Forskolin-resistant Y1 adrenal cell mutants are deficient in adenylyl cyclase type 4

Molecular and Cellular Endocrinology 214 (2004) 155–165

Forskolin-resistant Y1 adrenal cell mutants are deficient in adenylyl cyclase type 4 Abdallah Al-Hakim a , Xianliang Rui b , Jennivine Tsao b , Paul R. Albert c , Bernard P. Schimmer a,b,∗ a

b

Department of Pharmacology, University of Toronto, Toronto, Ont., Canada M5G 1L6 Banting and Best Department of Medical Research, University of Toronto, 112 College St., Toronto, Ont., Canada M5G 1L6 c Ottawa Health Research Institute (Neuroscience), University of Ottawa, Ottawa, Canada K1H 8M5 Received 3 September 2003; accepted 21 October 2003

Abstract Four mutant clones independently derived from the Y1 mouse adrenocortical tumor cell line have adenylyl cyclase (AC) activities that are resistant to forskolin, a direct activator of AC. In this study the AC isoform composition of the forskolin-resistant mutants was examined in order to explore the underlying basis for the resistance to forskolin. As determined by Western blot and RT–PCR analysis, the four forskolin-resistant mutants all were deficient in AC-4; the levels of other AC isoforms (AC-1, AC-3 and AC-5/6) were comparable to the levels in parent Y1 cells. Transfection of one of the mutant clones with an AC-4 expression vector increased forskolin-stimulated cAMP signaling, and restored forskolin-induced changes in cell morphology and growth. Taken together, these observations indicate that AC-4 deficiency is a hallmark of the forskolin-resistant phenotype of these mutants and suggest that AC-4 is an important target of forskolin action in the Y1 adrenal cell line. © 2003 Elsevier Ireland Ltd. All rights reserved. Keywords: Adenylyl cyclase type 4; Forskolin; Y1 mouse adrenocortical tumor cells

1. Introduction To date, nine membrane-bound adenylyl cyclase (AC) isoforms have been identified in mammalian cells. These isoforms have tissue-specific patterns of distribution and are distinguished by their responses to G protein subunits, calcium and protein kinases A and C—properties that are thought to contribute to the orchestrated actions of a diverse group of hormones, neurotransmitters and drugs on their respective target tissues (see D’Aquila et al., 1991; Defer et al., 2000; Onda et al., 2001, for review). Specific functions, however, have been definitively assigned to only a few of the mammalian isoforms. Based largely on the phenotypes of AC-disrupted mice, critical roles have been demonstrated for: AC-3 in olfaction (Wong et al., 2000); AC-5 in motor function (Iwamoto et al., 2003); AC-1 and AC-8 Abbreviations: AC, adenylyl cyclase; IPTG, isopropyl-␤-d-thiogalactoside; mc2r, ACTH-receptor; MEM, minimal essential medium ∗ Corresponding author. Tel.: +1-416-978-6088; fax: +1-416-978-8528. E-mail address: [email protected] (B.P. Schimmer).

in aspects of synaptic plasticity and memory (Wong et al., 1999); AC-8 in modulation of anxiety (Schaefer et al., 2000) and in Ca2+ -stimulated cAMP accumulation in the parotid (Watson et al., 2000). Overall, the nine AC isoforms are topologically similar. They have two transmembrane domains, each comprised of six helical clusters, and a catalytic core formed by the two major cytoplasmic domains of the enzyme (Sunahara et al., 1996; Tang and Hurley, 1998). Forskolin activates eight of the nine AC isoforms by binding to the catalytic core of each enzyme and stabilizing the enzyme in a catalytically favorable conformation (Tesmer et al., 1997); AC-9 fails to respond to forskolin but can be converted to a forskolin-responsive form by a single amino acid substitution within the catalytic core (Yan et al., 1998). Because of its ability to activate most AC isoforms, forskolin has been widely used to examine the roles of cAMP in hormone action and other aspects of cell regulation. As a model for cAMP-mediated signaling in the adrenal cortex, we have been investigating forskolin action in the Y1 mouse adrenocortical tumor cell line and in derivative mutants with specific defects in the cAMP-signaling

0303-7207/$ – see front matter © 2003 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.mce.2003.10.066

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pathway. Our investigations of these mutants have led to the appreciation of the obligatory roles of adenylyl cyclase and cAMP in many aspects of adrenocortical function (reviewed in Schimmer, 1989, 1995). One family of mutants isolated from Y1 cells is resistant to the inhibitory effects of forskolin on cell proliferation and morphology (Schimmer and Tsao, 1984; Qiu et al., 1996). The resistance to forskolin was shown to arise with a frequency consistent with single mutational events (Schimmer et al., 1985) and was due to the inability of forskolin to activate AC optimally (Schimmer and Tsao, 1984; Schimmer et al., 1987). In this study, we examine the possibility that forskolin resistance results from a defect affecting a specific AC isoform. We report that forskolin-responsive Y1 cells express principally AC types 1, 3, 4, 5 and 6. Four independently isolated forskolin-resistant mutants express AC types 1, 3, 5 and 6 but are deficient in AC-4. Transfection of one of our mutant clones with a rat AC-4 gene restored the growth-inhibitory and morphological responses to forskolin. On the basis of these observations, we suggest that AC-4 contributes to the growth-inhibitory and morphological effects of forskolin in the Y1 adrenal cell line.

2. Materials and methods 2.1. Cells and cell culture The forskolin-responsive cells referred to as Y1 in this study (clone Y1BS1; Schimmer, 1979) and the forskolinresistant OS3 mutant (Qiu et al., 1996; Schimmer, 1969) are stable subclones of the mouse adrenocortical tumor cell line originally isolated by Yasumura (1968). Mutant 10r6 and 10r9 cells were isolated from the Y1BS1 population by selective growth in 10 ␮M forskolin (Schimmer and Tsao, 1984). The forskolin-resistant mutant Y6 (Qiu et al., 1996) was cloned from the same mouse adrenocortical tumor that gave rise to the original Y1 isolate (Yasumura, 1968). Cells were propagated at 37 ◦ C under a humidified atmosphere of 95% air–5% CO2 in nutrient mixture F10 supplemented with 15% heat-inactivated horse serum, 2.5% heat-inactivated fetal bovine serum, penicillin G sodium and streptomycin sulfate as described previously (Schimmer, 1985). Tissue culture reagents were obtained from Invitrogen Canada (Burlington, Ont.). 2.2. Western blot analysis of AC isoforms Total cell lysates were prepared by scraping cell monolayers into a radioimmunoprecipitation assay buffer containing protease inhibitors (9.1 mM Na2 HPO4 , 1.7 mM NaH2 PO4 , 150 mM NaCl, pH 7.4, 1% Nonidet P-40, 0.5% deoxycholic acid, 0.1% SDS, 5 mM EDTA, 20 ␮g/ml leupeptin, 50 ␮g/ml aprotinin, 1 mM phenylmethanesulfonyl fluoride and 5 mM benzamidine). Samples were solubilized by trituration using a 21-guage needle and clarified by centrifugation at

20,800 × g for 20 min at 10 ◦ C. Plasma membrane-enriched fractions were prepared from cell homogenates using a combination of differential centrifugation and discontinuous sucrose density-gradient centrifugation as described in detail previously (Schimmer et al., 1979; Schimmer, 1983; Watt and Schimmer, 1981). Samples were boiled in SDS sample buffer (Laemmli, 1970), electrophoresed on 7% polyacrylamide gels in the presence of SDS and electroblotted onto nitrocellulose membranes. The membranes were blocked with 5% skim milk powder in 20 mM Tris–HCl, pH 7.6, 140 mM NaCl, 0.05% Tween-20, and incubated for 1 h with affinity-purified, isozyme-specific AC peptide antibodies (Santa Cruz Biotechnology Inc., Santa Cruz, CA) diluted 1:200 in blocking buffer. The identities of isozyme-specific peptide immunogens and the chemical modifications introduced for immunization are proprietary information and not available from the supplier. Membranes were rinsed three times in blocking buffer and then incubated with a 1:10,000 dilution of horseradish peroxidase-conjugated goat anti-rabbit immunoglobulin G (Amersham Bioscience, Que., Canada) for 1 h. Signals were developed using Renaissance Enhanced Luminol Reagent (NEN Life Science Products, Boston, MA) according to the manufacturer’s instructions. To quantitate the amount of each AC isoform present on the Western blots, a standard curve for each AC isoform was generated using the corresponding isoform-specific blocking peptide—i.e. the isoform-specific peptide immunogen. Each blocking peptide was dissolved in phosphate buffered saline containing 1% bovine serum albumin (BSA), and spotted onto nitrocellulose filters in varying amounts (0.4–25 pmol for AC-1, AC-3 and AC-5/6;1 0.045–0.60 pmol for AC-4) using a Minifold II slot blot apparatus (Schleicher and Schuell, Keene, NH). Blots were blocked and incubated with isozyme-specific AC antibodies together with the Western blotted membranes as described above. The chemiluminescent signals from the Western blots and slot blots were quantitated on the Fluor-S MultiImager System from Bio-Rad Laboratories (Canada) Ltd. (Missassauga, Ont.) using resident Quantity One software. 2.3. Detection of AC isoforms by RT–PCR RNA was isolated by extraction with guanidinium thiocyanate (Chirgwin et al., 1979), purified by density-gradient centrifugation through a 5.7 M CsCl cushion and chromatographed over oligo(dT) cellulose to obtain poly(A)+ RNA. The RNA (3 ␮g) was reverse-transcribed using SuperScript II RNase H− reverse transcriptase (Invitrogen Canada) and oligo(dT)18 (100 pmol) for 1 h at 37 ◦ C. AC isoform-specific cDNAs were amplified from the RT reaction by PCR using Platinum Taq DNA polymerase (Invitrogen Canada) and gene-specific primer pairs (1 pmol each/␮l). Each PCR reaction was initiated by the addition 1 The AC-5 and AC-6 isoforms are considered together because commercially available antibodies do not discriminate between the two.

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of the Taq polymerase with a hot start, and cycled through multiple rounds of amplification. Each cycle consisted of a 94 ◦ C incubation for 1 min, a specified annealing temperature for 1 min and a 1-min extension period at 72 ◦ C. The reaction was ended by incubating for 10 min at 72 ◦ C (final extension) and maintained at 4 ◦ C thereafter. The GenBank accession number, primer pairs, annealing temperature, amplification cycles and size of the expected PCR product for each isozyme were:

157

ing the cDNA into the NotI sites of the lac-switch vector pOPI3CAT (Stratagene, La Jolla, CA). Mutant 10r6 cells were transfected either with 6 ␮g of supercoiled pOPSV-AC-4 DNA alone for constitutive AC-4 expression or with 3 ␮g of supercoiled pOPSV-AC-4 DNA plus 7 ␮g of p3 SS (an expression vector encoding the lac repressor; Stratagene) for IPTG-inducible AC-4 expression. Gene transfers were carried out on monolayers of mutant 10r6 cells at 80% confluence using Lipofectamine 2000TM

AC-1

AF053980 (forward) 5 -AGCTATGAGCCAATCATGGC-3 , (reverse) 5 -GACATGAGGAAGTGCTGTGC-3 , 59 ◦ C, 37 cycles, 206 bp product

AC-3

NM 138305 (forward) 5 -CCTGTGTGCTATCATCGTGG-3 , (reverse) 5 -TCATCTAGGTAGTCGCAGCG-3 , 60 ◦ C, 30 cycles, 753 bp product

AC-4

AF442771 (forward) 5 -CGTTCTCTGTGGAGTCATTGG-3 , (reverse) 5 -CCTGGAAGAACTTAGCATCACC-3 , 60 ◦ C, 37 cycles, 503 bp product

AC-5

BC035550 (forward) 5 -ATGTCATGTGATGGAGTCGG-3 , (reverse) 5 -TCCTCCTTCTCTTCTGTGGC-3 , 60 ◦ C, 37 cycles, 450 bp product

AC-6

NM 007405 (forward) 5 -CTCCATGGAAGGACTGATGC-3 , (reverse) 5 -CCGCTGGTGTTAAGTTCAGC-3 , 60 ◦ C, 30 cycles, 623 bp product

The identities of the amplified cDNA fragments were confirmed by digestion with diagnostic restriction endonucleases and by sequencing PCR products cloned into pCR2.1 (Invitrogen Canada). 2.4. Southern blot hybridization analysis of the AC-4 gene High mol wt genomic DNA from parent and mutant cells (25 ␮g per sample) was digested with XhoI, KpnI or HindIII, electrophoresed on 0.5% agarose gels and blotted onto Nytran SuperCharge nylon membranes (Schleicher and Schuell). The membranes were probed by hybridization with a full length 3.4 kb AC-4 cDNA. The AC-4 cDNA for hybridization was isolated from the IMAGE Consortium mouse clone 4236007 (GenBank accession number BF782774) following digestion with XbaI and SalI restriction endonucleases and labeled by nick-translation with [␣-32 P]dCTP. 2.5. Immunocytochemical localization of AC-4

(Invitrogen Canada) in Alpha MEM without antibiotics, according to the manufacturer’s guidelines. Transformants were isolated using G418 (100 ␮g/ml; Invitrogen Canada) to select for the AC-4 vector and hygromycin (200 ␮g/ml; Invitrogen Canada) to select for the lac repressor vector. The cAMP-dependent reporter plasmid, pCRE-Luc (a gift from Dr. Christian Vaisse, Department of Medicine, University of California, San Franciso, CA) is a luciferase reporter gene under control of a modified mouse mammary tumor virus promoter with 16 copies of a cAMP-response element from the CRH gene replacing the glucocorticoid-responsive region (Spengler et al., 1993). Cells plated in 6-cm tissue culture dishes were transfected with 2 ␮g of pCRE-Luc using a high-efficiency calcium phosphate precipitation technique (Ausubel et al., 2001). Cells were incubated with DNA precipitates for 18–24 h at 37 ◦ C under a humidified atmosphere of 3% CO2 /97% air to facilitate DNA uptake. Cells were transferred to fresh medium and returned to a 5% CO2 environment for 48 h. Cells were stimulated with forskolin during the last 18 h of incubation.

Cells (2 × 105 ) were grown on poly-d-lysine-coated glass cover slips for 3 days, fixed in 100% methanol for 30 min at −20 ◦ C and then incubated at 4 ◦ C with a mixture of rabbit anti-AC-4 (1:400) and mouse anti-Na/K ATPase (1:2000; Upstate Biotechnology, Lake Placid, NY) in Tris-buffered saline containing 0.25% Triton X-100 and 10 ␮g/ml BSA. Coverslips were washed with Tris-buffered saline containing 10 ␮g/ml BSA and then incubated at room temperature for 1 h in the dark with a mixture of goat anti-rabbit Alexafluor 568 (1:2000) and goat anti-mouse Alexafluor 488 (1:2000) obtained from Molecular Probes (Eugene, OR). Specimens were visualized at 400× under a Leica SP confocal microscope.

2.7. Assay of luciferase activity

2.6. Plasmids and gene transfer

3.1. AC isoforms in Y1 and forskolin-resistant mutant adrenocortical tumor cells

The expression plasmid pOPRSV-AC-4 was prepared by excising rat AC-4 cDNA (Gao and Gilman, 1991) from pBluescript with XhoI and BamHI and blunt-end ligat-

Luciferase activity was assayed in a cocktail (250 ␮l) containing 9 mM magnesium acetate, 3 mM Na2 ATP and 60 ␮M luciferin as described previously (Frigeri et al., 2002). The reaction was initiated by the addition of luciferin to the reaction and followed by luminescence using a Berthold Lumat LB luminometer under conditions where signals obtained were proportional to the amount of supernatant protein added.

3. Results

The AC isoforms present in Y1 mouse adrenocortical tumor cells were investigated by Western blot analysis of

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AC-5/6 Immunogenic peptide

158

2000

r2=0.99

1500

1000

500

0 0

1000 2000 3000 4000 5000 6000

AC-5 C-terminal protein

Fig. 1. Identification of AC isoforms in Y1 and mutant cells. Western blots of lysates (50 ␮g protein) from Y1 and forskolin-resistant mutant cells together with slot blots containing various concentrations of the corresponding isozyme-specific synthetic immunizing peptides were probed for AC-5/6 (A), AC-1 (B), AC-3 (C) and AC-4 (D) as described in Section 2. The apparent masses of the AC isozymes were estimated using prestained protein markers from New England Biolabs (Beverly, MA). Bands corresponding to each specific AC isoform are noted with arrow heads.

whole cell lysates using AC isozyme-specific antisera. The major isoforms detected were AC-1, AC-3, AC-4 and AC-5/6 (Fig. 1). The relative molecular masses of the different isoforms ranged from 126 to 148 kDa (Table 1). Other isoforms were either undetectable or present only in trace amounts (data not shown). As shown in Fig. 1 and Table 1, the levels of AC-5/6, AC-1 and AC-3 in the mutants did not differ significantly from those in forskolin-responsive Y1 cells (P > 0.05), as determined using a multiple comparison statistical test (Harper, 1984). The levels of AC-4, however, were significantly reduced and fell below the limits of quantitation even when four-fold higher levels of protein were blotted (data not shown). The absolute levels of AC-5/6 also were estimated using a 26.4 kDa protein corresponding to the C-terminal cytoplasmic domain of AC-5 as the standard instead of the C-terminal immunopeptide. The levels of AC-5/6 in parent

Fig. 2. Immunoreactivity of the AC-5/6 synthetic peptide and a protein corresponding to the C-terminal cytoplasmic domain of AC-5. Western blots containing various concentrations (from 0.4 to 12.8 pmol) of a purified, bacterially expressed 26.4 kDa protein corresponding to C-terminal domain of canine AC-5 and slot blots containing equivalent concentrations of the AC-5/6 synthetic immunizing peptide (as in Fig. 1) were probed with anti-AC-5/6 antiserum. Signal intensities at each concentration of protein (x-axis) and peptide (y-axis) were quantitated (using arbitrary units) and fitted to a straight line by linear regression.

and mutant cells obtained by this estimate were closer to 16 pmol/mg protein and reflected a three-fold stronger reactivity of the antibody towards the epitope in the context of the cytoplasmic domain of AC-5/6 (Fig. 2). Closer estimates of the absolute levels of the other AC isoforms await the availability of purified proteins corresponding to their respective C-terminal domains. 3.2. AC transcripts in Y1 and forskolin-resistant mutant adrenocortical tumor cells Products corresponding to AC-1, AC-3, AC-4, AC-5 and AC-6 (based on fragment size and sequence; Section 2) were amplified from the poly(A)-enriched RNA of Y1 cells by RT–PCR (Fig. 3A); transcripts corresponding to AC-2 were not detected (data not shown). AC-6 appeared to represent the most abundant transcript in Y1 cells since much more AC-6 product was obtained with only a fraction (≤0.06) of the cDNA input, while AC-4 appeared to be the least

Table 1 Concentrations of AC isoforms in Y1 and mutant clones Isozyme

Mr (kDa)

AC-5/6a AC-1 AC-3 AC-4

148 129 148 121

± ± ± ±

2 2 2 6

Y1 (kDa) 47 14 1 0.6

± ± ± ±

20 3 0.2 0.1

10r6 (pmol AC/mg protein)

10r9 (pmol AC/mg protein)

Y6 (pmol AC/mg protein)

OS3 (pmol AC/mg protein)

31 ± 6 12 ± 4 1 ± 0.7 0b

40 ± 6 16 ± 3 2 ± 0.6 0b

38 ± 3 22 ± 4 2 ± 0.3 0b

32 ± 2 13 ± 2 2 ± 0.6 0b

The molecular masses (Mr ) of the AC isoforms were estimated based on their relative migration on Western blots (e.g. Fig. 1). Values for kDa are averages obtained from four to six experiments ± S.D. The levels of each AC isoform in parent and mutant cells were estimated by Western blot analysis of whole cell lysates using the corresponding immunizing peptide as standard (Section 2). Results are averages ± S.D. of three to five independent experiments. a Estimates of AC-5/6 concentrations using as standard a 26.4 kDa protein corresponding the C-terminal domain of AC-5 are three-fold lower than estimates using the immunizing peptide as standard (Fig. 2) and are approximate 16 ± 7 pmol/mg protein. b Values set to 0 were below the limits of detection.

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159

mouse est clone derived from a mouse kidney cDNA library (GenBank accession number BF782774) and showed that it corresponded to a complete AC-4 transcript (GenBank accession number AF442771). Using the cDNA sequence information, we determined the intron–exon organization and restriction map of the mouse AC-4 gene in silico (Fig. 4). Genomic DNA from Y1 and two forskolin-resistant mutants (clones 10r9 and OS3) was subjected to restriction endonuclease digestion with XhoI, HindIII or KpnI and analyzed by Southern blot hybridization using the full-length mouse AC-4 cDNA as probe. Following digestion with XhoI, HindIII or KpnI, genomic DNA from forskolin-responsive Y1 cells and the two forskolin-resistant mutants yielded restriction fragments that corresponded to the sizes predicted for the AC-4 gene (Fig. 4). Furthermore, the labeling intensities of undigested and digested DNA from Y1 and mutant cells were similar (Fig. 4). Together, these results indicate that the loss of AC-4 transcripts in the mutant clones was not the result of gene deletion or gross gene rearrangement. 3.4. Subcellular localization of AC isoforms

Fig. 3. Detection of AC transcripts in Y1 and mutant cells using RT–PCR. RT–PCR was performed on poly(A)+ RNA from Y1 and mutant cell lines. Products corresponding to each AC subtype were detected by staining with ethidium bromide following electrophoresis on agarose gels and are shown as negatives for clarity. In (A), AC-1, AC-3, AC-4, AC-5 and AC-6 were amplified from Y1 cell transcripts as described in Section 2. In (B), AC-4 was amplified from Y1 and mutant cells. A 100 bp DNA plus ladder (M) was used to size the RT–PCR products; the position of the 500 bp marker is noted. Gs␣ transcripts were amplified in separate reactions using the same RNA preparations (data not shown) to normalize for RNA input among cell lines (Schimmer et al., 2003).

abundant (Fig. 3A). These data are consistent with the AC isozyme content of Y1 cells and with the estimates of relative abundance obtained from Western blots (Fig. 1, Table 1). A similar comparison of the relative signal intensities for AC-5 and AC-6 transcripts in Y1 cells further suggested that AC-6 may be in greater abundance than AC-5. Consistent with a lack of detectable AC-4 in the mutant clones by Western blot analysis, we did not detect PCR products corresponding to AC-4 in the poly(A)+ RNA from mutant 10r6, 10r9, Y6 and OS3 cells, despite a clear signal for AC-4 in the Y1 sample (Fig. 3B). Thus the loss in AC-4 protein in the mutants was accompanied by a decrease in AC-4 transcripts. 3.3. Southern blot analysis of the AC-4 gene in forskolin-resistant mutants In order to assess the global integrity of the AC-4 genes in our forskolin-resistant mutants, we first sequenced a

As determined by immunofluorescence confocal microscopy, none of the four AC isoforms was concentrated at the plasma membrane; instead, each isoform appeared to be distributed throughout the cytoplasm. AC-4 (Fig. 5A), like the other three AC isoforms (data not shown), had a punctate distribution (red signal) throughout the cytoplasm, suggesting a vesicular localization. An AC-4 blocking peptide prevented the formation of the AC-4 signal (Fig. 5B) demonstrating the specificity of the antibody for the AC-4 epitope. The subcellular localization of the AC isoforms contrasted with that of the plasma membrane marker Na/K-ATPase (Fig. 5, green), which clearly decorated the cell surface. Others, using similar approaches, also have also reported the largely cytoplasmic distribution of AC isoforms (Parkinson and Bolsover, 2001; Cote et al., 2001). To test the validity of the immunohistochemistry experiments and to further explore the association of AC isoforms with the plasma membrane, plasma membrane fractions enriched 20-fold for Na/K-ATPase activity were Western blotted for AC isoform content. As shown in Fig. 6, only AC-5/6 was detected in the plasma membrane fraction. Despite the 20-fold enrichment of Na/K-ATPase activity, signals for AC-5/6 could only be detected on the Western blots when the concentrations of plasma membrane protein approached the concentrations of protein in the whole cell lysates. These observations support the conclusion that most of the AC in Y1 cells is cytoplasmic and indicate that only a small fraction of the total AC-5/6 associates with the plasma membrane. The other isoforms were not detected in the plasma membrane fraction suggesting that they were either absent or present in much lower abundance.

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Fig. 4. Estimation of the integrity of the AC-4 gene on Southern blots. Restriction sites for XhoI (X), KpnI (K) and HindIII (H) within the 32 kb mouse AC-4 gene (A) and the intron–exon organization of AC-4 (B) were determined in silico from genomic (accession number AC098877) and cDNA (accession number AF442771) sequences. The arrow and asterisk (B) represent translation start and stop sites, respectively, within the AC-4 transcript. AC-4 genomic DNA from parent and mutant cells was analyzed by Southern blot hybridization (C) as described in Section 2. Where indicated, high mol wt genomic DNA samples were digested with XhoI, KpnI or HindIII.

3.5. Effects of an AC-4 expression plasmid on forskolin resistance To determine if the AC-4 deficiency seen in the mutant clones contributed to forskolin-resistance, one of the mutants (10r6) was transfected with a rat AC-4 expression vector under control of the RSV promoter and lac operon. Three transformants were selected for analysis of AC-4 expression

and recovery of response to forskolin (Fig. 7). One of these transformants (AC 4-3) expressed AC-4 constitutively, producing 3 pmol AC-4/mg total protein as estimated using the AC-4 peptide as a standard. Two of the transformants (AC 4-1 and AC 4-2) expressed AC-4 in an IPTG-inducible manner. The IPTG-inducible transformants each produced two AC-4 products—i.e. a 126 ± 8 kDa (n = 4) product and a product of higher mass that may represent a glycosylated

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161

Fig. 6. Identification of AC isoforms in Y1 plasma membrane fractions. Whole cell lysates (50 ␮g, lane 1) and plasma membrane-enriched fractions (5 ␮g, lane 2; 25 ␮g, lane 3) from Y1 cells were Western blotted and probed for AC-5/6, AC-1, AC-3 and AC-4 as described in Fig. 1. The arrows identify the corresponding AC isoforms.

Fig. 5. Subcellular distribution of AC-4 in Y1 cells and in 10r6 transformants. Y1 cells (A and B), mutant 10r6 cells (C), the constitutive AC-4 transformant AC 4-3 (D) and the IPTG-inducible AC-4 transformant AC 4-1 (E and F) were grown on cover slips treated with poly-d-lysine and stained for AC-4 (red) and Na/K ATPase (green) using specific antibodies. Cells were examined by immunofluorescence under a confocal microscope as described in Section 2. In (A)–(E), cells were grown in regular growth medium before analysis; in (F), cells were treated with IPTG (100 ␮M) for 48 h to induce AC-4 expression and then incubated in regular growth medium for an additional 18 h before fixing and staining. In (B), Y1 cells were incubated with the Na/K-ATPase and AC-4 antibodies together with a 20-fold excess of the AC-4 blocking peptide to test the specificity of the AC-4 antibody.

form of the enzyme (Baker et al., 1999). The levels of AC-4 in the transformants AC 4-1 and AC 4-2 after treatment with IPTG were equivalent and were estimated to be 2 pmol/mg protein in each case. The percentage of cells expressing AC-4 was estimated by immunofluorescence under a confocal microscope, examining seven to eight fields, each containing approximately 40 cells. In the constitutive AC-4 transformant (clone AC 4-3, Fig. 5D), 43 ± 3% of the cells produced detectable levels of AC-4, generating signals that were clearly higher than those in the 10r6 parent (Fig. 5C). In the inducible clones AC 4-1 (Fig. 5E and F) and AC 4-2 (data not shown), treatment with

IPTG dramatically increased the intensities of the AC-4 signal in 42 ± 2% and 41 ± 1% of the cells, respectively. In order to determine if AC-4 expression in the mutants affected cellular responses to forskolin, we next examined the AC-4 transformants for forskolin-stimulated adenylyl cyclase activity, and forskolin-regulated growth and morphology—manifestations of cAMP accumulation in these cells (Schimmer and Tsao, 1984; Schimmer and Schulz, 1985). We could not detect recovery of forskolin-stimulated adenylyl cyclase activity by direct assay in intact cells or in cell homogenates (data not shown), and so exploited a sensitive cAMP-dependent, luciferase-based reporter gene assay (Spengler et al., 1993)

Fig. 7. AC-4 expression in 10r6 cells transfected with a rat AC-4 expression vector. The 10r6 transformants AC 4-1, AC 4-2 and AC 4-3 were incubated in the absence (-) or presence (+) of IPTG (100 ␮M) as described in Fig. 5. Cells were lysed and analyzed for AC-4 expression on Western blots as described in Fig. 1.

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A. Al-Hakim et al. / Molecular and Cellular Endocrinology 214 (2004) 155–165 Table 2 Contribution of AC-4 to forskolin resistance Clone

Plating efficiencya Control

10r6 AC 4-3 AC 4-1 AC 4-2

Fig. 8. Forskolin-stimulated cAMP accumulation in AC-4 transformants. The effects of forskolin on cAMP accumulation were assayed using the cAMP-responsive reporter gene pCRE-Luc as described in Section 2. To correct for differences in transfection efficiencies among cell lines and experiments, results in each experiment were normalized to the activities obtained from a luciferase reporter gene under control of the proximal promoter region of Rous sarcoma virus. Results were averaged from six to nine independent experiments and are expressed as means±S.D. Statistical significance was determined by ANOVA and the Bonferroni post hoc test. (A) Compares the effects of forskolin on cAMP accumulation in Y1 cells (䊏) and forskolin-resistant 10r6 cells (䊐). Asterisks (*) indicate values that differ significantly between Y1 and 10r6 cells (P < 0.05). (B) Compares the effects of forskolin on cAMP accumulation in 10r6 cells and in the 10r6 transformants expressing AC-4 constitutively (clone AC 4-3) or inducibly (clone AC 4-1). Clone AC 4-1 was incubated in the absence (−IPTG) or presence (+IPTG) of 100 ␮M IPTG for 48 h to regulate AC-4 expression as in Fig. 5. Asterisks (*) indicate values that are significantly different from those obtained with 10r6 cells at the same concentrations of forskolin.

that had been used previously to measure cAMP accumulation in mutant derivatives of the Y1 cell line (Fluck et al., 2002). In the Y1 cell line, forskolin maximally increased luciferase activity 100 ± 8-fold from the cAMP reporter gene; forskolin was considerably less effective in the 10r6 mutant, and maximally stimulated luciferase activity only 4.5 ± 2.8-fold (Fig. 8A). In clone AC 4-3, which expresses the transfected AC-4 constitutively, forskolin at 10 and 30 ␮M significantly increased luciferase activity three- to four-fold over the levels obtained in untransfected controls (P < 0.05). In clone AC 4-1, forskolin also increased the activity of the reporter gene, but this increase reached significance (P < 0.05) only after induction of AC-4 with IPTG (Fig. 8B). The recovery of forskolin-stimulated AC activity correlated with the recovery of growth inhibitory and morphological responses to forskolin in the AC-4 transformants. We showed previously that the plating efficiency of Y1 cells in the absence of forskolin is 25% and following treatment with 10 ␮M forskolin is reduced to 0.01% (Schimmer

25 15 16 19

± ± ± ±

0.5 0.5† 0.5† 1†

IPTG

Forskolin

ndb ndb 16 ± 0.5† 18 ± 1†

29 11 18 22

± ± ± ±

1† 0.4†,∗ 4† 1†

IPTG + forskolin ndb ndb 10 ± 11 ±

4†,∗ 1†,∗

Cells (103 per dish) were plated in 100 mm tissue culture dishes containing F10 growth medium (control) or F10 growth medium supplemented with forskolin (10 ␮M), incubated at 37 ◦ C for 48 h in order to permit cell attachment and then transferred to fresh growth medium without forskolin and propagated for 2 weeks. Where indicated, cells were incubated with IPTG (100 ␮M) for 48 h before plating. Dishes were stained with 2% methylene blue in 50% ethanol to visualize surviving colonies. a The plating efficiency, defined as the percentage of plated cells that grow into colonies, is presented as means of at least four separate experiments ± S.D. Values marked with dragger (†) are significantly different (P < 0.05) from untreated 10r6 cells; values marked with asterisk (*) are significantly different (P < 0.05) from the corresponding untreated controls. Statistical significance was determined using the Peritz’ F-test (Harper, 1984). For reference, the plating efficiency of parent Y1 cells (25% in the absence of forskolin) is reduced to 0.01% following treatment with 10 ␮M forskolin (Schimmer et al., 1985). b nd: not determined.

et al., 1985). In contrast, the plating efficiency of the 10r6 mutant is not reduced by the diterpene (e.g. Table 2). The three AC-4 transformants grown in regular growth medium all had lower plating efficiencies than the10r6 mutant from which the AC-4 transformants were generated (P < 0.05; Table 2); forskolin further reduced the plating efficiency of clone AC 4-3, which expresses AC-4 constitutively (P < 0.05). Forskolin also reduced the plating efficiencies of clones AC 4-1 and AC 4-2 (P < 0.05), but only after induction of AC-4 with IPTG. Although forskolin did not reduce the plating efficiencies of the AC-4 transformants to the levels seen in Y1 cells, the levels of reduction achieved were consistent with the expression of AC-4 only in 41–43% of the transformants (Fig. 5). The 10r6 transformants expressing rat AC-4 also recovered the ability to respond to forskolin with changes in cell shape (Fig. 9). Y1 cells, in the absence of forskolin, were stretched and adhered tightly to the plastic of tissue culture dish. When treated with forskolin, all the cells retracted and exhibited a rounded morphology. The10r6 mutant, on the other hand, resisted these morphological effects of forskolin and retracted only slightly in the presence of the diterpene. In the transformant AC 4-3, which expresses AC-4 constitutively, approximately half the cells were rounded in the absence of forskolin, possibly reflecting the heterogeneously elevated expression of AC-4. Rounding became more pronounced after treatment with 10 ␮M forskolin (Fig. 9). In the inducible clones, AC 4-1 and AC 4-2, rounding was evident after induction of AC-4 with IPTG and became more pronounced after treatment with forskolin (e.g. Fig. 9).

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Fig. 9. Effects of forskolin on cell shape. Parent Y1 cells, forskolinresistant 10r6 cells and the 10r6 transformants expressing rat AC-4 (clones AC 4-3 and AC 4-1) were plated in 60 mm plastic tissue culture dishes, grown for 3 days in regular growth medium and treated with 10 ␮M forskolin for 1 h. Clone AC 4-1 was treated with IPTG (100 ␮M) for 48 h prior to the addition of forskolin. Cells were photographed under phase contrast at the magnification indicated.

4. Discussion In the present study, we demonstrate that Y1 cells express five AC isoforms in measurable amounts—AC-5 and AC-6, isoforms that are inhibited by calcium, AC-1 and AC-3, two calcium-activated AC isoforms, and AC-4, which is activated by G␤/␥ subunits. The concentration of AC-5/6 appears to be 16 pmol/mg protein (estimated using the 26.4 kDa protein corresponding to the C-terminal cytoplasmic domain of AC-5 as standard). These estimates of AC levels in Y1 cells are higher than those estimated in other cell types using more indirect methods based on forskolin binding assays or assays of catalytic activity. Typically, AC levels measured using forskolin-binding assays are substantially lower than the levels of Gs␣ which are estimated by immunoassay to be in the range of 10–20 pmol/mg protein (see Milligan, 1996, for review). The forskolin-binding assay, however, only measures high-affinity forskolin-binding sites and therefore reflects only those forskolin-sensitive AC isoforms that are in activated complexes with Gs␣. AC levels also have been estimated from measures of catalytic activity. Purified preparations of AC have the capacity to produce approximately 10 ␮mol cAMP/min/mg protein (i.e. 1.3 nmol cAMP/min/pmol protein, assuming a molecular

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mass of 130 kDa) when maximally activated (Smigel, 1986; Pfeuffer et al., 1985; Taussig et al., 1993). The specific activity of AC may, in fact, be more than 10-fold higher since AC loses considerable activity upon purification (Sunahara et al., 1997). We have measured the adenylyl cyclase activity in Y1 and mutant cells extensively in previous studies and have observed NaF-stimulated activities in total homogenates of 0.3 nmol cAMP/min/mg protein (Schimmer and Tsao, 1984), or 0.23 pmol of AC/mg protein as calculated using the lowest estimates of specific activity (i.e. 1.3 nmol cAMP/min/pmol protein). This estimate of the AC level in Y1 cells based on AC enzymatic activity reflects <1.5% of the AC content estimated on Western blots. Since NaF stimulates AC activity through Gs, these results suggest that a large fraction of the AC in Y1 cells may sequestered away from Gs␣ and may be represented by the cytoplasmic pool of the enzymes (e.g. Fig. 5). Intriguingly, this report shows that our four forskolinresistant adrenal cell mutants were specifically deficient in AC-4, These observations raise the possibility of a linkage between AC-4 and forskolin action in Y1 cells. Supporting this connection was the finding that transfection of one of the mutant clones with an AC-4 expression vector led to a partial recovery of forskolin-dependent cAMP accumulation (Fig. 8) and a partial recovery of the growth inhibitory and morphological effects of forskolin (Table 2 and Fig. 9). Although the levels of AC-4 in the transformants were several fold higher than the levels observed in Y1 cells, AC-4 appeared to represent a small proportion of the total AC population based on Western blot (Table 1) and RT–PCR (Fig. 3A) experiments. Thus, a three- to five-fold increase in expression of AC-4 in the transformants would not have a large impact on the total AC concentration in these cells. Taken together, these findings strongly suggest that AC-4 makes a significant contribution to the forskolin-responsive pathways affecting proliferation and morphology in Y1 mouse adrenocortical tumor cells. Although we did not systematically evaluate the contributions of the other AC isoforms to forskolin action in these cells, the mutants retain 40–50% of the forskolin-responsive adenylyl cyclase activity seen in parent Y1 cells in direct assays of activity despite the absence of AC-4 (Schimmer and Tsao, 1984; Schimmer et al., 1987) and it is thus likely that one or more of the other isoforms also contribute to the forskolin response. The AC-4 deficiency in the forskolin-resistant mutants is accompanied by a decrease in AC-4 transcripts (Fig. 3), while the AC-4 gene is otherwise present and grossly intact as judged from Southern blot hybridization analysis (Fig. 4). Furthermore, the activity of a proximal promoter/regulatory region of the mouse AC-4 gene is impaired when assayed in these mutants (Rui et al., 2003) indicating that the AC-4 deficiency results from the impaired expression of the AC-4 gene. The mutation to forskolin resistance also is accompanied by a mc2r deficiency that is the consequence of impaired expression of the mc2r gene.

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Since transfection of the mutant cells with the AC-4 expression vector (unpublished observations) or treatment of the mutant cells with cAMP analogs (Qiu et al., 1996) fails to restore mc2r expression, we suggest that the primary mutation in these cells is not in AC-4 itself; rather, the AC-4 deficiency more likely is downstream of the major defect in these clones. In previous studies, we demonstrated that G␤/␥ was impaired in the mutant clones (Mitchell et al., 1992) and that transfection of the mutants with G␤/␥ expression vectors restored mc2r and ACTH-responsive AC activity (Qiu et al., 1998; Frigeri et al., 2000). More recently, we demonstrated that G␤/␥ also restored AC-4 expression in these mutants together with an appreciable level of forskolin-stimulated AC activity (Rui et al., 2003). Taken together, these results demonstrate that the AC-4 deficiency in the mutant clones is a hallmark of the forskolin-resistant phenotype and that AC-4 likely is an important target of forskolin. It is nevertheless unclear why AC-4 should have a significant role in forskolin action given its relatively low abundance and there is no precedent in the AC literature for this observation. Preliminary results (Al-Hakim and Schimmer, unpublished observations) indicate that treatment of Y1 cells with forskolin results in the redistribution of AC-4 from the cytoplasm to the cell surface. It is possible that this redistribution brings AC-4 in closer proximity to Gs␣, which in turn, potentiates the response of AC-4 to forskolin. We have not yet examined the isozyme-selectivity of this effect of forskolin on AC redistribution nor have we tested the contribution of AC-4 redistribution to forskolin-stimulated AC activity, so inferences regarding the contribution of AC-4 redistribution to forskolin response are highly speculative. Alternatively, since AC-4 is the only AC isozyme expressed in Y1 cells that is activated by G␤/␥, it is possible that this feature preferentially targets forskolin action to AC-4 in the Y1 cell line. Our findings regarding the composition and distribution of AC isoforms in Y1 mouse adrenocortical tumor cells contrast with those in two previous reports describing the AC isoform compositions of rat and human adrenal glands, respectively (Cote et al., 2001; Shen et al., 1997). Based on in situ hybridization data, AC-9 and possibly AC-6 appear to be the principal AC isozymes of the rat adrenal cortex; however, in situ hybridization is a relatively insensitive method and does not provide reliable estimates of protein levels. Based on qualitative immunohistochemical data, AC types 1, 2, 3, 4 and 5/6 appear to be present in the human adrenal cortex; only AC-1 was proximal to the plasma membrane, AC-2 and AC-3 appeared to be distributed between membrane and cytoplasm and AC-4 and AC-5/6 were mainly localized to the cytoplasm. The differences in adrenal AC isozyme composition reported for rat and human glands and for mouse Y1 cells may have reflected species-specific differences, differences between glands and cell lines or differences in assays and experimental approaches.

Acknowledgements We thank Drs. Alfred Gilman and Roger Sunahara for rat AC-4 cDNA, Roger Sunahara for the AC-5 C-terminal protein and for helpful discussions, Dr. Christian Vaisse for the CRE-Luc reporter plasmid and Waldemar Czerwinski and Mohammad H. Ghahremani for technical assistance. This work was supported by research grants from the Canadian Institutes of Health Research and the National Cancer Institute of Canada with funds from the Canadian Cancer Society to B.P.S. and P.R.A. Abdallah Al-Hakim received studentship support from the National Science and Engineering Research Council of Canada and from the University of Toronto.

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