Division of Gastroenterology and Nutrition, Departments of 1 Medicine and 3 Biochemistry and Molecular Biology, George Washington University Medical Center, Washington, District of Columbia 20037; and 2 Laboratory of Molecular Biology and Biochemistry, Howard Hughes Medical Institute, Rockefeller University, New York, New York 10021
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ABSTRACT |
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The modulation of glucagon receptor (GR) expression and biological response was investigated in human embryonic kidney cell (HEK-293) clones permanently expressing the GR with different densities. The GR mRNA expression level in these clones was upregulated by cellular cAMP accumulation and presented a good correlation with both the protein expression level and the maximum number of glucagon binding sites. However, the determination of glucagon-induced cAMP accumulation in these cell lines revealed that the enhancement of receptor expression did not lead to a proportional increase in cAMP formation. Under these conditions, the maximum cAMP production induced by NaF and forskolin was not significantly different among selected clones, regardless of the receptor expression level. High receptor-expressing clones showed the greatest susceptibility for agonist-induced desensitization compared with clones with lower GR expression levels. The results of the present study suggest that the GR can recruit non-GR-specific desensitization mechanism(s). Furthermore, the partial inhibition or alteration of the overall cAMP synthesis pathway at the receptor level may be a necessary adaptive step for a cell in response to a massive increase in membrane receptor expression level.
G protein-coupled receptor; adenosine 3',5'-cyclic monophosphate; desensitization; HEK-293 cells
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INTRODUCTION |
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ADENOSINE 3',5'-CYCLIC MONOPHOSPHATE (cAMP) is widely recognized as a second messenger that regulates intracellular metabolism and cell proliferation and differentiation, as well as gene expression in mammalian cells. A large number of neurotransmitters and hormonal agonists act through membrane receptors coupled to heterotrimeric GTP-binding (G) proteins to regulate adenylyl cyclase (AC) activity and cAMP synthesis.
Glucagon is specifically processed from proglucagon in the pancreatic
-cells and is secreted in response to low blood glucose levels.
Through cAMP production, one of the main physiological functions of
glucagon is to maintain glucose homeostasis, primarily by both
stimulating glycogenolysis and gluconeogenesis and by inhibiting
glycolysis (14). Recent reports showed that the glucagon receptor (GR) signaling pathway is altered in certain diseases such as
hepatic cirrhosis in humans (6) and cholestatic liver disease in an animal model (15). The clarification of the
mechanism responsible for this alteration would increase the
understanding of the complications of cirrhosis and aid in developing
novel methods to improve long-term survival and quality of life for the patients.
The GR is closely related to the glucagon-like peptide-1, secretin, vasoactive intestinal peptide, and gastric inhibitory polypeptide receptors, and belongs to the superfamily of G protein-coupled receptors (GPCRs) (13). To achieve its intracellular effects, glucagon must bind to a GR, which has seven putative transmembrane domains. Although the regulatory mechanism of GR expression and its structure have been studied intensively in the past 5-10 yr (1, 8), few studies have focused on the complex GR/G protein/AC, despite the physiological importance of the GR signaling pathway. Manipulation of the cellular GR expression in relation to the glucagon-induced signal transduction will provide information critical to the understanding of impaired glucagon signaling response under pathological conditions.
As a part of this work, the rat GR cDNA was expressed in human embryonic kidney cells (HEK-293) under the control of the cytomegalovirus (CMV) promoter/enhancer system (9). We have subcloned this cell line according to the steady-state expression level and biological activity of GR. In this study, we show that agents that stimulate cAMP production upregulate the GR expression level. In addition, this compulsory expression led to greater susceptibility for receptor desensitization and lowered responsiveness to agonist stimulation of cAMP production.
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MATERIALS AND METHODS |
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Cell culture. HEK-293 cells were transfected with the rat GR (HEK-GR) (9). This cell clone was established by transfection with the synthetic rat GR cDNA (1.5 kb) inserted into a eukaryotic cell expression vector, pcDNA3 (Invitrogen, Carlsbad, CA). The HEK-GR cells were maintained in Dulbecco's modified Eagle's medium (Mediatech, Herndon, VA) supplemented with 10% fetal bovine serum (FBS), 100 U/ml penicillin G and streptomycin, 10 mM HEPES (pH 7.2), L-glutamine, and nonessential amino acids at 37°C in a humidified atmosphere of 95% air-5% CO2.
Identification of GR-expressing clones. For cloning, the HEK-293 cells were suspended and diluted at a concentration of 10 cells/ml in culture medium, and each well of a 96-well plate was inoculated with 100 µl of cell suspension. Only wells having a single cell were selected microscopically and cultured. HEK-293 cell clones were dissolved in 500 µl of a 3 M guanidium-thiocyanate solution containing 50% DMSO, 12.5 mM sodium citrate, and 0.125% sarkosyl. Cell lysates were denatured by incubation at 65°C for 1 h and applied to a dot-blotting apparatus (Bio-Dot Apparatus; Bio-Rad, Hercules, CA) assembled with a nylon membrane (Hybond-N; Amersham Pharmacia, Piscataway, NJ). The blotted membrane was rinsed with 20× saline sodium citrate (SSC) and applied for hybridization as described below.
Total RNA was obtained from cells incubated in 60-mm culture plates by the method of Chomczynski and Sacchi (7) with minor modification using RNA-Zol B reagent (Tel-Test, Friendswood, TX). Ten micrograms of denatured total RNA were electrophoresed in 1× MOPS/EDTA buffer on a 1.2% agarose gel. RNA was transferred to a nylon membrane in 20× SSC and immobilized by ultraviolet cross-linking. The RNA was hybridized with a randomly [32P]dCTP-labeled cDNA probe for rat GR or glyceraldehyde-3-phosphate dehydrogenase (GAPDH), respectively. Hybridization was carried out in Hybrisol I (Intergen, Purchase, NY) at 42°C for 8-16 h. The membrane was then washed twice with 2× SSC/0.5% SDS for 30 min (25°C) and twice with 0.2× SSC/0.5% SDS for 30 min each (65°C). After overnight exposure to a PhosphorImager screen, the membrane was scanned and analyzed using Image Quant software (Molecular Dynamics, Sunnyvale, CA). Except where otherwise indicated, the GR mRNA was quantitated relative to that of GAPDH. Western blot, ligand binding assay, and cAMP determination were performed as previously described (4, 15).Membrane preparation and determination of AC
activity.
Cell membranes from the HEK-293 clones were prepared by passage
10 times through a 26-gauge needle in ice-cold lysis buffer [50 mM
Tris · HCl (pH 8.0), 2.5 mM MgCl2, 1 mM EDTA, 1 mM
phenylmethylsulfonyl fluoride, 1 mM dithiothreitol, and 20 µg/ml
leupeptin]. Nuclei and undisrupted cells were sedimented at 500 g for 5 min. The supernatants were centrifuged at 50,000 rpm
for 30 min in a Beckman TL100-3 centrifuge (Beckman, Palo Alto, CA) and
washed with lysis buffer twice. AC activity was determined by measuring
the production of cAMP from [-32P]ATP as described by
Salomon et al. (19). In these experiments, [3H]cAMP was used to correct for differences in recovery rate.
Statistical analyses. Except where otherwise indicated, results were expressed as means ± SE. The statistical significance of the mean was determined by either one-way analysis of variance or Student's paired t-test.
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RESULTS |
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To identify the GR-expressing cell (HEK-GR) clones, >40 clones
were screened by dot blotting using radiolabeled GR cDNA as a probe.
The steady-state mRNA expression levels of GR, quantified as a ratio to
those of GAPDH, ranged from 34:1 for the maximum (Fig.
1, clone 1-4B) to <0.6:1 for the minimum
(clone 2-5D). Among 10 clones selected, the expression level of GR mRNA
determined by Northern blotting is shown in Fig. 1. Clones 2-5D, 2-10F,
4-4E, and 5-5D had low or no detectable GR mRNA level, and clone 2-5D was selected as a control for the subsequent studies. In addition, one
highly (1-4B) and three moderately (2-2C, 2-10E, and 3-8D) expressing
clones were also selected. The GR mRNA expression level among the
different clones was well conserved over at least 10 passages. The
proliferation rate for each clone was determined by successive cell
number counting and calculation of doubling time. There were no
significant differences among the doubling times for each clone
(average 23.9 h).
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The effects of various cAMP synthesis stimulatory agents were tested on
the GR mRNA expression level. The expression level of the GR mRNA in
clone 2-2C was upregulated by ~70% 24 h after incubation of
cells with glucagon at the concentration of either 1 µM (Fig.
2A, lanes 3 and
4) or 100 nM (Fig. 2B, lane 4) in the presence of IBMX. Similar upregulation of the GR mRNA expression was
observed with either 10 µM forskolin (Fig. 2B, lane
2), 100 µM 8-bromo-cAMP (lane 3), or 5 µM
isoproterenol (lane 5) in the presence of 100 µM IBMX
(Fig. 2B). To support a glucagon-induced cAMP-dependent
regulation of the GR mRNA expression level in our clones, we incubated
the cells with either 5 or 20 µM of H-89 (Fig. 2C,
lanes 4 and 5), a known cAMP-dependent protein
kinase inhibitor, and determined the GR mRNA level 24 h after
addition of 100 nM glucagon (Fig. 2C). H-89 inhibited, in a
dose-dependent manner, the increased expression of the GR mRNA induced
by glucagon from 30% with 5 µM to >80% with 20 µM. However, this
inhibitor did not significantly inhibit either the basal GR or the
GAPDH mRNA level (Fig. 2C, lane 2). These levels
of GR mRNA, as modulated by cAMP synthesis stimulatory agents, were
seen among all selected clones except 2-5D, which had no detectable GR
mRNA. The possible involvement of either growth factors or other
hormones in this GR mRNA expression level was excluded by observing
similar upregulation of GR mRNA expression 24 h after incubation
of the cells with glucagon and in the presence and absence of FBS.
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To assess whether the cAMP-induced upregulation was due to modulation
of the GR gene transcription, HEK-GR cells were cultured with 4 µg/ml
actinomycin D (AMD), which is known to be a strong inhibitor of gene
transcription. AMD suppressed the induction of the GR mRNA expression
by glucagon (Fig. 3). This inhibition was
gene specific and was not due to a toxic effect of this drug, since
under the same conditions, the GAPDH mRNA expression level was
unaffected.
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As shown in Fig. 4A, the GR
protein expression level in each selected clone was determined by
Western blotting using a specific polyclonal antibody raised against
the synthetic rat GR peptide (21). The protein expression
level presented a significant correlation with that of the GR mRNA
(r = 0.974, P = 0.0002).
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The binding of 125I-glucagon to membranes from the different HEK-GR clones is shown in Fig. 4B. Membranes from HEK-GR were incubated with radiolabeled glucagon and increasing concentrations of unlabeled glucagon. The required concentration of unlabeled glucagon to inhibit the 125I-glucagon binding by 50% (IC50) was similar among the clones tested with an average value of ~23 nM, as determined from the fit of the respective sigmoidal curve. These values were in the same range as that previously reported for the GR cloned in COS-1 cells (9). However, under these conditions, the maximum number of glucagon binding sites varied from a minimum of 534 fmol (clone 2-10E) to a maximum of 996 fmol (clone 1-4B). It should be mentioned that the use of the agonist to determine the glucagon binding characteristics may lead to an underestimation of the maximum binding. The number of GR receptor binding sites presented a significant correlation with both the GR mRNA and protein expression level for each respective clone (binding site vs. mRNA: r = 0.868, P = 0.005; binding site vs. protein: r = 0.902, P = 0.005).
The basal cellular cAMP concentration, determined by radioimmunoassay,
was not significantly different among the different HEK-GR clones. The
cellular cAMP production was studied after 15 min of incubation with
increasing concentrations (1011-10
7 M) of
glucagon in the presence of IBMX. When expressed per milligram of
protein, while the concentration necessary to elicit 50% of the
maximum effect was similar among the clones (EC50
7.0 nM), the maximum cAMP synthesis varied from a minimum of 1,607 fmol/mg protein for clone 1-4B to a maximum of 3,735 fmol/mg protein for clone
3-8D (Fig. 5 and Table
1). The order of the maximal effect of
glucagon among clones was the following: 3-8D > 2-10E
2-2C > 1-4B
2-5D (Fig. 5 and Table 1). The glucagon-induced
cAMP production did not parallel that of either the expression level of
the GR mRNA or the maximum number of glucagon binding sites. The
femtomole of cAMP produced per femtomole of GR, i.e., receptor density,
varied from 1.6-6.2 (Table 1).
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To assess whether other sites between the receptor and the AC could be
altered and, therefore, responsible for the differential induction of
cAMP by glucagon between clones, we compared the effect of NaF and
forskolin, known to directly activate the G protein and AC,
respectively, on the cell membrane AC activity and cAMP synthesis. As
shown in Fig. 6, 20 mM NaF and 100 µM
forskolin stimulated AC activity approximately two- and sixfold,
respectively, without significant differences among the selected
clones, whereas under these conditions (Fig. 6B), there was
a differential stimulatory effect of glucagon on AC activity between
clones 1-4B and 3-8D. Ten nanomolar glucagon maximally activated AC in
both clones. However, while concentrations of glucagon >10 nM
activated AC by approximately threefold in clone 1-4B, it increased to
approximately fivefold in clone 3-8D. These results are in agreement
with those of glucagon-induced cAMP production reported in these clones
(Fig. 5).
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The membrane-associated GR expression level was increased over time
with a maximum increase observed 24 h following the addition of 1 µM glucagon to the culture medium in both 1-4B and 3-8D clones (Fig.
7A). In addition, these newly
expressed receptors were able to bind glucagon with a similar affinity
(data not shown). The respective maximum glucagon binding for clones
1-4B and 3-8D was 6.1 ± 1 pmol/mg of protein and 5.4 ± 1.5 pmol/mg of protein and was not significantly different in the absence
and presence of glucagon for 4 h (Fig. 7B). However,
the maximum glucagon binding increased by 46% and 270% to 15.1 ± 1.6 pmol/mg of protein and 20.4 ± 5.1 pmol/mg of protein for
clones 1-4B and 3-8D, respectively, after 24 h of incubation with
glucagon. This increased membrane GR expression level, and maximum
glucagon binding did not result in a parallel increased ability for
glucagon to stimulate the AC (Fig. 7C). Furthermore, the
1-4B clone completely lost the AC responsiveness to glucagon 4 h
after treatment with the agonist, and this decrease was still
noticeable 24 h after glucagon treatment. This phenomenon was much
more limited with clone 3-8D (Fig. 7C). In this clone, the
decreased activation of AC following incubation of cells with
glucagon was significant only after 4 h and disappeared after
24 h. The respective recovery of the glucagon signal after 24 h of incubation is probably due to the limited stability of the
hormone. However (not shown), the glucagon-induced cAMP production returned to control levels for both clones after 48 h of
incubation with glucagon.
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DISCUSSION |
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The diversity of expression level of transfected gene products in different clones can be the result of a single transfection procedure. In the present study, the steady-state GR mRNA expression levels varied among selected HEK-293 clones. The mRNA and protein expression levels increased in a parallel manner up to two- and fourfold, respectively. Furthermore, while the maximum number of binding sites differed from clone to clone, the affinity of the receptor for glucagon was not significantly affected and was in the same range as what has previously been reported in the liver and in GR-transfected cell lines (3, 9). Therefore, it can be concluded that the overexpression of GR in HEK-293 cells does not affect the receptor-ligand interaction.
In the present study, HEK-293 cells, which do not naturally possess GR, were transfected with the coding domain of the rat GR gene ligated into the expression vector pcDNA3 (9). One of the original components of the pcDNA3 vector, the CMV promoter, was employed as a promoter for the constitutive expression of GR. This transfected HEK-293 cell line showed a glucagon-induced, increased GR mRNA transcription, at least in part, in a cAMP-dependent manner. Several groups have suggested that the basal activity of a major immediate early enhancer of human CMV was cell cycle dependent (5) and could be augmented considerably by elevated levels of intracellular cAMP in a cell type-specific manner (20). Therefore, according to the result of the consensus sequence search, since the CMV promoter contains several activator protein-1 transcription factor sites as well as the cAMP-response element, the positive transcriptional regulation by cAMP is one of the possible explanations for the upregulation of GR mRNA following cAMP production in this system.
There was no correlation between the steady-state number of GRs and the
associated cAMP production in the present study. Indeed, the clone with
the highest receptor density (1-4B) was the least effective, as far as
cAMP production was concerned. The relationship between receptor number
and the associated cAMP production has been previously discussed,
mainly for the -adrenergic receptor (
-AR) (18). In
both cultured myocytes and transgenic mice, overexpression of the
-AR protein 20- to 200-fold resulted in a <2-fold increase in
isoproterenol-induced cAMP production (10, 17). The
conclusion from these studies was that the AC expression level was the
limiting factor in the receptor-mediated cAMP production (12,
18). In the present study, it is supposed that the AC activity
itself was identical between each clone since forskolin-induced cAMP
production was not different between selected clones having different
GR expression levels. Furthermore, the glucagon-induced maximum cAMP
production was <80% of that induced by 100 µM forskolin, thus
eliminating in our model any suggestion of the AC as a limiting factor.
The glucagon-induced complete inhibition of the glucagon response, also named homologous desensitization (2), persisted for up to 24 h in the 1-4B clone with a high GR expression level and modest glucagon responsiveness. Glucagon had a more limited desensitization effect in the 3-8D clone, with a more modest GR expression level and high glucagon responsiveness (Fig. 7B). The loss of hormonal responsiveness in GPCRs induced by the agonist can be observed in seconds to minutes following ligand binding and can be mediated by various mechanisms including activation of GPCR kinases (see Ref. 11 for review). The ability of cAMP to stimulate GR mRNA and protein expression in the present study excludes any possible transcriptional downregulation of GR in our system (Fig. 7A). In addition, since there was no significant change in either forskolin- or NaF-induced AC activity in selected clones (Fig. 6), this loss of responsiveness exclusively implicates the GR. Therefore, we speculate that this greater susceptibility of the receptor to desensitization might be an adaptive step for a cell in response to a massive increase in membrane receptor expression level. Our hypothesis is supported by the conclusion of Bohm et al. (2) that this desensitization mechanism is important to prevent any uncontrolled GPCR stimulation of signaling pathways. Furthermore, our findings underline a broad compatibility of the desensitization system for the GR even if this receptor is not naturally expressed in the cell. Finally, these clones will be useful tools to clarify the molecular mechanism(s) of GR desensitization.
This adaptation of the overall cAMP synthesis cascade to a net increase
in receptor is physiologically relevant and can, to a certain extent,
explain the results reported by Michel et al. (16). These
authors observed in the spontaneously hypertensive rat, and as a
function of age, that while the renal -AR number was significantly
increased, the associated cAMP production remained unchanged.
Furthermore, under these conditions the forskolin-induced cAMP
production remained unaffected and was 10-fold greater than that
induced by the adrenergic receptor. Together, these results suggest
that under pathophysiological conditions the net increase in receptor
number is not always consequently associated with an increased cAMP production.
In conclusion, the differential response of desensitization can explain, at least in part, the lack of a relationship between receptor number and hormonal responsiveness in these clones. It is also possible that the GR shares desensitization mechanism(s) already present in HEK-293 cells that do not naturally possess the GR.
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ACKNOWLEDGEMENTS |
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The valuable contribution of Dr. Thomas P. Sakmar, Rockefeller University, during the study and preparation of this manuscript is greatly appreciated. The authors also thank Zaheer Arastu and Rachel Weston for skillful technical assistance, Dr. Susan Ceryak, George Washington University Medical Center, for thoughtful discussions during the preparation and editing of this manuscript, and the Center for Microscopy and Image Analysis for the use of equipment.
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FOOTNOTES |
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This work was supported in part by National Institutes of Health Grants DK-46954 (to B. Bouscarel) and DK-54718 and GM-07739 (to T. P. Sakmar).
Address for reprint requests and other correspondence: B. Bouscarel, 2300 Eye St. N. W., Ross Hall, Rm. 523, Washington, DC 20037 (E-mail: dombeb{at}gwumc.edu).
The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
Received 29 December 2000; accepted in final form 11 June 2001.
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