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Endocrinology Vol. 140, No. 1 29-36
Copyright © 1999 by The Endocrine Society

ARTICLES

Epidermal Growth Factor-Induced Heterologous Desensitization of the Luteinizing Hormone/Choriogonadotopin Receptor in a Cell-Free Membrane Preparation Is Associated with the Tyrosine Phosphorylation of the Epidermal Growth Factor Receptor1

Marilyn L. G. Lamm2, Rajsree M. Rajagopalan-Gupta and Mary Hunzicker-Dunn

Department of Cell and Molecular Biology, Northwestern University Medical School, Chicago, Illinois 60611

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
 
Epidermal growth factor (EGF) attenuated hCG-stimulated adenylyl cyclase activity in rat luteal and follicular membranes. H7, an equipotent serine/threonine protein kinase inhibitor of cAMP-dependent protein kinases, cGMP-dependent protein kinases, and lipid-dependent protein kinase C, did not effect the ability of EGF to decrease hCG-responsive adenylyl cyclase activity, suggesting that a serine/threonine phosphorylation event catalyzed by these kinases was not critically involved in EGF-induced desensitization. Likewise, pertussis toxin-catalyzed ADP-ribosylation of a 40-kDa luteal membrane protein, which exhibited immunoreactivity with an antibody against Gi{alpha}, did not hinder the ability of EGF to attenuate hCG-stimulated adenylyl cyclase activity, indicating that Gi did not mediate EGF-induced desensitization. Rather, EGF-induced heterologous desensitization of LH/CG receptor in ovarian membranes was closely associated with the specific and prominent tyrosine phosphorylation of the 170-kDa EGF receptor. Both EGF-stimulated autophosphorylation of EGF receptor and EGF-induced LH/CG receptor desensitization were attenuated by genistein, a tyrosine kinase inhibitor. These results suggest that tyrosine phosphorylation of the 170-kDa EGF receptor is a necessary component of the signaling pathway in EGF-induced heterologous desensitization of the LH/CG receptor.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
THE LH/CG receptor belongs to a family of receptors characterized by the presence of seven membrane-spanning clusters of hydrophobic amino acids and by the ability to functionally couple to guanine nucleotide-binding (G) proteins to activate effectors such as adenylyl cyclase and phospholipase C (PLC) (1, 2). LH or its agonist hCG is well established to activate adenylyl cyclase in LH/CG receptor target cells (3). Also characteristic of G protein-coupled receptors is their ability to exhibit desensitization (4, 5). In the presence of saturating concentrations of LH or hCG, the ability of the LH/CG receptor to stimulate adenylyl cyclase becomes impaired, and cAMP production is reduced (6). Homologous LH/CG receptor desensitization in follicular membranes under cell-free conditions faithfully mimics the desensitization response of adenylyl cyclase in intact ovarian follicles to the preovulatory surge of LH or an ovulatory dose of hCG (6, 7, 8, 9, 10, 11, 12, 13).

The mechanism(s) that mediates homologous desensitization of the LH/CG receptor is not completely understood. Although the rat LH/CG receptor in a heterologous expression system is readily phosphorylated in an agonist-dependent manner (14, 15, 16), truncation of the cytoplasmic tail of the receptor to remove phosphorylated serine and threonine residues abolishes the rapid phase of adenylyl cyclase desensitization (t1/2 = <5 min) (14), but does not affect the slower component (t1/2 = ~20 min) of the desensitization response (14, 17). A similar delay in the rate of desensitization was observed when phosphorylated serine and threonine residues in the carboxyl-terminal tail of the LH/CG receptor were mutated to alanines (18). Desensitization in intact follicles (9, 11) and in ovarian follicular membranes (19, 20) exhibits a t1/2 of 15–20 min, consistent with the phosphorylation-independent slower phase of desensitization, and we have demonstrated that the LH/CG receptor in porcine follicular membranes is not phosphorylated under conditions that promote this slower phase of homologous adenylyl cyclase desensitization (20). We and others have established, however, that cell-free homologous LH/CG receptor desensitization exhibits a stringent requirement for micromolar concentrations of GTP (7, 12, 21, 22).

The LH/CG receptor has also been reported to undergo heterologous desensitization in response to epidermal growth factor (EGF) in rat luteal and MA-10 Leydig tumor cells and membranes (23, 24). The mechanism by which EGF attenuates hCG-stimulated adenylyl cyclase activity is not known, although it does not appear to involve changes in the activity of either the stimulatory G protein (Gs) or adenylyl cyclase, both of which remain fully responsive to direct activators such as sodium fluoride and forskolin, respectively (23, 24). EGF has also been shown to modulate signaling pathways to cAMP production in other cells. EGF enhances adenylyl cyclase activity in various cells, including bovine luteal cells (25) and myocytes (26, 27). In cardiac membranes, EGF increases adenylyl cyclase activity by activating Gs{alpha} (27) via EGF receptor-catalyzed tyrosine phosphorylation of Gs{alpha} (27, 28). Stoichiometric association between the EGF receptor and Gs{alpha} has been demonstrated both by yeast two-hybrid assays and with purified proteins, and this interaction is diminished with Gs{alpha} activation (26). Conversely, recent studies in various cells have shown that some G protein-coupled receptor agonists (such as thrombin and lysophosphatidic acid) promote ligand-independent EGF receptor activation and resulting signaling to downstream effectors (29, 30, 31, 32). Conceivably, homologous LH/CG receptor desensitization could be mediated by ligand-independent activation of the EGF receptor to attenuate hCG-stimulated adenylyl cyclase activity, although only phosphoserine has been detected in the phosphorylated rat LH/CG receptor upon agonist stimulation (16). In our continuing efforts to understand the mechanism(s) that mediates homologous LH/CG receptor desensitization in ovarian tissues, we investigated possible signaling pathways by which EGF promotes heterologous LH/CG receptor desensitization.


   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. Materials were purchased from the following sources: Pregnyl (hCG used for injections) from Organon, Inc. (West Orange, NJ); creatine phosphokinase from Calbiochem (La Jolla, CA); genistein and H7 from Alexis Laboratories (Woburn, MA); pertussis toxin (PTX) from List Biological Laboratories, Inc. (Campbell, CA); antiphosphotyrosine mouse monoclonal antibody, rabbit antiserum against Gi{alpha} subunit, and phosphorylated A431 proteins from Upstate Biotechnology, Inc. (Lake Placid, NY); electrophoresis purity reagents from Bio-Rad Laboratories, Inc. (Richmond, CA); prestained mol wt markers from Diversified Biotech (Newton Center, MA); Rainbow mol wt markers, enhanced chemiluminescence (ECL) Western blotting detection reagents, Hybond-C extra membrane, and [2,8-3H]cAMP (41 Ci/mmol) from Amersham (Arlington Heights, IL); [{alpha}-32P]ATP (10–50 Ci/mmol) and [32P]NAD (30 Ci/mmol) from New England Nuclear Research Products (Boston, MA); and human recombinant EGF (used in experiments with porcine follicular membranes), murine (m) EGF [purified to >=98% purity from submaxillary glands and containing 5 U endotoxin (and correspondingly 1–7 ng lipopolysaccharide)/100 µg EGF, according to specifications from Sigma Chemical Co., St. Louis, MO), PMSG, goat antimouse and antirabbit IgG peroxide conjugate, nucleotides, and most other reagents from Sigma Chemical Co.

Preparation of ovarian membranes
Female 21-day-old rats were obtained from Charles River Laboratories, Inc. (Portage, MI) and were maintained in accordance with the NIH Guide for the Care and Use of Laboratory Animals at the Northwestern University Animal Care Facility. All protocols were approved by the Northwestern University animal care and use committee. Superovulation in rats was induced via sc injection with 50 IU PMSG followed 54 h later with 50 IU hCG. Rats were killed 7 days after hCG injection. To obtain preovulatory follicles, rats were injected sc with 25 IU PMSG and killed 48 h later. Ovaries were homogenized in 1 mM EDTA and 10 mM Tris-HCl, pH 7.2, and a 20,000 x g membrane fraction enriched in adenylyl cyclase activity was prepared as previously described (12). 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 adenylyl cyclase activity was prepared (12). 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 (33).

Desensitization reaction and adenylyl cyclase assay
All incubations were performed 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), and 5 mM MgCl2. Unless otherwise stated, concentrations of reagents in the final reaction volume (of 50 µl) are given for this and subsequent reactions. Ovarian membranes were subjected to a two-stage reaction. In the desensitization reaction (stage 1), membranes (40–60 µg) were preincubated either at 30 C for varying periods of time (1–30 min) or at 4 C for 60 min in a 40-µl volume of IMRS containing 10 or 100 µM GTP, 2 mM ATP, and either 1 µg/ml BSA or hCG or the indicated concentrations of EGF. Immediately after the stage 1 preincubation, assay for adenylyl cyclase (stage 2) was preformed at 30 C for 5 min (10 min when preincubation was at 4 C) with the addition of a 10-µl volume containing the following components: 100 µM GTP, [{alpha}-32P]ATP (100–200 cpm/pmol), 1 mM [3H]cAMP (~10,000 cpm), and 10 µg/ml BSA or hCG, or, when indicated, 10 µM forskolin or ~20 µM AlF-4, the latter formed by adding 10 mM NaF and 20 µM AlCl2 (34). 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 (6). All determinations were run in triplicate. The presence of BSA in stages 1 and 2 was a measure of basal adenylyl cyclase activity, BSA in stage 1 and hCG in stage 2 was a measure of full hCG-stimulated adenylyl cyclase activity, hCG in stages 1 and 2 was a measure of hCG-induced desensitization of adenylyl cyclase activity, and EGF in stage 1 and hCG in stage 2 was a measure of EGF-induced desensitization of hCG-stimulated adenylyl cyclase activity. The percent reduction of full hCG-stimulated adenylyl cyclase activity above basal adenylyl cyclase activity, expressed as the percent desensitization, was used as a measure of the extent of LH/CG receptor desensitization.

Treatment with kinase inhibitors
Rat luteal membranes (40 µg) were preincubated at 30 C for 1 min in a 40-µl volume of IMRS containing 100 µM GTP and 2 mM ATP with or without 5 µg/ml EGF in the presence of 10 µM genistein [or 0.01% dimethylsulfoxide (DMSO) as vehicle control]. Thereafter, an assay for adenylyl cyclase activity was performed as described above. When membranes were analyzed for phosphotyrosine immunoreactivity, membranes were resuspended in electrophoresis sample buffer immediately after preincubation. In other experiments, rat luteal membranes (50 µg) were preincubated at 4 C for 60 min in a 40-µl volume of IMRS containing 100 µM GTP and 2 mM ATP with or without 10 µg/ml EGF in the absence and presence of 60 µM H7. Thereafter, an assay for adenylyl cyclase activity was performed as described above.

ADP-ribosylation with PTX
PTX (50 µg) was activated at 30 C for 30 min in a 100-µl volume of 25 mM Tris-HCl (pH 7.4), 0.5 mM EDTA, 25 mM sodium chloride, and 25 mM dithiothreitol. The activated toxin mixture was diluted 2.5-fold with 25 mM Tris-HCl (pH 7.4) and 0.1% BSA. Rat luteal membranes (0.3–1 mg) were preincubated for 15 min at 30 C in a 500-µl volume of buffer containing 25 mM 1,3-bis[Tris(hydroxymethyl)-methylamino]propane-HCl (pH 7.2), 0.5 mM EDTA, 1 mM EGTA, 1 µg/ml BSA, 100 µM GTP, 1 mM ATP, 10 mM thymidine, 10 or 20 µM NAD, and activated PTX (3 µg/ml). The reaction was stopped by dilution with cold 10 mM Tris-HCl (pH 7.2), and 1 mM EDTA, 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. Aliquots (20 µl; ~20–30 µg) of PTX-treated membranes were added to 20 µl IMRS containing 100 µM GTP and 2 mM ATP with or without 10 µg/ml EGF, and the stage 1 reaction was performed at 4 C for 60 min. Thereafter, the assay for adenylyl cyclase was performed as described above. In some experiments, [32P]NAD (~15 µCi) was used to ADP-ribosylate proteins.

SDS-PAGE and immunoblot procedures
SDS-PAGE was performed under reducing conditions using 5% stacking and 7.5% or 10% resolving polyacrylamide slab gels (35). Equal amounts of protein were loaded into gel lanes. For immunoblot analyses, proteins were transferred onto Hybond-C Extra (nitrocellulose) paper overnight at 4 C using 0.1-A constant current in buffer consisting of 25 mM Tris, 192 mM glycine (pH 8.3), and 20% methanol. After transfer, blots were incubated as follows: 1 h at 40 C in blocking buffer (PBS, pH 7.4, containing 0.1% Tween-20 and 10% nonfat milk), 1 h at room temperature with antiphosphotyrosine mouse monoclonal antibody or anti-Gi{alpha} rabbit antiserum, and 1 h at room temperature with horseradish peroxidase-conjugated goat antimouse or antirabbit antibodies. In between incubations, blots were washed several times with PBS-0.1% Tween-20 buffer. Blots were finally incubated with detection reagents for ECL according to the manufacturer’s instructions (Amersham). Blots were exposed to Kodak film (Eastman Kodak Co., Rochester, NY) for 0.5–5 min for autoradiographic analysis.

Statistics
Results (mean ± SEM) were analyzed using Student’s t test or Duncan’s one-way ANOVA (P <= 0.05) (36).


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
EGF-induced desensitization of hCG-stimulated adenylyl cyclase activity
Dose response. Adenylyl cyclase activity in rat luteal membranes was increased about 4-fold when membrane LH/CG receptors were activated by addition of a saturating concentration of hCG to a 5-min adenylyl cyclase assay (Table 1Go). Preincubation of membranes for 1 min at 30 C with hCG did not affect hCG-stimulated adenylyl cyclase activity (Table 1Go). However, as shown in Fig. 1Go, the increase in hCG-stimulated adenylyl cyclase activity above basal levels was significantly reduced (P < 0.05) when membranes were preincubated for 1 min at 30 C with EGF.1 Desensitization of hCG-stimulated adenylyl cyclase activity occurred with the lowest dose of EGF used with this experimental paradigm, and the extent of desensitization did not vary significantly with increasing concentrations of EGF, suggesting that the maximal effect on hCG-stimulated adenylyl cyclase activity was achieved with 100 ng/ml EGF. In agreement with previous reports (23, 24), EGF did not activate adenylyl cyclase activity (not shown) and did not affect the ability of aluminum fluoride (which directly activates Gs) or forskolin (which directly activates adenylyl cyclase) to stimulate cAMP production in luteal membranes (not shown).


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  		Table 1. Effect of preincubation with EGF on hCG-stimulated adenylyl cyclase (AC) activity in rat luteal membranes

 


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  		Figure 1. Effect of preincubation at 30 C with varying concentrations of EGF on hCG-stimulated adenylyl cyclase activity in rat luteal membranes. The experimental protocol is described in Table 1Go. BSA, hCG, or the indicated concentrations of EGF were added to stage 1 preincubation (1 min at 30 C); BSA or hCG was added to stage 2 adenylyl cyclase assay (5 min at 30 C) to measure basal and hCG-stimulated adenylyl cyclase activities. Basal adenylyl cyclase activities are shown in Table 1Go. Results represent hCG-stimulated (hCG added to stage 2 of incubation) minus basal (BSA added to stages 1 and 2) adenylyl cyclase activities and are the mean of triplicate determinations of four independent experiments ± SEM. *, Significantly different (P < 0.05) from other values. Numbers above bars represent the percent desensitization of hCG-stimulated adenylyl cyclase activity and were calculated as described in Table 1Go.

 
When rat luteal membranes were preincubated at 4 C for 60 min instead of at 30 C for 1 min, EGF promoted a dose-dependent increase in desensitization of hCG-stimulated adenylyl cyclase activity (Fig. 2Go, closed triangles). The requirement for higher doses of EGF and a longer incubation time (60 min) to attain maximal desensitization when the incubation was performed at 4 C compared with 30 C has been previously reported (24). EGF also promoted the dose- dependent desensitization of hCG-stimulated adenylyl cyclase activity in rat preovulatory follicular membranes (Fig. 2Go, open squares). By comparison, EGF did not promote desensitization of hCG-stimulated adenylyl cyclase activity in porcine preovulatory follicular membranes even at a dose of 10 µg/ml using human (Fig. 2Go, open circles) or murine (data not shown) EGF. EGF-induced desensitization of hCG-stimulated adenylyl cyclase activity in porcine luteal membranes was not examined, because porcine luteal membranes possess relatively few hCG-binding sites and exhibit diminished LH/hCG-stimulated adenylyl cyclase activity and minimal desensitization under cell-free conditions (13). Based on results presented in Figs. 1Go and 2Go, a saturating EGF concentration of 1 or 5 µg/ml was used in subsequent experiments.



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  		Figure 2. Effect of preincubation at 4 C with varying concentrations of EGF on hCG-stimulated adenylyl cyclase activity in rat follicular, rat luteal, and porcine follicular membranes. Membranes from rat large (preovulatory) follicles (rLF), rat corpora lutea (rCL), and porcine large (preovulatory) follicles (pLF) were preincubated (stage 1) with the indicated concentrations of murine EGF (rat tissues) or hEGF (porcine tissues) at 4 C for 60 min and then assayed for hCG-stimulated adenylyl cyclase activity (stage 2; 10 min for 30 C). The percent desensitization of hCG-stimulated adenylyl cyclase activity was calculated as described in Table 1Go. Results are the mean ± SEM from three or four independent experiments. *, Significantly different (P < 0.05) from 0.1 and 0.2 µg/ml EGF. **, Significantly different (P < 0.05) from all other values.

 
Time dependency. When the preincubation was performed at 30 C for 1 min, hCG-stimulated adenylyl cyclase activity was significantly (P < 0.05) reduced in membranes preincubated with 5 µg/ml EGF compared with that in control membranes preincubated without EGF (Fig. 3AGo). When preincubation was continued for 2, 5, or 10 min, hCG-stimulated adenylyl cyclase activity in EGF-preincubated membranes did not differ significantly (P > 0.05) from that in control membranes. In contrast to hCG-stimulated adenylyl cyclase activity in EGF-treated membranes, hCG-stimulated adenylyl cyclase activity in control membranes did not change significantly with increasing time of preincubation (Fig. 3AGo, open bars). Thus, EGF-induced desensitization of hCG-stimulated adenylyl cyclase activity in rat luteal membranes occurred very rapidly (within 1 min) at 30 C, but was not sustained (Fig. 3BGo). The rapid recovery of hCG-stimulated adenylyl cyclase activity in luteal membranes preincubated with EGF also indicates that EGF does not reduce LH/CG receptor numbers.



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  		Figure 3. Effect of preincubation at 30 C with EGF for varying periods of time on hCG-stimulated adenylyl cyclase activity in rat luteal membranes. Rat luteal membranes were preincubated without or with EGF (5 µg/ml) for 1–10 min at 30 C (stage 1). The assay for hCG-stimulated adenylyl cyclase activity (stage 2) was described in Table 1Go and Fig. 1Go. Results are the mean of triplicate determinations of three independent experiments ± SEM. *, Significantly different (P < 0.05) from other values. A, Values represent hCG-stimulated minus basal adenylyl cyclase activity. B, The percent desensitization of hCG-stimulated adenylyl cyclase activity was calculated as described in Table 1Go.

 
EGF-stimulated tyrosine phosphorylation
The binding of EGF to its receptor results in stimulation of the intrinsic tyrosine kinase activity of the receptor, which leads to autophosphorylation of the EGF receptor and phosphorylation of downstream signaling proteins such as PLC-{gamma} to activate protein kinase C (PKC) (37, 38, 39). Therefore, it was of interest to determine whether EGF-induced desensitization of the LH/CG receptor in rat luteal membranes involved the tyrosine kinase activity of the EGF receptor. To identify EGF-stimulated tyrosine phosphorylation of protein substrates, luteal membranes were incubated with or without EGF, and membrane proteins were separated by SDS-PAGE and analyzed for tyrosine phosphorylation in immunoblots with an antiphosphotyrosine antibody.

Preincubation with 5 µg/ml EGF for 60 min at 4 C increased the tyrosine phosphorylation predominately of a 170-kDa protein band (denoted by arrow) in rat luteal membranes (Fig. 4Go, compare lanes 1 and 2) and rat follicular membranes (lanes 3 and 4), but not in porcine follicular membranes (compare lanes 5 and 6). The migration of the 170-kDa protein was coincident with that of the tyrosine-phosphorylated EGF receptor obtained from A431 cells (Fig. 4Go, lane 7). Identical results were obtained when preincubation was conducted at 30 C for 1 min with 5 µg/ml EGF (not shown). Tyrosine phosphorylation of the EGF receptor was observed upon preincubation of rat luteal membranes at 30 C for 1 min with the lowest dose of EGF used (0.1 µg/ml; Fig. 5Go, lane 3), and the extent of phosphorylation did not differ with increasing concentrations of EGF (Fig. 5Go, lanes 4–7). Tyrosine phosphorylation of the EGF receptor was not stimulated when incubations were performed in the absence of EGF (Fig. 5Go, lane 2). Inclusion of hCG to activate the LH/CG receptor also did not stimulate the tyrosine phosphorylation of the EGF receptor (Fig. 5Go, lane 1; see Fig. 7BGo, lane 4). Unlike EGF-induced desensitization of hCG-stimulated adenylyl cyclase activity, which abated after 1 min, the extent of tyrosine phosphorylation of the EGF receptor did not differ with increasing time of incubation of luteal membranes with EGF at 30 C (Fig. 6Go), suggesting that the loss of the effect on hCG-stimulated adenylyl cyclase activity was not due to EGF receptor down-regulation or inactivation.



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  		Figure 4. Comparison of tyrosine phosphorylation of ovarian membrane proteins after incubation with EGF. For lanes 1–6, membranes (50 µg) from rat corpora lutea, rat large preovulatory follicles, and porcine large preovulatory follicles were incubated without or with 5 µg/ml EGF for 60 min at 4 C, mixed with SDS stop buffer, and loaded onto gels for SDS-PAGE. A431 cellular membranes containing phosphorylated EGF receptor (Upstate Biotechnology, Inc.) were added to lane 7. After SDS-PAGE and transfer of proteins to Hybond, membrane proteins were analyzed for tyrosine phosphorylation by immunoblot using antiphosphotyrosine antibody, as described in Materials and Methods. Molecular mass markers in kilodaltons are shown on the right. The arrow denotes the migration position of the 170-kDa EGF receptor. Identical results were obtained when membranes were incubated at 30 C for 1 min. Results are representative of four independent experiments.

 


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  		Figure 5. Tyrosine phosphorylation of rat luteal membrane proteins after incubation with varying concentrations of EGF. Rat luteal membranes (40 µg) were incubated at 30 C for 1 min (stage 1) in the absence (lane 2) or presence (lanes 3–7) of the indicated concentrations of EGF or in the presence of 1 µg/ml hCG (lane 1). Membrane proteins were separated by SDS-PAGE and analyzed for tyrosine phosphorylation as described in Fig. 4Go. The arrow denotes migration of the EGF receptor. Results are representative of three independent experiments.

 


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  		Figure 7. Effect of genistein on EGF-induced desensitization of hCG-stimulated adenylyl cyclase activity and EGF-stimulated tyrosine phosphorylation. A, Rat luteal membranes were preincubated at 30 C for 1 min (stage 1) with 5 µg/ml EGF in the presence of 0.01% DMSO (vehicle control) or 10 µM genistein (in 0.01% DMSO). The assay for hCG-stimulated adenylyl cyclase activity was described in Fig. 1Go. The percent desensitization was determined as described in Table 1Go. Results are the mean of triplicate determinations from three independent experiments ± SEM. *, Significantly different (P < 0.05) from other values. B, Rat luteal membranes were incubated without or with 5 µg/ml EGF, 1 µg/ml hCG, and 10 µM genistein, as indicated, for 60 min at 4 C. For details, see Fig. 4Go. The arrow denotes the migration position of EGF receptor. Results are representative of three independent experiments.

 


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  		Figure 6. Tyrosine phosphorylation of rat luteal membrane proteins after incubation with EGF for varying periods of time. Rat luteal membranes (40 µg) were incubated without or with 5 µg/ml EGF at 30 C for the indicated times. For details, see Fig. 5Go. Results are representative of three independent experiments.

 
Effect of the tyrosine kinase inhibitor, genistein
The following experiment was designed to determine whether the tyrosine kinase activity of the EGF receptor was required for EGF to attenuate hCG-stimulated adenylyl cyclase activity. As shown in Fig. 7AGo, inclusion of 10 µM genistein, a tyrosine kinase inhibitor (40), in the stage 1 incubation significantly decreased EGF-induced desensitization of hCG-stimulated adenylyl cyclase activity in rat luteal membranes. Treatment with genistein, however, did not affect either basal adenylyl cyclase activity (45.0 ± 4.2 and 45.2 ± 3.9 pmol cAMP formed/min·mg protein in the absence and presence of genistein, respectively; mean ± SEM; n = 3 experiments) or full hCG-stimulated adenylyl cyclase activity (202.3 ± 4.9 and 195.2 ± 15.5 pmol cAMP formed/min·mg protein in the absence and presence of genistein, respectively; mean ± SEM; n = 3 experiments). Correspondingly, genistein treatment abolished EGF-stimulated tyrosine phosphorylation of the EGF receptor (Fig. 7BGo).

Effect of the serine/threonine kinase inhibitor, H7
EGF-stimulated tyrosine phosphorylation may also lead to activation of serine/threonine kinases such as PKC via PLC-{gamma} (39, 41). To determine whether EGF-induced desensitization of hCG-stimulated adenylyl cyclase activity was mediated by serine/threonine phosphorylation, rat luteal membranes were incubated with 10 µg/ml EGF at 4 C for 60 min in the absence or presence of 60 µM H7, an equipotent inhibitor of cAMP-dependent protein kinase (PKA), cGMP-dependent protein kinases (PKG), and PKC (42). H7 did not inhibit the ability of EGF to induce desensitization of hCG-stimulated adenylyl cyclase activity (32.0 ± 1.9% and 35.9 ± 2.1% desensitization in the absence and presence of H7, respectively; mean ± SEM; n = 3 experiments).

Effect of ADP-ribosylation by PTX
PTX-catalyzed ADP-ribosylation of Gi family proteins inactivates receptor coupling to these G proteins (43, 44). Several EGF receptor-mediated actions involve PTX-sensitive G proteins, based on the ability of PTX to block EGF-stimulated responses (45, 46, 47, 48). Therefore, we determined whether PTX-sensitive G proteins also mediated EGF-stimulated heterologous desensitization of LH/CG receptor-stimulated adenylyl cyclase activity in rat luteal membranes. We first determined whether rat luteal membranes contained a G protein that could be ADP-ribosylated by PTX. Results show that treatment of luteal membranes with activated PTX in the presence of [32P]NAD caused the specific ADP-ribosylation of a protein that migrated at 40 kDa (Fig. 8AGo) and exhibited immunoreactivity with an antibody against Gi{alpha} on immunoblot analysis (Fig. 8BGo). However, treatment of rat luteal membranes with 3 µg/ml PTX did not inhibit the ability of EGF to induce desensitization of hCG-stimulated adenylyl cyclase activity (Fig. 8CGo). PTX treatment did not affect either basal adenylyl cyclase activity (42.3 ± 12.4 and 41.3 ± 7.5 pmol cAMP formed/min·mg protein in the absence and presence of PTX, respectively; mean ± range; n = 2 experiments) or full hCG-stimulated adenylyl cyclase activity (188.7 ± 48.6 and 184.0 ± 40.4 pmol cAMP formed/min·mg protein in the absence and presence of PTX, respectively; mean ± range; n = 2 experiments).



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  		Figure 8. Effect of PTX on EGF-induced desensitization of hCG-stimulated adenylyl cyclase activity. Rat luteal membranes were preincubated at 30 C for 30 min in the absence and presence of 3 µg/ml activated PTX in the presence of [32P]NAD (A) or NAD (B and C), as described in Materials and Methods. After reaction, membranes were washed and either resuspended in sample buffer and processed for SDS-PAGE (A and B) or preincubated at 4 C for 60 min (stage 1) with 10 µg/ml EGF and then assayed for hCG-stimulated adenylyl cyclase activity (C). A, Autoradiograph of gel containing [32P]NAD-incubated samples exposed to film at room temperature for 2 days. The arrow indicates the migration position of 40-kDa protein. B, Immunoblot analysis of samples incubated with NAD using antibody against Gi{alpha}; the arrow denotes the 40-kDa protein. Molecular mass markers are shown on the left. C, Percent desensitization of hCG-stimulated adenylyl cyclase activity in membranes preincubated with or without PTX, incubated with EGF, and assayed for adenylyl cyclase activity. The assay for hCG-stimulated adenylyl cyclase activity was described in Fig. 2Go. The percent desensitization was determined as described in Table 1Go. Results are the mean of triplicate determinations from two independent experiments ± range.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
EGF can promote an increase (25, 26, 27, 49) or a decrease (23, 24, 48, 50) or have no effect on agonist-stimulated adenylyl cyclase activity, depending on the cellular model. We have shown that EGF not only attenuates hCG-stimulated adenylyl cyclase activity in rat luteal membranes, in agreement with an earlier report (24), but also attenuates hCG-stimulated adenylyl cyclase activity in rat, but not porcine, preovulatory follicular membranes. EGF-induced desensitization of hCG-responsive adenylyl cyclase does not appear to involve changes in the activity of the adenylyl cyclase enzyme itself, as the direct activation of adenylyl cyclase by forskolin is not impaired by EGF (24) (see text).

We provide evidence that demonstrates that the tyrosine phosphorylation of the 170-kDa EGF receptor is necessary for EGF to induce heterologous LH/CG receptor desensitization. Both EGF-stimulated tyrosine phosphorylation of the EGF receptor and desensitization of hCG-responsive adenylyl cyclase activity in rat luteal membranes are inhibited by the tyrosine kinase inhibitor genistein. In rat follicular membranes where EGF also attenuates hCG-stimulated adenylyl cyclase activity, EGF promotes the tyrosine phosphorylation of the EGF receptor, whereas in the porcine follicular membrane model where EGF does not modulate adenylyl cyclase activity, EGF does not promote detectable tyrosine phosphorylation of the EGF receptor. The requirement for EGF receptor tyrosine phosphorylation is consistent with an earlier report demonstrating the requirement for ATP in the presence of EGF to promote heterologous adenylyl cyclase desensitization (24).

It is not known how the tyrosine kinase activity of the EGF receptor is involved in heterologous LH/CG receptor desensitization in a cell-free membrane model. Conceivably, the activated EGF receptor tyrosine kinase directly or indirectly via a cascade of phosphorylation events phosphorylates either the LH/CG receptor and attenuates its interaction with Gs or phosphorylates Gs{alpha} to attenuate its activation of adenylyl cyclase. The intracellular carboxyl-terminal region of the LH/CG receptor contains, in addition to serine and threonine residues, tyrosine residues that are potential sites for phosphorylation (1, 2). However, agonist-dependent LH/CG receptor activation has not been shown to promote detectable tyrosine phosphorylation of the LH/CG receptor based on phosphoamino acid analysis (16). In this report we show that hCG does not promote the ligand-independent activation of the EGF receptor in rat luteal membranes. These results suggest that homologous desensitization of the LH/CG receptor is not mediated by ligand-independent activation of the EGF receptor. However, it is not known whether EGF-dependent activation of the EGF receptor promotes tyrosine phosphorylation of the LH/CG receptor. It is unlikely that a serine/threonine phosphorylation event catalyzed by a second messenger protein kinase is involved in EGF receptor-mediated heterologous LH/CG receptor desensitization, because H7, an equipotent inhibitor of PKA, PKG, and PKC, does not inhibit the ability of EGF to decrease hCG-stimulated adenylyl cyclase activity. This idea is consistent with our earlier data showing that cell-free desensitization of hCG-stimulated adenylyl cyclase is unaffected by activation or inhibition of endogenous PKA, PKG, or PKC (51). Several studies have linked EGF actions with PTX-sensitive Gi proteins (43, 47). Based upon our data, however, Gi proteins do not appear to mediate the effect of EGF on LH/CG receptor desensitization. PTX-mediated ADP ribosylation of a 40-kDa luteal membrane protein that cross-reacts with an antibody against Gi{alpha} does not hinder the ability of EGF to attenuate hCG-stimulated adenylyl cyclase activity in rat luteal membranes. Likewise, PTX failed to inhibit the ability of EGF to attenuate the hCG-stimulated accumulation of cAMP in MA-10 Leydig cells (23).

Although EGF receptor activation leads to the apparent direct tyrosine phosphorylation and activation of Gs{alpha} in cardiac membranes (27, 28) and to activation of the type V adenylyl cyclase (52), the lack of an effect of EGF on fluoride-stimulated (Ref. 24 and this report) or on CTX-stimulated (24) adenylyl cyclase activities in rat luteal membranes suggests that the EGF effect in rat luteal membranes to attenuate hCG-stimulated adenylyl cyclase activity is upstream of Gs{alpha}. Thus, EGF may have interfered with the ability of the LH/CG receptor to interact fully with Gs, possibly by catalyzing phosphorylation of the LH/CG receptor.

To summarize, EGF attenuates the hCG-responsive adenylyl cyclase activity in rat luteal and rat follicular, but not porcine follicular, membranes. Although EGF-induced heterologous LH/CG receptor desensitization is not mediated by a serine/threonine phosphorylation event catalyzed by PKC, PKA, or PKG or by the inhibitory G protein Gi, the tyrosine phosphorylation of the EGF receptor is a necessary component of the signaling pathway in LH/CG receptor desensitization.


   Footnotes
 
1 This work was supported by USDA Grant NRICGP-9401432 (to M.H.D.). Back

2 Recipient of National Research Service Award HD-07348 from the NICHHD, NIH. Current address: Department of Urology, Northwestern University Medical School, Chicago, Illinois 60611. Back

Among all experiments with rat luteal membranes, EGF promoted a 30.6 ± 2.0% (n = 22; mean ± SEM of 22 independent experiments) attenuation (i.e. desensitization) of hCG-stimulated adenylyl cyclase activity above basal activity.

Received March 25, 1998.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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