J. Tiss. Cult. Meth. 15:82-86, 1993 © 1993 Tissue Culture A~meiation 0271-8057/93 $01.50+0.00

ACCELERATED IDENTIFICATION OF TRANSFORMED CELL CLONES EXPRESSING RECEPTORS COUPLED TO ADENYLYL CYCLASE

Stephanie Gilbert and Mariel Birnbaumer

Department of Cell Biology, Baylor College of Medicine, One Baylor Plaza, Houston, Texas 77030

SUMMARY: Protocols and techniques are described for the transfection of DNA into eucaryotic cells. Both transient and stable transfection protocols are described. The modifications of the methodology to obtain and analyze stable transfected clones are designed to accelerate the isolation of clones and as a consequence reduce the effort and cost involved in carrying several cell lines simultaneously. Plating the transfected cells for selection in 96-well microtitra- tion plates facilitates the expansion of the clones and minimizes mixing of cells derived from different ones. An in situ assay for binding and adenylyl cyclase activity reduces the number of cells needed for preliminary identification of cell lines expressing receptors that either stimulate or inhibit adenylyl cyclase.

Key words: receptor characterization; stable transformants; in situ assay; expression cloning; transfection; adenylyl cyclase.

". I ° INTRODUCTION

The first intracellular messenger identified was cyclic AMP. This finding stimulated the study of adenylyl cy- clase, the enzyme that converts ATP to cAMP, and the regulation of this pathway. The activity of this plasma membrane effector is regulated by a variety of hormones that bind receptors coupled to G proteins, which in turn modify the activity of the enzyme. This system is still the object of many studies to elucidate the mechanism of ac- tion of hormone receptors (9). In addition to the receptors coupled to adenylyl cyclase that were identified in tissues for the last 20 yr, the isolation of receptor cDNAs uncov- ered the existence of receptor subtypes for which the ef- fector needs to be identified. The recent discovery of sub- types of adenylyl cyclase (6) has revived interest in this signaling pathway and increased the complexity of possi- ble interactions.

The cloning of cDNAs encoding plasma membrane re- ceptors requires the characterization of hormone binding and effector coupling activities of known and new recep- tors by expressing them in transfected cells. The cells provide a uniform background to test receptor activities and overcome the problems that arise when the receptor is present in tissues that are scarce or difficult to obtain. Additionally, expression cloning in eucaryotic cells has been widely applied to clone new receptors that were usually identified by binding. Both transient and stable transfections have been applied successfully.

While establishing a methodology to clone receptors that stimulate adenylyl cyclase activity by genomic DNA expression in eucaryotic cells, we modified existing meth- ods to reduce the cost of tissue culture materials and obtain, with minimal manipulation, a good number of stably transformed clones that were replicated and as-

82

sayed for modification of adenylyl cyclase activity. To ac- celerate the process of identification of the clones of inter- est we decided to measure stimulation and inhibition of adenylyl cyclase activity as well as binding in intact cells. The adenylyl cyclase activity can be measured in the cells contained in one well of a microtitration plate, anywhere between 10 000 and 20 000 cells are the minimum num- ber needed. Cells expressing stimulatory as well as inhibi- tory receptors are assayed in the presence of saturating concentrations of hormone, and, in the case of the inhibi- tory receptors, in the presence of saturating concentra- tions of forskolin to stimulate adenylyl cyclase activity and thus facilitate the detection of inhibition. This methodol- ogy was applied to clone the human antidiuretic hormone (ADH) receptor (4), and to characterize the dual coupling activity of the murine luteinizing hormone receptor (8). We are presently examining the receptor functions af- fected by the mutations in the ADH receptor coding region that we identified in patients suffering nephrogenic dia- betes insipidus (14). For this purpose we have modified the methodology to identify cells that express a receptor able to bind hormone even if it is unable to stimulate adenylyl cyclase. Cells grown in 12-well plates are tested in a binding assay for the ability to express receptors, one well is used to determine total hormone binding, another to determine non-specific binding. The wells contain ap- proximately 1 × 106 cells at the time of the assay and the positives are assayed again within 2 days. These in situ assays require fewer cells than those done with cells in suspension, and even less than assays that require the preparation of plasma membranes, a very costly proce- dure when several cell lines need to be studied.

Other methods have been proposed to measure adeny- lyl cyclase activity in intact cells, such as labeling the cellular nucleotide pool with [3H]adenine followed by

GILBERT AND BIRNBAUMER 83

II .

A.

B.

chaUenging the eells with hormones in the presence of a phosphodiesterase inhibitor (1). We found the measure- ment of adenylyl cyclase in permeabilized cells to be supe- rior in sensitivity, a factor of particular importance for the detection of the inhibition of adenylyl cyclase when devel- oping eell lines expressing novel serotonin reeeptors (11).

After extensive work in various aspects of ceil transfec- tion protocols, we found that coexistence of the selectable marker with the cDNA encoding the protein in the same plasmid does not increase the proportion of stable clones that express the receptor of interest. In out experimental conditions, typically 30 to 50% of the clones express the protein encoded by the exPression vector. We describe here our modifications of the methods of Graham and van der Eb (7) and from Luthman and Magnusson (12) to transfect DNA into eucaryotic eells, as weil as a modifica- tion of the adenylyl cyclase assay (5,15).

For the cloning of receptors by genomic expression the cell clones were replicated and the replicate cells assayed for adenylyl cyclase aetivity in the presence of hormones. A detailed description of these approach can be found in Birnhaumer et al. (3). The positive clone assays were assayed a second time to confirm their identity. When expression vectors containing receptor cDNAs were used for transfection we aimed at identifying quickly 4 to 10 cell clones, and then chose among them those that will best serve the purpose of our studies. When cell lines expressing different levels of receptor protein were de- sired, more clones were chosen, 10 to 15 for example, selecting those that displayed different degrees of hor- monal response in the in situ test. We later eharacterized their activity using celi homogenates or membranes for the adenylyl cyelase assay in conjunction with binding studies. This information was used to select the clones we wished to expand and study. In all cases this approaeh identified suffieient clones of interest to have a broad choice be- tween them.

MATERIALS

MgC12* 6H20, no. M-875 NazHPO4* 7H20, no. $471-3 » Bovine Serum Albumin, no. A-80227

C. Supplies lO0-mm Tissue cuhure (TC) dishes, no. 25025-100

Corninb O 96-Weil plates, Falcon, no. 3072 s 12-Well plates, Costar, no. 3512 » 6-Weil plates, Falcon, no. 3046 s Filter units, 0.22 #m, Corning, no. 25942-500 » 10-ml Pipette, sterile, Falcon, no. 75515 15-tal Sterile conical tubes, no. 62.553.002 PS, Sar-

stedt lo 50-ml Sterile eonical tubes no. 62.547.044 pplo Pasteur pipettes, no. 13-678-20A 5

D. Tissue culture media/serum Minimum essential medium (MEM)-alpha modified, free

of ribonucleosides and deoxy-ribonucleosides, no. 410-2000 EG, Gibco il

Pennicillin/streptomycin (penn/strap), no. 600-5140 AG n

Fetal bovine serum (FBS), no. 200-6140 AJ 11 HAT supplement (100×), no. 320-1062 AS Il

G-418 sulfate, no. 860-1811 IJ n 0.05% Trypsin/0.53 mM EDTA, no. 610-5300 AG H Dulbecco's modified Eagle's medium (DMEM), high glu-

cose, no. 430-2100 EG n • Sodium bicarbonate, no. 670-5080 AG Il

NuSerum, no. 55000, Collaborative Research Inc 12 Hanks' balanced salt solution HBSS, no Mg/Ca, no. 310-

4180 H E. Selection markers

pHSV106, a pBR322 plasmid containing a short version of the herpes simplex virus thymydine kinase gene, stably transfeeted Ltk- cells become tk +, purchased from BRL. a pKNH, an eucaryotic expression vector, it also encodes the lactamase gene that renders stably transfected cells resistant to G418 or genet- icin, available from Dr. Numa, Kyoto, Japan.

Equipment Air/COz controlled incubator, model no. 3326/3336,

FORMA 1 Centrifuge, model no. T J-6, Beckman 2 Water bath, model no. 184, Precision 3 Laminar-flow hood, model no. 11281 CO 2 tanks, type G Inverted microseope, Nikon TMS 4 Chemicals CaCl» no. C79-500, Fisher » HEPES, no. 242-608, Boehringer 6 NaC1, no. S-3014, Sigma 7 KCI, no.P-217 » Na2HPOg, no. S-374-1 » Dextrose, no. D16-500 » Glycerol, no. 5514UA, BRL s Trizma-base, Si no. T-15037 DEAE Dextran, Si no. D-98857 DMSO, no. 20684, Pierce 9 Chloroquine, no. C-66287

l l I .

A.

PROCEDURES

Ca/PO 4 stable transfection 1. Solutions

a. 2X CaCI 2, autoclaved: dissolve 3.68 g CaCI 2 • 2H20 in 100 ml ddH20. Autoclave 40 min on liquid cycle. Mix While hot. When cool store at 4" C.

b. 20× Phosphate/HEPES buffer: 10 g HEPES, 16 g NaC1, 0.74 g KC1, 0.22 g Na2HPO4, 2 g Dex- trose. Bring to 100 ml with ddH20. Adjust pH to 7.05. Dilute 1 ml 1 to 20 in H20, adjust the pH again if necessary to obtain 7.05. This is critical for the formation of the preeipitate. Filter sterilize through a 0.22-#m filter unit.

c. 25% Glycerol: Add 25 ml glycerol to 75 ml of HBSS. Autoclave 40 min on liquid cycle. Mix while bot. When cool, store at 4 ° C.

d. Growth media: MEM-alpha modified media 450

84 GILBERT AND BIRNBAUMER

2.

tal, FBS heat inactivated 50 ml, penn/strep 5 rel. Make sure all components are kept sterile. If they are, you may mix them together and use immedi- ately. Otherwise, filter sterilize before use.

Preparation of FBS: defrost sera completely. In° activate by placing the bottle in a 56 ° C water bath for 30 min. Swirl oceasionally. When serum has cooled, filter it through a 0.22-gm filter unit. Most L cell clones grow better and have good viability after thawing when the particulate matter is re- moved from the serum.

e. Selection media: Same as growth medium with 5 ml HAT supplement per 500 ml total medium, or G-418 at a final concentration of 400 gg/tal of active product (the active product is usually 40 to 50% of the commercial powder).

f. DNA: linearized plasmids containing the selectable markers yield twice as many stable transformants, we use pHSV106 linearized with HindIlI. Complex expression plasmids such as pKNH must be used supercoiled; they rarely contain a restriction site that will not alter function when cut.

If genomic DNA is used it must be larger than 50 kb after isolation and very clean, with a 260:280 absorbance ratio of 1.7 or more. If it nucleates the CaPO,, it can be sheared through a 22-gauge needle and small syringe. However, clean DNA rarely does.

Make all DNA solutions with sterile buffers and use a laminar flow hood. To check the sterility, incubate your DNA with subconfluent eells for a couple of days.

Transfection a. Cell Cuhure: The Ltk- ceils (10) should always be

kept 70% subconfluent in preparation for trans- fection. To cuhure these cells, remove medium from a dish that is 70% confluent. Wash with 10 ml of HBSS. Add 2 ml of trypsin/EDTA while rocking the plate gently. After 2 min, start detach- ing the cells with gentle pipetting, add the cell sus- pension to 8 ml of growth media previously added to a 15-ml tube. Add 1:20 of the total volume (0.5 ml) into dishes containing fresh media. Incubate for 3 days and subcuhure again. Cells are grown in 5% CO~:95% air, 90% humidity, at 37 ° C; they may need more or less dilution once they adapt to your laboratory.

b. Transfection: all reagents are at 37 ° C. Day 1: Not later than noon trypsinize ceUs as for

euhure. Plate 1 to 2 × 10 6 cells into 100-mm TC plates (as many as needed).

Day 2: At 3 to 5 p.m., prepare the Ca-PO, precipi- tate in the TC hood. Use a 15-ml sterile poly- styrene tube, and mix in this order: 100 ng of plasmid containing the selection marker, 10 to 40 #g genomic or 3 to 5 gg plasmid DNA in no more than 900 gl of TE. Add sterile H20 to bring the volume to 900 gl if neces- sary. Mix gently by tapping on the tube; do not vortex.

Add dropwise and slowly (wirb a Pipetman and sterile tip), 1 ml 2× CaCl» with gentle mixing after each addition. Add very slowly, 100/.tl of the 20)< phosphate-HEPES buffer with a 200-#1 Pipetman. Mix gently after each addition. You should see a faint, whitish cloudiness. Cover the tube and let it stand at room temperature for 5 to 10 min. The precipitate should not become floeeulent. If it does, prepare a fresh tube and incubate the CaPO4H precipitate for shorter time. Add 1 ml of this sus- pension to each plate, dropwise and swirling gently to mix. Two plates are sufficient for stable trans- fections. Return cells to the incubator for 18 h. Day 3: Carefully remove all the media by suction.

Add 2 ml of 25% glycerol/HBSS. Cover the plate and swirl slowly. Start the timer. After 60 s, start adding 10 ml of HBSS slowly and with continuous mixing. The cells burst if the glycerol is diluted too fast. After it is weil mixed and no more diffraction lines are seen, suction the HBSS and rinse the cells again with HBSS. Add 10 ml fresh growth media to the plate and return the cells to the incubator until the next day.

Day 4: The selection of stable transformants ean be done in 96-weil mierotitration or 100-mm tissue culture plates.

(1) Selection in 96-weil plates: The next morning trypsinize the cells as usual. Dilute the cells into selection media containing HAT or G-418, depending on what plasmid has been used. Plate 2000 to 4000 cells/well using a COSTAR transplate device to distribute the cells. We plate 4 plates for the plasmid trans- fections; 12 plates for the genomic DNA trans- fections. The cells should be fed every 3 to 4 days by suctioning the old media and adding fresh. After 10 days, the clones should be visi- bie. After 16 to 18 days the cell density should be sufficient to expand the clones and plate them for in situ cyclase assay to identify posi- tive clones. Twenty-five to fifty microliters of trypsin solution are sufficient to trypsinize cells from each microwell. Plate four wells per clone for an assay minus and plus hormone. To test receptor expression by binding assay, expand the cells until they fill the weil of a 24-weil plate. At this time plate cells in rephcate wells of a 12-weil plate. For screening purposes the determination need not be in replicate.

(2) Selection in lO0-mm dishes: The next morning trypsinize the cells as usual. Distribute the cells from one dish into two to four dishes con- taining the appropriate selection media. Feed the plates every 3 to 4 days, but do not disturb the clones until they are big enough to expand into 24-well plates. Draw a circle on the bot- tom of the dish to identify the colonies that you wish to expand. Generally, there should be 50 to 100 cells per colony when they can be seen

GILBERT AND BIRNBAUMER 85

B.

easily. Remove the medium, wash the plate with 10 ml HBSS, suction the liquid very weil, and add 10 gl of the trypsin/EDTA solution to the marked clones. After 3 min, add 10/.tl of complete medium to the trypsin solution and detach the clone by pipetting and scratching the plate if necessary. Transfer the 20 #1 ob- tained from each clone to a weil of a 24-weU plate containing 1 ml of medium. Once the clones are confluent in the 24-weil plate, plate them in a 96-weil plate for in situ cyclase as- say. Plate four wells per clone for an assay minus and plus hormone.

Note: Clean plasmid preparations yield 60 to 80 clones from 1 × 106 transfected cells by either method of selection. If fewer than 20 clones are obtained, clean the DNA further and repeat the experiment. In out experience, at least 30% of the clones express the protein of interest. Usuaily we assay 30 clones to pick the 5 to 6 we want for future tests. We provide below a brief description of the in situ adenylyl cyclase assay, more details can be found in Salomon et al. (14). The in situ binding assay was described in (2).

DEAE-dextran transient transfection 1. Solutions:

a. Tris buffered saline (TBS). Solution A: NaCI 80 g, KCI 3.8 g, Na2HPO4 2 g,

tris-base 30 g. Dissolve in 900 ml water, ad- just pH to 7.5, bring to 1 liter. Filter sterilize. Store frozen.

Solution B: CaCI2 15 g, MgCI2 10 g, dissolve, bring to 1 liter. Filter sterilize. Store frozen.

TBS: For 1 liter add 100 ml solution A to 890 ml ddH20. While stirring rapidly, add 10 ml so- lution B, dropwise. Filter sterilize and store at 4 ° C.

b. NuSerum: Store frozen aliquots in 15-ml sterile tubes.

c. Chloroquine stock: 10 mMis prepared in 140 mM NaCI/50 mM Tris-HCl, pH 7.4.

d. DMEM with 100/xM chloroquine: To DMEM with 2% FBS, add 1:100 vol of 10 mM chloroquine stock. Prepare it fresh every time.

e. DEAE-dextran: Prepare a solution of 5 mg/ml in TBS, fresh every time.

f. Growth media: 450 ml DMEM, high glucose, 50 ml FBS (heat inactivated, filtered), 5 ml penn/ strep. Make sure all components are kept sterile. If not, sterilize media through a 0.22-#m filter unit before usage.

g. Dimethylsulfoxide (DMSO): Bought sterile from Pierce 9 and diluted 1:10 in HBSS.

2. Transfeetion a. Cell cuhure

The COS.M6 ceils grow rapidly in culture, wash the ceils with 10 ml HBSS and add 2 ml trypsin/ EDTA. Treat the cells 2 to 5 min with the trypsin solution, then detach them from the plate, disperse

them weil by pippetting, and add them to 8 ml growth media. The time of trypsinization is crucial to preserve efficient transfection. The eells should not be in clumps, rather they should attach as sin- gle eells evenly distributed in the dish. The cells are then seeded at a density of 0.5 to 1.0 × 106 cells/ 100-mm dish. Do not let them get over 70% con- fluenee. Grow them in 5% CO 2, 90% humidity at 37 ° C.

b. Transfecüon Day 1: Before noon, trypsinize the cells as usual

and plate 1.0 to 2.0 X 106 cells/100-mm dish. Make sure the cells are evenly distrib- uted in the dish.

Day 2: Prepare the 5 mg/tal DEAE-dextran in TBS in a 15-ml sterile tube. Aliquot the DNA (1 to 10/.tg/plate) in a sterile tube and bring it to 60 #1 with TBS. Mix weil. Heat both solu- tions at 56 ° C for 15 min. Periodically vortex the DEAE-dextran while heating to help dis- solufion.

Mix 60 #1 of DNA-TBS with 120 #1 of DEAE-dex- tran solution and incubate at room tempera- ture for 15 to 30 min.

Make 10% Nuserum in TBS and bring to 37 ° C. In a 15-tal sterile tube, mix 4 ml of 10% Nu- serum-TBS with the DNA-dextran mixture. Wash the eeLls twiee with HBSS removing all the solution by suetion. Transfer the DNA/ Dextran Nuserum-TBS to the plate, lncubate ceLls at 37 ° C for 4 h. Make sure all cells are eovered by the solution, watch out for tihed incubator shelves.

Prepare 10% DMSO in HBSS and DMEM with 100 #M chloroquine/2% FBS. Suction oft all of the DNA solution, do not wash, and add 5 ml of 10% DMSO/HBSS. Time for 2 min. suction the liquid, do not wash. Add 5 ml of the ehloroquine/DMEM solution. Return the plate to the ineubator for 3 to 4 h.

Wash the eeils twice with 10 ml HBSS. Add 10 ml growth media and incubate for 48 h at 37 ° C.

Day 3: For binding in intact ceUs they can be tr?.p- sinized and seeded at the desired density of about 0.5 to 1.0 × 106 eeils/well in 12- or 24-weil plate. If you used an iodinated ligand there will be suffieient ceils in the smaller weil. It may not be so for tritiated ligands of lower specific activity.

Day 4: Harvest the cells, lyse them, and test them for hormonally sfimulated adenylyl cyclase ac- tivity. The lysed eells ean also be used to pre- pare membranes for binding assays.

C. Measurement of adenylyl cyclase activity in attached cel[s Diseard the growth medium from the 96-weil plates.

rinse them once with 200 •l/weil of 10 mM Tris-HCl/ 150 mM NaCI, pH 7.4 (TBS), and add 50 #1 of incubation medium to each weil. The ineubation medium contains 0.1 mM [a-32P]ATP (5 × 106 cpm/well), 2 mM MgCI 2,

86 GILBERT AND BIRNBAUMER

IV.

10 #M GTP, 1 mM EDTA, 1 mM cAMP, 2 mM methyl- isobutyLxanthine, a nucleotide triphosphate regenerating system composed of 20 mM creatine phosphate, 0.2 mg/ ml (2000 U/mg) creatine phosphokinase, 0.02 mg/tal myokinase, and 25 mM Tris-HC1, pH 7.4. In addition, the incubation medium contains 10 000 cpm/well of [aH]- cAMP to determine the recovery of this nucleotide. In- clude in the reaetion 5 mMp-nitrophenylphosphate which serves as substrate for the assessment of phosphatase ac- tivity. Ineubate the plates at 32 ° C for 2 h in a covered water bath, avoiding water entry into the plates. Add the hormone(s) and compounds of interest diluted in 0.1% bovine serum albumen. Stop the reaction by adding 100 /.tl/weR of the foUowing solution: 40 mM ATP, 10 mM cAMP, and 1% sodium dodeeylsulfate.

The phosphatase assay is performed simuhaneously with the adenylyl eyclase assay for the ceUs assayed in situ. After adding the stop solution, measure the absor- banee at 405 nm in a Titertek Ehsa reader to determine the extent of hydrolysis of p-nitrophenylphosphate. Wells containing a similar number of ceUs will yield similar val- ues. The adenylyl eyelase aetivity should be expressed relative to phosphatase activity in arbitrary units (3). Iso- laie the eAMP formed in the reaction by a modification (5,15) of the standard double Dowex-alumina chromato- graphie procedure (14).

DISCUSSION

The methodology described here has been in use in our laboratory for the last 4 yr. We started selecting our clones in microtitration plates while cloning by expression from transfected genomic DNA. Later we found the tech- nique superior to the cloning rings when isolating stably transfeeted clones. The use of the microtitration plate for the isolation of individual eell clones, as weil as for the in situ adenylyl eyelase assay, offers a great simphfieation for the deteetion of enzymatie aetivities. The in situ assay ehminated the need to harvest cells, or make homoge- nates, therefore a large number of clones ean be assayed easily. The use of high specific acüvity [a~P]ATP as sub- strate, ahhough more cosfly perhaps than the tritiated compounds, increases the sensiavity of the assay done in a small number of cells and thus reduces the eost of cell culture materials. The use of the simultaneous phospha- rase assay in the identißcation of the cell clones of interest helps to evaluate whether the high activities obtained in

particular weUs are due to higher cell number or to higher density of receptors. The phosphatase assay is reliable, easy to perform simultaneously with the adenylyl cyclase assay, and ehminates the need to do either DNA or protein determinations that become problematic with small quan- tities of material. We hope to help the readers simplify their methodology by applying these techniques that we constanfly apply with success in our laboratory.

REFERENCES

1. Alvarez, R.; Daniels, D. V. A separation method for the assay of adenylyl cyclase, intracellular cAMP, and cyelie AMP phosphodiesterase using tritium labelled substrates. Aoal. Biochem. 203:76-82; 1992.

2. Birnbaumer, M.; Antaramian, A.; Themmen, A. P. N., et al. Desensitizaüon of the human V2 vasopressin receptor. Homologous effects in the ab- sence of heterologous desensitization. J. Biol. Chem. 267:11783- 11788; 1992.

3. Birnbaumer, M.; Hinrichs, V.; Themmen, A. P. N. Development and char- acterization of a mouse eell Iine expressing the human V2 vasopressin receptor gene. Mol. Endocrinol. 4:245-254; 1990.

4. Birnbaumer, M.; Seibold, A.; Gilbert, S., et al. Molecular cloning of the human antidiuretie hormone receptor. Nature 357:333-335: 1992.

5. Bockaert, J.; Hunzicker-Dunn, M.; Birnbaumer, L. Hormone-sümulated desensitization of hormone-dependent adenylyl eyclase. Dual aetion of luteinizing hormone on pig graafian folliele membranes. J. Biol. Chem. 251:2653-2663; 1976.

6. Gao, B.; Gilman, A. G. Cloning and expression ofa widely distributed (type IV) adenylyl cyclase. Proc. Natl. Aead. Sei. USA 88:10178-10182: 1991.

7. Graham, F. L.; Van der Eh, A. J. A new technique for the assay ofinfectiv- ity of human adenovirus S DNA. Virology 52:456-467; 1973.

8. Gudermann, T.; Birnbaumer, M.; Birnbaumer, L. Evidence for dual cou- pling of the murine LH receptor to adenylyl eyclase and phosphoinosi- tide hreakdown/Ca z+ mobilization. J. Biol. Chem. 267:4479-4488; 1992.

9. lyengar, R.; Birnbaumer, L. Signal transduction by G proteins. ISI atlas of science. Pharmaeology 1:213-221; 1987.

10. Kit, S.; Dubhs, D.; Piekarski, L., et al. Deletion of thymidine kinase activity from L ceUs resistant to bromodeoxyuridine. Exp. Cell. Res. 31:297- 312; 1963.

11. Levy, F. O.; Gudermann, T.; Perez-Reyes, E., et al. Moleeular eloning of a human serotonin reeeptor (S12) with a pharmacological profile resem- bling that of the 5-HTm subtyp¢. FEBS Lett. 296:201-206; 1992.

12. Luthman, H.; Magnusaon, G. High efliciency polyoma DNA transfection of chloroquine treated cells. Nueleic Acids Res. 11:1295-1308; 1983.

13. Rosenthsl, W.; Antaramiam A.; Arthus, M-F., et al. Molecular identißea- tion of the gene respomible for eongenital nephrogenie diabetes insipi- dus. Nature 359:233-235; 1992.

14. Salomon, Y.; Londes, C.; Rodbell, M. A highly aensitive adenylate cyclase assay. Anal. Biochem. 58:541-548; 1974.

15. Themmen, A. P. N.; Hin,ichs, V.; Bimbaumer, M. In situ a&say of hor- mone-sümulated adenylyl cyclase in 96-weU mierotitration plates: an aid to rapid identißeaäon of transformed eell clones. J. Recept. Res. 13:69- 78: 1993.

1 Forma, Marietta, OH 2 Beckman, Dallas, TX

Preeision, Chicago, IL * Nikon, Tokyo, Japan s Fisher, Houston, TX s Boehringer, Indianapolis, IN

7 Sigma. St. Louis, MO s BRL/GIBCO, Grand Island, NY 9 Pieree. Rockford, IL

Lo Sarstedt, Princeton, NJ N GIBCO/BRL, Grand Island, NY 12 Collaborative Research, Bedford, MA