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Endocrinology Vol. 138, No. 4 1471-1475
Copyright © 1997 by The Endocrine Society

ARTICLES

Constitutive Activity of Native Thyrotropin-Releasing Hormone Receptors Revealed Using a Protein Kinase C-Responsive Reporter Gene

Arti Jinsi-Parimoo and Marvin C. Gershengorn

Division of Molecular Medicine, Department of Medicine, Cornell University Medical College and The New York Hospital, New York, New York 10021

Address all correspondence and requests for reprints to: Marvin C. Gershengorn, Cornell University Medical College, 1300 York Avenue, New York, New York 10021. E-mail: mcgersh{at}mail.med.cornell.edu


   Abstract
Top
 Abstract
Introduction
 Experimental Procedures
 Results and Discussion
 References
 
The native TRH receptor (TRH-R), which is a G protein-coupled receptor that signals via the phosphoinositide transduction pathway, has been assumed to be inactive in the absence of agonist. In contrast, a mutant mouse TRH-R (C335Stop TRH-R) was shown previously to exhibit constitutive (or agonist-independent) signaling activity. In this report, we measured signaling activity of TRH-Rs using a protein kinase C-responsive reporter gene instead of formation of inositol phosphate second messenger molecules. Using this more sensitive system, we show that native mouse TRH-Rs exhibit agonist-independent signaling activity that is directly proportional to the number of receptors expressed in COS-1 cells and is inhibited by negative antagonist benzodiazepine drugs. As expected, the basal signaling activity of native TRH-Rs is lower than C335Stop TRH-Rs. Constitutive activity of native TRH-Rs is not peculiar to COS-1 cells in which receptor density is markedly elevated, because it can also be demonstrated in Madin Darby canine kidney cells stably expressing mouse TRH-Rs and GH4C1 cells endogenously expressing rat TRH-Rs. These findings support the thesis that native TRH-Rs oscillate between active and inactive states. We suggest that demonstration of constitutive activity of native receptors may depend on the sensitivity of the signaling assay employed.


   Introduction
Top
 Abstract
 Introduction
Experimental Procedures
 Results and Discussion
 References
 
CONSTITUTIVE (or agonist-independent) signaling activity of G protein-coupled receptors (GPCRs) has been well documented (1, 2). The finding that certain receptors exhibit constitutive activity has led to a modification of traditional receptor theory (3, 4). It is now thought that receptors can exist in at least two conformations (or populations of conformations), inactive (R) and active (R*), and that an equilibrium exists between these two states that markedly favors R over R* in the majority of receptors in the absence of agonist. It has been proposed that there is a shift in equilibrium in some GPCRs that allows a sufficient number of receptors to be in the active R* state and initiate signaling in the absence of agonist. In most instances, these have been mutated receptors that were produced in the laboratory or found naturally in certain disease states (5). A number of wild-type (or native) GPCRs have been shown to exhibit agonist-independent activity (6, 7, 8). Even with mutant GPCRs, constitutive activity has been more readily shown with receptors that couple to the adenylyl cyclase-cAMP cascade than with receptors that couple to the phosphoinositide-inositol 1,4,5-trisphosphate (IP3)/diacylglycerol (DAG) pathway (9). In several instances, for example, receptors for thyroid-stimulating hormone that can signal via both the cAMP and IP3/DAG pathways, the majority of constitutively active mutant receptors studied have exhibited agonist-independent activity with regard to cAMP but not via IP3/DAG signaling (10).

TRH-R is a GPCR that signals via the phosphoinositide transduction pathway (11, 12). In previous studies (13, 14), we found no evidence for constitutive activity of native TRH-R in experiments in which we measured generation of inositol phosphate (IP) second messengers. In this report, we present evidence of agonist-independent signaling activity via the IP3/DAG pathway of native TRH-Rs using a sensitive reporter system in which we measure induction of firefly luciferase via a protein kinase C (PKC)-responsive promoter (15).


   Experimental Procedures
Top
 Abstract
 Introduction
 Experimental Procedures
Results and Discussion
 References
 
Materials
DMEM, HBBS, FBS, and lipofectamine were purchased from GIBCO/BRL (Grand Island, NY), Nu-serum was from Collaborative Research (Bedford, MA). [3H]Methyl-TRH was purchased from DuPont New England Nuclear (Boston, MA), and myo-[3H]inositol was from Amersham (Arlington Heights, IL). COS-1 were obtained from American Type Culture Collection (Rockville, MD) and Madin-Darby canine kidney (MDCK) type II cells were a gift from Dr. Enrique Rodriguez-Boulan (Cornell University Medical College). Plasmid containing an activating protein-1 (AP-1)-fos-Luc reporter gene construct was a gift from Dr. Paul Deutsch (previously of Cornell University Medical College). All other chemicals were obtained from Sigma Chemical Co. (St. Louis, MO).

Cell culture and transfection
COS-1 cells, GH4C1 cells endogenously expressing rat pituitary TRH-RS, and MDCK type II cells stably expressing mouse TRH-Rs were grown in DMEM supplemented with 5% Nu-serum (COS-1 and GH4C1 cells) or calf serum (MDCK cells) in a humidified atmosphere of 5% CO2 at 37 C. Cells were seeded in six-well plates at a density of 2–3 x 105/well on the day before transfection. Cells were transfected using either the diethylaminoethyl-dextran (12) (COS-1 cells) or lipofectamine (GH4C1 and MDCK cells) methods with 5 µg/ml of a PKC-responsive reporter gene construct (AP-1-fos-Luc) (15) without or with varying amounts of wild-type mouse TRH-R plasmid (pCDM8-mTRHR) (12) or a mutant pCDM8-C335Stop (13) at concentrations ranging from 0.2–2000 ng/ml.

Measurement of TRH-R binding
Receptor binding was determined using a high affinity analog of TRH, [3H]methyl-TRH, in intact cell monolayers under equilibrium conditions in HBSS containing 25 mM HEPES, pH 7.4, at 4 C for 2 h. The concentration of [3H]methyl-TRH was 2 nM; the equilibrium dissociation constant for methyl-TRH binding to mouse TRH-R is 5.0 nM at 4 C (14).

Assay of luciferase activity
Cells transfected with plasmid containing AP-1-fos-Luc, without or with the indicated amount of pCDM8-mTRHR or pCDM8-C335Stop mTRHR, were incubated in DMEM containing 10% serum. To test the effect of negative TRH-R antagonists on basal luciferase activity, midazolam, chlordiazepoxide, or diazepam was added at the indicated concentration immediately at the end of transfection for the entire length of the experimental incubation. Luciferase activity reached its maximum at 48 h posttransfection in either the AP-1-fos-Luc alone group or receptor cotransfected groups when the number of receptor sites also achieved a maximum level (data not shown). Agonist was added 4 h before the end of the experiment. Cells were washed with PBS and lysed with 0.4 ml/10 cm well of lysis buffer (25 mM GlyGly, pH 7.8, 15 mM MgSO4.6H2O, 4 mM EGTA, 1 mM dithiothreitol, 1% Triton X-100) 48 h posttransfection. Cell supernatant was separated and combined with reaction buffer (25 mM GlyGly, pH 7.8, 15 mM MgSO4.6H2O, 4 mM EGTA, 1 mM dithiothreitol, 15 mM KH2PO4, 2 mM ATP). The substrate luciferin (0.4 mM) was made up in reaction buffer. Luminescence was measured using a Monolight 2010 (Analytical Luminescence Laboratory, San Diago, CA) for 10 sec following semiautomated addition of equi-volume amount of substrate to the cell supernatant.

Measurement of IP formation
Following transfection, cells were labeled with myo-[3H]inositol for 48 h. Stimulation of IP formation was determined as previously described (12) in absence or presence of 10 mM LiCl or midazolam.

Data analysis
All data were analyzed using GraphPad Prism (GraphPad Software, San Diego, CA). Statistical significance was determined using paired Student’s t test with a probability criterion of P <= 0.05.


   Results and Discussion
Top
 Abstract
 Introduction
 Experimental Procedures
 Results and Discussion
References
 
To determine whether native mouse TRH-Rs exhibit constitutive activity, we cotransfected COS-1 cells with both plasmid encoding AP-1-fos-Luc and various amounts of plasmid encoding wild-type mouse TRH-R. Figure 1Go illustrates the results of such an experiment in which the level of TRH-Rs, the basal rate of formation of IPs, and basal effects on PKC-responsive gene induction were measured. As expected, the number of cell surface receptors increased with increasing dose of plasmid between 0.7–200 ng/ml (Fig. 1AGo). The maximum level of expression was 3 x 106 TRH-Rs per cell assuming 17% transfection efficiency (our unpublished results). The measured basal rates of IP formation were not different in any cell population (Fig. 1BGo). Addition of TRH, however, caused the rate of IP formation to increase in all cells in which TRH-R expression was measurable. TRH stimulated IP formation 6 ± 1-fold in cells transfected with 70 ng/ml pCDM8mTRHR or greater (data not shown). In marked contrast to the lack of measurable effect on IP second messengers, there was a direct relationship between the amount of plasmid used for transfection and the basal level of luciferase reporter gene activity (Fig. 1CGo). Luciferase activity increased from 1800 ± 260 in cells transfected with pAP-1-fos-Luc alone to 25,000 ± 4,400 relative luciferase units (RLU)/well in cells transfected with pAP-1-fos-Luc and maximally effective doses of pCDM8-mTRHR. TRH (10 µM for 4 h) elevated luciferase activity 3-fold above basal levels (cells transfected with 2000 ng/ml plasmid; data not shown). There was no effect on luciferase activity in cells transfected with 2000 ng/ml plasmid without a sequence encoding a receptor and no effect of expression of receptors, for example FSH receptors, that couple to the adenylyl cyclase-cAMP cascade (data not shown). These data suggest that expression of the TRH-R results in agonist-independent activation of luciferase gene transcription that is mediated by activation of PKC (16). Threshold induction of basal luciferase activity could be detected at 100 x 103 receptor sites per cell. This value is similar to the level of TRH-Rs expressed endogenously in GH cells [50–200 x 103 sites per cell (16)]. This finding suggests that basal (agonist-independent) signaling activity may be present in cells that express TRH-Rs endogenously (see below).



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  		Figure 1. Effect of transfecting COS-1 cells with various doses of pCDM8-mTRHR on TRH-R expression, basal inositol phosphate formation, and basal luciferase gene transcription. COS-1 cells were transiently transfected with various amounts of pCDM8-mTRHR and a fixed amount (5 µg) of pAP-1-fos-Luc and incubated in medium containing myo-[3H]inositol. Two days after transfection, cells were analyzed for receptor expression by measuring [3H]methyl-TRH binding (A), [3H]inositol phosphate formation (B), and luciferase activity (C). Results are expressed as mean ± SEM of three independent experiments, each performed in duplicate.

 
AP-1 has been shown to confer responsivity to genes by both the PKC and protein kinase A pathways (15). Although it appears unlikely that TRH-R would activate the adenylyl cyclase-cAMP pathway (17), we determined whether the basal activity of TRH-R was inhibited by calphostin C, a selective PKC inhibitor (18). Basal induction of luciferase activity was blocked by calphostin C with an IC50 = 0.3 µM (data not shown), which is consistent with the reported value for calphostin C inhibition of PKC (18). Thus, it appears that the agonist-independent signaling activity of TRH-R is via the phosphoinositide signaling cascade (19).

We have previously described a mutant TRH-R (C335Stop TRH-R), which is truncated at position-335 in its carboxy-terminal cytoplasmic tail, that exhibits constitutive activity when assessed by measuring IP second messengers (13, 14). The basal rate of IP formation in cells expressing C335Stop TRH-Rs was 32% higher, on average, than in cells not expressing TRH-Rs or in cells expressing wild-type TRH-Rs (WT TRH-Rs). To further characterize basal signaling activity of WT TRH-Rs, we compared its activity to that of C335Stop TRH-Rs using the AP-1-responsive luciferase activity reporter system. Figure 2Go illustrates the linear relationship between number of receptors expressed (up to transfection with 200 ng plasmid/ml) and the level of basal luciferase activity in COS-1 cells expressing native or C335Stop TRH-Rs. As expected, C335Stop TRH-Rs, like WT TRH-Rs, exhibit basal stimulation of luciferase activity that is stimulated by TRH to the same extent as with WT TRH-Rs (data not shown). Basal stimulation of luciferase is directly proportional to the number of C335Stop TRH-Rs expressed over the range of 100-4000 x 103 per cell. Importantly, there was significantly greater basal activity exhibited by C335Stop TRH-R than WT TRH-R. This distinction is evident in the differences in the slopes of the lines in a plot of luciferase activity vs. receptor binding: for C335Stop TRH-R of 5.5 ± 0.79 (r2 = 0.82) and for WT TRH-R of 1.2 ± 0.25 (r2 = 0.71). That is, the basal activity of C335Stop TRH-R was 4-fold higher than that of WT-TRH-R. These results provide further support for the idea that the basal activity measured with the luciferase reporter system is valid. They also provide insight into why the basal activity of WT TRH-R is not apparent in the standard assay for generation of IP second messengers. C335Stop TRH-R exhibited only a 1.3-fold increase in IP formation, whereas it was four times more active than WT TRH-R when basal luciferase activity was measured. Thus our results provide strong evidence for signal amplification capability of PKC-responsive (AP-1) reporter plasmid. The use of the functional expression system (reporter plasmid) with the receptor is artificial, however changes in receptor-mediated IP3/DAG levels although below the detection of the assay can sufficiently activate and produce measurable PKC-dependent luciferase expression. These results may also suggest the potential functional significance of TRH-R mediated enhancement of gene expression as has been proposed for PRL (20).



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  		Figure 2. Comparison of basal signaling activity of WT and C335Stop TRH-Rs expressed in COS-1 cells. Cells were transiently transfected with either various amounts of pCDM8mTRHR or pCDM8C335Stop and a fixed amount (5 µg) of pAP-1-fos-Luc. Two days after transfection, cells were analyzed for receptor expression and luciferase activity. Results are expressed as mean ± SEM of three independent experiments, each performed in duplicate.

 
To show more definitively that the activity of WT TRH-R is independent of agonist, we measured the effect of expression of WT TRH-R in cells incubated in mediums of different serum composition and in serum-free medium. In cells incubated in defined medium supplemented with Nu-serum, fetal calf, horse, and charcoal-stripped horse serums, expression of WT TRH-R induced luciferase activity more than 10-fold above cells transfected with pAP-1-fos-Luc alone (data not shown). Most importantly, induction of luciferase gene transcription occurred in cells incubated in serum-free, defined medium. In serum-free medium, luciferase activity was lower than in cells incubated in serum-containing medium. In a representative experiment, luciferase activity was 420 ± 1 or 1160 ± 74 RLU/well in cells transfected with pAP-1-fos-Luc alone and incubated in serum-free or serum-containing mediums, respectively, and was 5660 ± 450 and 108,000 ± 17,000 RLU/well in cells transfected with pCDM8TRHR and pAP-1-fos-Luc and incubated in serum-free or serum-containing mediums, respectively. These findings strongly support the idea that the basal activity we measured was agonist independent.

To show that the basal signaling activity is specifically caused by TRH-R in transfected COS-1 cells, we tested the effects of the negative antagonists (or inverse agonists), midazolam, chlordiazepoxide, and diazepam (21). These benzodiazepine drugs were shown to be negative antagonists of the constitutively active C335Stop TRH-R (14). Figure 3Go illustrates that these benzodiazepines inhibit agonist-independent induction of luciferase activity in cells expressing WT TRH-Rs. There was no effect of these drugs in cells expressing luciferase but not TRH-R nor in cells activated by another GPCR, and benzodiazepines did not affect TRH-R number under these conditions (data not shown). Benzodiazepines inhibited basal luciferase activity in a dose-dependent manner with a rank order of potency midazolam > diazepam >= chlordiazepoxide; IC50 values were 7, 20, and 30 µM, respectively, and complete inhibition was achieved at 100 or 250 µM. These values compare well with TRH binding inhibitory constants (Ki) in cells in culture (21). These results show that basal activation of luciferase gene transcription is specifically a TRH-R-mediated effect.



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  		Figure 3. Effect of benzodiazepines on basal luciferase activity in COS-1 cells expressing WT TRH-Rs. The negative antagonist benzodiazepine drugs, midazolam (Mid), diazepam (Dzp), and chlordiazepoxide (CDE), were added to incubation medium of COS-1 cells immediately after transfection. Basal luciferase activity in the control group (pAP-1-fos-Luc alone) was 380 ± 40 RLU. Results are expressed as mean ± SEM of three independent experiments, each performed in duplicate.

 
It has been suggested that constitutive activity of native and mutant receptors may only be demonstrable in situations in which the receptor is markedly over-expressed (22), such as in COS-1 cells. To address the possibility that the observed constitutive activity of the WT TRH-R was peculiar to COS-1 cells, we studied this phenomenon in MDCK II cells stably expressing WT mouse TRH-R (MDCK-mTRHR) (23) and in GH4C1 cells that endogenously express native rat TRH-Rs (24). We transiently transfected each of these cell lines with pAP-1-fos-Luc reporter plasmid. The receptor level was approximately 100 x 103 per cell in both cell lines. Figure 4Go shows the negative antagonistic effect of midazolam on basal luciferase activity in MDCK-mTRHR and GH4C1 cells. Midazolam inhibited luciferase activity in both MDCK-mTRHR and GH4C1 cells in similar dose-dependent manner; the IC50s were 7 and 17 µM in MDCK-mTRHR and GH4C1 cells, respectively. This value is similar to that in COS-1 cells (Fig. 3Go). Thus, constitutive activity of WT TRH-R is not limited to expression in COS-1 cells and is exhibited by both mouse and rat TRH-Rs.



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  		Figure 4. Effect of midazolam on basal luciferase activity in MDCK-mTRHR and GH4C1 cells. Midazolam (Mid) was added to incubation medium of cells immediately after transfection with pAP-1-fos-Luc. After 48 h, cells were lysed, and luciferase activity was measured. Basal luciferase activity in control groups (pAP-1-fos-Luc alone) was 246 ± 71 (MDCK-mTRHR) and 126 ± 7 (GH4C1) RLUs. Results are expressed as mean ± SEM of three independent experiments, each performed in duplicate.

 
In summary, we have shown that native TRH-Rs are constitutively active by demonstrating that they induce transcription of a reporter gene (luciferase) in COS-1 cells transiently expressing native mouse TRH-Rs, MDCK-mTRHR cells stably expressing native mouse TRH-Rs and GH4C1 cells that endogenously express native rat TRH-Rs in the absence of agonist. Benzodiazepine drugs, which were shown previously to be negative antagonists (or inverse agonists) of a constitutively active TRH-R mutant (14), exhibited specific inhibition of the agonist-independent activity of native TRH-Rs also. We think that the constitutive activity of native TRH-Rs became evident when a more sensitive assay, which took advantage of an amplified downstream signaling response (transcription of a catalytic reporter enzyme during a prolonged period of action) rather than measuring a proximal event in signal transduction over a short period of time (IP second messenger formation over 1 h) was employed. We suggest that many native GPCRs may exhibit constitutive activity when studied using sensitive reporter systems. We are aware of the controversy regarding whether receptors found to be constitutively active in vitro would exhibit this activity in vivo, especially when expressed at physiological levels (22). Although our data do not resolve this conflict, we think it is possible that a native GPCR, which is minimally active in the absence of agonist, may exert a regulatory function on cellular responses that are regulated chronically, for example, modulation of neurotransmitter action in the central nervous system (25). Control of this regulation would occur, not at the level of ligand expression, but at the level of receptor expression.

Received November 11, 1996.


   References
Top
 Abstract
 Introduction
 Experimental Procedures
 Results and Discussion
 References
 

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Inhibition of Constitutive Signaling of Kaposi's Sarcoma-associated Herpesvirus G Protein-Coupled Receptor by Protein Kinases in Mammalian Cells in Culture
J. Exp. Med., March 2, 1998; 187(5): 801 - 806.
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A. M. Thomson, J. T. Rogers, and P. J. Leedman
Thyrotropin-releasing Hormone and Epidermal Growth Factor Regulate Iron-regulatory Protein Binding in Pituitary Cells via Protein Kinase C-dependent and -independent Signaling Pathways
J. Biol. Chem., October 6, 2000; 275(41): 31609 - 31615.
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K. M. Kroeger, A. C. Hanyaloglu, R. M. Seeber, L. E. C. Miles, and K. A. Eidne
Constitutive and Agonist-dependent Homo-oligomerization of the Thyrotropin-releasing Hormone Receptor. DETECTION IN LIVING CELLS USING BIOLUMINESCENCE RESONANCE ENERGY TRANSFER
J. Biol. Chem., April 13, 2001; 276(16): 12736 - 12743.
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