Luteinizing Hormone/Choriogonadotropin-Dependent, Cholera Toxin-Catalyzed Adenosine 5'-Diphosphate (ADP)-Ribosylation of the Long and Short Forms of Gs{alpha} and Pertussis Toxin-Catalyzed ADP-Ribosylation of Gi{alpha}

Rajsree M. Rajagopalan-Gupta1, Mark M. Rasenick and Mary Hunzicker-Dunn

Department of Cell and Molecular Biology (R.M.R.-G., M.H.-D) Northwestern University Medical School Chicago, Illinois 60611
Department of Physiology and Biophysics and Psychiatry (M.M.R.) University of Illinois College of Medicine Chicago, Illinois 60680


    ABSTRACT
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Although it is well established that activated LH/human (h) CG receptor stimulates adenylyl cyclase activity (via the heterotrimeric stimulatory guanine nucleotide-binding protein, Gs) and in some cells stimulates phospholipase C activity, there is no evidence for a direct physical interaction between the LH/CG receptor and Gs or any other G protein(s). We conducted studies using cholera toxin (CTX) and pertussis toxin (PTX) to determine which G{alpha} proteins were associated with the LH/CG receptor in ovarian follicular membranes. Since hormone-dependent, CTX-catalyzed ADP ribosylation (AR) constitutes evidence that a G{alpha} protein is specifically associated with a receptor, CTX-catalyzed AR of membrane proteins was examined both in the presence and absence of guanine nucleotides to determine which G proteins exhibit hCG-dependent labeling by [32P]NAD. Results demonstrated the time- and hCG-dependent AR of both a 45-kDa protein and a 48/50-kDa doublet as well as a 40-kDa protein that was also sensitive to AR by PTX in a time- and hCG-dependent manner. Using anti-G protein antisera to specifically immunoprecipitate photoaffinity-labeled G proteins, we were able to identify the 45- and 48/50 kDa proteins as the short and long forms of Gs{alpha} and the 40-kDa protein as Gi{alpha}. A monoclonal anti-hCG antibody immunoprecipitated the activated LH/CG receptor along with the long and short forms of Gs{alpha} and Gi. These results suggest that a portion of Gi along with the long and short forms of Gs{alpha} are associated physically with the LH/CG receptor in ovarian follicular membranes.


    INTRODUCTION
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Members of the seven transmembrane-spanning domain receptor family, including the LH/CG receptor, transduce information from extracellular signals, such as hormones, neurotransmitters, and sensory stimuli, to cellular effector enzymes or ion channels via membrane-associated heterotrimeric GTP-binding proteins (G proteins) (1). These G proteins are composed of {alpha}-, ß-, and {gamma}-subunits. In the inactive heterotrimeric conformation, GDP is bound to the {alpha}-subunit. Agonist stimulation of receptor promotes the rate-limiting release of GDP from the {alpha}-subunit and subsequent binding of GTP. The GTP-bound {alpha}-subunit, in its active conformation, activates the appropriate effector. Then, through the intrinsic GTPase activity of the {alpha}-subunit, GTP is hydrolyzed to GDP resulting in the formation of the inactive heterotrimeric G protein. This cycle continues as long as agonist is available and able to activate receptor and as long as G protein can stimulate effector activity.

Based upon their differential ability to covalently modify guanine nucleotide-binding proteins, bacterial toxins have become useful as biochemical tools in the study and identification of heterotrimeric G proteins. The {alpha}-subunit of G proteins contains a site that may be covalently modified by the NAD-dependent ADP ribosylation catalyzed by the bacterial toxins, cholera toxin (CTX) and/or pertussis toxin (PTX). PTX catalyzes the transfer of ADP-ribose from NAD to a cysteine residue four amino acids from the carboxyl terminus of Gi{alpha}, Go{alpha}, and Gt{alpha} (2, 3, 4, 5, 6). Because it is the carboxyl terminus of the G protein that interacts with receptor, ADP ribosylation prevents this interaction and results in uncoupling of the G protein from receptor (7, 8).

CTX catalyzes the ADP ribosylation of an internal arginine residue (9) of Gs{alpha}, Golf{alpha}, and Gt{alpha}. The modified arginine residue is located close to the GTP-binding domain of the {alpha}-subunit (10), and ADP ribosylation of this residue results in a decreased rate of GTP hydrolysis (11, 12), leading to constitutive activation of G proteins. Of the G proteins that are widely expressed, Gs{alpha} is the only well characterized G protein that undergoes CTX-catalyzed ADP ribosylation (Arg 201 in the long form of Gs{alpha}, Arg 187 in the short form of Gs{alpha}). However, since all G proteins contain the substrate site for CTX-catalyzed ADP-ribosylation (13), it has been predicted that G proteins other than Gs{alpha} might also be covalently modified by CTX. Indeed, as detailed below, various laboratories have shown that some PTX-sensitive G proteins (Gi2{alpha}, Gi3{alpha}, Go{alpha}) also exhibit hormone-dependent ADP ribosylation by CTX under specific conditions. The site of CTX-catalyzed ADP ribosylation on Gi{alpha} has been identified as an arginine residue that corresponds to Arg 201 of Gs{alpha} (14, 15, 16, 17). When membranes from Rat 1 fibroblasts transfected with the human {alpha}2-C10 adrenergic receptor were incubated with CTX and [32P]NAD in the absence of added guanine nucleotides, Milligan and co-workers (13) demonstrated the incorporation of an ADP-ribose moiety into Gi2{alpha} and Gi3{alpha} in the presence of agonist as well as the agonist-independent ADP ribosylation of the long and short forms of Gs{alpha} (13).

Although it has been well characterized that agonist stimulation of the LH/CG receptor stimulates adenylyl cyclase activity (18), presumably via Gs, and in some cells stimulates phospholipase C (PLC) (19, 20, 21, 22, 23) via a PTX-sensitive G protein (24), there is no reported evidence that indicates direct physical interaction between the LH/CG receptor and any associated G protein(s). In this study, CTX-catalyzed ADP ribosylation of porcine ovarian follicular membranes was examined, both in the presence and absence of guanine nucleotides, to determine which G proteins exhibit hCG-dependent ADP ribosylation. Using anti-G protein antisera to specifically immunoprecipitate radiolabeled G proteins as well as a monoclonal anti-hCG antibody, which immunoprecipitates the activated LH/CG receptor (25) and presumably any receptor-associated G proteins, we were able to identify conclusively that both the long and short forms of Gs{alpha} interact with the LH/CG receptor. Our results also show that the LH/CG receptor also couples to a portion of the Gi{alpha} expressed in ovarian follicular membranes.


    RESULTS
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Hormone-Dependent ADP Ribosylation of G Proteins
Immunoblot analysis showed that Gs{alpha}2, Gq/11{alpha}, Gi{alpha}, G13{alpha}, and ras are present in porcine ovarian follicular membranes while Go{alpha} and Gz{alpha} are absent (Table 1Go). Antibodies used in immunoblot analyses and immunoprecipitation studies are listed in Table 2Go. In an effort to identify the G proteins in porcine follicular membranes that are physically associated with the LH/CG receptor, we used the ability of bacterial toxins to catalyze the hormone-dependent ADP ribosylation of those G{alpha} proteins that are functionally coupled to LH/CG receptors.


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Table 1. Expression of G{alpha} Proteins in Porcine Ovarian Follicular Membranes

 

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Table 2. Source and Specificity of Antibodies

 
Incubation of follicular membranes under ADP ribosylation conditions using [32P]NAD and PTX in the absence and presence of hCG and in the absence or presence of GTP resulted in the ADP ribosylation of a single protein that migrated on SDS-PAGE at 40 kDa, consistent with the molecular mass of Gi (Fig. 1AGo, lanes 5–8). This result was expected since the only PTX-sensitive G protein in porcine follicular membranes is Gi (Table 1Go). Control studies indicated that the 40-kDa band was specifically labeled by PTX since samples that were incubated in the presence of excess cold NAD (400 µM; not shown) or in the absence of PTX (Fig. 1BGo, lanes 5–8) did not exhibit the 40-kDa radiolabeled band. On immunoprecipitation using anti-Gi{alpha} antibody (antiserum 117), the 40-kDa protein ADP-ribosylated by PTX was immunoprecipitated (Fig. 2AGo, lane 2). These data lead us to conclude unambiguously that the PTX-sensitive, radiolabeled 40-kDa band in porcine follicular membranes is Gi{alpha}.



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Figure 1. CTX- and PTX-Catalyzed ADP Ribosylation of Follicular Membrane Proteins

A, SDS-PAGE of membranes incubated with[32P]NAD in the presence of either CTX or PTX. Membranes (100 µg protein) were incubated at 30 C for 30 min in the presence of either CTX (lanes 1–4) or PTX (lanes 5–8), in the presence (lanes 1, 2, 5, 6) or absence (lanes 3, 4, 7, 8) of 5 mM GTP, and in the absence (lanes 1, 3, 5, 7) or presence (lanes 2, 4, 6, 8) of 10 µg/ml hCG in a reaction mix as described in Materials and Methods. The gels were exposed to x-ray film with an intensifying screen. Molecular masses of labeled proteins (kDa) are indicated at the left and were calculated based on the migration of protein standards (shown at right). Coommassie staining of the gel showed that equal levels of proteins were loaded in each lane. Equivalent results were obtained in three separate experiments. B, SDS-PAGE of membranes indicating specificity of PTX-catalyzed ADP ribosylation of membrane proteins. Membranes (100 µg protein) were incubated in the presence (lanes 1–4) or absence (lanes 5–8) of PTX (2. 5 µg/ml) at 30 C for 30 min in the presence of 10 µg/ml BSA (lanes 2, 4, 6, 8) or hCG (lanes 1, 3, 5, 7) and in the presence (lanes 1, 2, 5, 6) or absence (lanes 3, 4, 7, 8) of 5 mM GTP in a reaction mix prepared as described in Materials and Methods. For the remainder of details, see legend in panel A. Equivalent results were obtained in two separate experiments.

 


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Figure 2. Immunoprecipitation of Bacterial Toxin-Catalyzed ADP-Ribosylated Membrane Proteins

Membranes (100 µg in lanes 1 and 3; 250 µg in lanes 2, 4, and 5) were incubated at 30 C for 30 min in the presence of either PTX (2. 5 µg/ml, lanes 1, 2, and 5) or CTX (50 µg/ml CTX, lanes 3 and 4) and in the presence of 10 µg/ml hCG in a reaction mix prepared as described in Materials and Methods. Membranes were then either pelleted immediately (lanes 1 and 3) or incubated with anti-Gi (antiserum 117) (lane 2) or anti-hCG (B105) (lanes 4 and 5) for subsequent immunoprecipitation as described in Materials and Methods. Samples were subjected to 10.5% SDS-PAGE, and this figure represents an autoradiogram of the dried gel. For the remainder of details see legend to Fig. 1Go. Equivalent results were obtained in three separate experiments. B, [32P]AAGTP-labeled membrane proteins immunoprecipitated with anti-LH/CG receptor monoclonal antibody. Membranes (200 µg) were incubated with 1.0 µg/ml hCG for 20 min in a reaction mix as described in Materials and Methods. Membrane proteins were immunoprecipitated using either anti-LH/CG receptor antibody, LHR 38 (lane 1), or normal mouse serum (lane 2) 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. Molecular masses of labeled proteins are indicated at the left and were calculated based on the migration of protein standards, which are shown at the right. This result is representative of two experiments.

 
It has been established that in order for Gi to serve as an optimal substrate for ADP ribosylation by PTX, it should be in its heterotrimeric conformation (26, 27). Upon receptor activation, receptor-coupled Gi subunits uncouple, rendering {alpha}i a poorer substrate for ADP ribosylation by PTX and resulting in decreased labeling of {alpha}i with [32P]NAD. Incubation of follicular membranes in the presence of GTP resulted in an approximately 2-fold increase in the ADP ribosylation of Gi when compared with incubations in the absence of guanine nucleotides (Fig. 1AGo, lanes 5–8; Fig. 1BGo, lanes 1–4). However, PTX-catalyzed ADP ribosylation of Gi did not appear to be affected, i.e., was not reduced, by LH/CG receptor activation in this 30-min incubation (Fig. 1AGo, lanes 5–8; Fig. 1BGo, lanes 1–4). A time course of PTX-sensitive ADP ribosylation was also performed in the presence or absence of hCG. We observed that ADP ribosylation of Gi was decreased in the presence of agonist, especially at the earliest times of incubation, whether or not exogenous GTP was included in the incubations (Fig. 3Go). This result is consistent with LH/CG receptor activation of a portion of the Gi in follicular membranes.



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Figure 3. Time Course of PTX-Catalyzed ADP Ribosylation of Membrane Proteins

Membranes (100 µg protein) were incubated at 30 C for varying time periods (1–30 min) in the presence of PTX (2. 5 µg/ml) and 5 mM GTP, in the presence or absence of 10 µg/ml hCG (as indicated), in a reaction mix as described in Materials and Methods. The dried gel was exposed to x-ray film with an intensifying screen for 2 days. Equivalent results were obtained in two separate experiments.

 
When membranes were incubated with CTX and [32P]NAD in the presence of GTP, we observed the hCG-independent ADP ribosylation of a 45-kDa band as well as a 48/50-kDa doublet (Fig. 1AGo, lanes 1 and 2). Equivalent results (i.e., hCG-independent CTX-catalyzed ADP ribosylation of the 48/50 and 45-kDa bands) were obtained when membranes were incubated with GDP (10 µM, not shown). Immunoblotting of membrane proteins using anti-Gs{alpha} (U-584 antiserum) showed immunoreactive bands that also migrated at 45 and 48 kDa (Fig. 4Go, lane 2). When incubations with CTX were performed in the absence of added guanine nucleotides, we detected a slight hormone-dependent increase in ADP ribosylation (~1.4-fold over incubation performed in the absence of hCG or basal levels) of the 45-kDa band as well as a more distinct increase in labeling (~2.5-fold over basal levels) of the 48/50-kDa doublet in the presence of hCG (Fig. 1AGo, lanes 3 and 4). A 40-kDa band was also ADP-ribosylated by CTX in the absence, but not in the presence, of GTP (Fig. 1AGo, lanes 3 and 4). CTX-catalyzed ADP ribosylation of the 40-kDa protein was often slightly increased by hCG, consistent with coupling of the activated LH/CG receptor to this protein.



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Figure 4. Migration of CTX-Catalyzed ADP-Ribosylated Membrane Proteins Compared with Migration of Immunoreactive Gs{alpha}, Gq/11{alpha}, and Gi{alpha} in Follicular Membranes

For lane 1, membranes (100 µg) were incubated at 30 C for 30 min in the presence of 10 µg/ml hCG and 50 µg/ml CTX, as described in Materials and Methods, pelleted, and boiled in SDS sample buffer. For lanes 2–4, the same set of membranes was boiled in SDS sample buffer. Sample proteins were resolved on a 10.5% SDS-polyacrylamide gel and transferred onto nytran. Lanes 2–4 were then subjected to immunoblot analysis using either anti-Gs{alpha} (antiserum U-584), anti-Gq/11{alpha} (antiserum B6T), or anti-Gi{alpha} (UBI) as the primary antibodies. Antiserum U-584 is consistently immunoreactive with the 45-kDa short form and 48/50 kDa-long form of Gs{alpha}, antiserum B6T immunoreacts with the 42/43 kDa Gq/11 proteins, and UBI’s anti-Gi is immunoreactive to the 40 kDa Gi band. Note that the 48/50-kDa doublet and the 40-kDa band are faint in lane 1; however, these bands are usually distinctly radiolabeled (see Fig. 1AGo, lane 4, and Fig. 6BGo, lanes 4, 6, 8, 10, 12). Molecular masses of the proteins are indicated at the left and were calculated from migration of protein standards. Equivalent results were obtained in two separate experiments.

 
This smaller CTX-catalyzed ADP-ribosylated band migrated at the same molecular mass as PTX-sensitive Gi (Fig. 1AGo, compare lanes 3 and 4 to lanes 5–8), which suggests that this CTX-labeled 40 kDa protein most likely represents Gi. To confirm the identity of the 40-kDa band, membranes were incubated with CTX and immunoprecipitated using anti-Gi{alpha} antibody. As shown in Fig. 5AGo, the 40-kDa CTX-catalyzed ADP-ribosylated band was immunoprecipitated by anti-Gi antibody (as indicated by the arrow in lane 3). This anti-Gi{alpha} antibody also immunoprecipitates a portion of the CTX-labeled 45-kDa band (shown below to be the short form of Gs{alpha}3).



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Figure 5. Immunoprecipitation of CTX-Catalyzed ADP-Ribosylated Membrane Proteins Using G Protein-Specific Antisera

A, Immunoprecipitation of CTX-catalyzed ADP-ribosylated membrane proteins using anti-Gi{alpha}. Membranes (100 µg in lane 1; 250 µg in lanes 2 and 3) were incubated at 30 C for 30 min in the presence of CTX (50 µg/ml CTX) and 10 µg/ml hCG using [32P]NAD as described in Materials and Methods. Membranes were then either pelleted immediately (lane 1) or immunoprecipitated using anti-Gi (antiserum 117) or preimmune (PI) sera (lanes 2 and 3). Samples were boiled and subjected to 10.5% SDS-PAGE, and this figure represents an autoradiogram of the dried gel. Molecular masses of labeled proteins are indicated at the left and were calculated based on the migration of protein standards shown at right. Equivalent results were obtained in two separate experiments. B, Immunoprecipitation of CTX-catalyzed ADP-ribosylated membrane proteins using anti-Gs{alpha}. Membranes (100 µg in lane 1; 250 µg in lanes 2–4) were incubated at 30 C for 30 min in the presence of CTX (50 µg/ml CTX) and 10 µg/ml hCG using [32P]NAD. Membranes were then either pelleted immediately (lane 1) or immunoprecipitated using anti-Go (antiserum 9072), preimmune (PI) sera, or anti-Gs (antiserum 1190) (lanes 2–4). For the remainder of details, see legend in panel A. Equivalent results were obtained in three separate experiments.

 
Because the CTX-catalyzed ADP-ribosylated 45- and 48/50 kDa proteins migrated at the same molecular mass as bands that were immunoreactive with anti-Gs{alpha} (antiserum U-584) (Fig. 4Go), we determined whether the CTX-catalyzed radiolabeled bands indeed represented the long and short forms of Gs{alpha}. Membranes were incubated with [32P]NAD and CTX in the absence of GTP, since these conditions appeared to be optimal for the ADP ribosylation of the 45-, 48/50-, and 40-kDa proteins in the same sample, and Gs{alpha} was then immunoprecipitated with anti-Gs{alpha} antisera (antiserum 1190). Both 45- and 48/50-kDa CTX-catalyzed ADP-ribosylated bands, representing the short and long forms of Gs{alpha}, were immunoprecipitated using the anti-Gs{alpha} antiserum (Fig. 5BGo, lane 4). Anti-Go antibody (antiserum 9072), as well as preimmune sera, served as negative controls (Fig. 5BGo, lanes 2 and 3).

In view of the clear dependence on hCG of CTX-catalyzed ADP ribosylation of the long form Gs{alpha} in a 30-min reaction, a time course of CTX-catalyzed ADP ribosylation was performed. When GTP was added to the incubations, both the short and long forms of Gs{alpha} were increasingly ADP-ribosylated with increasing time of incubation, exhibiting 2.3- and 4-fold increases at 30 min relative to levels at 1 min of incubation, respectively, but in a hormone-independent manner (Fig. 6AGo). When incubations were conducted in the absence of GTP (Fig. 6BGo), a slight hCG-dependent increase in the ADP ribosylation of the 45-kDa short form of Gs{alpha} was detectable as early as 1 min (1.5-fold over basal levels) which persisted for at least 30 min (1.4-fold over basal levels). However, more dramatic hCG-dependent labeling of the 48/50 kDa long form of Gs{alpha} was noted by 1 min (3.6-fold over basal levels) and lasted at least 30 min (2.5-fold over basal levels). Like labeling of the Gs{alpha} forms, CTX-catalyzed ADP ribosylation of Gi at 40 kDa increased with incubation time (Fig. 6BGo). Human CG promoted a slight increase in ADP ribosylation of Gi, especially at the early incubation times. When the same study was performed using the hCG antagonist, deglycosylated hCG (dhCG), instead of hCG, no change was observed in the level of ADP ribosylation of the short or long forms of Gs{alpha} or Gi{alpha} between membranes incubated in the absence of dhCG compared with membranes incubated for the same amount of time with dhCG (not shown). This result confirmed that ADP ribosylation of Gs{alpha} and Gi catalyzed by CTX in the absence of GTP was specific for hCG-dependent receptor activation.



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Figure 6. Time Course of CTX-Catalyzed ADP Ribosylation of Membrane Proteins

Membranes (100 µg protein) were incubated at 30 C for varying time periods (1–30 min) in the presence of CTX (50 µg/ml), in the presence (A) or absence (B) of 5 mM GTP, and in the presence or absence of 10 µg/ml hCG, as indicated and as described in Materials and Methods. Molecular masses of labeled proteins are indicated at the left and were calculated based on the migration of protein standards (shown at right). Equivalent results were obtained in three separate experiments.

 
We next determined whether CTX-catalyzed ADP ribosylation of the long and short forms of Gs{alpha} in the absence of GTP was dependent on the concentration of hCG in incubations. As shown in Fig. 7AGo (lanes 7–12), increased labeling of both short and long forms of Gs{alpha} was observed with increasing concentrations of hCG while labeling of Gi was hCG-independent in this 30-min incubation. Membranes incubated with CTX in the presence of GTP (Fig. 7AGo, lanes 1–6) exhibited only hormone-independent labeling of Gs as predicted from results shown in Figs. 1Go and 6AGo. The graphs in Fig. 7BGo quantify the level of ADP ribosylation of the long and short forms of Gs{alpha} from three separate hCG dose-response studies performed in the absence of GTP.



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Figure 7. Human CG Dose-Dependence of CTX-Catalyzed ADP-Ribosylated Follicular Membrane Proteins

A, hCG dose-dependence of CTX-catalyzed ADP-ribosylated membrane proteins. Membranes (100 µg protein) were incubated at 30 C for 30 min in the presence of either 10 µg/ml BSA (lanes 1 and 7) or indicated concentration of hCG in the presence or absence of 10 µM GTP, as indicated, in a reaction mix containing CTX (50 µg/ml) as described in Materials and Methods. Molecular masses of labeled proteins are indicated at the left and were calculated based on the migration of protein standards. Equivalent results were obtained in three separate experiments. B, Quantification of levels of ADP-ribosylated Gs(long) and Gs(short) upon incubation with increasing levels of hCG. Levels of radiolabel incorporated into the long and short forms of Gs from autoradiograph in panel A were quantified by PhosphoImager (Molecular Dynamics, Sunnyvale, CA) and graphed as means ± SEM.

 
As it is not known whether activated LH/CG receptor stimulates PLC in physiologically relevant cellular models via a PTX-sensitive G protein, as is the case with LH/CG receptor transfected into L cells (24), or via a PTX-insensitive G protein (19), which is likely Gq/11 (see Discussion), we also performed an immunoprecipitation study using an anti-Gq/11 antibody to determine whether Gq/11 is ADP-ribosylated by CTX in an hCG-dependent manner in porcine ovarian follicular membranes. Although Gq/11{alpha} protein at 42/43 kDa was detected in immunoprecipitates, we were unable to detect any ADP ribosylation of this protein in incubations conducted in the absence or presence of hCG and/or GTP (not shown). Thus, we hypothesized that Gq/11 was not a good substrate for CTX under the conditions used in our assay. To further investigate this, membranes from SF9 cells that overexpress both Gq and the muscarinic acetylcholine receptor (mAChR), which signals via Gq/11, were incubated in the presence of CTX and in the absence or presence of carbachol. Despite the presence of Gq/11 protein in the membranes (Fig. 8Go, lane 1), no CTX-catalyzed ADP-ribosylated protein at 42/43 kDa was immunoprecipitated by the anti-Gq/11 antibody from SF9 cell membranes (Fig. 8Go, lanes 2 and 3). From these results we conclude that Gq/11 is not an efficient substrate for CTX-catalyzed ADP ribosylation under the conditions used in these studies.



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Figure 8. Immunoprecipitation of CTX-Catalyzed ADP-Ribosylated SF9 Cell Lysate Proteins Using anti-Gq/11{alpha}

Lysate (250 µg in lanes 2 and 3) was incubated at 30 C for 30 min in the presence of CTX (50 µg/ml) and in the presence or absence of 1 mM carbachol using [32P]NAD in a reaction mix prepared as described in Materials and Methods. Proteins were immunoprecipitated using anti-Gq/11 (C-19; Santa Cruz Biotechnology) as described in Materials and Methods. For lane 1, cell lysate (50 µg) was boiled in SDS sample buffer and run with samples in lanes 2 and 3 on the same 10.5% SDS-polyacrylamide gel. Proteins were transferred onto nytran, and immunoblot analysis was performed on lane 1 as described in Materials and Methods using anti-Gq/11. Nytran containing lanes 2 and 3 was dried and exposed to film. Molecular mass of the labeled protein is indicated between lane 1 and lanes 2 and 3.

 
Coimmunoprecipitation of Gs{alpha} and Gi{alpha} with the LH/CG Receptor
Although our results suggest that the majority of the long form of Gs{alpha} and a portion of the short form of both Gs{alpha} and Gi are functionally associated with the LH/CG receptor, based on the hCG dependence of the CTX-catalyzed ADP ribosylation of Gs{alpha} and Gi and the PTX-catalyzed ADP ribosylation of Gi, these results do not provide any direct evidence that the LH/CG receptor physically interacts with Gs or Gi. To obtain more direct evidence for an association of the LH/CG receptor with G proteins, we used an anti-hCG monoclonal antibody, B105, which has previously been shown by our laboratory to immunoprecipitate hormone-activated LH/CG receptor (25). B105 was used to immunoprecipitate the LH/CG receptor from membranes with the idea that if receptor is physically associated with one or more G protein(s), then B105 should be able to immunoprecipitate the receptor/G protein(s) complex, as was shown for the muscarinic acetylcholine receptor (28). Follicular membranes were incubated with hCG in the absence of GTP and in the presence of CTX and [32P]NAD, since these conditions were optimal for the ADP ribosylation of both forms of Gs and of Gi, or in the presence of PTX. Both the 45-kDa and 48/50-kDa Gs{alpha} forms were immunoprecipitated by B105 (Fig. 2AGo, lane 4). Based on the amount of membrane protein that was labeled with CTX (see legend to Fig. 2Go) and the ability of the anti-hCG antibody to immunoprecipitate 50% of activated LH/CG receptor (25), approximately 40% of the CTX-labeled Gs{alpha} is being immunoprecipitated with the LH/CG receptor. ADP-ribosylated Gi from membranes incubated with either CTX or PTX was also immunoprecipitated with the activated LH/CG receptor (Fig. 2AGo, lanes 4 and 5). Based on the amount of membrane protein that was labeled with CTX or PTX and the efficiency of immunoprecipitation of the LH/CG receptor by the anti-hCG antibody (25), approximately 7% of Gi{alpha} is immunoprecipitated with the activated LH/CG receptor.

Because the amount of Gi{alpha} that was physically associated with the LH/CG receptor was low (7%), a similar immunoprecipitation experiment was performed using the anti-LH/CG receptor antibody, LHR38 (29), after membrane proteins were incubated with the radiolabeled, nonhydrolyzable, photoaffinity GTP analog, P3-(4-azidoanilido)-P1 5'-GTP ([32P]AAGTP) (Fig. 2BGo, lane 1). Indeed, anti-LH/CG receptor antibody immunoprecipitated Gi{alpha} (and Gs{alpha}) along with the LH/CG receptor. Normal mouse serum was used as a negative control and was unable to immunoprecipitate any [32P]AAGTP-bound proteins (Fig. 2BGo, lane 2).


    DISCUSSION
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Although it is well established that stimulation of the LH/CG receptor by agonist results in the subsequent activation of its effector enzyme, adenylyl cyclase (18), several groups have recently shown that agonist-stimulated LH/CG receptor can also activate PLC in a number of cellular models (20, 21, 22, 23, 30). Davis and co-workers (20, 21, 22, 23) reported that hCG can mobilize phosphoinositides and increase [Ca2+]i in luteal and granulosa cells (22, 31). Recent studies using the murine LH/CG receptor transfected into L cells have shown that activated LH/CG receptor stimulates both adenylyl cyclase (leading to increases in cAMP) and PLC (leading to the formation of inositol phosphates and elevations in [Ca2+]i) (19, 24). The cloned dog TSH receptor has also been shown to stimulate the generation of both cAMP and inositol phosphates, so the ability of the LH/CG receptor to stimulate both adenylyl cyclase and PLC is not unique (32).

Because the agonist-occupied LH/CG receptor has been shown to activate two distinct effectors, it is possible that this receptor is coupled to more than one G protein. Indeed, Herrlich et al. (24) have recently shown that the LH/CG receptor transfected into L cells is capable of coupling both to Gi to activate PLC and to inhibit adenylyl cyclase activities and to Gs to stimulate adenylyl cyclase activity. Receptor coupling to more than one G protein is not an unprecedented phenomenon since various investigators have demonstrated multiple G proteins coupled to a single receptor based upon immunoprecipitation of receptor-G protein complexes using G protein peptide-specific antisera (33), agonist-dependent bacterial toxin labeling of G proteins (34), and agonist-dependent stimulation of effector activity (24, 35). In light of the potential ability of the LH/CG receptor to couple to more than one G protein in a physiological cell model, CTX was used under specific conditions as a means of identifying LH/CG receptor-associated G proteins in porcine ovarian follicular membranes.

Many studies designed to detect receptor association to specific G proteins have used transfected cell systems or proteins reconstituted into phospholipid vesicles. Although these studies have been invaluable in identifying with which G proteins a receptor may couple, it is difficult to determine physiological interactions utilizing these methods because overexpressing receptors or G proteins into a cell system can skew the actual associations taking place between these signaling proteins. We performed our toxin studies using porcine ovarian follicular membranes, with no exogenous signal-transducing molecules introduced to the system, with the expectation of identifying physiological interactions between G proteins and the LH/CG receptor.

Studies were performed first to confirm LH/CG receptor association with Gs. Our results provide evidence, for the first time, that the LH/CG receptor is directly associated with both the long and short forms of Gs in ovarian follicular membranes. Under conditions used in our studies, ADP ribosylation of the long form of Gs{alpha} requires agonist-dependent activation of the LH/CG receptor and is dependent on the concentration of agonist bound to the receptor.

Next we wanted to determine whether the LH/CG receptor in porcine follicular membranes was associated with any G proteins other than Gs, possibly to activate an effector distinct from adenylyl cyclase such as PLC. Early studies reported that hCG-dependent activation of PLC appeared to be PTX-insensitive in L cells transfected with the murine LH/CG receptor (19). As PLC can be activated by Gq{alpha} (36, 37, 38, 39), ß{gamma} from Gi or potentially Gs (40, 41, 42, 43), or by both Gq{alpha} and ß{gamma} from Gi (44) and because G proteins that modulate PLC in physiological cell models have not been elucidated, we determined whether LH/CG receptor activation promoted CTX-catalyzed ADP-ribosylation of Gq/11. However, we did not detect CTX-catalyzed ADP-ribosylation of Gq/11 in porcine follicular membranes under any of the labeling conditions that we used (± GTP, ± hCG) or in SF9 cells (± carbachol) overexpressing mAChR and Gq. This result does not eliminate the possibility that Gq/11 proteins are activated by the LH/CG receptor but rather indicates that Gq/11 proteins are not good substrates for CTX-catalyzed ADP ribosylation under the conditions used.

Recent studies have demonstrated that the murine LH/CG receptor transfected into L cells couples both to PLC and to adenylyl cyclase via Gi2{alpha} (24). In this model, PTX treatment augmented hCG-stimulated cAMP production and greatly reduced PLC activity. Our results provide evidence that the endogenous LH/CG receptor is also coupled to Gi in follicular membranes, albeit a relatively small percentage of the total Gi. This conclusion is based on the following observations. First, some of the Gi is ADP ribosylated in a hormone-dependent manner by CTX in the absence of added GTP (see Figs. 1AGo and 6BGo). Second, we consistently observed a distinct decrease of agonist-induced PTX-catalyzed ADP ribosylation of Gi in follicular membranes consistent with agonist-dependent activation of Gi (seen in Fig. 3Go). Third, the anti-hCG antibody, B105, was able to immunoprecipitate both CTX- and PTX-catalyzed ADP-ribosylated Gi from membranes, albeit at very low levels relative to the amount of Gi in membranes labeled with PTX or CTX and relative to the amount of Gs in membranes that was immunoprecipitated with this antibody. Fourth, anti-LH/CG receptor antibody, LHR38, immunoprecipitated [32P]AAGTP-bound Gi from membranes. Taken together, these data show that a portion of Gi in ovarian follicular membranes is functionally and physically associated with the LH/CG receptor. However, it is likely that only a small percentage of the membrane Gi is coupled to the LH/CG receptor, based on the following observations: hCG-dependent CTX-catalyzed ADP ribosylation of Gi was not observed in every experiment (compare Figs. 1Go and 6BGo with Fig. 7AGo), and when it was observed, it was not robust; the hCG-dependent reduction in PTX-catalyzed ADP ribosylation of Gi was consistent but weak; only a small percentage of membrane Gi (labeled with PTX or CTX) immunoprecipitated with the LH/CG receptor.

The ability of the anti-hCG antibody (B105) to precipitate LH/CG-activated receptor (25) and Gi ADP-ribosylated by PTX appears contradictory because PTX-catalyzed ADP ribosylation functionally uncouples Gi from the receptor. At this time we are unable to explain the mechanism behind this observation; however, it is possible that although most of the ADP-ribosylated Gi is uncoupled from the LH/CG receptor, a small percentage remains bound and is precipitated along with the activated LH/CG receptor using B105. Alternatively, PTX-stimulated Gi uncoupling from LH/CG receptor may be only functional and may not abolish association of the G protein with receptor. That Gi is a poorer substrate for PTX in the presence of hCG during the time course of ADP-ribosylation (seen in Fig. 3Go) supports the observed interaction between LH/CG receptor and Gi.

Several studies have been performed using CTX to ADP ribosylate PTX-sensitive G proteins in the absence of GTP (13, 16, 45). Investigators have observed that in order for {alpha}-subunits other than {alpha}s to serve as optimal substrates for CTX-catalyzed ADP ribosylation, the guanine nucleotide-binding pocket must be devoid of nucleotide. Consequently, this technique can be used to demonstrate specific receptor/G protein associations since GDP is released from the {alpha}-subunit upon receptor activation. When incubations are conducted in the absence of exogenous guanine nucleotides, the guanine nucleotide-binding domain then remains empty and the arginine substrate site for ADP ribosylation in at least {alpha}i proteins is accessible to CTX. Gs also has been shown to be an optimal substrate for CTX when coupled to an agonist-activated receptor in the absence of GTP (45). In porcine ovarian follicular membranes, hormone-dependent CTX-catalyzed ADP ribosylation of the long form of Gs was observed in the absence but not in the presence of either GTP or GDP. In the presence of guanine nucleotides, ADP ribosylation of the long form of Gs appears to be independent of hCG, suggesting that in the presence of GTP the CTX-sensitive arginine is equally accessible for ADP ribosylation in the absence or presence of hCG. In the absence of GTP, however, when LH/CG receptor is activated by hCG, GDP is released from the G protein and because GTP is not available to bind, the site is open and accessible for CTX-catalyzed ADP ribosylation. It appears that guanine nucleotides eliminate the hCG requirement for CTX-catalyzed ADP ribosylation. In contrast to the long form of Gs{alpha}, the 45-kDa short form of Gs{alpha} appears to be a good substrate for CTX-catalyzed ADP ribosylation whether or not the guanine nucleotide-binding site is filled with GTP or GDP or is empty. Gi also is a substrate for CTX-catalyzed ADP ribosylation in the absence of GTP. It appears that like the long form of Gs, the arginine substrate site of Gi is optimally accessible to CTX only when the guanine nucleotide-binding site is empty (seen in Fig. 1AGo, lanes 1–4).

It has long been recognized that the LH/CG receptor, unlike most other G protein-linked receptors, does not exhibit the typical guanine nucleotide dependence on agonist affinity (46, 47, 48). Thus, in the absence or presence of guanine nucleotides, the LH/CG receptor always exhibits high affinity for the agonist. Perhaps the basis for this observation lies in the sustained coupling, in the absence or presence of GTP and hormone, of the majority of the short form of Gs to the LH/CG receptor. Perhaps it is the long form of Gs that specifically couples to adenylyl cyclase. Further studies are required to elucidate potentially distinct roles for the long vs. the short forms of Gs in follicular membranes.


    MATERIALS AND METHODS
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Materials
Purified hCG (CR-127) was provided by the Center for Population Research, NICHHD. Materials were purchased from the following sources: [32P]NAD (30 Ci/mmol), Dupont-New England Nuclear (Boston, MA); [125I]donkey anti-rabbit IgG (100 µCi/ml), Amersham (Arlington Heights, IL); anti-Gi{alpha} (06–190; recognizes Gi{alpha}, Go{alpha} and Gt{alpha}) used in immunoblot analysis, UBI (Lake Placid, NY); anti-Gq/11{alpha} (C-19, specific for Gq{alpha} and G11{alpha}) used in immunoprecipitation studies, Santa Cruz Biotechnology, Inc. (Santa Cruz, CA); LHR38, Transbio; coarse Sephadex G25, Pharmacia Biotechnology Inc. (Piscataway, NJ); pertussis toxin, List Biological Inc. (Campbell, CA) ; electrophoresis purity reagents, Bio-Rad Laboratories (Richmond, CA); prestained mol wt markers, Diversified Biotech; nytran, Schleicher & Schuell, Inc. (Keene, NH); other reagents, Sigma (St. Louis, MO). Radiochemicals were used without further purification.

Preparation of Ovarian Follicular Membranes
Walls from follicles that were 6–12 mm in diameter were dissected from ovaries of nonpregnant pigs. A 10,000 x g membrane fraction was then prepared as described previously (49). Protein was determined by the method of Lowry et al. (50).

ADP Ribosylation of Porcine Large Follicle Membranes by CTX or PTX
Before addition to membrane preparations, CTX (2 mg/ml) was activated by incubation at 37 C for 15 min with an equal volume of 40 mM dithiothreitol (DTT). The sample was then desalted on a coarse Sephadex G25 column using 1 mg/ml BSA as the elution buffer. PTX (50 µg) was dissolved in 250 µl distilled water (GIBCO, Grand Island, NY) and activated by incubation with an equal volume of 50 mM DTT for 30 min at 30 C. Membranes (100 µg) were then incubated for 30 min at 30 C for standard assays (varying incubation times for time course studies) in a final volume of 100 µl containing 1 mM ATP, 15 mM thymidine, 5 mM ADP-ribose, 20 mM L-Arg-HCl, 5 mM DTT, 25 mM Tris-HCl, pH 7. 5, 20 µM [32P]NAD (1 Ci/mmol), 5 mM GTP, in the absence or presence of 10 µg/ml hCG, and in the presence of either 50 µg/ml CTX or 2. 5 µg/ml PTX. Membranes were then washed with 1 ml wash buffer (10 mM Tris-HCl, pH 7. 5, 1 mM EDTA) and resuspended in 50 µl SDS sample buffer. The samples were run on a 10.5% SDS-polyacrylamide gel (51). The gel was then either stained, destained, dried, and exposed to Kodak X-Omat AR film (Eastman Kodak, Rochester, NY) or transferred onto nytran overnight (4 C, 0.1 A).

Solubilization
Membranes (500 µg) were pelleted and resuspended to a final concentration of 5 µg/µl (100–150 µl per Eppendorf tube) in solubilization buffer (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 phenylmethylsulfonylfluoride, 50 mM benzamidine, 100 µM leupeptin, 5 µg/ml aprotinin, and 50 µg/ml soybean trypsin inhibitor). Membranes were allowed to stir slowly in solubilization buffer at 4 C for 60 min at which time they were diluted 10-fold in solubilization buffer in the absence of Triton so that the Triton concentration in the final volume was 0.1%. Nonsolubilized material was removed by centrifugation (100,000 x g, 60 min, 4 C). Supernatant was either used in Western blot analyses or immunoprecipitation studies.

Immunoprecipitation
Protein A-Sepharose (33%) (30 µl) was added to solubilized membranes and rotated at 4 C for 2 h to bind any nonspecific solubilized proteins. Sepharose was then pelleted and discarded, and the immunoprecipitating antibody was added (1:50 dilution of anti-G protein antibodies and preimmune sera, 75 µg B105) and allowed to rotate at 4 C overnight. If B105 was the primary antibody used, 100 µl rabbit anti-mouse IgG were added the next morning and the mixture was rotated for 2 h at 4 C. If a polyclonal antibody was used as the immunoprecipitating antibody (G protein antibodies and preimmune sera), no secondary antibody was added. Subsequently, 30 µl Protein A-Sepharose (33%) were added, and the incubation was allowed to continue for 2 h. Immunocomplexes were collected as Protein-A Sepharose pellets by centrifugation (10,000 x g, 10 min, 4 C). 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. The Sepharose pellet was then resuspended in 50–100 µl of 3-fold SDS sample buffer, vortexed briefly, and allowed to sit at room temperature for at least 30 min. Samples were placed in a boiling water bath (5 min) and centrifuged (10,000 x g, 10 min), and supernatant was subjected to SDS-PAGE (10.5% acrylamide, 50 mA).

Western Blot Analysis
To detect G proteins in porcine follicular membranes, 50–75 µg membranes (and up to 300 µg membranes to detect Gz) in SDS sample buffer were routinely run on a 10.5% SDS-polyacrylamide gel, after which the gel was transferred onto nytran (0.2-µm pore size) overnight (4 C, 0.1 A). Nytran was blocked in blocking buffer (50 mM Tris-HCl, pH 8. 5, 150 mM NaCl, 3% BSA, 0. 1% NaN3) for 3 h and then incubated with primary antibody in blocking buffer (overnight, 4 C, rotating). The next day, nytran was washed sequentially in wash buffer (50 mM Tris-HCl, pH 8. 5, 150 mM NaCl) for 10 min, detergent wash buffer (wash buffer, 0. 5% BSA, 0.1% Triton X-100) twice for 10 min, and wash buffer again for 10 min. Nytran was then incubated with [125I]donkey anti-rabbit IgG for approximately 3 h, rotating at room temperature. Then the wash sequence was repeated and nytran was air dried and exposed to Kodak X-Omat AR film.

[32P]AAGTP-Labeling of Porcine Ovarian Large Follicle Membrane Proteins
Porcine ovarian large follicle membranes were incubated in a volume of 40 µl incubation medium containing 0.5 µM [32P]AAGTP, 1.0 µg/ml hCG, 10 µM GDP, 31.25 mM 1,3-bis[tris(hydroxymethyl)-methylamino]propane, pH 7.2, 6.25 mM MgCl2, 0.5/1.25 mM EDTA/EGTA, 25 mM creatine phosphate, and 0.2 mg/ml creatine phosphokinase. Incubation was allowed to proceed at 30 C for 20 min, and 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 ß-mercaptoethanol. Samples were then centrifuged (20,000 x g, 5 min, 4 C), and membrane pellets were resuspended in 40 µl incubation medium that 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. Samples were then pelleted again and resuspended in solubilization buffer followed by immunoprecipation with LHR38 or normal mouse sera. This method is adapted from that of Rasenick et al. (61). Baculovirus expression of Gq SF9 cells were infected with viruses encoding mAChR (from Elliott Ross) and Gq (from James Garrison). Membranes were prepared and photolabeled as described by Popava et al. (62).


    ACKNOWLEDGMENTS
 
We would like to thank the following investigators for their generous gifts of antibodies, which were crucial to the studies performed in this manuscript: anti-hCG (monoclonal B105) from Dr. John O’Connor (Irving Center for Clinical Research, Department of Medicine, College of Physicians and Surgeons of Columbia University, New York, NY); anti-LH/CG receptor (LHR38) from Dr. Edwin Milgrom (Hormones et Reproduction, Hopital de Bicetre, Inserm Unite 135, Kremlin-Bicetre-France); anti-Gs{alpha} (antiserum 1190), anti-Gi{alpha} (antiserum 117), anti-Go{alpha} (antiserum 9072), and corresponding preimmune sera from Dr. David Manning (Department of Pharmacology, University of Pennsylvania School of Medicine, Philadelphia, PA); anti-Gs{alpha} (antiserum U-584) used for immunoblot analysis from Drs. Alfred Gilman and Susanne Mumby (University of Texas Southwestern Medical Center, Dallas, TX); anti-Gq/11{alpha} (antiserum B6T) used for immunoblot analysis from Dr. Tom Martin (University of Wisconsin, Madison, WI); anti-Gz{alpha} (antiserum P-961) from Dr. Patrick Casey (Department of Biochemistry, Duke University Medical Center, Durham, NC); anti-ras (antiserum Y13–259) from Dr. Frank McCormick (Onyx Pharmaceuticals, Richmond, CA); deglycosylated hCG was a gift from Dr. Robert Ryan (Mayo Medical School, Rochester, MN).


    FOOTNOTES
 
Address 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.

This research was supported by the US Department of Agriculture Grant NRICGP 9401432 (to M.H.D.) and USPHS Grant MH 39595 and The Council for Tobacco Research Grant 4089 (to M.M.R.).

1 Predoctoral appointee to the NIH Training Program in Reproductive Biology (T32-HD 07068–17). Back

2 The long form of Gs{alpha} usually is seen to resolve as a doublet upon CTX-catalyzed ADP-ribosylation but as a single band in immunoblots. Back

3 This anti-Gi antibody also immunoprecipitates [32P]-azidoanilido-GTP-labeled Gs{alpha} from porcine follicular membranes (R. M. Rajagopalan-Gupta, M. M. Rasenick, and M. Hunzicker-Dunn, manuscript in preparation). Back

Received for publication September 3, 1996. Revision received January 13, 1997. Accepted for publication February 18, 1997.


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