Development and Characterization of a Mouse Cell Line Expressing the Human V2 Vasopressin Receptor Gene
Mariel Birnbaumer, Victoria Hinrichs, and Axel P. N. Themen Department of Cell Biology Baylor College of Medicine Houston, Texas 77030
Human genomic DNA and the HSV tk gene were cotransfected into mouse Ltk" cells and assayed for the acquisition of a Gs-coupled receptor to obtain cell lines expressing human receptors that are so far unavailable. The transfected cells were distributed into 96-well microtitration plates at a density such that after HAT (100 MM hypoxanthine, 1 MM aminopterine, and 10 MM thymidine) selection each well contained, on the average, two to three tk+ cell clones. After replication, half of them were tested for expression of a new phenotype: an adenylyl cyclase stimulatory receptor not normally expressed in the Ltk" recipient cell. The screen yielded a positive result on testing cells arising from the third transfection, the newly expressed receptor is that for arginine vasopressin, commonly referred to as type 2 or V2. DNA from primary transformants (HTB1 cells) served to obtain secondary transformants by the same technique (HTB-2 cells). Pharmacological properties confirmed that this new receptor, which stimulates adenylyl cyclase activity 7- to 10fold, is the human V2 receptor and not the activated homologous murine gene. The new cell line provides a permanent accessible source to study the human receptor, by-passing the need for human kidneys. The V2 receptor was susceptible to homologous down-regulation in the HTB-2 cell, but no downregulation of the cell authentic prostaglandin E1 receptor was observed. The vasopressin receptor did not modify phospholipase-C activity in these cells as expected from V2 receptors. Thus, we successfully applied genomic DNA-mediated gene transfer and were able to develop a cell line expressing a Gs-coupled human receptor of low abundance and poor accessibility. (Molecular Endocrinology 4: 245254, 1990)
INTRODUCTION It has been shown that hormone cell surface receptors may exhibit species specificity in their interaction with 0888-8809/90/0245-0254$02.00/0 Molecular Endocrinology Copyright © 1990 by The Endocrine Society
ligands. As a result, properties of receptors such as ligand binding, complexity of cellular signalling and tachyphylactic responses determined in animal model systems need to be confirmed using human tissues. While this is possible for a few cases, such as for receptors expressed on blood-borne cells, immortalized skin fibroblasts, and a few discards of surgical (organ transplant) procedures, in most cases the proper models for studying human cell surface receptors in the laboratory are still lacking. One approach to this problem, which is yielding positive results, is purification and cloning of selected receptors from animal sources, followed by the cloning of the human homolog and their expression in tissue culture cells, where they now become accessible to biochemical and pharmacological characterization. Human /V, ftr and, more recently, /33-adrenergic receptors are examples of this kind (1-3). Another approach to the study of human receptors is to obtain their expression in tissue culture cells by genomic DNA-mediated gene transfer (4, 5) which obviates the need of purification. The expression in murine cells of the human transferrin and nerve growth factor receptors by Kuhn ef a/. (6) and Chao et al. (7), respectively, are examples of this kind. In these examples the transformed cell clones expressing the acquired cell surface receptor were identified with highly specific antibodies using fluorescent-activated cell sorting (6) or a hemagglutination technique (7). In the present work we have focussed on the possibility of obtaining by genomic DNA-mediated gene transfer the expression of human cell surface receptors that are coupled to their effector by G-proteins of the Gs type (8). Many of these receptors are unavailable for purification because they are either of low abundancy and/or from tissues of which large quantities cannot be obtained. We report below the development of a new strategy for identifying transformed cell lines that acquired a Gscoupled receptor and the successful isolation of one such cell line. This approach is independent of the availability of antibodies, for it uses as a rapid and very sensitive assay for receptor function the measurement
245
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MOL ENDO-1990 246
of ligand-stimulated adenylyl cyclase activity. Applying this methodology we obtained primary and secondary transformants that express the human type 2 vasopressin receptor (V2 receptors) (9) which mediates the antidiuretic action of the hormone arginine vasopressin (AVP).
RESULTS Our objective was to develop a strategy for the expression of Gs-coupled human receptors according to the general scheme shown in Fig. 1. We wished to do so using genomic DNA-mediated gene transfer to insert human genes into a mouse recipient cell and a screening technique that measured appearance of a new hormonal response in the transformed clones. For this, the recipient cell needed to fulfill two conditions: 1) to be an effective acceptor of foreign genes and 2) to express normally as few as possible of the type of receptors we were interested in. Ltk" cells fulfilled both requirements. Wigler et al. (5) and other laboratories had shown Ltk" cells to be versatile recipients of foreign genes (6, 7,10). We found the adenylyl cyclase system of these cells to be unresponsive to any one of the 12 polypeptide and glycoprotein hormones listed in Table 1 as well as to /3-adrenergic receptor ligands. The cells responded, however, to prostaglandin Ei (PGEi) (11), forskolin, and manipulations that activate Gs, such as addition of NaF and GTP analogs [guanosine 5'-(3thiotriphosphate) (GTP7S) and (Guanyl-5'yl imidodiphosphate GMP-P(NH)P)]. These results indicated that Ltk" cells have a normal adenylyl cyclase system susceptible to stimulation by Gs-coupled stimulatory receptors). The presence of at least one positively coupled receptor (PGE^ served to determine whether receptor-
Cloning Strategy for Isolation and Identification of Receptor Genes 'Human Genomic ONA (50kb) HSV tk Gene CaPC>4 Precipitate, 20hr. Glycerol Shock. Wait 24hr. .Trypsinize Cells
Subdivide into 96 Well Microlitration Plates at 5,000 cells/well into HAT Medium
Select Ik + Cells 1 Clone/3,000 Cells
2-3 clones/well after 3 Weeks
Culture Further Pick Putative Positive Clones Subculture. Confirm.
Assay: Adenylyl Cyclase Phosphatase Analyze AC/Phosphatase Ratio
Fig. 1. Schematic Representation of the Strategy Employed for the Selection and Replication of a Large Number of Independent Transformant Cell Clones
Table 1. Hormonal effects on adenylyl cyclase of Recipient Ltk" and Transformed HTB Cells Additions to Assay
None AVP ACTH PTH Calcitonin Glucagon Secretin VIP CRF GRF LH FSH
TSH PGE, Forskolin
Adenylyl Cyclase Activity in (picomol cAMP/min • mg) Ltk" cells
3.14 ±0.08 3.22 ± 0.40 3.06 ± 0.08 2.89 ± 0.40 2.89 ± 0.08 2.97 ± 0.08 2.89 ± 0.08 2.97 ± 0.08 2.81 ± 0.08 3.14 ±0.08 2.89 ± 0.24 3.55 ± 0.40 3.39 ± 0.08 7.56 ± 0.08 9.42 ±0.16
HTB cells
2.89 13.05 3.14 3.06 3.22 3.06 3.22 3.30 2.89 2.97 3.06 2.97 3.22 12.80 17.10
± 0.08 ±0.16 ±0.08 ±0.16 ± 0.08 ± 0.24 ± 0.08 ±0.16 ± 0.08 ±0.16 ±0.16 ± 0.24 ±0.16 ±0.16 ±0.08
Cell homogenates were prepared and assayed for adenylyl cyclase as described in Materials and Methods. Each assay (final volume 50 /J) received 18 or 21 ng protein of Ltk" or HTB cell homogenate, respectively. Peptide and protein hormone additions were at 100 nM each; PGE! and forskolin were used at 100 ^M each. VIP, Vasoactive intestinal peptide.
mediated stimulation of the adenylyl cyclase activity in the Ltk" cells could be measured in situ (data not shown). Cells were plated in 96-well microtitration plates at a density of 30,000-60,000 cells/well and assayed the following day, as described in Materials and Methods. The hypotonicity of the medium was sufficient to alter the permeability of the cell membrane and allow the entry of [a-32P]ATP into the cells, since it was possible to detect newly formed [32P]cAMP after incubation for 60 min at 32 C in the absence of saponin. Nevertheless, addition of saponin to the incubation medium increased the production of cAMP by 3-fold without interfering with the stimulatory effect of PGET (not shown). For the remainder of the experiments the incubations were carried out in the presence of 50 Mg/m' saponin. Higher concentrations of saponin had an inhibitory effect on the stimulation of the enzyme by PGE^ In other experiments, in which we plated cells at different densities, we found that the minimum number of cells necessary to detect enzymatic activity was 5000/well. Since it was expected that the different cell clones that arise after HAT (100 HM hypoxanthine, 1 /XM aminopterine, and 10 HM thymidine) selection might grow at very different rates, it was necessary to measure a normalizing parameter to relate adenylyl cyclase activity per well to the number of cells present. As shown in Fig. 2, phosphatase activity of the cells provided such information. This activity was assessed by hydrolysis of p-nitrophenyl phosphate occurring under the same assay conditions used for assessment of adenylyl cy-
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247
Human V2 Vasopressin Receptor in Mouse Cell
Proportionality Between Number of Cells Assayed and Adenylyl Cyclase Activity
"0 10 20 30 Assayed Cells/well (x10~3)
Phosphatase Activity (OD4
Fig. 2. Correlation between Number of Cells, Phosphatase Activity, and Adenylyl Cyclase Activity Ltk~ cells were plated in 96-well plates, and the assays carried out as described in Materials and Methods. All determinations were performed in replicate. Cells from replicate wells to the ones assayed were trypsinized and counted to determine the number of cells on the day of the assay. A, Proportionality between phosphatase activity and number of cells per well. S, Correlation between adenylyl cyclase activity and phosphatase activity measured simultaneously in 96-well plates.
clase activity and quantified in an enzyme-linked immunosorbent assay reader. Indeed, within the desired range of cell density (1,000-30,000 cells/well), phosphatase and adenylyl cyclase activities were linearly correlated and proportional to cell number.
Ligand Dependence of the HTB Cell Adenylyl Cyclase Activity
Since screening of the plates was carried out by assaying for adenylyl cyclase activity only in the presence of stimulatory ligands, the elevated enzymatic activity detected in HTB cells could have been due to a constitutively elevated basal activity induced by insertional mutagenesis and not necessarily to the presence of a new plasma membrane receptor that the L cells normally do not express. To distinguish between these two possibilities the HTB cells were expanded, homogenates were prepared as described in Materials and Methods and assayed for basal and ligand-stimulated adenylyl cyclase activity. As shown in Table 1 the elevated adenylyl cyclase activity of HTB cells was due to the appearance of a new hormonal response: the ability to respond to AVP. The cells were cloned by limiting dilution. All clones obtained showed the same extent of stimulation in the presence of saturating concentrations of AVP. One clone (HTB-1) was selected for further studies. Southern blots, using as probe radioactively labeled human genomic DNA that assays for the presence of repetitious Alu\ sequences (12), confirmed the presence of human DNA in HTB-1 cells (Fig. 4).
Transfection, Selection and Assay for the New Phenotype
Secondary Transformants
The transfection and subsequent selection of Ltk cells was carried out as described in Materials and Methods. Twelve plates were used per experiment. Approximately 95% of the wells contained two or three independent clones, while the remainder were empty. Thus, on incubating all wells of the 12 plates, we assayed simultaneously the activities of 2000-3400 independent cell clones. Incubations were carried out in the presence of the mixture of 12 stimulatory peptide hormones listed in Table 1 at the concentrations mentioned in Materials and Methods. The values of adenylyl cyclase activity were plotted as a function of the values of phosphatase activity per well, as shown for one such experiment in Fig. 3. Each dot represents the enzymatic activity of a different well due to two or three surviving cell clones. Those wells with adenylyl cyclase activities that deviated significantly from the average were subcultured and assayed again in the presence of the mixture of 12 peptide and protein hormones. As the inset in Fig. 3 depicts, one of the subcultured cells continued to exhibit higher adenylyl cyclase activity than the others. These cells were designated HTB cells. They were identified during the analysis of the third set of 12 96well plates of transfected and HAT-selected cells.
Genomic DNA was extracted from the cloned HTB-1 cell line, and secondary transfections were carried out using this preparation as the source of human DNA containing the vasopressin receptor gene. After selection in HAT medium, the cells were assayed in situ for AVP-stimulated adenylyl cyclase activity in the presence of saturating concentrations of peptide. Clones with significantly higher enzymatic activity than the average were treated as described above. Figure 5 and its inset summarize the experimental data obtained when the second set of 12 96-well plates of the secondary transfections was assayed. The transformant cell clone identified by the arrow in fig. 5 expressed the AVP receptor commonly referred to as V2 or type 2. A clonal cell line (HTB-2) was obtained by limiting dilution. DNA dot assay measurements using human repetitive sequences as probes allowed us to estimate that our primary transformants contain about 12 x 106 basepairs of human genomic DNA. For the secondary transformant it was not possible to estimate this value due to the weakness of the signal. Nevertheless, four or five bands that hybridize to human genomic DNA could be seen in the Southern blot of HTB-2 cells (Fig. 4).
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MOL ENDO-1990 248
Vol 4 No. 2
Hormone-Stimulated Adenylyl Cyclase of L Cells Transfected with Human Genomic DNA (Primary Transfection) 0.26 _ 0.24
0.4
> '>
*
m
•
~ o 0.3 _
> c
°E
•Comp. M:4000nM OT: 1320nM 25
Potency Ratios: Comp. I/Comp. I 2350; AVP/LVP: 10 I I I I I I I I I 11 10 8 7 6 Ligand (-logM)
Fig. 6. Dose-Response Curves of Stimulation of Adenylyl Cyclase by AVP and Analogs Comparison of the mouse vasopressin receptor to the vasopressin receptor of HTB-2 cell. Upper panel, The adenylyl cyclase dose-response curves were obtained with partially purified HTB-2 cell membranes prepared and assayed as described in Materials and Methods. Lower panel, The enzymatic assay was carried out using partially purified mouse kidney plasma membranes prepared as described in Materials and Methods.
EC50 (nM; n = 3)8 AVP LVP OT dDAVP desGly(NH2)LVP Potency Ratios AVP/LVP (ratio I) dD-AVP/desGly(NH2)LVP (ratio II) Mouse kidney vs. HTB-2 Cell Mouse kidney ratio I/HTB2 ratio I Mouse kidney ratio 11/ HTB-2 ratio II Difference
HTB-2 cells
13±4 111 ±43 1350 ±284 4 ±1.4 4633 ± 279
18±5 57 ±17 465 ±17 14±6 1128 ±279
8.8 ± 3.5 1121 ±360
3.5 ±1.6 78.0 ± 6.2
2.5 ±1.5 14.3 ±2.4 11.8 ± 2 . 8 "
" Results are from three independent repeat experiments (mean ± SE). For details, see Fig. 6. b P < 0.025, by Student's t test.
Table 3. Phosphoinositide Hydrolysis by Ltk" and HTB-2 Cells in Response to AVP and ATP Addition to Wells
A. Ltk" cells Control AVP ATP
clones, with very few having either a single or four clones. For the reported experiments the seeding density had been adjusted to give no more than 10% empty wells. With the transfection protocol described in Materials and Methods this required seeding 4000-5000 cells/well at the time of placing the cells into HAT medium. Some general considerations are in order about the expected frequency of success in cloning a given phenotype by the method developed here. Each 12-plate transfection experiment screens about 3000 clones. If one assumes an average DNA uptake per cell of 3 x 106 basepairs (5, 6), then one transfection experiment screens about two thirds of the human genome. Thus, if integration is such that expression can occur if the gene is not accidentally broken during DNA manipulation and the assay is adequate, a given receptor has a maximum cloning likelihood of once in every three transfection experiments. However, this number is not realistic because of a number of factors. Probably the most important is the site of integration of the human DNA
Mouse kidney
B. HTB-2 cells Control AVP ATP
[3H]lnositol Phosphates Accumulated (cpm/ Well)
1530 ± 37 1400 ± 26 3444 ± 41
818 ± 23 816 ± 12 1834 ± 35
Cells were grown in 35-mm wells in the presence of [3H] inositol to a density of 2.1 x 106 cells/well, washed, exposed to 10 mM LiCI for 10 min, and then incubated in duplicate for 30 min in the absence (control) or presence of 100 nM AVP or 100 MM ATP as described in Materials and Methods. Reactions were terminated, and fractions corresponding to total inositol phosphates were isolated by Dowex 1 chromatography.
in the mouse genome, with at least 90% of the human DNA ending up in nonexpressed areas. Another factor is the dissimilar growth rate among the transformed cell lines. About 20% of them do not grow sufficiently so as to be assayed, as can be seen by the low values along the phosphatase assay axis in Figs. 3 and 5. A
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251
Human V2 Vasopressin Receptor in Mouse Cell
Control
AVP Treated
PGE. Treated
Fig. 7. Homologous Desensitization of the Vasopressin Receptor but not of the PGE, Receptor Replicate dishes containing confluent HTB-2 cells were treated for 2 h at 37 C with 100 nM AVP or 27 MM PGE,. Homogenates were prepared in Dounce homogenizers as described in Materials and Methods and assayed for adenylyl cyclase activity in the absence of any added hormone (•) and in the presence of 100 nM AVP (H) or 27 HM PGE, (•).
third identifiable factor is DNA breakage. The probability of interrupting a coding sequence by random breaks is proportional to the size of the genes to be isolated. If there are no hot spots in a gene of about 5 kilobases (Kb), the probability of random breaking will be about 1 in 20 if the fragments of genomic DNA are 100 kb long. With these numbers in mind, the expected number of transfections necessary to efficiently screen the human genome once for any given receptor increases by a factor of 10 (wrong place of integration) times 1.25 (insufficient growth) times 1.05 (DNA breakage if gene is 5 kb or larger), i.e. to 40 or 50. This number may indeed be higher. The finding of the vasopressin receptor as one of the 12 possible hormone receptors simultaneously tested for within the first three transfections agrees with this number. The recovery of the vasopressin receptor gene in the secondary transformants within the first two transfections was, therefore, somewhat surprising, unless one assumes a linkage with the thymidine kinase gene or that the site of integration plays a role only in the first transformation such that in subsequent transfections the gene may be already linked to a strong promoter that can override other cellular controls and insure better expression. Overall, it would appear that this may be a method of choice for the cloning of receptors that are very difficult to purify or for which there are no cell lines that express them. Furthermore, the logistics used to maintain and replicate up to 3000 individual transformed cell clones could be useful for the expression cloning of other functions, provided the phenotype resides in a single gene and they can be assayed in a single well. These transformed cells provide the only available cell line expressing the human V2 vasopressin receptor.
Until now only the pig-derived LLCPK, cells expressed a related receptor that recognizes lysine vasopressin (LVP) instead of AVP as its physiological ligand. The ability of these cells to support ligand-induced desensitization of the acquired vasopressin receptor while failing to desensitize the normal PGE, receptor may be an indication of great susceptibility of the vasopressin receptor to be regulated by its ligand. It also suggests that in these cells increases in intracellular cAMP are not sufficient to reduce the activity of this Gs-coupled receptor. We observed differences in the rate of desensitization of the AVP and LVP receptors as well as in the effect of high salt concentrations in this phenomenon. These will remain a puzzle until cDNAs coding for both receptors are available. At that time expression of both proteins in the same cell should allow us to distinguish between receptor or cell line differences. The studies of Guillon et al. (13) and Butlen et al. (14) had established, by measuring both adenylyl cyclase activation and competition in binding assays, that rat and human kidney AVP receptors displayed different rank order of potencies and apparent Ka toward natural and synthetic agonists. Our data confirmed those results. More recently, the cloning of human and chicken epidermal growth factor receptors has provided another example of species-related heterogeneity (17) in receptor-ligand interaction, strengthening the case for the need to evaluate ligands intended for humans with receptors of the same species. The apparent cosegregation of the V2 vasopressin receptor gene and a small number oM/ul-positive bands in the HTB-2 cells makes them an appropriate source of DNA to construct genomic libraries from which to isolate the gene coding for the vasopressin receptor using established molecular biology techniques (6,7,18).
MATERIALS AND METHODS Radiochemicals [a-32P]ATP was obtained from the NICHHD Center for Population Research and Studies in Reproductive Biology at Baylor College of Medicine; [«-32P]dCTP was from Amersham (Arlington Heights, IL); [3H]AMP was from ICN Biochemicals (Irvine, CA); myo-[3H]inositol (40 Ci/mmol) was from DuPont-New England Nuclear (Wilmington, DE). Materials and Reagents Minimum Essential Medium (MEM) a, Hanks' Buffered Salt Solution, penicillin-streptomycin, 0.5% trypsin 5 miwi EDTA and fetal bovine serum were obtained from Gibco (Grand Island, NY). Tissue culture plasticware was purchased from Costar (Cambridge, MA). The hormones and synthetic peptides were obtained from Cambridge Research Biochemicals (Atlantic Beach, NY), except for glucagon, vasoactive intestinal peptide, AVP, LVP, dDAVP, and PGE,, which were from Sigma (St. Louis, MO). Desglycinamide-LVP (des-Gly(NH2)9LVP) was obtained from Peninsula Laboratories (Belmont, CA). Human TSH, ovine LH and ovine follicle stimulating hormone FSH were provided by Dr. Salvatore Raiti from the NIDDK NIH National Pituitary Agency (Baltimore, MD). Forskolin was pur-
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MOL ENDO-1990 252
chased from Calbiochem (San Diego, CA). Agarose gel electrophoresis supplies were obtained from BRL (Bethesda, MD). Oligonucleotides for random priming were purchased from Pharmacia (Piscataway, NJ). Restriction endonculeases were obtained from Boehringer-Mannheim Biochemicals (Indianapolis, IN). All other reagents were purchased from Sigma. Cell Line Ltk" aHT, a clone of the mouse fibroblast cell line originally established by Kit ef al. (19) was a generous gift from Dr. Frank Ruddle, Department of Biology, Yale University. This subclone had been selected for high frequency of transformation. The cells were cultured at 5% CO2 in MEMa containing 10% heat-inactivated fetal bovine serum, penicillin (50 U/ml), and streptomycin (50 M9/ml). Cell Transformation The calcium phosphate precipitation technique was used (4). Cells, which had been kept subconfluent for all passages preceding the transformation, were plated 24 h before transfection (1.5x10 6 /100-mm dish), and exposed to a precipitate of calcium phosphate formed in the presence of 20 ^g human genomic DNA (>50 kb) and 50 ng pHSV-106 plasmid linearized with H/ndlll. After 18 h of exposure to the precipitate the cells were treated with 25% glycerol in Hanks' Buffered Salt Solution for 60 sec at room temperature, thoroughly rinsed with Hanks' solution, and returned to the incubator with fresh medium. Twenty-four hours later the cells were trypsinized, counted, and diluted to a concentration of 25,000 cells/ml in the culture medium containing HAT. Cells were then distributed into 96-well plates using a Transtar 96 pipettor (Costar), delivering 200 ^I/well (-5,000 cell/well). After 2-3 weeks in the selective medium the plates were examined for the presence of clones. Wells containing one to three clones were trypsinized to obtain replicate plates, one of which was assayed when cells had reached confluency. After their isolation, the transformed cells were cultured always in the presence of HAT. Purification of Plasmid DNA and Human Genomic DNA The plasmid pHSV106, pBR322 containing the herpes simplex thymidine kinase gene, was obtained from Bethesda Research Laboratories and amplified in E. coli AG1 following the protocol of Hanahan (20). Human genomic DNA was prepared from white blood cells obtained from normal volunteers following the technique of DiLella and Woo (21). The genomic DNA was precipitated in the presence of sodium acetate and ethanol and redissolved in sterile 1 mM Tris-HCI and 0.1 mM EDTA, pH 7.4. The size of the genomic DNA was analyzed in a 0.3% agarose gel run in 89 mM Tris-boric acid and 0.2 mM EDTA, pH 8.8. Only fragments larger than intact Xphage were used. Measurement of Adenylyl Cyclase Activity Attached Cells Growth medium was discarded, the 96-well plates were rinsed once with 200 ^I/well 10 mM Tris-HCI and 150 mM NaCI, pH 7.4 (TBS), and 50 fi\ incubation medium were added to each well. The incubation medium contained 0.1 mM [«-32P]ATP (5 x 106 cpm/well), 2 mM MgCI2, 10 IXM GTP, 1 mM EDTA, 1 mM cAMP, 2 mM methylisobutylxanthine, and a nucleotide triphosphate-regenerating system composed of 20 mM creatine phosphate, 0.2 mg/ml (2,000 U/mg) creatine phosphokinase, 0.02 mg/ml myokinase, and 25 mM Tris-HCI pH 7.4. In addition, the incubation medium contained 50 ng/ ml saponin to permeabilize the cells to the reagents and [3H] cAMP (10,000 cpm/well) to determine recoveries of this nucleotide. Also included in the reaction was 5 mM p-nitrophenylphosphate, which served as substrate for assessment of phosphatase activity. The incubations were carried out at 32
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C for 2 h. Hormones (diluted in 0.1% BSA) were present at concentrations of 100 nM. The reaction was stopped by the addition of 100 ^I/well of a solution containing 40 mM ATP, 10 mM cAMP, and 1 % sodium dodecyl sulfate. The phosphatase assay was simultaneous with the adenylyl cyclase assay for the cells assayed in situ. The extent of hydrolysis of p-nitrophenylphosphate was determined by measuring the absorbance at 405 nm in a Titertek enzyme-linked immunosorbent assay reader, and the activity was expressed in arbitrary units. The cAMP formed in the reaction was isolated by a modification (22) of the standard double Dowex-alumina chromatographic procedure (23). Activity in Cell Homogenates and Partially Purified Membranes The same incubation conditions described above were used, omitting the saponin and the p-nitrophenylphosphate. The incubations were carried out for 10 min at 32 C. The activities were expressed as picomoles of cAMP per min/mg homogenate or membrane protein. Protein was determined by the method of Lowry ef al. (24). Homogenate Preparation After withdrawing the growth medium the cells were rinsed once with TBS, scraped off the dishes with a rubber policeman, and centrifuged. The supernatant was discarded, and the pellet resuspended in approximately 10 vol ice-cold homogenization buffer consisting of 27% (wt/vol) sucrose, 1 mM EDTA, and 20 mM Na-HEPES, pH 7.8. For small volumes (0.5-2 ml) the suspension was transferred to a 75-ml capacity Parr bomb, and the cells were broken by N2 cavitation. If the homogenate was assayed directly, it was allowed to sit on ice for 20 min before the assay. Alternatively, the cells were homogenized in a 7-ml Dounce homogenizer (Kontes Co., Vineland, NJ) with 10 strokes of the tight (B) pestle. For membrane isolation larger quantities of cells were required. They were homogenized in a 15-ml Corex tube using four 10-sec bursts of a Brinkmann PT-10 Polytron (Westbury, NY) at a setting of 6, for this method yielded a homogenate more suitable for membrane isolation than the cavitation technique. The homogenate was centrifuged at 800 rpm for 5 min at 4 C, the pellet discarded, and the supernatant centrifuged further at 15,000 rpm for 15 min. The pellet obtained after the second centrifugation was significantly enriched in plasma membranes. The membranes were resuspended in homogenization buffer containing 20 mM MgCI2 (11), aliquoted, quick frozen, and stored at - 7 0 C until used. Membranes from Mouse Kidney Retired breeder female mice were obtained from Harlan (Angleton, TX). They were killed by cervical dislocation, and the kidneys were quickly excised, freed of adherent tissues, and immersed in an icecold solution of 0.25 M sucrose containing 1 mM EDTA. All subsequent steps were carried out at 4 C. The kidneys were minced and homogenized in 20 vol 1 mM KHCO3 with a Polytron homogenizer (25). The homogenate was filtered through silk screen, and the filtrate was centrifuged for 5 min at 1500 rpm. The pellet was discarded, and the supernatant centrifuged for 20 min at 10,000 rpm. The second membrane pellet was subjected to discontinuous sucrose gradient purification (26), and membranes with the highest stimulation of adenylyl cyclase by vasopressin were collected at the interphase between 37% and 41.5% (t/wt) sucrose. They were diluted to approximately 40 Mg/ml, fractionated in 0.2-ml aliquots, and kept frozen at - 7 0 C until used. DNA Analysis DNA isolated from the positive clones was subjected to Southern blot analysis (27) for detection of human repeated sequences (11). Genomic DNA (20) was digested to completion with restriction endonucleases following the manufacturer's recommendations, applied to a horizontal 0.8% agarose gel, electrophoresed at 4 V/cm, and blotted onto nitrocellulose filters. Nitrocellulose filters were baked at 68 C for 3 h, prehybridized for 2 h, and hybridized for 20 h with human genomic DNA that was labeled with 32P by extension of oligonucleotide primers (28) to specific activities of 1 x 109 . Prehybridization and hybridization were carried out
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Human V2 Vasopressin Receptor in Mouse Cell
at 45 C under the conditions described by Kuhn et al. (6) using for the prehybridization 50% formamide. 4 x SSC (1 x SSC = 150 mM NaCI and 15 mM Na-citrate), 5 x Denhardt's solution (1 x Denhardt = 0.02% Ficoll, 0.02% polyvinylpyrrolidone, and 0.02% BSA), and 0.1% sodium dodecyl sulfate. The same mixture with the addition of 0.1 mg/ml sheared herring sperm DNA, 10% dextran sulfate, and 8 x 106 cpm/ml labeled human DNA was used to hybridize the filter. Phosphoinositide Hydrolysis Receptor stimulation of cellular phosphoinositide-specific phospholipase-C was tested for in intact cells by an adaptation (14) of the procedures described by Bone et al. (29) and Kirk et al. (30), in which cells were labeled with [3H]/nyo-inositol and incubated with agonist in the presence of LiCI, and the free 3H-labeled inositol phosphates accumulated during the incubation are extracted, separated on Dowex-1 columns, and quantified by liquid scintillation counting. Typically, about 0.5 x 106 LtK" or HTB-2 cells were seeded into 35-mm wells of six-well plates in 2.5 ml HAT selection medium (see above) supplemented with 2 ^Ci/ml [3H]inositol and grown for 48 h to a density of about 2 x 106 cells/well. Labeled cells were then placed into 12.5 ml fresh HAT selection medium without fetal calf serum or labeled inositol, and incubated for 1 h at 37 C. The cells were then rinsed twice at room temperature with 2.0 ml PBS supplemented with 5.5 mM glucose, 0.5 mM CaCI2, and 0.5 mM MgCI2. After the addition of 2 ml PBS with glucose, Ca2+, and Mg 2+ , cells were placed again at 37 C for 20 min, at which time 10 mM LiCI was added. Test agents were then added after a further 10 min. Unless indicated otherwise the incubations were stopped 30 min after the additions of stimulatory agents by placing the plates on ice, removing and discarding the incubation medium, and adding 1 ml ice-cold 5% perchloric acid and 20 /zl 10% BSA.
Acknowledgments The authors wish to acknowledge Dr. Lutz Birnbaumer for helpful discussions and invaluable advice on the execution of the large number of adenylyl cyclase assays; Karen Thiessen for expert technical assistance, and Betty Kilday for help in the preparation of the manuscript.
Received October 17, 1989. Revision received November 20,1989. Accepted November 20,1989. Address requests for reprints to: Dr. Mariel Birnbaumer, Department of Cell Biology, Baylor College of Medicine, Houston, Texas 77030. This work was supported in part by NIH Grant HD-09581 and Center Grants DK-27685 and HD-07945.
REFERENCES 1. Frielle T, Collins S, Daniel KW, Caron MG, Lefkowitz RJ, Kobilka BK 1987 Cloning of the cDNA for the human 0,adrenergic receptor. Proc Natl Acad Sci USA 84:79207924 2. Emorine LJ, Marullo S, Delavier-Klutchko C, Kaveri SV, Durieu-Trautmann O, Strosberg AD 1987 Structure of the gene for human /32-adrenergic receptor: expression and promoter characterization. Proc Natl Acad Sci USA 84:6995-6999 3. Emorine LJ, Marullo S, Briend-Sutren M-M, Patey G, Tate K, Delavier-Klutchko C, Strosberg AD 1989 Molecular characterization of the human /33-adrenergic receptor. Science 245:1118-1121 4. Graham FL, Van der Erb AJ 1973 A new technique for the assay of infectivity of human adenovirus S DNA. Virology 52:456-467
5. Wigler M, Sweet R, Sim GK, Wold B, Pellicer A, Lacy E, Maniatis T, Silverstein S, Axel R 1979 Transformation of mammalian cells with genes from prokaryote and eukaryotes. Cell 16:777-785 6. Kuhn LC, McClelland A, Ruddle FH 1984 Gene transfer, expression, and molecular cloning of the human transferrin receptor gene. Cell 37:95-103 7. Chao MV, Bothwell MA, Ross AH, Koprowski H, Lanahan AA, Buck CR, Sehgal A1986 Gene transfer and molecular cloning of the human NGF receptor. Science 232:518521 8. Birnbaumer L, Codina J, Mattera R, Yatani A, Scherer N, Toro M-J, Brown AM 1987 Signal transduction by G proteins. Kidney [Suppl 23] 32:S-14-S-37. 9. Michell RH, Kirk CJ, Billah MM 1979 Hormonal stimulation of phosphatidylinositol breakdown, with particular reference to the hepatic effects of vasopressin. Biochem Soc Trans 7:861-865 10. Mendelsohn CL, Johnson B, Lionetti KA, Nobis P, Simmer E, Racaniello VR 1986 Transformation of a human poliovirus receptor gene into mouse cells. Proc Natl Acad Sci 83:7845-7849 11. Strader CD, Sigal IS, Blake AD, Cheung AH, Register RB, Rands E, Zemcik BA, Candelore MR, Dixon RAF 1987 The carboxyl terminus of the hamster 0-adrenergic receptor expressed in mouse L cells is not required for receptor sequestration. Cell 49:855-863 12. Jelinek WR, Toomey TP, Leinwand L, Duncan CH, Biro PA, Choudary PV, Weissman SM, Rubin CM, Houck CM, Deininger PL, Schmid CW 1980 Ubiquitous, interspersed repeated sequences in the mammalian genome. Proc Natl Acad Sci USA 77:1398-1402 13. Guillon G, Butlen D, Cantau B, Barth T, Jard S 1982 Kinetic and pharmacological characterization of vasopressin membrane receptors from human kidney medulla: relation to adenylate cyclase activation. Eur J Pharmacol 85:291-304 14. Butlen D, Guillon G, Rajerison RM, Jard S, Sawyer WH, Manning M 1978 Structural requirements for activation of vasopressin sensitive adenylate cyclase, hormone binding and antidiuretic action: effects of highly potent analogues and competitive inhibitors. Mol Pharmacol 14:1006-1017 15. Liao C-F, Themmen APN, Joho R, Barberis C, Birnbaumer M, Birnbaumer L 1989 Molecular cloning and expression of a fifth muscarinic acetylcholine receptor (M5-mAChR). J Biol Chem 264:7328-7337 16. Roy C, Hall D, Karish M, Ausiello DA 1981 Relationship of (8-lysine) vasopressin receptor transition to receptor functional properties in a pig kidney cell line (LLCPK,). J Biol Chem 256:3423-3427 17. Lax I, Bellot F, Howk R, Ullrich A, Givol D, Schlessinger J 1989 Functional analysis of the ligand binding site of EGF receptor utilizing chimeric chicken/human receptor molecules. EMBO J 8:421-427 18. Mendelsohn CL, Simmer E, Racaniello VR 1989 Cellular receptor for polio-virus: molecular cloning, nucleotide sequence, and expression of a new member of the immunoglobulin superfamily. Cell 56:55-865 19. Kit S, Dubbs D, Piekarski L, Hsu T 1963 Deletion of thymidine kinase activity from L cells resistant to bromodeoxyuridine. Exp Cell Res 31:297-312 20. Hanahan D 1985 Techniques for transformation of E. coli. In: Glover D M (ed) DNA Cloning. IRL Press, Oxford, Vol 1:109-135 21. DiLella AG, Woo SLC 1987 Cloning large segments of genomic DNA using cosmid vectors. Methods Enzymol 152:199-212 22. Bockaert J, Hunzicker-Dunn M, Birnbaumer L 1978 Hormone-stimulated desensitization of hormone-dependent adenylyl cyclase. Dual action of luteinizing hormone on pig graafian follicle membranes. J Biol Chem 251:26532663 23. Salomon Y, Londos C, Rodbell M 1974 A highly sensitive adenylate cyclase assay. Anal Biochem 58:541 -548
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24. Lowry OH, Rosebrough NJ, Farr OL, Randall RJ 1951 Protein measurement with the Folin phenol reagent. J Biol Chem 193:265-275 25. Kaumann AJ, Bimbaumer L 1974 Studies on receptormediated activation of adenylyl cyclases. IV. Characteristics of the adrenergic receptor coupled to myocardial adenylyl cyclase. Stereospecificity for ligands and determination of apparent affinity constants for 0-blockers. J Biol Chem 249:7874-7885 26. Bimbaumer L, Yang P-Ch 1974 Studies on receptormediated activation of adenylyl cyclases. I. Preparation and description of general properties of an adenylyl cyclase system in beef renal medullary membranes sensitive to neurohypophyseal hormones. J Biol Chem 249:78487856
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27. Southern EM 1975 Detection of specific sequences among DNA fragments separated by gel electrophoresis. JMol Biol 98:503-517 28. Feinberg AP, Vogelstein B 1983 A technique for radiolabeling DNA restriction endonuclease fragments to high specific activity. Anal Biochem 132:6-13 29. Bone EA, Fretten P, Palmer S, Kirk CJ, Michell RH 1984 Rapid accumulation of inositol phosphates in isolated rat superior cervical sympathetic ganglia exposed to V,-vasopressin and muscarinic cholinergic stimuli. Biochem J 221:803-811 30. Kirk CJ, Guillon G, Balestre MN, Jard S 1986 Stimulation by vasopressin and other agonists of inositol-lipid breakdown and inositol phosphate accumulation in WRK1 cells. Biochem J 240:197-204
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Mariel Birnbaumer, Victoria Hinrichs, and Axel P. N. Themen Department of Cell Biology Baylor College of Medicine Houston, Texas 77030
Human genomic DNA and the HSV tk gene were cotransfected into mouse Ltk" cells and assayed for the acquisition of a Gs-coupled receptor to obtain cell lines expressing human receptors that are so far unavailable. The transfected cells were distributed into 96-well microtitration plates at a density such that after HAT (100 MM hypoxanthine, 1 MM aminopterine, and 10 MM thymidine) selection each well contained, on the average, two to three tk+ cell clones. After replication, half of them were tested for expression of a new phenotype: an adenylyl cyclase stimulatory receptor not normally expressed in the Ltk" recipient cell. The screen yielded a positive result on testing cells arising from the third transfection, the newly expressed receptor is that for arginine vasopressin, commonly referred to as type 2 or V2. DNA from primary transformants (HTB1 cells) served to obtain secondary transformants by the same technique (HTB-2 cells). Pharmacological properties confirmed that this new receptor, which stimulates adenylyl cyclase activity 7- to 10fold, is the human V2 receptor and not the activated homologous murine gene. The new cell line provides a permanent accessible source to study the human receptor, by-passing the need for human kidneys. The V2 receptor was susceptible to homologous down-regulation in the HTB-2 cell, but no downregulation of the cell authentic prostaglandin E1 receptor was observed. The vasopressin receptor did not modify phospholipase-C activity in these cells as expected from V2 receptors. Thus, we successfully applied genomic DNA-mediated gene transfer and were able to develop a cell line expressing a Gs-coupled human receptor of low abundance and poor accessibility. (Molecular Endocrinology 4: 245254, 1990)
INTRODUCTION It has been shown that hormone cell surface receptors may exhibit species specificity in their interaction with 0888-8809/90/0245-0254$02.00/0 Molecular Endocrinology Copyright © 1990 by The Endocrine Society
ligands. As a result, properties of receptors such as ligand binding, complexity of cellular signalling and tachyphylactic responses determined in animal model systems need to be confirmed using human tissues. While this is possible for a few cases, such as for receptors expressed on blood-borne cells, immortalized skin fibroblasts, and a few discards of surgical (organ transplant) procedures, in most cases the proper models for studying human cell surface receptors in the laboratory are still lacking. One approach to this problem, which is yielding positive results, is purification and cloning of selected receptors from animal sources, followed by the cloning of the human homolog and their expression in tissue culture cells, where they now become accessible to biochemical and pharmacological characterization. Human /V, ftr and, more recently, /33-adrenergic receptors are examples of this kind (1-3). Another approach to the study of human receptors is to obtain their expression in tissue culture cells by genomic DNA-mediated gene transfer (4, 5) which obviates the need of purification. The expression in murine cells of the human transferrin and nerve growth factor receptors by Kuhn ef a/. (6) and Chao et al. (7), respectively, are examples of this kind. In these examples the transformed cell clones expressing the acquired cell surface receptor were identified with highly specific antibodies using fluorescent-activated cell sorting (6) or a hemagglutination technique (7). In the present work we have focussed on the possibility of obtaining by genomic DNA-mediated gene transfer the expression of human cell surface receptors that are coupled to their effector by G-proteins of the Gs type (8). Many of these receptors are unavailable for purification because they are either of low abundancy and/or from tissues of which large quantities cannot be obtained. We report below the development of a new strategy for identifying transformed cell lines that acquired a Gscoupled receptor and the successful isolation of one such cell line. This approach is independent of the availability of antibodies, for it uses as a rapid and very sensitive assay for receptor function the measurement
245
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MOL ENDO-1990 246
of ligand-stimulated adenylyl cyclase activity. Applying this methodology we obtained primary and secondary transformants that express the human type 2 vasopressin receptor (V2 receptors) (9) which mediates the antidiuretic action of the hormone arginine vasopressin (AVP).
RESULTS Our objective was to develop a strategy for the expression of Gs-coupled human receptors according to the general scheme shown in Fig. 1. We wished to do so using genomic DNA-mediated gene transfer to insert human genes into a mouse recipient cell and a screening technique that measured appearance of a new hormonal response in the transformed clones. For this, the recipient cell needed to fulfill two conditions: 1) to be an effective acceptor of foreign genes and 2) to express normally as few as possible of the type of receptors we were interested in. Ltk" cells fulfilled both requirements. Wigler et al. (5) and other laboratories had shown Ltk" cells to be versatile recipients of foreign genes (6, 7,10). We found the adenylyl cyclase system of these cells to be unresponsive to any one of the 12 polypeptide and glycoprotein hormones listed in Table 1 as well as to /3-adrenergic receptor ligands. The cells responded, however, to prostaglandin Ei (PGEi) (11), forskolin, and manipulations that activate Gs, such as addition of NaF and GTP analogs [guanosine 5'-(3thiotriphosphate) (GTP7S) and (Guanyl-5'yl imidodiphosphate GMP-P(NH)P)]. These results indicated that Ltk" cells have a normal adenylyl cyclase system susceptible to stimulation by Gs-coupled stimulatory receptors). The presence of at least one positively coupled receptor (PGE^ served to determine whether receptor-
Cloning Strategy for Isolation and Identification of Receptor Genes 'Human Genomic ONA (50kb) HSV tk Gene CaPC>4 Precipitate, 20hr. Glycerol Shock. Wait 24hr. .Trypsinize Cells
Subdivide into 96 Well Microlitration Plates at 5,000 cells/well into HAT Medium
Select Ik + Cells 1 Clone/3,000 Cells
2-3 clones/well after 3 Weeks
Culture Further Pick Putative Positive Clones Subculture. Confirm.
Assay: Adenylyl Cyclase Phosphatase Analyze AC/Phosphatase Ratio
Fig. 1. Schematic Representation of the Strategy Employed for the Selection and Replication of a Large Number of Independent Transformant Cell Clones
Table 1. Hormonal effects on adenylyl cyclase of Recipient Ltk" and Transformed HTB Cells Additions to Assay
None AVP ACTH PTH Calcitonin Glucagon Secretin VIP CRF GRF LH FSH
TSH PGE, Forskolin
Adenylyl Cyclase Activity in (picomol cAMP/min • mg) Ltk" cells
3.14 ±0.08 3.22 ± 0.40 3.06 ± 0.08 2.89 ± 0.40 2.89 ± 0.08 2.97 ± 0.08 2.89 ± 0.08 2.97 ± 0.08 2.81 ± 0.08 3.14 ±0.08 2.89 ± 0.24 3.55 ± 0.40 3.39 ± 0.08 7.56 ± 0.08 9.42 ±0.16
HTB cells
2.89 13.05 3.14 3.06 3.22 3.06 3.22 3.30 2.89 2.97 3.06 2.97 3.22 12.80 17.10
± 0.08 ±0.16 ±0.08 ±0.16 ± 0.08 ± 0.24 ± 0.08 ±0.16 ± 0.08 ±0.16 ±0.16 ± 0.24 ±0.16 ±0.16 ±0.08
Cell homogenates were prepared and assayed for adenylyl cyclase as described in Materials and Methods. Each assay (final volume 50 /J) received 18 or 21 ng protein of Ltk" or HTB cell homogenate, respectively. Peptide and protein hormone additions were at 100 nM each; PGE! and forskolin were used at 100 ^M each. VIP, Vasoactive intestinal peptide.
mediated stimulation of the adenylyl cyclase activity in the Ltk" cells could be measured in situ (data not shown). Cells were plated in 96-well microtitration plates at a density of 30,000-60,000 cells/well and assayed the following day, as described in Materials and Methods. The hypotonicity of the medium was sufficient to alter the permeability of the cell membrane and allow the entry of [a-32P]ATP into the cells, since it was possible to detect newly formed [32P]cAMP after incubation for 60 min at 32 C in the absence of saponin. Nevertheless, addition of saponin to the incubation medium increased the production of cAMP by 3-fold without interfering with the stimulatory effect of PGET (not shown). For the remainder of the experiments the incubations were carried out in the presence of 50 Mg/m' saponin. Higher concentrations of saponin had an inhibitory effect on the stimulation of the enzyme by PGE^ In other experiments, in which we plated cells at different densities, we found that the minimum number of cells necessary to detect enzymatic activity was 5000/well. Since it was expected that the different cell clones that arise after HAT (100 HM hypoxanthine, 1 /XM aminopterine, and 10 HM thymidine) selection might grow at very different rates, it was necessary to measure a normalizing parameter to relate adenylyl cyclase activity per well to the number of cells present. As shown in Fig. 2, phosphatase activity of the cells provided such information. This activity was assessed by hydrolysis of p-nitrophenyl phosphate occurring under the same assay conditions used for assessment of adenylyl cy-
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247
Human V2 Vasopressin Receptor in Mouse Cell
Proportionality Between Number of Cells Assayed and Adenylyl Cyclase Activity
"0 10 20 30 Assayed Cells/well (x10~3)
Phosphatase Activity (OD4
Fig. 2. Correlation between Number of Cells, Phosphatase Activity, and Adenylyl Cyclase Activity Ltk~ cells were plated in 96-well plates, and the assays carried out as described in Materials and Methods. All determinations were performed in replicate. Cells from replicate wells to the ones assayed were trypsinized and counted to determine the number of cells on the day of the assay. A, Proportionality between phosphatase activity and number of cells per well. S, Correlation between adenylyl cyclase activity and phosphatase activity measured simultaneously in 96-well plates.
clase activity and quantified in an enzyme-linked immunosorbent assay reader. Indeed, within the desired range of cell density (1,000-30,000 cells/well), phosphatase and adenylyl cyclase activities were linearly correlated and proportional to cell number.
Ligand Dependence of the HTB Cell Adenylyl Cyclase Activity
Since screening of the plates was carried out by assaying for adenylyl cyclase activity only in the presence of stimulatory ligands, the elevated enzymatic activity detected in HTB cells could have been due to a constitutively elevated basal activity induced by insertional mutagenesis and not necessarily to the presence of a new plasma membrane receptor that the L cells normally do not express. To distinguish between these two possibilities the HTB cells were expanded, homogenates were prepared as described in Materials and Methods and assayed for basal and ligand-stimulated adenylyl cyclase activity. As shown in Table 1 the elevated adenylyl cyclase activity of HTB cells was due to the appearance of a new hormonal response: the ability to respond to AVP. The cells were cloned by limiting dilution. All clones obtained showed the same extent of stimulation in the presence of saturating concentrations of AVP. One clone (HTB-1) was selected for further studies. Southern blots, using as probe radioactively labeled human genomic DNA that assays for the presence of repetitious Alu\ sequences (12), confirmed the presence of human DNA in HTB-1 cells (Fig. 4).
Transfection, Selection and Assay for the New Phenotype
Secondary Transformants
The transfection and subsequent selection of Ltk cells was carried out as described in Materials and Methods. Twelve plates were used per experiment. Approximately 95% of the wells contained two or three independent clones, while the remainder were empty. Thus, on incubating all wells of the 12 plates, we assayed simultaneously the activities of 2000-3400 independent cell clones. Incubations were carried out in the presence of the mixture of 12 stimulatory peptide hormones listed in Table 1 at the concentrations mentioned in Materials and Methods. The values of adenylyl cyclase activity were plotted as a function of the values of phosphatase activity per well, as shown for one such experiment in Fig. 3. Each dot represents the enzymatic activity of a different well due to two or three surviving cell clones. Those wells with adenylyl cyclase activities that deviated significantly from the average were subcultured and assayed again in the presence of the mixture of 12 peptide and protein hormones. As the inset in Fig. 3 depicts, one of the subcultured cells continued to exhibit higher adenylyl cyclase activity than the others. These cells were designated HTB cells. They were identified during the analysis of the third set of 12 96well plates of transfected and HAT-selected cells.
Genomic DNA was extracted from the cloned HTB-1 cell line, and secondary transfections were carried out using this preparation as the source of human DNA containing the vasopressin receptor gene. After selection in HAT medium, the cells were assayed in situ for AVP-stimulated adenylyl cyclase activity in the presence of saturating concentrations of peptide. Clones with significantly higher enzymatic activity than the average were treated as described above. Figure 5 and its inset summarize the experimental data obtained when the second set of 12 96-well plates of the secondary transfections was assayed. The transformant cell clone identified by the arrow in fig. 5 expressed the AVP receptor commonly referred to as V2 or type 2. A clonal cell line (HTB-2) was obtained by limiting dilution. DNA dot assay measurements using human repetitive sequences as probes allowed us to estimate that our primary transformants contain about 12 x 106 basepairs of human genomic DNA. For the secondary transformant it was not possible to estimate this value due to the weakness of the signal. Nevertheless, four or five bands that hybridize to human genomic DNA could be seen in the Southern blot of HTB-2 cells (Fig. 4).
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MOL ENDO-1990 248
Vol 4 No. 2
Hormone-Stimulated Adenylyl Cyclase of L Cells Transfected with Human Genomic DNA (Primary Transfection) 0.26 _ 0.24
0.4
> '>
*
m
•
~ o 0.3 _
> c
°E
•Comp. M:4000nM OT: 1320nM 25
Potency Ratios: Comp. I/Comp. I 2350; AVP/LVP: 10 I I I I I I I I I 11 10 8 7 6 Ligand (-logM)
Fig. 6. Dose-Response Curves of Stimulation of Adenylyl Cyclase by AVP and Analogs Comparison of the mouse vasopressin receptor to the vasopressin receptor of HTB-2 cell. Upper panel, The adenylyl cyclase dose-response curves were obtained with partially purified HTB-2 cell membranes prepared and assayed as described in Materials and Methods. Lower panel, The enzymatic assay was carried out using partially purified mouse kidney plasma membranes prepared as described in Materials and Methods.
EC50 (nM; n = 3)8 AVP LVP OT dDAVP desGly(NH2)LVP Potency Ratios AVP/LVP (ratio I) dD-AVP/desGly(NH2)LVP (ratio II) Mouse kidney vs. HTB-2 Cell Mouse kidney ratio I/HTB2 ratio I Mouse kidney ratio 11/ HTB-2 ratio II Difference
HTB-2 cells
13±4 111 ±43 1350 ±284 4 ±1.4 4633 ± 279
18±5 57 ±17 465 ±17 14±6 1128 ±279
8.8 ± 3.5 1121 ±360
3.5 ±1.6 78.0 ± 6.2
2.5 ±1.5 14.3 ±2.4 11.8 ± 2 . 8 "
" Results are from three independent repeat experiments (mean ± SE). For details, see Fig. 6. b P < 0.025, by Student's t test.
Table 3. Phosphoinositide Hydrolysis by Ltk" and HTB-2 Cells in Response to AVP and ATP Addition to Wells
A. Ltk" cells Control AVP ATP
clones, with very few having either a single or four clones. For the reported experiments the seeding density had been adjusted to give no more than 10% empty wells. With the transfection protocol described in Materials and Methods this required seeding 4000-5000 cells/well at the time of placing the cells into HAT medium. Some general considerations are in order about the expected frequency of success in cloning a given phenotype by the method developed here. Each 12-plate transfection experiment screens about 3000 clones. If one assumes an average DNA uptake per cell of 3 x 106 basepairs (5, 6), then one transfection experiment screens about two thirds of the human genome. Thus, if integration is such that expression can occur if the gene is not accidentally broken during DNA manipulation and the assay is adequate, a given receptor has a maximum cloning likelihood of once in every three transfection experiments. However, this number is not realistic because of a number of factors. Probably the most important is the site of integration of the human DNA
Mouse kidney
B. HTB-2 cells Control AVP ATP
[3H]lnositol Phosphates Accumulated (cpm/ Well)
1530 ± 37 1400 ± 26 3444 ± 41
818 ± 23 816 ± 12 1834 ± 35
Cells were grown in 35-mm wells in the presence of [3H] inositol to a density of 2.1 x 106 cells/well, washed, exposed to 10 mM LiCI for 10 min, and then incubated in duplicate for 30 min in the absence (control) or presence of 100 nM AVP or 100 MM ATP as described in Materials and Methods. Reactions were terminated, and fractions corresponding to total inositol phosphates were isolated by Dowex 1 chromatography.
in the mouse genome, with at least 90% of the human DNA ending up in nonexpressed areas. Another factor is the dissimilar growth rate among the transformed cell lines. About 20% of them do not grow sufficiently so as to be assayed, as can be seen by the low values along the phosphatase assay axis in Figs. 3 and 5. A
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251
Human V2 Vasopressin Receptor in Mouse Cell
Control
AVP Treated
PGE. Treated
Fig. 7. Homologous Desensitization of the Vasopressin Receptor but not of the PGE, Receptor Replicate dishes containing confluent HTB-2 cells were treated for 2 h at 37 C with 100 nM AVP or 27 MM PGE,. Homogenates were prepared in Dounce homogenizers as described in Materials and Methods and assayed for adenylyl cyclase activity in the absence of any added hormone (•) and in the presence of 100 nM AVP (H) or 27 HM PGE, (•).
third identifiable factor is DNA breakage. The probability of interrupting a coding sequence by random breaks is proportional to the size of the genes to be isolated. If there are no hot spots in a gene of about 5 kilobases (Kb), the probability of random breaking will be about 1 in 20 if the fragments of genomic DNA are 100 kb long. With these numbers in mind, the expected number of transfections necessary to efficiently screen the human genome once for any given receptor increases by a factor of 10 (wrong place of integration) times 1.25 (insufficient growth) times 1.05 (DNA breakage if gene is 5 kb or larger), i.e. to 40 or 50. This number may indeed be higher. The finding of the vasopressin receptor as one of the 12 possible hormone receptors simultaneously tested for within the first three transfections agrees with this number. The recovery of the vasopressin receptor gene in the secondary transformants within the first two transfections was, therefore, somewhat surprising, unless one assumes a linkage with the thymidine kinase gene or that the site of integration plays a role only in the first transformation such that in subsequent transfections the gene may be already linked to a strong promoter that can override other cellular controls and insure better expression. Overall, it would appear that this may be a method of choice for the cloning of receptors that are very difficult to purify or for which there are no cell lines that express them. Furthermore, the logistics used to maintain and replicate up to 3000 individual transformed cell clones could be useful for the expression cloning of other functions, provided the phenotype resides in a single gene and they can be assayed in a single well. These transformed cells provide the only available cell line expressing the human V2 vasopressin receptor.
Until now only the pig-derived LLCPK, cells expressed a related receptor that recognizes lysine vasopressin (LVP) instead of AVP as its physiological ligand. The ability of these cells to support ligand-induced desensitization of the acquired vasopressin receptor while failing to desensitize the normal PGE, receptor may be an indication of great susceptibility of the vasopressin receptor to be regulated by its ligand. It also suggests that in these cells increases in intracellular cAMP are not sufficient to reduce the activity of this Gs-coupled receptor. We observed differences in the rate of desensitization of the AVP and LVP receptors as well as in the effect of high salt concentrations in this phenomenon. These will remain a puzzle until cDNAs coding for both receptors are available. At that time expression of both proteins in the same cell should allow us to distinguish between receptor or cell line differences. The studies of Guillon et al. (13) and Butlen et al. (14) had established, by measuring both adenylyl cyclase activation and competition in binding assays, that rat and human kidney AVP receptors displayed different rank order of potencies and apparent Ka toward natural and synthetic agonists. Our data confirmed those results. More recently, the cloning of human and chicken epidermal growth factor receptors has provided another example of species-related heterogeneity (17) in receptor-ligand interaction, strengthening the case for the need to evaluate ligands intended for humans with receptors of the same species. The apparent cosegregation of the V2 vasopressin receptor gene and a small number oM/ul-positive bands in the HTB-2 cells makes them an appropriate source of DNA to construct genomic libraries from which to isolate the gene coding for the vasopressin receptor using established molecular biology techniques (6,7,18).
MATERIALS AND METHODS Radiochemicals [a-32P]ATP was obtained from the NICHHD Center for Population Research and Studies in Reproductive Biology at Baylor College of Medicine; [«-32P]dCTP was from Amersham (Arlington Heights, IL); [3H]AMP was from ICN Biochemicals (Irvine, CA); myo-[3H]inositol (40 Ci/mmol) was from DuPont-New England Nuclear (Wilmington, DE). Materials and Reagents Minimum Essential Medium (MEM) a, Hanks' Buffered Salt Solution, penicillin-streptomycin, 0.5% trypsin 5 miwi EDTA and fetal bovine serum were obtained from Gibco (Grand Island, NY). Tissue culture plasticware was purchased from Costar (Cambridge, MA). The hormones and synthetic peptides were obtained from Cambridge Research Biochemicals (Atlantic Beach, NY), except for glucagon, vasoactive intestinal peptide, AVP, LVP, dDAVP, and PGE,, which were from Sigma (St. Louis, MO). Desglycinamide-LVP (des-Gly(NH2)9LVP) was obtained from Peninsula Laboratories (Belmont, CA). Human TSH, ovine LH and ovine follicle stimulating hormone FSH were provided by Dr. Salvatore Raiti from the NIDDK NIH National Pituitary Agency (Baltimore, MD). Forskolin was pur-
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MOL ENDO-1990 252
chased from Calbiochem (San Diego, CA). Agarose gel electrophoresis supplies were obtained from BRL (Bethesda, MD). Oligonucleotides for random priming were purchased from Pharmacia (Piscataway, NJ). Restriction endonculeases were obtained from Boehringer-Mannheim Biochemicals (Indianapolis, IN). All other reagents were purchased from Sigma. Cell Line Ltk" aHT, a clone of the mouse fibroblast cell line originally established by Kit ef al. (19) was a generous gift from Dr. Frank Ruddle, Department of Biology, Yale University. This subclone had been selected for high frequency of transformation. The cells were cultured at 5% CO2 in MEMa containing 10% heat-inactivated fetal bovine serum, penicillin (50 U/ml), and streptomycin (50 M9/ml). Cell Transformation The calcium phosphate precipitation technique was used (4). Cells, which had been kept subconfluent for all passages preceding the transformation, were plated 24 h before transfection (1.5x10 6 /100-mm dish), and exposed to a precipitate of calcium phosphate formed in the presence of 20 ^g human genomic DNA (>50 kb) and 50 ng pHSV-106 plasmid linearized with H/ndlll. After 18 h of exposure to the precipitate the cells were treated with 25% glycerol in Hanks' Buffered Salt Solution for 60 sec at room temperature, thoroughly rinsed with Hanks' solution, and returned to the incubator with fresh medium. Twenty-four hours later the cells were trypsinized, counted, and diluted to a concentration of 25,000 cells/ml in the culture medium containing HAT. Cells were then distributed into 96-well plates using a Transtar 96 pipettor (Costar), delivering 200 ^I/well (-5,000 cell/well). After 2-3 weeks in the selective medium the plates were examined for the presence of clones. Wells containing one to three clones were trypsinized to obtain replicate plates, one of which was assayed when cells had reached confluency. After their isolation, the transformed cells were cultured always in the presence of HAT. Purification of Plasmid DNA and Human Genomic DNA The plasmid pHSV106, pBR322 containing the herpes simplex thymidine kinase gene, was obtained from Bethesda Research Laboratories and amplified in E. coli AG1 following the protocol of Hanahan (20). Human genomic DNA was prepared from white blood cells obtained from normal volunteers following the technique of DiLella and Woo (21). The genomic DNA was precipitated in the presence of sodium acetate and ethanol and redissolved in sterile 1 mM Tris-HCI and 0.1 mM EDTA, pH 7.4. The size of the genomic DNA was analyzed in a 0.3% agarose gel run in 89 mM Tris-boric acid and 0.2 mM EDTA, pH 8.8. Only fragments larger than intact Xphage were used. Measurement of Adenylyl Cyclase Activity Attached Cells Growth medium was discarded, the 96-well plates were rinsed once with 200 ^I/well 10 mM Tris-HCI and 150 mM NaCI, pH 7.4 (TBS), and 50 fi\ incubation medium were added to each well. The incubation medium contained 0.1 mM [«-32P]ATP (5 x 106 cpm/well), 2 mM MgCI2, 10 IXM GTP, 1 mM EDTA, 1 mM cAMP, 2 mM methylisobutylxanthine, and a nucleotide triphosphate-regenerating system composed of 20 mM creatine phosphate, 0.2 mg/ml (2,000 U/mg) creatine phosphokinase, 0.02 mg/ml myokinase, and 25 mM Tris-HCI pH 7.4. In addition, the incubation medium contained 50 ng/ ml saponin to permeabilize the cells to the reagents and [3H] cAMP (10,000 cpm/well) to determine recoveries of this nucleotide. Also included in the reaction was 5 mM p-nitrophenylphosphate, which served as substrate for assessment of phosphatase activity. The incubations were carried out at 32
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C for 2 h. Hormones (diluted in 0.1% BSA) were present at concentrations of 100 nM. The reaction was stopped by the addition of 100 ^I/well of a solution containing 40 mM ATP, 10 mM cAMP, and 1 % sodium dodecyl sulfate. The phosphatase assay was simultaneous with the adenylyl cyclase assay for the cells assayed in situ. The extent of hydrolysis of p-nitrophenylphosphate was determined by measuring the absorbance at 405 nm in a Titertek enzyme-linked immunosorbent assay reader, and the activity was expressed in arbitrary units. The cAMP formed in the reaction was isolated by a modification (22) of the standard double Dowex-alumina chromatographic procedure (23). Activity in Cell Homogenates and Partially Purified Membranes The same incubation conditions described above were used, omitting the saponin and the p-nitrophenylphosphate. The incubations were carried out for 10 min at 32 C. The activities were expressed as picomoles of cAMP per min/mg homogenate or membrane protein. Protein was determined by the method of Lowry ef al. (24). Homogenate Preparation After withdrawing the growth medium the cells were rinsed once with TBS, scraped off the dishes with a rubber policeman, and centrifuged. The supernatant was discarded, and the pellet resuspended in approximately 10 vol ice-cold homogenization buffer consisting of 27% (wt/vol) sucrose, 1 mM EDTA, and 20 mM Na-HEPES, pH 7.8. For small volumes (0.5-2 ml) the suspension was transferred to a 75-ml capacity Parr bomb, and the cells were broken by N2 cavitation. If the homogenate was assayed directly, it was allowed to sit on ice for 20 min before the assay. Alternatively, the cells were homogenized in a 7-ml Dounce homogenizer (Kontes Co., Vineland, NJ) with 10 strokes of the tight (B) pestle. For membrane isolation larger quantities of cells were required. They were homogenized in a 15-ml Corex tube using four 10-sec bursts of a Brinkmann PT-10 Polytron (Westbury, NY) at a setting of 6, for this method yielded a homogenate more suitable for membrane isolation than the cavitation technique. The homogenate was centrifuged at 800 rpm for 5 min at 4 C, the pellet discarded, and the supernatant centrifuged further at 15,000 rpm for 15 min. The pellet obtained after the second centrifugation was significantly enriched in plasma membranes. The membranes were resuspended in homogenization buffer containing 20 mM MgCI2 (11), aliquoted, quick frozen, and stored at - 7 0 C until used. Membranes from Mouse Kidney Retired breeder female mice were obtained from Harlan (Angleton, TX). They were killed by cervical dislocation, and the kidneys were quickly excised, freed of adherent tissues, and immersed in an icecold solution of 0.25 M sucrose containing 1 mM EDTA. All subsequent steps were carried out at 4 C. The kidneys were minced and homogenized in 20 vol 1 mM KHCO3 with a Polytron homogenizer (25). The homogenate was filtered through silk screen, and the filtrate was centrifuged for 5 min at 1500 rpm. The pellet was discarded, and the supernatant centrifuged for 20 min at 10,000 rpm. The second membrane pellet was subjected to discontinuous sucrose gradient purification (26), and membranes with the highest stimulation of adenylyl cyclase by vasopressin were collected at the interphase between 37% and 41.5% (t/wt) sucrose. They were diluted to approximately 40 Mg/ml, fractionated in 0.2-ml aliquots, and kept frozen at - 7 0 C until used. DNA Analysis DNA isolated from the positive clones was subjected to Southern blot analysis (27) for detection of human repeated sequences (11). Genomic DNA (20) was digested to completion with restriction endonucleases following the manufacturer's recommendations, applied to a horizontal 0.8% agarose gel, electrophoresed at 4 V/cm, and blotted onto nitrocellulose filters. Nitrocellulose filters were baked at 68 C for 3 h, prehybridized for 2 h, and hybridized for 20 h with human genomic DNA that was labeled with 32P by extension of oligonucleotide primers (28) to specific activities of 1 x 109 . Prehybridization and hybridization were carried out
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253
Human V2 Vasopressin Receptor in Mouse Cell
at 45 C under the conditions described by Kuhn et al. (6) using for the prehybridization 50% formamide. 4 x SSC (1 x SSC = 150 mM NaCI and 15 mM Na-citrate), 5 x Denhardt's solution (1 x Denhardt = 0.02% Ficoll, 0.02% polyvinylpyrrolidone, and 0.02% BSA), and 0.1% sodium dodecyl sulfate. The same mixture with the addition of 0.1 mg/ml sheared herring sperm DNA, 10% dextran sulfate, and 8 x 106 cpm/ml labeled human DNA was used to hybridize the filter. Phosphoinositide Hydrolysis Receptor stimulation of cellular phosphoinositide-specific phospholipase-C was tested for in intact cells by an adaptation (14) of the procedures described by Bone et al. (29) and Kirk et al. (30), in which cells were labeled with [3H]/nyo-inositol and incubated with agonist in the presence of LiCI, and the free 3H-labeled inositol phosphates accumulated during the incubation are extracted, separated on Dowex-1 columns, and quantified by liquid scintillation counting. Typically, about 0.5 x 106 LtK" or HTB-2 cells were seeded into 35-mm wells of six-well plates in 2.5 ml HAT selection medium (see above) supplemented with 2 ^Ci/ml [3H]inositol and grown for 48 h to a density of about 2 x 106 cells/well. Labeled cells were then placed into 12.5 ml fresh HAT selection medium without fetal calf serum or labeled inositol, and incubated for 1 h at 37 C. The cells were then rinsed twice at room temperature with 2.0 ml PBS supplemented with 5.5 mM glucose, 0.5 mM CaCI2, and 0.5 mM MgCI2. After the addition of 2 ml PBS with glucose, Ca2+, and Mg 2+ , cells were placed again at 37 C for 20 min, at which time 10 mM LiCI was added. Test agents were then added after a further 10 min. Unless indicated otherwise the incubations were stopped 30 min after the additions of stimulatory agents by placing the plates on ice, removing and discarding the incubation medium, and adding 1 ml ice-cold 5% perchloric acid and 20 /zl 10% BSA.
Acknowledgments The authors wish to acknowledge Dr. Lutz Birnbaumer for helpful discussions and invaluable advice on the execution of the large number of adenylyl cyclase assays; Karen Thiessen for expert technical assistance, and Betty Kilday for help in the preparation of the manuscript.
Received October 17, 1989. Revision received November 20,1989. Accepted November 20,1989. Address requests for reprints to: Dr. Mariel Birnbaumer, Department of Cell Biology, Baylor College of Medicine, Houston, Texas 77030. This work was supported in part by NIH Grant HD-09581 and Center Grants DK-27685 and HD-07945.
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24. Lowry OH, Rosebrough NJ, Farr OL, Randall RJ 1951 Protein measurement with the Folin phenol reagent. J Biol Chem 193:265-275 25. Kaumann AJ, Bimbaumer L 1974 Studies on receptormediated activation of adenylyl cyclases. IV. Characteristics of the adrenergic receptor coupled to myocardial adenylyl cyclase. Stereospecificity for ligands and determination of apparent affinity constants for 0-blockers. J Biol Chem 249:7874-7885 26. Bimbaumer L, Yang P-Ch 1974 Studies on receptormediated activation of adenylyl cyclases. I. Preparation and description of general properties of an adenylyl cyclase system in beef renal medullary membranes sensitive to neurohypophyseal hormones. J Biol Chem 249:78487856
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27. Southern EM 1975 Detection of specific sequences among DNA fragments separated by gel electrophoresis. JMol Biol 98:503-517 28. Feinberg AP, Vogelstein B 1983 A technique for radiolabeling DNA restriction endonuclease fragments to high specific activity. Anal Biochem 132:6-13 29. Bone EA, Fretten P, Palmer S, Kirk CJ, Michell RH 1984 Rapid accumulation of inositol phosphates in isolated rat superior cervical sympathetic ganglia exposed to V,-vasopressin and muscarinic cholinergic stimuli. Biochem J 221:803-811 30. Kirk CJ, Guillon G, Balestre MN, Jard S 1986 Stimulation by vasopressin and other agonists of inositol-lipid breakdown and inositol phosphate accumulation in WRK1 cells. Biochem J 240:197-204
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