGTP in their transfected cellular model or with bovine luteal membranes. We do not know the basis for the apparent discrepancy between our results and those of the Herrlich group, but it may be attributable to different levels of G{alpha}q/11 and G{alpha}13 in the different cellular models and/or to slow binding of [32P]AAGTP to G{alpha}q/11. We do not know whether the slow binding of [32P]AAGTP to G{alpha}q/11 in ovarian follicular membranes reflects the true time-course for receptor activation of this G protein, which would be considerably slower than that for other receptors to activate Gq/11 in cellular models where receptor coupling to Gq/11 is the predominant response (32, 48, 49), or whether it reflects relatively lower levels of this G protein in our membranes. Although we have been unable to measure hCG-stimulated PLC activity in our membrane preparations, and therefore cannot address the functional roles for Gq/11 and Gi, it is likely that one or the other may play a role in signaling to PLC. We also cannot disregard the possibility that both G proteins might act synergistically to stimulate PLC, because distinct binding regions for the {alpha}-subunit of Gq/11 and the ß{gamma}-subunit of Gi have been identified on PLC-ß2 (50).

G{alpha}13 is a ubiquitously distributed member of the G12 family of G proteins (51). Overexpression of constitutively active G{alpha}13 mutants does not lead to activation of adenylyl cyclase or PLC (52), but it promotes activation of phospholipase D (53), C-Jun protein kinase (54), and the Na+-H+ exchanger (52, 55). G{alpha}13 has recently been shown to be an intermediate in lysophosphatidic acid-induced Rho-dependent stress fiber formation in Swiss 3T3 cells (56). The functional significance of LH/CG-receptor activation of G{alpha}13 in follicular membranes is yet to be established, because none of these G{alpha}13 effectors has been linked to LH/CG receptor activation.

The ability of the LH/CG receptor to associate with at least one member of each of the four subfamilies of G proteins (3, 4) is reminiscent of the TSH receptor. Activation of the human TSH receptor in thyroid gland membranes promotes activation of G{alpha}s’s, G{alpha}i’s, G{alpha}o, G{alpha}q/11, G{alpha}12, and G{alpha}13 (39). Like the TSH receptor, no subfamilies of LH/CG receptors have been identified (1, 2, 57), suggesting that a single receptor species is capable of activating multiple G proteins. Also like the TSH receptor (58, 59), whereas LH/CG receptor-stimulated activation of adenylyl cyclase represents the predominant signaling pathway (12, 13, 47, 60, 61), the LH/CG receptor also signals to activate PLC (as reviewed above), and the 42- and 44-kDa mitogen activated protein kinases5 (62) in follicles via unidentified G proteins. Although activation of G proteins by the FSH receptor has not been strictly investigated, available evidence indicates that at least the LH/CG and TSH receptors are capable of activating multiple G proteins in a natural membrane environment, perhaps to regulate a variety of cellular effectors. Additional studies with the FSH receptor will be required to determine whether this is a common feature of this subfamily of G protein-coupled receptors.

In summary, we have shown that agonist-dependent LH/CG receptor activation promotes the activation of G{alpha}s, G{alpha}q/11, G{alpha}13, and G{alpha}i in porcine ovarian follicular membranes. Because these studies were carried out in a physiological membrane model system, the G protein/LH/CG receptor interactions observed are believed to be representative of natural associations between these proteins. These results further demonstrate that LH/CG receptor activation does not promote a rapid, detectable uncoupling of LH/CG receptor and Gs activation under the experimental conditions used in these studies.


   Footnotes
 
1 This research was supported by the U.S. Department of Agriculture Grant NRICGP-9401432 (to M.H.D.) and the PHS through MH-39595 and AG-15482 and the Council for Tobacco Research (to M.M.R.). Back

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

3 Current address: Department of Urology, Northwestern University Medical School, 303 East Chicago Avenue, Chicago, Illinois 60611. Back

4 Rajagopalan-Gupta and Hunzicker-Dunn, unpublished observation. Back

5 Peters, Cottom, Salvador, and Hunzicker-Dunn, unpublished results. Back

Received April 24, 1998.


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S. Mukherjee, V. V. Gurevich, J. C. R. Jones, J. E. Casanova, S. R. Frank, E. T. Maizels, M.-F. Bader, R. A. Kahn, K. Palczewski, K. Aktories, et al.
The ADP ribosylation factor nucleotide exchange factor ARNO promotes beta -arrestin release necessary for luteinizing hormone/choriogonadotropin receptor desensitization
PNAS, May 23, 2000; 97(11): 5901 - 5906.
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