A Role for Guanyl Nucleotide-Binding Regulatory Protein ß- and {gamma}-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


    ABSTRACT
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
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/{gamma} 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/{gamma} 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{gamma}2 increased ACTH receptor expression and restored ACTH-responsive adenylyl cyclase activity. Interestingly, either Gß2 or G{gamma}2 alone was effective. These results thus support the hypothesis that the impairment in Gß/{gamma} 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/{gamma} defect compromised transcriptional activity at the proximal promoter region of the ACTH receptor gene.


    INTRODUCTION
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
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/{gamma} defect may be responsible for the complex phenotype of these mutants since the ß/{gamma}-subunits from mutant cells were impaired in functional reconstitution assays with the {alpha}-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/{gamma}-dependent signaling process. In the present study, we tested the role of Gß/{gamma} 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{gamma}, thus demonstrating a role for Gß/{gamma} 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/{gamma} effect.


    RESULTS
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
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. 1Go, 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. 1Go). 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. 1Go). 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 {phi}X174 a standard. The signals corresponding to ACTH receptor (ACTH-R) and SF-1 cDNAs are indicated.

 
Effects of Gß/{gamma} on ACTH Receptor Expression and Function
We next tested the hypothesis that the defects in G protein ß/{gamma} 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{gamma}2 cDNAs or encoding human Gß2 and bovine G{gamma}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/{gamma}2 and Gß2/{gamma}2 transformants rounded after ACTH treatment, suggesting a ß/{gamma}-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/{gamma} genes was unstable and rapidly lost.

The recovery of ACTH-induced cell rounding after transfection with Gß/{gamma} cDNA was accompanied by an increase in ACTH receptor gene expression (Fig. 2Go). RNA from six of the selected Gß/{gamma} 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. 2AGo). These findings were confirmed in a ß1/{gamma}2 transformant and a ß2/{gamma}2 transformant using oligo-dT as primer for first-strand cDNA synthesis (Fig. 2BGo). The PCR products generated in each case contained a diagnostic PstI site that is present in ACTH receptor cDNA between the amplification primers (Fig. 2CGo). Amplification of the oligo-dT-derived cDNA with SF-1 primers indicated that each transformant contained equivalent amounts of the SF-1 transcript (Fig. 2BGo). One transformant, ß2/{gamma}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. 2AGo). Upon reexamination, this clone lost its morphological response to ACTH, indicating that the influence of ß/{gamma} 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/{gamma} cDNAs

Mutant 10r-9 cells were transfected with cDNA expression vectors encoding bovine Gß1 and bovine G{gamma}2 cDNAs (10 µg each) or encoding human Gß2 and bovine G{gamma}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/{gamma}2 and Gß2/{gamma}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. 1Go. B, cDNAs were synthesized from RNA derived from parent Y1 cells, mutant 10r-9 cells, a Gß1/{gamma}2 transformant, and a Gß2/{gamma}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/{gamma}2 and Gß2/{gamma}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 {phi}X174 as a standard.

 
The recovery of ACTH-induced cell rounding in the 10r-9 mutant after transfection with Gß1/{gamma}2 or Gß2/{gamma}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 1Go). In four of the Gß/{gamma} 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/{gamma}2#3, ACTH stimulated adenylyl cyclase activity to levels approaching those in ACTH-stimulated parent Y1 cells (Table 1Go). 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. 3Go) and on cell shape (data not shown).


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Table 1. cAMP Accumulation in Forskolin-Resistant 10r-9 Cells Transfected with Gß/{gamma} cDNA

 


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Figure 3. Stability of ACTH-Stimulated cAMP Accumulation in Gß/{gamma}-Transfected Mutant 10r9 Cells

The ß/{gamma}-transformants described in Table 1Go were thawed from frozen stocks and then plated out for the assay of adenylyl cyclase activity after different intervals in culture. Cells were prelabeled with [8-3H] adenine for 2 h to label the endogenous pool of ATP and assayed for cAMP accumulation in the absence or presence of ACTH (24 nM) as described in Materials and Methods. Results are presented as the mean fold increases in ACTH-stimulated cAMP accumulation among the four transformants at the different time intervals ± SEM. The fold increases in cAMP accumulation among the transformants at day 41 were not significantly different from unstimulated controls.

 
The induction of ACTH receptor transcripts seemed to be a specific effect of Gß/{gamma}. 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. 1Go). Similarly, transfection of 10r-6 or 10r-9 cells with an epitope-tagged Gs{alpha} 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{alpha} 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/{gamma}, 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{gamma}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. 4Go, transfection of mutant 10r-9 cells with the ß2 and {gamma}2 expression vectors induced the accumulation of ACTH receptor transcripts in a time-dependent manner.



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Figure 4. ACTH Receptor Transcripts in 10r-9 Cells Transiently Transfected with Expression Vectors Encoding Gß- and G{gamma}-Subunits

Mutant 10r-9 cells were transiently transfected with 5 µg each of human Gß2 and bovine G{gamma}2 cDNAs using Lipofectin Reagent (Canadian Life Technologies). RNA was prepared from the transformants at different intervals after transfection using a Qiagen RNA purification kit; cDNA was synthesized using the ACTH receptor gene-specific primer P29 and amplified by PCR using primers P30 and P31 (Fig. 1Go). Results are shown for two transfected samples at each time interval.

 
Transfection of mutant 10r-9 cells with either the Gß2 or G{gamma}2 expression vector alone was sufficient for recovery of the rounding response to ACTH and for expression of the ACTH receptor gene (Fig. 5Go). The levels of ACTH receptor transcripts seen after transfection with Gß2 or G{gamma}2 varied among clones and, as seen after transfection with combinations of Gß and G{gamma} cDNAs, were unstable. For example, the Gß2 transformant 1 was ACTH receptor deficient (Fig. 5Go) 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.



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Figure 5. ACTH Receptor Transcripts from Mutant Cells Transfected with Expression Vectors Encoding either Gß2 or G{gamma}2

Mutant 10r-9 cells were transfected with either the Gß2 or G{gamma}2 expression vector (5 µg) and pSV2-neo (2 µg). Transformants were isolated by selective growth in G418 and screened for recovery of morphological responses to ACTH. A, RNA was extracted from parent Y1 cells, mutant 10r-9 cells, and the 10r-9 transformants; cDNA was synthesized using the oligo-dT primer; ACTH receptor transcripts were amplified using the primers P30 and P31 (Fig. 1Go). B, SF-1 transcripts were amplified from the same cDNA samples. HaeIII-digested {phi}X174 served as a standard to estimate the sizes of the PCR products.

 
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. 6Go) 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. 6Go). 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.

 

    DISCUSSION
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
In the present study, we show that transfection of the ACTH receptor-deficient 10r-9 mutant with expression vectors encoding Gß and G{gamma} increases ACTH receptor expression (Figs. 2Go, 4Go, and 5Go) and restores ACTH-responsive adenylyl cyclase activity (Table 1Go). These observations, together with our previous study indicating that Gß/{gamma} function is impaired in this mutant (14), support the hypothesis that the altered Gß/{gamma} activity is responsible for the decreased expression of the ACTH receptor gene and establish a role for Gß/{gamma} in ACTH receptor expression. Although Gß/{gamma} 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/{gamma} 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/{gamma}2 or ß2/{gamma}2 but also with ß2 or {gamma}2 alone. The ß- and {gamma}-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 {gamma}-subunits appear to be tightly coupled (18). Therefore, it is likely that the effects of ß2 or {gamma}2 alone resulted from the de novo synthesis and assembly of ß/{gamma} complexes using endogenous partners. The finding that either ß or {gamma} 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 {gamma} alone is able to rescue ß{gamma} function in the ACTH receptor-deficient mutant. We have not yet identified the spectrum of ß/{gamma}-isoforms present in Y1 and 10r-9 cells; however, it should be noted that ß1 and ß2 each can form functional complexes with different {gamma} isoforms including {gamma}2, {gamma}5, and {gamma}7 (22, 23, 24). Therefore, the restoration of ACTH receptor expression after transfection of mutant 10r-9 cells with ß2 or {gamma}2 does not necessarily reflect the specific contribution of these isoforms to the mutant phenotype.

The ß/{gamma}-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 3Go). 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 {gamma} 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. 6Go), 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/{gamma} may have resulted from an effect of Gß/{gamma} on the ACTH receptor promoter. Unfortunately, we have not been able to demonstrate a direct effect of Gß/{gamma} on ACTH receptor promoter function since the Gß and G{gamma} 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/{gamma} regulates expression of the ACTH receptor gene (this study) which, in turn, stabilizes Gs{alpha} and Gi{alpha} 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{alpha} (26), our results indicate that the mutant phenotype does not result from Gs{alpha} or adenylyl cyclase mutations. Although Gs{alpha} levels are reduced in these mutants (27), restoration of Gs{alpha} levels either by transfection with G protein-coupled receptors (13) or with wild-type Gs{alpha} (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{alpha} and Gi{alpha} levels (13). Thus, the underlying mutation leading to forskolin resistance remains undefined. It is tempting to speculate that the altered Gß/{gamma} activity is also responsible for the forskolin resistance seen in this mutant; however, the instability of the ß/{gamma} transformants and resultant population heterogeneity precluded a definitive conclusion regarding the effects of ß/{gamma} on forskolin-stimulated adenylyl cyclase activity or forskolin-dependent growth inhibition.


    MATERIALS AND METHODS
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Plasmids and Oligonucleotides
DNAs encoding bovine Gß1 and G{gamma}2 and human Gß2 were in the expression plasmid pCDM8–1 (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. 1Go) 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 manufacturer’s instructions. For first-strand cDNA synthesis, RNA (1–10 µg) was mixed with 10–50 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 50–100 pmol of oligonucleotide primers in a total volume of 50–100 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 25–35 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 {alpha}-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 {alpha}-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/{gamma} on ACTH receptor gene expression. In one protocol, cells at 30–40% saturation density were transfected with Gß and/or G{gamma} 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 2–3 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{gamma} 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 48–72 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{gamma}-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.


    REFERENCES
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 

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