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Endocrinology Vol. 143, No. 4 1310-1317
Copyright © 2002 by The Endocrine Society

RECEPTORS

Regulation of RGS3 and RGS10 Palmitoylation by GnRH

Cecilia Castro-Fernández, Jo Ann Janovick, Shaun P. Brothers, Rory A. Fisher, Tae H. Ji and P. Michael Conn

Oregon Regional Primate Research Center and Department of Physiology and Pharmacology (C.C.-F., J.A.J., S.P.B., P.M.C.), Oregon Health and Science University, Portland, Oregon 97201; Department of Pharmacology (R.A.F.), University of Iowa College of Medicine, Iowa City, Iowa 52242; and Department of Chemistry (T.H.J.), University of Kentucky, Lexington, Kentucky 40506

Address all correspondence and requests for reprints to: P. Michael Conn, 505 NW 185th Avenue, Beaverton, Oregon 97006. E-mail: .


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Regulators of G protein signaling (RGS) play a pivotal role in cellular signal transduction. RGS3 or RGS10 were overexpressed in GGH3 cells [GH3 cells stably expressing the GnRH receptor (GnRHR)]. Responsiveness to a GnRH agonist was assessed because RGS proteins attenuate production of inositol phosphates (IP) and/or cAMP, molecules believed to be involved in GnRH signaling. In addition, site-directed mutagenesis of a potentially palmitoylated Cys60 residue of RGS10 was used to assess the significance of this site. We observed maximum inhibition of GnRH-stimulated IP responses by RGS3 and by the conserved domain of RGS10 at both 48 and 72 h after transfection, indicating their involvement in Gq{alpha} mediated signaling. Significantly diminished cAMP production was observed at all times when cells overexpressed the conserved domain of RGS10; no effect was observed with RGS3 on Gs{alpha}-mediated signaling. Palmitic acid incorporation into RGS3 was dependent on agonist occupancy of GnRHR, whereas palmitoylation of RGS10 was constitutive. Mutation of the conserved Cys60 residue of RGS10 obviated its negative regulatory action on GnRH-stimulated responses, indicating that this site is crucial for its activity on this system. This study is the first demonstration of a role for palmitoylation of this conserved Cys60 in mammalian G protein signaling.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
THE GnRH RECEPTOR (GnRHR) is a G protein-coupled receptor (GPCR) (1, 2, 3, 4). Inactive G proteins are heterotrimers; the active (monomeric) form of G{alpha}-subunit contains bound GTP (5, 6). G{alpha}-subunits have slow intrinsic GTPase activity, resulting in hydrolysis of GTP to GDP, and promoting reassociation of this subunit with the ß{gamma} dimers, reforming inactive heterotrimers (7, 8). Regulators of G protein signaling (RGS) negatively regulate G protein activity by accelerating GTP hydrolysis (9, 10, 11, 12) and thereby the duration and magnitude of signals generated through activation of heterotrimeric G proteins (13). Although RGS proteins vary in size, they share a common conserved domain of 120 amino acids (11). Previous studies demonstrated that RGS4, RGS10, and GAIP serve as GTPase-activating proteins (GAPs) for Gi{alpha}- and Gq{alpha}-subunits (but not for Gs{alpha}; 14) and that RGS3 is capable of negatively regulating Gq{alpha}, Gi{alpha} and Gs{alpha} by unknown mechanisms (15, 16, 17).

Many RGS proteins are membrane-associated (18, 19, 20) and have hydrophobic domains (21), but others are not always membrane bound, and it is unclear what governs this association. One attractive possibility is palmitoylation because both RGS4 and RGS10 contain a conserved Cys residue within the conserved RGS domain capable of becoming palmitoylated (22). Although mutation of two potential palmitoylation sites within the N-terminal domain of RGS4 (Cys2 and Cys12) does not alter the ability to inhibit signaling in yeast (19), and mutation of the conserved Cys within the conserved RGS domain of RGS10 affects the ability of this protein to inhibit G{alpha} signaling in Sf9 cells (22), this possibility has not been examined in mammals.

In GGH3 cells [GH3 cells, a lactotrope-derived cell line, stably expressing the rat GnRHR (23)], agonist occupancy of GnRHR activates PLC and IP turnover, as well as production of cAMP (4, 24, 25, 26).

In this study, we assessed the role of RGS3 and RGS10 on GnRH signaling pathways in GGH3 cells using overexpression, mutagenesis, palmitoylation, as well as the effect of NaF on regulation of RGS3. RGS3 and RGS10 incorporate [3H] palmitate in response to a GnRH agonist, this being the first demonstration that RGS3 can become palmitoylated. To examine if the conserved Cys60 (a potential site of palmitoylation) within the conserved RGS domain influences RGS10 signaling, we mutated this residue (RGS10, Cys60) to Ala, an amino acid incapable of being palmitoylated. The substitution totally abolished palmitoylation and RGS activity. Taken together, our observations provide novel mechanisms of RGS regulation.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Materials
Human RGS3 (hRGS3) and human RGS10 (hRGS10) (full length) cDNAs were prepared in the laboratory of one of the co-authors (Rory A. Fisher, University of Iowa College of Medicine, Iowa City, IA), and the conserved domain hRGS10 (amino acids 23–141) was generously provided by Thomas L. Wilkie (University of Texas Southwestern Medical Center, Dallas, TX).

DMEM was purchased from Irvine Scientific (Santa Ana, CA), and OPTI-MEM and Lipofectamine were purchased from Life Technologies, Inc. (Grand Island, NY). A GnRH agonist, Buserelin (D-tert-butyl-Ser6-des-Gly10-Pro9-ethylamide-GnRH), was a kind gift from Hoechst-Roussel Pharmaceuticals (Somerville, NJ); a GnRH antagonist, Antide, was obtained from the NIH Contraceptive Development Branch (Bethesda, MD). PCR reagents were purchased from Life Technologies, Inc. Restriction enzymes were purchased from Promega Corp. (Madison, WI). The RGS3 polyclonal antibody was made in our laboratory by immunizing rabbits with a keyhole limpet hemocyanin conjugate of the amino acid sequence for amino acids 361–373 from human RGS3. RGS10 polyclonal antibody RGS10 (C-20) (epitope mapping at the carboxyl terminus of RGS10 of human origin) was purchased from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA). All antibodies showed specificity for target proteins, showing a single band corresponding to the protein molecular weight by Western blots. Other reagents were obtained at the highest grade available from commercial sources.

Generation of the conserved domain RGS10-C60A and RGS10-C60A (full length) mutant constructs
Mutants of the conserved domain RGS10-C60A (amino acids 23–141) and RGS10-C60A (full length) were amplified by PCR using specific primers. For the RGS10 (full length) in pcDNA3.1 vector, the outside flanking primers used were T7, corresponding to the sequence within the T7 polymerase promoter of the pcDNA3.1 vector and the BGH reverse primer (BGH-rev), complementary to the sequence within the BGH polyadenylation signal of the pcDNA3.1 vector. For the conserved domain RGS10 in pQE60 vector, the outside flanking primers used were KY3 (5') TGC TTT GTG AGC GGA TAA CAA and KY4 (3') GCG TTC TGA ACA AAT CCA GAT. The codon 60 (TGT) of both RGS10 (full length) and conserved domain RGS10, was substituted for GCT using the sense primer G TTT TGG CTA GCA GCT GAA GAT TTT AAG and the antisense primer CTT AAA ATC TTC AGC TGC TAG CCA AAA C. The two separate fragments obtained from the two RGS10 (full-length and conserved domain) were gel purified and used as templates in a second PCR reaction with only the two outer primers, T7 and BGH-rev for RGS10 (full length); and KY3 and KY4 for the conserved domain RGS10. The mutant protein cDNAs were cut with KpnI- XbaI and NcoI-BamHI for RGS10-C60A (full length) and conserved domain RGS10-C60A, respectively; and subcloned into the same sites of pcDNA3.1 (full-length RGS10-C60A) and pQE60 (conserved domain RGS10-C60A) vectors. The cDNA clones were sequenced; the purity and identity of cDNAs were further verified by restriction enzyme analysis.

Western blots
SDS polyacrylamide gels (10% acrylamide) and Western transfers to nitrocellulose papers (Hoefer Scientific Instruments, San Francisco, CA) were performed as previously described (27). Polyclonal antibodies were used at a 1:1000 titer. Color was developed on Western blots using 4-chloro-1-naphthol (horseradish peroxidase) color development reagent (Bio-Rad Laboratories, Inc., Richmond, CA). Standards were color-stained proteins (rainbow markers, Amersham Pharmacia Biotech, Arlington Heights, IL) with the following molecular weights: myosin, 220 K; phosphorylase b, 97.4 K; BSA, 66 K; ovalbumin, 46 K; carbonic anhydrase, 30 K; trypsin inhibitor, 21.5 K; lysozyme, 14.3 K.

Overexpression of RGS proteins in GGH3 cells
Cells were maintained in growth medium [DMEM containing 10% FCS (Life Technologies, Inc.) and 20 µg/ml gentamicin (Gemini Bioproducts, Calabasas, CA)] in an atmosphere of 5% CO2 at 37 C. A total of 105 GGH3 cells/0.5 ml growth medium were plated in 24-well plates (Costar, Cambridge, MA). Twenty-four hours later, cells were transfected with 0.8 µg of cDNA for the sequences coding for hRGS3, hRGS10 (full length), conserved domain RGS10, RGS10-C60A (full length), conserved domain RGS10-C60A, or Lac Z (a common reporter gene, in this case employed as a negative control because its product, ß-galactosidase, is a very stable enzyme that does not interfere with the GnRHR-mediated signaling) using 2 µl of Lipofectamine in 0.25 ml OPTI-MEM/well (23, 28) and allowed to express for 48, 72, or 96 h following transfection.

Quantification of IP accumulation
Thirty, 54, or 78 h after transfection, cells were washed twice with DMEM/0.1% BSA containing 20 µg/ml gentamicin (DBG) to remove serum, and incubated in 0.5 ml/well DMEM (inositol free) containing 4 µCi/ml of [3H] inositol for 18 h at 37 C; after this period, the cells were washed twice with 0.5 ml DMEM (inositol free) containing 5 mM LiCl and stimulated with 0, 10-11, 10-9, or 10-7 M Buserelin/0.5 ml of the same DMEM (inositol free)/LiCl in the presence or absence of 10 mM NaF or 10-7 M Antide, for 2 h at 37 C. The treatment solutions were removed, and 1 ml of 0.1 M formic acid was added to each well. The cells were frozen, then thawed to disrupt cell membranes, and the IP accumulation was determined by Dowex anion exchange chromatography and liquid scintillation spectroscopy (29).

Quantification of cAMP release
Twenty-four, 48, or 72 h after transfection, cells were washed twice with DBG then stimulated with 0, 10-11, 10-9, or 10-7 M Buserelin in 0.5 ml DBG containing 0.2 mM methylisobutylxanthine (to prevent degradation of cAMP) in the presence or absence of 10-7 M Antide for 24 h at 37 C. After stimulation, the medium from each well was collected in tubes containing 50 µl of 10 mM theophylline. The samples were heated at 95 C for 5 min and RIA of cAMP was determined as described previously (1, 30).

Palmitoylation of RGS3 and RGS10 (full length) in GGH3 cells
A total of 500,000 cells/2 ml growth medium were plated in 6-well plates (Costar). Twenty-four hours later, cells were washed twice with DMEM, and incubated for another 3 h. Cells were labeled with [9,10-3H] palmitic acid (specific activity 30–60 Ci/mmol, 0.5 mCi/ml of DMEM) and stimulated with 10-7 M Buserelin for 40, 60, 90, 120, or 180 min. Subsequently, the labeling medium was aspirated and the cells were washed once with cold PBS, then 500 µl of cold radioimmune-precipitation assay buffer [50 mM HEPES, pH 7.4, 150 mM NaCl, 1% (vol/vol) Igepal (Sigma, St. Louis, MO), 0.5% (wt/vol) sodium deoxycholate, 1 mM EDTA, and 2.5 mM MgCl2] containing 2 µg/ml leupeptin and 2 µg/ml aprotinin. Insoluble material was removed by centrifugation at 12,000 x g for 3 min. Nonspecific binding was removed by rocking the cell extract in Eppendorf tubes containing 75 µl of protein A-Sepharose 6MB (Amersham Pharmacia Biotech, Piscataway, NJ), previously coupled to IgG from normal rabbit serum (for RGS3 samples) or normal goat serum (for RGS10 samples), for 30 min at 4 C. The cell extract was transferred to new tubes containing 75 µl protein A-Sepharose pre-coupled to our rabbit polyclonal antibody specific for RGS3 or to the goat polyclonal antibody specific for RGS10 and immunoprecipitated overnight at 4 C. The next day, the supernate was discarded, and the Sepharose beads were washed three times with cold radioimmune-precipitation assay buffer, and resuspended in 75 µl of SDS-PAGE sample buffer (in absence of reducing agents) and heated for 2 min at 100 C. The immunoprecipitates were resolved by 10% SDS-PAGE, fixed, and prepared for fluorography with Fluoro-Hance (RPI, Mt. Prospect, IL). The gels were dried and exposed to Kodak X-OMAT autoradiography film (Eastman Kodak Co., Rochester, NY) for 6 wk at -80 C (4).

Data analysis
Data are presented as the means of triplicate assay wells ± SEM of replicates in each experiment. The data were analyzed using one-, two-, and/or three-way ANOVA followed by Tukey’s HSD test, and by t test; P < 0.05 was considered as significant.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Time course for the effect of RGS3 or conserved domain RGS10 overexpression on IP accumulation
The effect of overexpression of RGS3 or conserved domain RGS10 on IP production in GGH3 cells in response to various concentrations of Buserelin (0, 10-11, 10-9, or 10-7 M) is shown (Fig. 1Go). Cells overexpressing RGS proteins responded to Buserelin stimulation in a dose-response manner. Either 48 or 72 h after transfection, RGS3 or conserved domain RGS10 significantly diminished IP production compared with control (Lac Z) values (P <= 0.005). After 96 h, there was not a significant diminished response with RGS3 or RGS10 compared with control values at the three doses examined (P > 0.05). The IP profiles for both RGS proteins and Lac Z had similar EC50 values (~0.1 µM). Full-dose response curves for RGS3 showed similar EC50 values (data not shown). When the RGS protein transfectants (72 h after transfection) were treated with 10-7 M Antide, this GnRH antagonist oblated IP production in response to all concentrations of Buserelin except 10-7 M, which was inhibited by 92% (data not shown). These results suggest that RGS proteins have an optimal expression 48 or 72 h after transfection. Expression of each of the proteins was confirmed by Western blot. All the bands obtained were of similar density, indicating that they all express at the same levels (Fig. 2Go). GnRHR expression in GGH3 cells is approximately 30% in transient transfections (31).



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  		Figure 1. Time course of the effect of RGS3 or conserved domain RGS10 overexpression on IP accumulation. Time course and dose-response curves for Buserelin-stimulated IP production from GGH3 cells overexpressing RGS3 or conserved domain RGS10, assayed as described in Materials and Methods. Forty-eight hours (upper panel), 72 h (middle panel), or 96 h (lower panel) after transfection, cells were stimulated for 2 h with the indicated concentrations of Buserelin, and IP production was measured. Data are presented as the mean ± SE of triplicate transfections. Three different experiments showed similar results.

 


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  		Figure 2. Expression of RGS3, RGS10 and RGS10-C60A. Expression of RGS3, RGS10, and RGS10-C60A was confirmed by Western blot, assayed as described in Materials and Methods. Three different experiments were performed and showed similar results.

 
Time course for the effect of RGS3 or conserved domain RGS10 overexpression on cAMP release
The time course for the effect of overexpression of RGS3 or conserved domain RGS10 on cAMP release from GGH3 cells is shown in Fig. 3Go. Control cells and those expressing RGS3 or conserved domain RGS10 responded to Buserelin stimulation in a dose-response manner. cAMP production from cells overexpressing conserved domain RGS10 was significantly diminished (compared with the control values) at all Buserelin doses examined and at the three different times after transfection (48 h, P < 0.002; 72 h, P < 0.05; 96 h, P < 0.03). In contrast, RGS3 did not show significant differences from the control in any dose or time studied (P > 0.05). On cells overexpressing the conserved domain RGS10, attenuation of cAMP accumulation occurred even in the absence of the agonist. The cAMP profiles for both RGS proteins and Lac Z had similar EC50 values (~0.1 µM). Seventy-two hours after transfection 10-7 M Antide totally inhibited cAMP release in response to Buserelin except at 10-7 M, in which case it was inhibited by a 20% (data not shown).



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  		Figure 3. Time course of the effect of RGS3 or conserved domain RGS10 overexpression on cAMP accumulation. Time course and dose- response curves for Buserelin-stimulated cAMP accumulation from GGH3 cells overexpressing RGS3 or RGS10 conserved domain, assayed as described in Materials and Methods. Twenty-four hours (upper panel), 48 h (middle panel), or 72 h (lower panel) after transfection, cells were stimulated for 24 h with the indicated concentrations of Buserelin, and cAMP accumulation was assayed by RIA. Data are presented as the mean ± SE of triplicate transfections. Three different experiments showed similar results.

 
Effect of NaF on RGS3 regulation
We transfected GGH3 cells with RGS3 and assessed the effect of pretreatment with 10 mM NaF, an activator of G proteins (Fig. 4Go). Cells responded to Buserelin stimulation in a dose-response manner 72 h after transfection, showing a significant diminution of IP production compared with Lac Z either with or without NaF (P <= 0.003). Activation of the G proteins in the presence of NaF showed a pronounced augmentation of the RGS3 protein activity by diminishing IP production more substantially when the cells were treated with NaF compared with untreated cells in response to 10-9 or 10-7 M Buserelin (Table 1Go). The IP profiles for RGS3 and Lac Z in absence or presence of NaF showed similar EC50 values (~0.1 µM). These results suggest that activation of G proteins with NaF leads to enhanced RGS3 activity.



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  		Figure 4. Effect of NaF on RGS3 regulation. Dose-response curves for Buserelin-stimulated IP production in GGH3 cells overexpressing RGS3, as described in Materials and Methods. Seventy-two hours after transfection, cells were stimulated for 2 h with the indicated concentrations of Buserelin in absence (A) or in presence (B) of 10 mM NaF; and IP production was measured. Data are presented as the mean ± SE of triplicate transfections. Three different experiments showed similar results.

 

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  		Table 1. Effect of NaF on hRGS3 regulation

 
Palmitoylation of RGS3 and RGS10 (full length) in GGH3 cells
The time-course of palmitoylation of RGS3 and RGS10 (full length) in GGH3 cells is shown in Figs. 5Go and 6Go, respectively. [3H] Palmitic acid incorporation into RGS3 (~57 kDa) was measurable only when the cells were stimulated with 10-7 M Buserelin (Fig. 5Go). [3H] palmitic acid incorporation into RGS3 was measurable at 10-11 M Buserelin and was not increased at doses up to 10-7 M. In contrast, for RGS10 (~20 kDa), incorporation is significantly enhanced by Buserelin only at the 120-min time point (Fig. 6Go). RGS10 migrates very close to the end of the gel due to its low molecular weight, placing the corresponding band near the dye front of the gel. Palmitoylation occurs after 40 min of incubation with [3H] palmitic acid for RGS3 and RGS10; shorter time points did not show measurable incorporation (data not shown). For both proteins, incorporation of [3H] palmitic acid was observed up to 180 min, the longest period of time studied. RGS3 showed a maximum label incorporation 60 and 90 min after stimulation, whereas for RGS10 the largest amount of incorporation was observed at 120 and 180 min after stimulation.



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  		Figure 5. Palmitoylation of RGS3. Time course of Buserelin-stimulated palmitoylation of RGS3 in GGH3 cells, assayed as described in Materials and Methods. Cells were treated with medium (M) or with 10-7 M Buserelin (B) for the indicated times in presence of [3H] palmitic acid. A, RGS3 was immunoprecipitated and visualized by autoradiography. B, Data are presented as the band intensity in arbitrary optical density units. Three different experiments were performed and showed similar results.

 


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  		Figure 6. Palmitoylation of RGS10. Time course of Buserelin-stimulated palmitoylation of RGS10 in GGH3 cells, assayed as described in Materials and Methods. Cells were treated with medium (M) or with 10-7 M Buserelin (B) for the indicated times in presence of [3H] palmitic acid. A, RGS10 was immunoprecipitated and visualized by autoradiography. B, Data are presented as the band intensity in arbitrary optical density units. Two different experiments were performed and showed similar results.

 
Effect of RGS10-C60A (full length) and the conserved domain RGS10-C60A mutants on IP and cAMP production
The effect of overexpression of RGS10-C60A (full length) and conserved domain RGS10-C60A on IP and cAMP production in GGH3 cells is shown in Fig. 7Go. All cells responded to Buserelin stimulation in a dose-dependent manner. Cells overexpressing both RGS10-C60A (full length) and conserved domain RGS10-C60A had similar IP responses compared with the control values (P > 0.05) but were significantly different from those measured with cells overexpressing RGS10 (full length) (P < 0.001) and conserved domain RGS10 (P = 0.034), when cells were stimulated with 10-7 M Buserelin. These results indicate that mutation of this conserved Cys60 limits the ability of RGS10 to negatively regulate this signaling pathway. cAMP release from cells overexpressing the conserved domain RGS10 was significantly diminished compared with the control values and with that from cells overexpressing RGS10 (full length) with all the Buserelin doses examined (P < 0.05). In contrast, cells overexpressing RGS10 (full length) and RGS10-C60A (full length) did not show significant differences from controls (P > 0.05), except for basal values (P = 0.01). Further, cells overexpressing only the conserved domain RGS10 had significantly diminished cAMP release compared with the mutated form of the conserved domain (RGS10-C60A) basal values (P = 0.024) and when cells were stimulated with 10-11 M Buserelin (P = 0.002). For the RGS10 (full length) and RGS10-C60A (full length), there were no significant differences with any dose studied (P > 0.05).



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  		Figure 7. Effect of RGS10-C60A (full length) or conserved domain RGS10-C60A on IP and cAMP production. Dose-response curves for Buserelin-stimulated IP production (upper panel) and cAMP release (lower panel) from GGH3 cells overexpressing Lac Z, RGS10 (full length), conserved domain RGS10, RGS10-C60A (full length) or conserved domain RGS10-C60A, assayed as described in Materials and Methods. Seventy-two or 48 h after transfection, cells were stimulated for 2 or 24 h with the indicated concentrations of Buserelin, and IP or cAMP production respectively, were measured. Data are presented as the mean ± SE of triplicate transfections. Three different experiments showed similar results.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
Previous studies demonstrated that RGS proteins negatively regulate G protein activity by accelerating the hydrolysis of GTP to GDP and thereby inactivating G{alpha} (9, 10, 11, 12, 13). The present study supports a role for two RGS proteins (RGS3, RGS10) in signaling pathways controlled by GnRHR. RGS3 protein involvement in regulation of GnRHR has been proposed previously (15). The present work shows that signaling is altered by RGS proteins, resulting in a significantly lower production of IP and/or cAMP and suggesting that the RGS proteins examined potentially play a role in GnRH-mediated signaling.

When cells overexpressed RGS proteins for 48 or 72 h, they showed significantly diminished IP production compared with control cells, suggesting that these periods of time allow significant RGS protein expression. RGS3 and RGS10 do not appear to require long periods of time to be expressed because they significantly inhibited IP responses even when overexpression times were as short as 48 h. Allowing the cells to express for longer periods (96 h) uncoupled RGS activity because there is not a significant diminution of GnRH- mediated IP production with any dose studied. This effect is probably due to degradation or inactivation of RGS proteins bound to Gq{alpha} after long periods of expression. These results also suggest that the RGS proteins studied suppress GnRH-stimulated Gq{alpha}-subunit mediated signaling (14, 15, 16). Activation of G proteins with NaF also showed significant augmentation of RGS3 protein action on the Gq{alpha} signaling pathway by diminishing IP production more substantially, compared with cells that were not similarly treated. This effect could represent PLC effector antagonism (32, 33). It has been demonstrated that fluoride plays a role in stabilizing the transition state for hydrolysis of protein-bound GTP, by formation of a high-affinity complex between GTPases and GAPs (34, 35).

It is well known that GnRH agonist stimulates modest cAMP release from GGH3 cells (36, 37), with the characteristic 3–4 log dose-response curve (36, 37, 38). Previous studies have shown that the highest cAMP response is observed 24 h after agonist stimulation in these cells (39). When cells were allowed to overexpress the conserved domain of RGS10, cAMP release in response to all Buserelin doses studied was significantly diminished compared with the control values. However, there is attenuation of cAMP accumulation even without Buserelin stimulation, suggesting that, although there is a probable action of RGS10 on the Gs{alpha}-subunit, this protein is inhibiting this signaling pathway with little effect on GnRH regulation. It could be acting as an effector antagonist, or it could be interacting with this subunit by some other mechanisms, which are independent on receptor activation. There is no previous evidence for an action of this RGS protein on the Gs{alpha}-subunit (14, 40).

In contrast, for RGS3 protein there is no measurable effect on the Gs{alpha} signaling pathway because there is only a very modest diminution of cAMP release that is not significantly different from control values. These results are similar to those reported previously, in which the RGS3 protein and a truncated form of RGS3 (RGS3T) containing the common conserved domain of the protein were examined. It was demonstrated that RGS3 does not have an effect on the Gs{alpha} signaling pathway in contrast to the truncated form, which had an inhibitory action on this subunit, probably due to a reduced level of expression of RGS3 compared with RGS3T (16).

RGS proteins are likely to be membrane-associated to regulate the GPCR systems. Some RGS proteins have hydrophobic domains that anchor them to the membrane. Other RGS proteins, like RGS10, are not membrane localized; however, they must maintain physical proximity to the membrane to perform optimal receptor dependent GAP activity (41). The mechanism by which this occurs is not well established. Previous studies have shown that some RGS proteins are capable of becoming palmitoylated (19, 20, 21, 22, 42). In this study, we demonstrate that immunoprecipitation of endogenous RGS3 and RGS10 showed [3H] palmitic acid incorporation. Palmitoylation of RGS3 only occurs in the presence of Buserelin, suggesting that palmitic acid turnover of RGS3 in GGH3 cells is dependent on GnRHR activation. In contrast, measurable palmitoylation of RGS10 occurs even in the absence of agonist. However, the incorporation is only slightly higher after stimulation with Buserelin and significantly higher only at the 120-min time point, suggesting that RGS10 palmitoylation is not totally dependent on stimulation with agonist. The role of palmitoylation in RGS protein function is not yet clear. It has been proposed that palmitoylation of some RGS proteins serve functions other than membrane anchoring, this being the case with RGS4 because mutants of this protein, unable to incorporate palmitic acid, are still membrane anchored (19). It is likely that palmitoylation of RGS proteins could be regulating the RGS GAP activity in a different manner, such as by interacting with G{alpha}-subunits (43), and this regulation seems to vary between RGS proteins.

A previous study demonstrated that a conserved Cys residue within the conserved RGS domain is palmitoylated in both RGS4 and RGS10 (22). Mutation of this site has been shown to effect the ability of RGS10 to inhibit G{alpha}-subunit signaling in Sf9 cells. Further, it has been demonstrated that mutation of two potential palmitoylation sites in RGS4 (Cys2 and Cys12) did not affect the ability to inhibit signaling in yeast (19). In the present study, mutational analysis of the conserved Cys60 residue was performed to assess the significance of this site on the function of RGS10 (full length) and conserved domain RGS10 on regulation of GnRHR signaling. Mutation of the palmitoylated Cys60 residue in RGS10 (full length) and conserved domain RGS10 prevented the inhibition of this RGS protein on Gq{alpha} signaling pathway. We observed no difference between IP levels when cells were overexpressed with the mutant, compared with control cells. These results suggest that the palmitoylation site of RGS10 plays an important regulatory role in this signaling pathway, probably the RGS10 has to be palmitoylated to perform an optimal GAP activity. This palmitoylation site also seems to play a role in the Gs{alpha} signaling pathway because the mutant form of the conserved domain RGS10 prevented the diminution of cAMP release but only in basal levels and when the cells were stimulated with the lowest Buserelin dose; this being further evidence of the little effect of conserved domain RGS10 on GnRH regulation of this signaling pathway.

Here we demonstrate that RGS proteins act as negative regulators of GnRH-stimulated G{alpha}-mediated signaling in a mammalian cell system. The ability of these proteins to regulate GnRHR signaling indicates their action on the different G{alpha} proteins involved in signaling through this receptor. We also show that palmitoylation of RGS3 is dependent on GnRHR activation, this being the first evidence for RGS3 palmitoylation. Further, we have shown that mutation of a conserved Cys60 residue of RGS10 plays a crucial role on mammalian GnRH-regulated G protein signaling. This is the first report of the significance of these likely palmitoylated Cys residues in mammalian G protein signaling in vivo and provides the basis of a mechanism of differential signal transduction.


   Acknowledgments
 
We thank Diane Ryles for helping with the manuscript.


   Footnotes
 
Sections of this study were presented in abstract form at the 83rd Annual Meeting of The Endocrine Society, June 2001, Toronto, Canada.

This work was supported by NIH Grants HD-19899, RR-00163, HD-18185, and Fogarty Grant TW/HD00668.

Abbreviations: DBG, DMEM/0.1% BSA containing 20 µg/ml gentamicin; GAP, GTPase activating protein; GGH3, GH3 cells stably expressing the rat GnRHR; GnRHR, GnRH receptor; GPCR, G protein-coupled receptor; IP, inositol phosphates; RGS, regulators of G protein signaling.

Received September 20, 2001.

Accepted for publication December 10, 2001.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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