A Role for Guanyl Nucleotide-Binding Regulatory Protein ß- and
-Subunits in the Expression of the Adrenocorticotropin Receptor
Rong Qiu,
Claudia Frigeri and
Bernard P. Schimmer
Banting and Best Department of Medical Research and Department
of Pharmacology University of Toronto Toronto, Ontario, Canada
M5G 1L6
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ABSTRACT
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Mutant Y1 mouse adrenocortical tumor cells,
isolated on the basis of their resistance to the growth-inhibitory
effects of forskolin, arise from single mutational events. These
mutants present complex phenotypes in which the activity of Gß/
is
impaired, ACTH receptor gene expression is markedly diminished, and
ACTH-responsive adenylyl cyclase activity is lost. In this study, we
have tested the hypothesis that the impairment in Gß/
activity is
responsible for the loss of ACTH receptor gene expression and
ACTH-responsive adenylyl cyclase activity. Transfection of one of the
mutant clones with expression vectors encoding either Gß1 or Gß2
together with G
2 increased ACTH receptor expression and restored
ACTH-responsive adenylyl cyclase activity. Interestingly, either Gß2
or G
2 alone was effective. These results thus support the hypothesis
that the impairment in Gß/
activity is responsible for the loss of
ACTH receptor expression. A luciferase reporter plasmid driven by the
proximal promoter region of the mouse ACTH receptor gene was expressed
poorly in the mutants compared with parental Y1 cells, suggesting that
the Gß/
defect compromised transcriptional activity at the
proximal promoter region of the ACTH receptor gene.
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INTRODUCTION
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The ACTH receptor is a member of the melanocortin receptor
subfamily of G protein-coupled receptors (1) and is expressed in a
cell-selective and ACTH-inducible manner. Transcripts encoding this
receptor are present at high levels in the adrenal cortex and at lower
levels in fat tissue and skin, but appear to be undetectable at other
sites (2, 3, 4). In human, bovine, and mouse adrenocortical cells, ACTH
receptor transcripts are up-regulated by ACTH via a
cAMP-dependent mechanism (5, 6, 7, 8). The up-regulation of ACTH receptor
transcripts in human cells may involve increases in ACTH receptor gene
expression (5), whereas ACTH receptor up-regulation in bovine adrenal
cells is thought to result principally from mRNA stabilization (6). The
proximal promoter region of the mouse ACTH receptor (
1800 bp) has
been isolated, partially characterized, and shown to direct the
cell-selective expression of the gene when transfected into
adrenocortical cell lines in culture. Analysis of the mouse ACTH
receptor promoter revealed two distinct regions that contribute to the
adrenal cell-selective expression of the gene: a positive control
region that includes a steroidogenic factor-1 (SF-1) regulatory element
at -25 (relative to the start of transcription) and a negative control
region from -1236 to -908 (4). The SF-1 element appears to restrict
ACTH receptor expression to the few cell types where SF-1 is expressed,
including the adrenal cortex and other steroidogenic cells (9), whereas
the negative control region seems to prevent ACTH receptor expression
in SF-1-containing cells other than the adrenal cortex (4). Promoter
analyses also indicate a role for SF-1 in the expression of the human
ACTH receptor gene (10).
To gain a better understanding of the factors that regulate ACTH
receptor gene expression, we have examined its expression in a family
of mutants isolated from the Y1 mouse adrenocortical tumor cell line.
These mutants appeared to result from single mutational events as
determined by fluctuation analysis (11), were impaired in their ability
to express the ACTH receptor gene and, as a consequence, were resistant
to the stimulatory effects of ACTH on adenylyl cyclase and cAMP
accumulation (8, 12, 13). As we reported previously, a G protein
ß/
defect may be responsible for the complex phenotype of these
mutants since the ß/
-subunits from mutant cells were impaired in
functional reconstitution assays with the
-subunit of transducin
(14). These observations thus raised the possibility that ACTH receptor
gene expression was impaired in the mutants because of an underlying
defect in a Gß/
-dependent signaling process. In the present study,
we tested the role of Gß/
in ACTH receptor expression in a
representative mutant from this family. We show that ACTH receptor
expression can be restored in the mutant after transfection with genes
encoding Gß and G
, thus demonstrating a role for Gß/
in ACTH
receptor gene expression. In addition, we demonstrate that the mutation
affects the transcriptional activity of the proximal promoter region of
the ACTH receptor gene, suggesting a possible target of the Gß/
effect.
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RESULTS
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Analysis of ACTH Receptor Transcripts in Y1, 10r6, and 10r9
Cells
In previous studies, we demonstrated that several mutant clones
independently isolated from the Y1 mouse adrenocortical tumor cell line
were resistant to ACTH and had undetectable levels of ACTH receptor
transcripts by Northern blot hybridization or ribonuclease (RNase)
protection assays (8, 13). Furthermore, the decreased accumulation of
ACTH receptor transcripts in the mutants resulted from impaired
expression of the ACTH receptor gene as determined in nuclear run-off
experiments (13). In Fig. 1
, the levels
of ACTH receptor transcripts in parental Y1 cells and two ACTH
receptor-deficient mutants, 10r-9 and 10r-6, were compared using an
RT-PCR assay. ACTH receptor cDNA was prepared from total RNA and
amplified using an ACTH receptor-specific primer pair that spanned the
second intron of the ACTH receptor gene (4). After 30 cycles of
amplification, a prominent 240-bp product corresponding to the ACTH
receptor cDNA fragment was detected in parent Y1 cells, but not in the
10r-9 and 10r-6 mutants. When the amplification reaction was extended
to 40 cycles, 240-bp products were detected in both mutant clones as
well. The identity of the 240-bp ACTH receptor cDNA fragment was
confirmed by the formation of restriction fragments consistent with the
expected size of 134 bp and 106 bp after digestion of the PCR product
with the restriction endonuclease PstI (Fig. 1
).
Amplification of the oligo(dT)-primed cDNA products with primers
corresponding to the nuclear transcription factor SF-1 indicated that
this transcript was present in equivalent amounts in the samples from
parent and mutant cells (Fig. 1
). These results, together with our
previous results obtained using nuclear run-off and Southern blot
hybridization assays (13), thus indicate that the structural gene
encoding the ACTH receptor is present and grossly intact but is poorly
transcribed in the mutant cells.

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Figure 1. Amplification of ACTH Receptor Transcripts by
RT-PCR
A, Schematic diagram showing the primers used for cDNA synthesis
(oligo-dT and P29) and PCR amplification (P30 and P31). Also shown are
the position of a 1.6-kb intron that distinguishes ACTH receptor
genomic DNA from ACTH receptor cDNA (4 ) and the location of a
diagnostic PstI restriction site. B, RNA was isolated
from parent Y1 cells, from forskolin-resistant 10r6 and 10r9 cells, and
from 10r-6 and 10r-9 cells transfected with the mouse
ß2-adrenergic receptor (ß2AR) gene. cDNA
was synthesized using an oligo-dT primer, and ACTH receptor cDNAs were
amplified by PCR at 94 C for 1 min, 60 C for 1 min, and 72 C for 1 min
for 30 cycles with a hot start as described in Materials and
Methods. cDNA for the transcription factor, SF-1, was also
amplified from the same samples. Under these PCR conditions, the amount
of PCR product obtained was proportional to the amount of input
transcript. C, ACTH receptor cDNAs were amplified by PCR for 40 cycles,
and the PCR products were digested with PstI to confirm
the specific amplification of ACTH receptor transcripts. Under these
extended reaction conditions, the amount of PCR product obtained with
the Y1 transcript was not proportional to the input transcript. All
samples were electrophoresed on 1.5% agarose gels in the presence of
ethidium bromide and visualized by fluorescence. Sizes of PCR products
were determined using HaeIII-digested X174 a
standard. The signals corresponding to ACTH receptor (ACTH-R) and SF-1
cDNAs are indicated.
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Effects of Gß/
on ACTH Receptor Expression and Function
We next tested the hypothesis that the defects in G protein
ß/
activity previously reported in these mutants (14) contributed
to the decreased expression of the ACTH receptor gene. Mutant 10r-9
cells were transfected with cDNA expression vectors encoding bovine
Gß1 and bovine G
2 cDNAs or encoding human Gß2 and bovine G
2
cDNAs. Transformants were screened for recovery of ACTH responsiveness
by testing for ACTH-induced changes in cell shape. This morphological
screen was based on the well documented observation that Y1 cells
retract from the cell monolayer and assume a rounded morphology when
stimulated with agents that raise intracellular levels of cAMP. This
screen has been used successfully to isolate transformants expressing G
protein-coupled receptors including the ACTH receptor, the
ß2-adrenergic receptor, and the dopamine D1 receptor (13, 15, 16). As estimated by microscopic examination under phase contrast,
approximately 15% of the Gß1/
2 and Gß2/
2 transformants
rounded after ACTH treatment, suggesting a ß/
-dependent recovery
of functional ACTH receptor activity. The morphological response of the
colonies to ACTH was heterogeneous, even at the earliest stages of
transformation when the colonies contained only several hundred cells,
suggesting that the influence of the ß/
genes was unstable and
rapidly lost.
The recovery of ACTH-induced cell rounding after transfection with
Gß/
cDNA was accompanied by an increase in ACTH receptor gene
expression (Fig. 2
). RNA from six of the
selected Gß/
transformants was evaluated for the presence of ACTH
receptor transcripts by RT-PCR. cDNA was synthesized using an ACTH
receptor-specific primer (P29) and amplified using the ACTH receptor
primers that spanned intron 2 (P30 and P31). Five of the six
transformants contained ACTH receptor transcripts at levels that were
significantly greater than those in the untransfected 10r-9 mutant but
far less than the levels seen in parental Y1 cells (Fig. 2A
). These
findings were confirmed in a ß1/
2 transformant and a ß2/
2
transformant using oligo-dT as primer for first-strand cDNA synthesis
(Fig. 2B
). The PCR products generated in each case contained a
diagnostic PstI site that is present in ACTH receptor cDNA
between the amplification primers (Fig. 2C
). Amplification of the
oligo-dT-derived cDNA with SF-1 primers indicated that each
transformant contained equivalent amounts of the SF-1 transcript (Fig. 2B
). One transformant, ß2/
2#4, which was isolated on the basis of
ACTH-induced cell rounding, did not appear to be ACTH-receptor positive
in the subsequent PCR analysis (Fig. 2A
). Upon reexamination, this
clone lost its morphological response to ACTH, indicating that the
influence of ß/
was unstable in this clone and that the ability to
express the ACTH receptor transcript was lost during the propagation of
the colony.

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Figure 2. Amplification of ACTH Receptor Transcripts from
10r-9 Cells Transfected with G Protein ß/ cDNAs
Mutant 10r-9 cells were transfected with cDNA expression vectors
encoding bovine Gß1 and bovine G 2 cDNAs (10 µg each) or encoding
human Gß2 and bovine G 2 cDNAs (10 µg each). The neomycin
resistance plasmid, pSV2-neo (4 µg), was included in the transfection
experiments to facilitate recovery of transformation-competent cells.
Cells were isolated based on their morphological response to ACTH as
described in Materials and Methods. A, RNA was isolated
from parent Y1 cells, mutant 10r-9 cells, and the Gß1/ 2 and
Gß2/ 2 transformants of 10r-9 cells, reverse transcribed into cDNA
using the gene-specific primer P29, and amplified using P30 and P31 as
described in Fig. 1 . B, cDNAs were synthesized from RNA derived from
parent Y1 cells, mutant 10r-9 cells, a Gß1/ 2 transformant, and a
Gß2/ 2 transformant using oligo-dT as primer. The cDNAs were
amplified using the ACTH receptor primers P30 and P31 or SF-1 primers.
The arrows indicate the positions of the amplified
240-bp ACTH receptor (ACTH-R) cDNA and the 550-bp SF-1 cDNA. C, ACTH
receptor cDNA fragments were synthesized and amplified from parent Y1
cells and representative Gß1/ 2 and Gß2/ 2 transformants as in
panels A and B, using oligo-dT or the gene-specific primer P29 for
first-strand cDNA synthesis. The PCR products were digested with
PstI restriction endonuclease and electrophoresed on
1.5% agarose gels in the presence of ethidium bromide. Sizes of PCR
products were determined using HaeIII-digested X174
as a standard.
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The recovery of ACTH-induced cell rounding in the 10r-9 mutant after
transfection with Gß1/
2 or Gß2/
2 cDNAs suggested that the
transcripts detected by RT-PCR indeed encoded ACTH receptors
functionally coupled to adenylyl cyclase. Direct measurements of
ACTH-responsive adenylyl cyclase activity confirmed this conclusion
(Table 1
). In four of the Gß/
transformants, ACTH increased cAMP accumulation 2.4- to 10-fold,
whereas the adenylyl cyclase of the untransfected 10r-9 mutant was
completely resistant to ACTH; in the transformant Gß2/
2#3, ACTH
stimulated adenylyl cyclase activity to levels approaching those in
ACTH-stimulated parent Y1 cells (Table 1
). Concordant with the
heterogeneity and instability of the morphological response to ACTH,
the recovery of ACTH-responsive adenylyl cyclase activity was unstable
and declined rapidly as the transformants were kept in culture for a
period of time. The transformants lost approximately 50% of their
ACTH-responsive adenylyl cyclase activity when grown in culture for 30
days; after 41 days, the transformants were completely resistant to the
effects of ACTH on adenylyl cyclase (Fig. 3
) and on cell shape (data not
shown).
The induction of ACTH receptor transcripts seemed to be a specific
effect of Gß/
. Transfection of mutant 10r-9 or 10r-6 cells with
expression vectors encoding the mouse ß2-adrenergic
receptor and the neomycin resistance gene (pSV2-neo) gave rise to
neomycin-resistant transformants that expressed
ß2-adrenergic receptors functionally coupled to adenylyl
cyclase (13). These mutants remained functionally resistant to ACTH
(13), and ACTH receptor transcripts remained below detectable levels
(Fig. 1
). Similarly, transfection of 10r-6 or 10r-9 cells with an
epitope-tagged Gs
expression vector and pSV2-neo gave rise to
G418-resistant transformants that were ACTH-resistant and ACTH receptor
deficient by RT-PCR analysis, but stably expressed the Gs
gene (data
not shown).
Although we did not detect the spontaneous appearance of ACTH-induced
cell rounding in 10r-9 cells in the absence of Gß/
, we were
concerned that the use of a screen for ACTH-induced cell rounding may
have biased the selection process for spontaneous revertants.
Therefore, we examined the ability of Gß2 + G
2 cDNAs to affect
ACTH receptor transcript accumulation in 10r-9 cells in transient
transfection assays without selection for ACTH-induced cell rounding.
As shown in Fig. 4
, transfection of
mutant 10r-9 cells with the ß2 and
2 expression vectors induced
the accumulation of ACTH receptor transcripts in a time-dependent
manner.
Transfection of mutant 10r-9 cells with either the Gß2 or G
2
expression vector alone was sufficient for recovery of the rounding
response to ACTH and for expression of the ACTH receptor gene (Fig. 5
). The levels of ACTH receptor
transcripts seen after transfection with Gß2 or G
2 varied among
clones and, as seen after transfection with combinations of Gß and
G
cDNAs, were unstable. For example, the Gß2 transformant 1 was
ACTH receptor deficient (Fig. 5
) and morphologically unresponsive to
ACTH (data not shown) at the time of the RT-PCR assay, even though the
clone was originally isolated based on its ability to round in response
to ACTH.
Impaired ACTH Receptor Promoter Activity in Mutant 10r-9 and 10r-6
Clones
To further explore the basis for the impaired transcription of the
ACTH receptor gene in the mutant clones, a luciferase expression
plasmid under control of the proximal promoter region of the ACTH
receptor (bases -1808 to +104, Ref. 4) was tested for activity in
parent Y1 cells and in the 10r-9 and 10r-6 mutants. The activity of the
ACTH receptor promoter/luciferase construct was compared with the
activity of a luciferase reporter plasmid under control of the SV40
promoter and enhancer (Fig. 6
) or the
Rous sarcoma virus promoter (data not shown) to control for differences
in transfection efficiencies and transcriptional activity among
experiments and among clones. The ACTH receptor promoter enhanced
luciferase expression 48 ± 14-fold over the promoterless vector
when transfected into parental Y1 cells, but was only 5% as effective
when transfected into mutant 10r-9 cells and 8% as effective when
transfected into mutant 10r-6 cells (P < 0.05; Fig. 6
). ACTH receptor promoter activity was similarly compromised in clones
Y6 and OS3, two other ACTH receptor-deficient Y1 mutants (data not
shown). These observations thus indicate that the defect in these cells
leading to reduced expression of the ACTH receptor gene affects the
activity of its proximal promoter region.

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Figure 6. ACTH Receptor Promoter Activity in Mutant 10r-6 and
10r-9 Cells
Parent Y1 and mutant 10r-6 and 10r-9 cells were transfected with a
luciferase reporter plasmid (1 µg) containing ACTH receptor promoter
sequences from -1808 to +104, harvested 48 h later in a lysis
buffer containing 50 mM
Tris-2-[N-morpholino]ethanesulfonic acid (pH 7.8),
1% Triton X-100, 4 mM EDTA, and 1 mM
dithiothreitol and clarified by centrifugation. Cell supernatants were
assayed for luciferase activity (41 ) using a Berthold Lumat LB 9501
Luminometer. Results are expressed as percentages of the activity
achieved with a luciferase reporter plasmid under control of the SV40
promoter and enhancer and are the means of three experiments ±
SEM.
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DISCUSSION
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In the present study, we show that transfection of the ACTH
receptor-deficient 10r-9 mutant with expression vectors encoding Gß
and G
increases ACTH receptor expression (Figs. 2
, 4
, and 5
) and
restores ACTH-responsive adenylyl cyclase activity (Table 1
). These
observations, together with our previous study indicating that Gß/
function is impaired in this mutant (14), support the hypothesis that
the altered Gß/
activity is responsible for the decreased
expression of the ACTH receptor gene and establish a role for Gß/
in ACTH receptor expression. Although Gß/
has not been
investigated previously for its effects on gene expression, the dimer
has been shown to regulate a number of signaling pathways that might
impact on this process. These include both positive and negative
effects on the cAMP-signaling cascade through regulation of different
adenylyl cyclase isoforms, activation of Ca2+-signaling
pathways and protein kinase C through effects on Ca2+ and
K2+ channels and through activation of phospholipases C and
A2, and activation of the mitogen-activated protein kinase cascade
(17). We think it unlikely that ß/
influenced ACTH receptor
expression in this mutant through an effect on cAMP accumulation or
Ca2+ signaling, since agents that raise intracellular cAMP
or Ca2+ levels failed to restore ACTH receptor expression
(Ref. 13 and R. Qiu and B.P. Schimmer, unpublished observations).
Restoration of ACTH receptor expression was achieved after transfection
not only with combinations of ß1/
2 or ß2/
2 but also with ß2
or
2 alone. The ß- and
-subunits form tight complexes that
function as heterodimers in the physiological state (18) and can fold
into a native structure only when cosynthesized (19). In addition, the
accumulation and assembly of ß- and
-subunits appear to be tightly
coupled (18). Therefore, it is likely that the effects of ß2 or
2
alone resulted from the de novo synthesis and assembly of
ß/
complexes using endogenous partners. The finding that either
ß or
was able to restore ACTH receptor expression was somewhat
surprising, given that the underlying defect results from single
mutational events (11), but was reminiscent of a similar phenomenon
seen in cAMP-resistant, protein kinase A-defective mutants (20, 21). In
the latter example, cAMP resistance caused by point mutations in the
regulatory subunit of the enzyme could be overcome by transfection with
expression plasmids encoding either the wild-type regulatory or
catalytic subunits of the enzyme. We suggest that models of subunit
interaction similar to those proposed for the protein kinase A
mutations (20, 21) may explain how either ß or
alone is able to
rescue ß
function in the ACTH receptor-deficient mutant. We have
not yet identified the spectrum of ß/
-isoforms present in Y1 and
10r-9 cells; however, it should be noted that ß1 and ß2 each can
form functional complexes with different
isoforms including
2,
5, and
7 (22, 23, 24). Therefore, the restoration of ACTH receptor
expression after transfection of mutant 10r-9 cells with ß2 or
2
does not necessarily reflect the specific contribution of these
isoforms to the mutant phenotype.
The ß/
-transformants were highly unstable and resulted in mixed
populations of ACTH-sensitive and -insensitive clones that rapidly
progressed to ACTH resistance in continuous culture (e.g.
Fig 3
). We have not encountered this degree of instability upon
transfection of Y1 cells with a variety of other genes (e.g.
Refs. 13, 15, 16, 25), suggesting that ß and/or
may be growth
inhibiting or otherwise toxic to these cells. Despite this population
heterogeneity, we were able to detect increased expression of ACTH
receptor transcripts and recovery of ACTH-responsive adenylyl cyclase
activity after transfection, probably because the background signals
for these measurements were very low. The loss of ACTH receptor
expression in the 10r-9 and 10r-6 mutants was accompanied by a defect
in the transcriptional activity of the proximal promoter region of the
ACTH receptor gene (Fig. 6
), suggesting that the loss of ACTH receptor
expression in the mutant cells resulted from a defect that impaired
promoter function. It thus follows that the restoration of ACTH
receptor expression after transfection of the mutant with Gß/
may
have resulted from an effect of Gß/
on the ACTH receptor promoter.
Unfortunately, we have not been able to demonstrate a direct effect of
Gß/
on ACTH receptor promoter function since the Gß and G
expression vectors nonspecifically squelched promoter activity in
transient transfection experiments (data not shown).
The ACTH receptor-deficient mutants 10r-9 and 10r-6 were isolated by
selection of cells resistant to the growth inhibiting effects of
forskolin and arose from mutational events at single genetic loci as
determined by fluctuation analysis (11). In an effort to identify
candidate genes responsible for the underlying mutation, we have
carried out an extensive analysis of these mutants and showed that they
exhibit a complex phenotype, much of which is secondary to the
underlying mutation. Intriguingly, the characterization of these
mutants has revealed a pathway of regulation not previously appreciated
in which Gß/
regulates expression of the ACTH receptor gene (this
study) which, in turn, stabilizes Gs
and Gi
in the plasma
membrane leading to optimal activation of adenylyl cyclase by ACTH and
other G protein-coupled receptors (13). As we have shown previously,
the resistance to the growth-inhibiting effect of forskolin results
from a failure of forskolin to maximally activate adenylyl cyclase and
enhance cAMP accumulation (12). Although forskolin activates adenylyl
cyclase by binding directly to its catalytic domain and acts
synergistically with Gs
(26), our results indicate that the mutant
phenotype does not result from Gs
or adenylyl cyclase mutations.
Although Gs
levels are reduced in these mutants (27), restoration of
Gs
levels either by transfection with G protein-coupled receptors
(13) or with wild-type Gs
(data not shown) does not reverse the
forskolin-resistant phenotype. Likewise, mutations in adenylyl cyclase
are not likely to account for the mutant phenotype since
fluoride-stimulated, Mg2+-stimulated, and
Mn2+-stimulated adenylyl cyclase activities are not
compromised in these mutants (28), and treatment of mutant cells with
cAMP or with agents that elevate cAMP fail to restore parts of the
phenotype including ACTH receptor expression and Gs
and Gi
levels
(13). Thus, the underlying mutation leading to forskolin resistance
remains undefined. It is tempting to speculate that the altered
Gß/
activity is also responsible for the forskolin resistance seen
in this mutant; however, the instability of the ß/
transformants
and resultant population heterogeneity precluded a definitive
conclusion regarding the effects of ß/
on forskolin-stimulated
adenylyl cyclase activity or forskolin-dependent growth inhibition.
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MATERIALS AND METHODS
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Plasmids and Oligonucleotides
DNAs encoding bovine Gß1 and G
2 and human Gß2 were in the
expression plasmid pCDM81 (29). The luciferase expression plasmid
under control of the proximal promoter region of the mouse ACTH
receptor gene (from -1808 to +104 relative to the start of
transcription, Ref. 4) and the expression plasmid containing the
neomycin resistance gene (pSV2-neo, Ref. 30) were described previously.
The luciferase expression plasmid containing SV40 promoter and enhancer
sequences (pGL3-control) was obtained from Promega (Madison, WI).
The oligonucleotides used for analysis of ACTH receptor transcripts by
RT-PCR (Fig. 1
) were derived from the GenBank sequence under accession
number D31952 and are:
P29, 5'-CCAACATGTCAGAAATGGCCAAACTGC-3'
P30, 5'-CAGGACAATCGGAGTTATTTCTTGCGG-3'
P31, 5'-CCAAGGAGAGGAGCATTATTGG-3'
The forward and reverse oligonucleotide primers used for amplification
of SF-1 transcripts were derived from the GenBank sequence under
accession number S65874 and are:
5'-GCATTACACGTGCACCGAG-3' and 5'-GGCTCTAGT- TGCAGCAGCTG-3'.
All oligonucleotides were obtained from Canadian Life Technologies Inc.
(Burlington, Ontario, Canada).
RT-PCR Analysis
In most instances, total cellular RNA was extracted from cells
using guanidinium thiocyanate and purified by centrifugation through a
cushion of CsCl (31). For transient transfection experiments, total
cellular RNA was isolated using an RNA-easy Mini kit (Qiagen Inc.,
Canada, Mississauga, Ontario, Canada) according to the manufacturers
instructions. For first-strand cDNA synthesis, RNA (110 µg) was
mixed with 1050 pmol of primer and heated for 10 min at 70 C. A
reaction solution containing 20 mM Tris-HCl (pH 8.4), 50
mM KCl, 3 mM MgCl2, 10
mM dithiothreitol, 400 µM deoxynucleoside
triphosphates (dATP, dCTP, dGTP, dTTP) and 200 U Superscript II
reverse transcriptase (Canadian Life Technologies) was added to RNA
samples, and the mixture was incubated for 1 h at 42 C. Reverse
transcriptase was inactivated by heating the samples for 15 min at 70
C. RNase H (Canadian Life Technologies) was added for 10 min at 55 C to
eliminate the RNA template. The cDNA was next added to a PCR mixture
containing 10 mM Tris-HCl, 50 mM KCl, 1.5
mM MgCl2, 0.1% gelatin, 200 µM
deoxynucleoside triphosphates, and 50100 pmol of oligonucleotide
primers in a total volume of 50100 µl. PCR was initiated by the
addition of Taq DNA polymerase (2.5 U, Boehringer Mannheim
Canada, Laval, Quebec, Canada) using a hot start (32). Samples were
incubated for 1-min intervals at 94 C, 60 C, and 72 C over 2535
cycles and then finally incubated at 72 C for 10 min to complete the
reaction. The amount of input cDNA and the number of cycles were
adjusted to ensure a linear relationship between the amount of starting
cDNA and PCR products.
cAMP Accumulation
Assays of cAMP accumulation were performed essentially as
described previously (33). Cell monolayers were incubated with
[2-3H]adenine (8 µCi) for 2 h in 2 ml of
-MEM
to label endogenous pools of ATP, and then were rinsed and incubated
with 1 ml of medium plus 0.1% trypsin inhibitor for 30 min at 36.5 C.
ACTH (ACTHar, 24 nM) was added to the incubation medium as
indicated. At the end of the incubation, medium was collected from the
dishes, 0.2 ml of a recovery mix containing 40 mM disodium
ATP and 12.5 mM cAMP was added, and labeled cAMP was
separated from other labeled compounds by chromatography on Dowex AG
50W-X4 ion exchange resin and treatment with BaSO4. Cell
protein was determined by the method of Lowry et al.
(34).
Cells, Cell Culture, and Gene Transfer
The Y1 mouse adrenocortical tumor cell line used in this study
is a stable subclone (35) of the population originally described by
Yasumura et al. (36). ACTH receptor-deficient Y1 mutants
(clones 10r-9 and 10r-6) were isolated on the basis of their resistance
to the growth-inhibiting effects of forskolin (12). The
forskolin-resistant mutants expressing the mouse
ß2-adrenergic receptor were described previously (13, 37). Cells were cultured as monolayers at 36.5 C under a humidified
atmosphere of 95% air-5% CO2 in nutrient mixture F-10
supplemented with 15% heat-inactivated horse serum, 2.5%
heat-inactivated FBS and antibiotics. Tissue culture reagents were
obtained from Canadian Life Technologies.
For transfection assays, cells were transferred to
-MEM supplemented
with serum and antibiotics as above and transformed with super-coiled
plasmid DNA using a calcium phosphate precipitation technique described
previously (38, 39). Two different transfection protocols were used to
assess the roles of Gß/
on ACTH receptor gene expression. In one
protocol, cells at 3040% saturation density were transfected with
Gß and/or G
cDNA expression vectors together with the neomycin
resistance plasmid, pSV2-neo (30). Cells were then transferred to
growth medium containing the neomycin analog G418 (100 µg/ml) and
cultured for an additional 23 weeks to selectively grow transformants
that incorporated and stably expressed the neo gene. G418-resistant
colonies were screened for changes in cell shape in response to ACTH
(24 nM); colonies that rounded up in the presence of the
hormone were isolated using cloning cylinders and maintained in
continuous culture in the presence of G418. In a second protocol, cells
were transfected with the Gß and/or G
cDNA expression vectors in
the absence of pSV2-neo using the lipid-mediated transfection procedure
of Felgner et al. (40). Cells then were incubated in growth
medium for 4872 h without selection and assayed for ACTH receptor
expression.
 |
ACKNOWLEDGMENTS
|
---|
We thank Adrian Clark for the ACTH receptor promoter-luciferase
construct, Harry Elsholtz for use of the lumino-meter, William
Simonds for the Gß- and G
-expression vectors, J. Bourgouin
(Rhône-Poulenc Rorer, Montreal, Quebec, Canada) for ACTHar, and
Keith L. Parker for helpful discussion.
 |
FOOTNOTES
|
---|
Address requests for reprints to: Professor Bernard P. Schimmer, Ph.D., Banting and Best Department of Medical Research, University of Toronto, 112 College Street, Toronto, Ontario M5G 1L6 Canada. E-mail:
bernard.schimmer{at}utoronto.ca
This work was supported by research grants from the Medical Research
Council of Canada.
Received for publication August 3, 1998.
Revision received September 9, 1998.
Accepted for publication September 14, 1998.
 |
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