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Endocrinology Vol. 139, No. 1 397-402
Copyright © 1998 by The Endocrine Society

ARTICLES

Redistribution of Gq/11{alpha} in the Pituitary Gonadotrope in Response to a Gonadotropin-Releasing Hormone Agonist1

Anda Cornea, Jo Ann Janovick, Dinesh Stanislaus and P. Michael Conn

Oregon Regional Primate Research Center (A.C., J.A.J., D.S., P.M.C.), Beaverton, Oregon 97006; and Department of Physiology and Pharmacology, Oregon Health Sciences University (D.S., P.M.C.), Portland, Oregon 97201

Address all correspondence and requests for reprints to: P. Michael Conn, Oregon Regional Primate Research Center, 505 Northwest 185th Avenue, Beaverton, Oregon 97006.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
In the present study, we took advantage of high-resolution multilaser confocal microscopy to examine the distribution of the {alpha}-subunit of the guanyl nucleotide binding protein subfamily Gq/11 (Gq/11{alpha}). Dispersed cultures of pituitary cells were prepared from female weanling rats, fixed, permeabilized, and then stained with monoclonal antiserum (mouse) to the gonadotrope-specific form of secretogranin (SIIp), which was then tagged with Texas Red. Accordingly, the subpopulation of gonadotropes (~15% of total cells) could be identified against a background of other pituitary cell types. Gq/11{alpha} was localized with antiserum made in rabbit, then tagged with fluorescein. Hoechst 33258 nuclear stain was also used in some experiments for topological reference. The data indicate localization of the Gq/11{alpha} in a cellular region near the plasma membrane and external to the border of the layer occupied by secretory granules. In the absence of activation, there were an average of six clusters of Gq/11{alpha} in a section 1 µm thick and through the center of the cell. This corresponds to an average of 60 clusters per cell, assuming a mean gonadotrope diameter of 10 µm. Following continuous treatment with 0.1 µg/ml Buserelin, a metabolically stable GnRH agonist, the average number of clusters increased to 200/cell after 40 min and remained approximately constant for 120 min. This increase was blocked by the protein synthesis inhibitor, cycloheximide. In response to Buserelin, there was an additional increase in the number of clusters inside the cell in the area occupied by the secretory granules and in the perinuclear area. Prolonged (24 h) treatment with Buserelin, sufficient to provoke the onset of desensitization, did not significantly change total numbers of Gq/11{alpha} clusters, although more were located in the peripheral compartment, an increase that occurred at the expense of the cytoplasmic compartment. Redistribution of the Gq/11{alpha} family may be functionally significant, because this moiety may be rate limiting at the site of regulation of signal transduction.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
ACTIVATION of the pituitary gonadotrope by GnRH and by its agonists begins with coupling of an agonist-liganded seven transmembrane segment receptor to the effector system (1). Such seven transmembrane receptors are typically coupled to G proteins (2), and there is a wealth of evidence to indicate a role for G proteins in the action of the GnRH receptor. The earliest studies relied on G protein activation by aluminum fluoride (3), and by toxins (4) that have direct effects on G proteins. More recent studies have taken advantage of specific antisera (5), genetic knockout animals lacking specific G proteins (6), or DNA sequences that encode particular receptor sequences that bind G proteins (5). When these sequences are expressed in cells, they serve as endogenous receptor inhibitors, because they compete with receptors for G proteins (7). Of particular interest is the observation that treatment of GGH3 cells (a PRL-derived cell line stably expressing the rat GnRH receptor) with a GnRH analog results in loss of immunoassayable Gq/11{alpha}. This treatment also results in palmitoylation of Gq/11{alpha} in a time- and dose-dependent fashion (5). Because this chemical modification is associated with increased hydrophobicity, the issue of redistribution from the soluble form to the membrane-associated form is raised. Redistribution of calmodulin (8) and protein kinase C (9) in the gonadotrope have been reported and could, in principle, be associated with either microaggregation (10, 11, likely heralding the onset of activation) or macroaggregation (12, 13, likely associated with the extinction of the response). To identify the locus of the Gq/11{alpha} moiety, laser scanning microscopy was used in this study, coupled with specific antisera and stains.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Preparation of pituitary cell cultures
Enzymatically dispersed pituitary cells from 28-day-old female weanling rats (Sprague-Dawley; B & K Universal, Kent, WA) were prepared as previously described (14). After dispersion, the cells were resuspended in medium 199 (M199; Irvine Scientific, Santa Ana, CA) containing 0.3% (wt/vol) BSA (fraction V; Irvine Scientific), 10% horse serum (Irvine Scientific), 2.5% FBS (Hyclone Labs., Logan, UT), 20 µg/ml gentamicin sulfate (Gemini Bio-products, Calabasas, CA), and 10 mM HEPES, pH 7.4. The cells were plated (800 µl/well; 2 pituitary equivalents/well) on a glass chamber slide (Nunc, Naperville, IL) previously treated 2–3 h with 12 N HCl, rinsed three times with distilled water, then washed with sterile M199/BSA. The plated cells were incubated in a water-saturated atmosphere maintained at 37 C for 3 days. The cells were washed with M199/BSA to remove dead cells and then treated with 0.1 µg/ml Buserelin (Hoechst, GnRH agonist) for 0, 20, 40, or 120 min or 24 h. The supernates were saved and assayed for LH by RIA. In some studies, the protein synthesis inhibitor cycloheximide was included at 1 mM during stimulation with Buserelin (or in controls).

Immunohistochemistry
After stimulation, the cells were fixed with 1 ml 4% paraformaldehyde in Dulbecco’s PBS (without Ca+2) at 4 C for 45 min, then washed three times in 1 ml Dulbecco’s PBS (without Ca+2). Cells were incubated for 30 min at 4 C in serum-buffer containing 4% normal goat serum (heat-inactivated), 0.2% Triton X-100, 50 mM Tris-HCl, pH 7.8, and 0.1% sodium azide. The primary antibodies were prepared in serum-buffer at a final titer of 1:400 for mouse monoclonal antisecretogranin (15) and 1:100 for rabbit anti-Gq/11{alpha} (5), then incubated with the cells for 45 min at room temperature. The cells were washed four times in 1 ml Dulbecco’s PBS (without Ca+2). The secondary antibody was prepared in serum-buffer at a final titer of 1:400 for both goat-antimouse TX Red (GAM-TR) and goat-antirabbit fluorescein isothiocyanate (GAR-FITC; both purchased from Jackson Immunoresearch Labs., West Grove, PA) and incubated with the cells for 60 min at room temperature in the dark. Thirty minutes after the second antibody incubation, 5 µg/ml Hoechst 33258 (nuclear stain; 16, 17, from Molecular Probes, Eugene, OR) was added to each well, and the incubation continued for an additional 30 min at room temperature in the dark. The cells were then washed three times with Dulbecco’s PBS (without Ca+2). Buffered glycerol solution (equal volume of 0.4 M potassium bicarbonate, pH 8.6, plus an equal volume of glycerol) was placed on the slide and covered with a coverslip. The slides were stored at 4 C before viewing.

Preabsorption of antiserum
For preabsorption studies, 200 µl antiserum (1:50) was incubated 16–20 h at 4 C with 10 µg peptide in the presence of 1 mM phenyl-methylsulfonylfluoride or 10 µg/ml aprotinin to inhibit degradation.

Confocal microscopy and image analysis
Fluorescently labeled cells from primary rat pituitary cultures were imaged using a Leica (Heidelberg, Germany) TCS-NT confocal laser scanning microscope. An argon UV laser (351 nm) was used for the excitation of Hoechst 33258, the 488-nm line of an argon laser was used to excite fluorescein labeled Gq/11{alpha}, and the 568-nm line of a Kr laser was used to image TX Red- labeled secretogranin. The section thickness was estimated to be 1 µm (pinhole aperture half open) with a x40, Plan APO numeric aperature 1.25 objective lens. Images were processed and printed using Adobe Photoshop 4.0 (Adobe Systems, San Jose, CA).

Gonadotropes were identified among the other cell types of the pituitary culture by their binding of antisecretogranin (SIIp) antibody. Isolated cells or cells at the periphery of larger clusters were selected for analysis.

To estimate the distribution of Gq/11{alpha} in a gonadotrope cell, the number of spots of fluorescein were counted for one section through the middle of a cell. Fluorescent clusters were defined as areas at least 25 pixels large with FITC intensity >=50% higher than immediately neighboring areas. Three subcellular compartments were considered: the plasma membrane (external layer adjacent to the ring of secretory granules), the cell periphery (occupied by the secretory granules labeled with TX Red), and the internal cytoplasm (the compartment between the nucleus and the secretory granules).

For each time point after activation, approximately 20 cells were analyzed. Averages and SEMs were calculated using Excel 7.0 (Microsoft Corp., Redmond, WA). The numbers of membrane-associated fluorescein clusters were normalized to the whole cell by approximating gonadotropes with spheres of 10 µm diameter and assuming a uniform distribution of G proteins over the cell surface.

Measurement of LH released
LH was measured by RIA using purified rat LH (NIDDK I-8) for iodination (18) and a reference preparation (RP-3) obtained from the NIDDK (Baltimore, MD). LH antiserum C102 was prepared and characterized as previously described (19). Bound and free hormone were separated with immobilized protein A (20).

Antiserum to Gq/11{alpha}
Antiserum was prepared as previously described (5).

Western blotting
Indicated tissues from the rat and from GGH31' cells (5) were electrophoresed in 10% polyacrylamide in SDS, then transferred onto nitrocellulose and immunoblotted with the polyclonal Gq/11{alpha} antiserum. Color was developed with antirabbit antiserum coupled to horseradish peroxidase (Bio-Rad, Richmond, CA) using 4-chloronaphthol as substrate.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Antibody to the common C-terminal sequence of rat Gq and G11{alpha} subunits recognized discrete punctate structures, seen as fluorescent spots or clusters, on most cells of the primary pituitary culture (Fig. 1Go). The labeling was very intense and, for cell clumps, could not be resolved by standard epifluorescence. Optical sectioning with a confocal microscope allowed the observation of gonadotropes buried in the cell cluster, and the identification of G proteins associated with a particular cell.



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  		Figure 1. Time course showing distribution of Gq/11{alpha} in gonadotropes of rat primary pituitary cultures. Gonadotropes were identified with antisecretogranin (SIIp) antibody that recognizes gonadotrope secretory granules (red). Nuclei (blue) were labeled with Hoechst 33258 DNA stain. Gq/11{alpha} was labeled with fluorescein (green) in absence of activation (A), after 20 min (B), 40 min (C), and 120 min (D) in presence of the GnRH agonist Buserelin. Bar in D = 10 µm (valid for all images).

 
In a Western blot, the antibody used recognized a single band of molecular weight 41–43 kDa (Fig. 2AGo). The fluorescent labeling was specific to the common peptide of the Gq{alpha} and G11{alpha} subunits (Fig. 2BGo). Preincubation of the antibody with the same peptide used in immunizations completely abolished FITC staining in gonadotropes in other pituitary cells.



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  		Figure 2. Specificity of anti Gq/11{alpha} subunit antibody. A, Western blot showing immunoreactivity of Gq/11{alpha} antisera in various rat tissue. Lane 1 shows molecular weight standards. Adjacent lanes contain homogenates of pituitary, GGH31' A cells, ovary, lung, spleen, adrenal, or liver. Antisera was diluted 1:4000 for immunoblot. B, Fluorescently labeled primary pituitary cell cultures in which gonadotropes are recognized by an antisecretogranin antibody (red) and anti Gq/11{alpha} (green). Cells were activated for 2 h with 0.1 µg/ml Buserelin before fixation. The primary antibody had been preabsorbed with C-terminus peptide of Gq/11{alpha} as described in Materials and Methods.

 
In unstimulated cells, gonadotropes (identified by antisecretogranin SIIp labeling) showed less Gq/11 staining than neighboring cells (Fig. 1AGo). A section through the middle of a gonadotrope at this stage showed, on average, 6.2 ± 0.9 fluorescent clusters on the plasma membrane, 3.4 ± 0.7 in the peripheral cytoplasmic compartment occupied by the secretory granules, and 1.3 ± 0.5 in the internal cytoplasm, or a total of 10.9 ± 1.0 clusters per section. Averages and SEMs were calculated for 22 cells, derived from six animals in three different experiments. If the gonadotrope was assumed to be a sphere and the distribution of fluorescent clusters was uniform throughout, there would be approximately 60–70 clusters per cell, of which a vast majority (~60) would be located at the plasma membrane level.

After incubation with 0.1 µg/ml Buserelin, a GnRH agonist, the number of clusters of Gq/11{alpha} proteins in gonadotropes increased in a time-dependent manner (Figs. 1Go, B–D and 3). A modest increase was observed in the average number of fluorescent spots in each compartment as early as 20 min after activation. A nonpaired Student’s t test comparing the populations of data before and 20 min after activation gave P values equal to 0.071, 0.047, and 0.011 for the membrane, peripheral, and cytoplasmic compartments, respectively.

After 40 min of stimulation with Buserelin, the number of G protein clusters were approximately three times larger than before activation in all compartments: 18.2 ± 1.6 at the plasma membrane level, 14.1 ± 1.8 in the peripheral cytoplasm, and 10.4 ± 2.4 in the internal cytoplasmic compartment. Averages and SEMs were calculated from 15 cells. Only a moderate additional increase in the total number of labeled clusters was observed after 120 min of activation, most of them occurring in the cytoplasm. For the 15 cells counted at this time point, the averages and SEMs were 18.1 ± 1.9, 16.2 ± 1.7, and 19.3 ± 1.9 for the membrane, periphery, and internal cytoplasm, respectively. The total number of fluorescent clusters associated with the plasma membrane in a gonadotrope after 120 min activation became approximately 200 (calculated under the same assumptions as before), whereas the total number in the three compartments increased to 350.

The increase of the total number of fluorescent clusters at the time of activation was not due to the dissociation of clusters into several components, because the total intensity of fluorescence per section increased after activation, and the size of individual clusters did not diminish.

After incubation with 0.1 µg/ml Buserelin, the supernate was assayed for released LH. The time course (Fig. 4Go) shows that LH was released as time increased. The LH at time zero was the basal level of LH after 120 min because all time points were started with medium alone; Buserelin was added in a negligible volume at appropriate times such that all time points were stopped at the same time.



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  		Figure 4. Time course of LH release from rat primary pituitary cells. Cells were stimulated with 0.1 µg/ml Buserelin for 0, 20, 40, and 120 min as described in Materials and Methods. LH level at time 0 is measurement of basal LH over 120 min, because all assays were stopped simultaneously. LH was assessed by RIA. Representative data from a single experiment are shown. Similar results were obtained in three experiments.

 
Activating gonadotropes with Buserelin for longer times, up to 24 h, did not significantly change the total number of Gq/11{alpha} clusters compared with 120 min. The distribution did, however, change the number found in the peripheral compartment occupied by the secretory granules, increased at the expense of the internal cytoplasmic compartment (Fig. 5Go).



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  		Figure 5. Desensitization, distribution, and level of expression of Gq/11{alpha} in gonadotropes after longer exposure to the GnRH agonist Buserelin as described for Fig. 2Go.

 
When cells were incubated with cycloheximide to inhibit synthesis of new proteins, activation with Buserelin failed to elicit an increase in the number of fluorescently labeled clusters of Gq/11{alpha} proteins (Fig. 6Go), and no consistent migration toward the outer compartments was observed. The values measured for cells activated in the presence of cycloheximide were similar to the values measured for cells not activated with Buserelin, either in the presence or absence of cycloheximide.



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  		Figure 6. Effect of cycloheximide on distribution of Gq/11{alpha} after activation by Buserelin. A, Cells activated by Buserelin. B, Control cells not activated by Buserelin. C, Cells treated with 1 mM cycloheximide and activated by Buserelin. D, Cells treated with cycloheximide but not activated. Total number of fluorescent clusters counted in a central section through a gonadotrope was, on average, 13.9 ± 1.5 at time 0 and 20.7 ± 9.7, 11.0 ± 5.5, and 10.7 ± 1.8 for 6, 12, and 24 h of activation, respectively. In same series of experiments, Buserelin increased number of fluorescent clusters from 17.0 ± 8.1 at time 0 to 38.0 ± 9.0 after 6 h, 41.3 ± 5.4 after 12 h, and 47.2 ± 4.0 after 24 h (A). Average number of clusters counted in cells exposed to cycloheximide and activated with Buserelin for 6, 12, and 24 h was 14.1 ± 4.6, similar to average for nonactivated, cycloheximide-exposed cells (16.0 ± 4.2) and average counted for cells at time 0 of their exposure to Buserelin (15.7 ± 1.3).

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
The results of the present study rely on identification of pituitary gonadotropes with a monoclonal antibody to a specific marker, secretogranin (15). Gq/11{alpha} was identified with a rabbit antiserum prepared against a common sequence in the C-terminus of this family of proteins. Use of confocal imaging reveals increased cluster formation throughout the cell, most notably at the periphery, where more than a 3-fold increase is measured; an increase is also seen in the cytoplasm. The time course of this is consonant with palmitoylation of Gq/11{alpha} in response to GnRH analog in this same system, and onset of responses measured by gonadotropin release (5). Distribution is blocked by the protein synthesis inhibitor, cycloheximide.

There is growing evidence for the significance of redistribution of G proteins in cell signaling. Dopamine, for example, causes a rapid but short-lived decrease in cytosolic Gq/11{alpha} in the anterior pituitary (21) and occupancy of the M1 acetylcholine receptor appears to promote redistribution of G proteins in target cells (22).

Other molecules involved in signaling pathways appear to redistribute to the gonadotrope periphery rapidly following stimulation by agonists. These include calmodulin (8) and protein kinase C (9). In addition, decreases in entropy of the GnRH receptors themselves appear associated with the onset of responsiveness (microaggregation, 10, 11) and the extinction of responsiveness (macroaggregation), patching, capping, and internalization (12, 13).

Both changes in total mass of specific G proteins and redistribution of these Gq/11{alpha} offer potential means of regulating the opportunity for interaction with the receptor and with Gq/11{alpha} proteins. Considering this report and another recent publication (5), both mechanisms appear to be in place for GnRH receptor signaling in different systems.

If a particular G protein is rate limiting in a signaling system, then loss of that G protein may result in regulation. In that regard, it is interesting to note that in several systems, particular G proteins are present in considerable (10- to 100-fold) excess compared with effector enzymes (23, 24, 25). Accordingly, a change in mass would have to be dramatic to exert a regulatory influence. Evidence indicates (26), however, that G proteins may be substantially compartmentalized, and estimates of total cellular mass likely do not reflect local concentrations at the level of regulatory enzymes. Accordingly, the ability to redistribute G proteins, presumably by way of altering hydrophobicity by palmitoylation, may provide an important means of controlling access to regulatory enzymes.

Previous studies (27) show that on activation, Gs{alpha} moves from a membrane-bound compartment to a soluble compartment. The present study shows the reverse for Gq/11{alpha}: the movement of the {alpha}-subunit from areas away from the plasma membrane to an area more closely associated with the plasma membrane. It is possible that the confocal approach measures the unveiling of the epitope for the antibody, rather than a translocation of G proteins from one site to another. This is possible because, after activation of G proteins, the {alpha}-subunit dissociates from the receptor and the ß-{gamma}-subunits, potentially exposing a C-terminal epitope. In either event, the current observation indicates the ability of the gonadotrope to redistribute Gq/11{alpha} and suggests a role for this event in hormone action.



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  		Figure 3. Effect of activation by Buserelin on level of expression and distribution of Gq/11{alpha} in gonadotropes. Number of fluorescent clusters recognized by antibody raised against C-terminus of {alpha}-subunits of Gq and G11 proteins were counted in central sections through gonadotropes in rat primary pituitary cultures after 0, 20, 40, and 120 min of activation. Cells were divided into three compartments: plasma membrane (mem.); peripheral compartment, occupied by secretory granules (per.); and cytoplasmic compartment (cyt.), defined as space bordered by secretory granules at exterior and nucleus. Inset shows change in total number of clusters per section. Bars = SEMs.

 

   Footnotes
 
1 This work was supported by NIH Grants HD-19899, HD-00163, HD-18185, and RR-0011257. Back

Received July 7, 1997.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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  2. Probst WC, Snyder LA, Schuster DI, Brosius J, Sealfon SC 1992 Sequence alignment of the G-protein coupled receptor superfamily. DNA Cell Biol 11:1–20[Medline]
  3. Hawes BE, Conn PM 1992 Sodium fluoride provokes gonadotrope desensitization to GnRH and gonadotrope sensitization to A23187: evidence for multiple G proteins in GnRH action. Endocrinology 130:2465–2475[Abstract/Free Full Text]
  4. Hawes BE, Barnes S, Conn PM 1993 Cholera toxin and pertussis toxin provoke differential effects on luteinizing hormone release, inositol phosphate production, and gonadotropin-releasing hormone (GnRH) receptor binding in the gonadotrope: evidence for multiple guanyl nucleotide binding proteins in GnRH action. Endocrinology 132:2124–2130[Abstract/Free Full Text]
  5. Stanislaus D, Janovick JA, Brothers S, Conn PM 1997 Regulation of Gq/11{alpha} by the gonadotropin-releasing hormone receptor. Mol Endocrinol 6:738–746
  6. Stanislaus D, Janovick JA, Wilkie T, Ji TH, Conn PM G11 knockout mice show altered response to GnRH agonist: evidence for a role of G11 in pituitary regulation. Program of the 79th Annual Meeting of The Endocrine Society, June 1997, Minneapolis, MN P1–136:168 (Abstract)
  7. Luttrell LM, Ostrowski J, Cotecchia S, Kendall H, Lefkowitz RJ 1993 Antagonism of catecholamine receptor signaling by expression of cytoplasmic domains of the receptors. Science 259:1453–1457[Abstract/Free Full Text]
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  12. Hazum E, Cuatrecasas P, Marian J, Conn PM 1980 Receptor-mediated internalization of fluorescent gonadotropin-releasing hormone by pituitary gonadotropes. Proc Natl Acad Sci USA 77:6692–6695[Abstract/Free Full Text]
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  15. Conn PM, Janovick JA, Braden TD, Maurer RA, Jennes L 1992 SIIp: a unique secretogranin/chromogranin of the pituitary released in response to gonadotropin-releasing hormone. Endocrinology 130:3033–3040[Abstract/Free Full Text]
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  20. Gupta R, Morton DL 1979 Double-antibody method and the protein-A-bearing Staphylococcus aureus cells method compared for separating bound and free antigen in radioimmunoassay. Clin Chem 25:752–756[Abstract/Free Full Text]
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  25. Alousi AA, Jasper JR, Insel PA, Motulsky HJ 1991 Stoichiometry of receptor-Gs-adenylate cyclase interactions. FASEB J 5:2300–2303[Abstract]
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Endocrinology Endocrine Reviews J. Clin. End. & Metab.
Molecular Endocrinology Recent Prog. Horm. Res. All Endocrine Journals