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Binding and signal transduction of the cloned vascular angiotensin II (AT1a) receptor cDNA stably expressed in Chinese hamster ovary cells
Regulatory Peptides, 44 (1993) 131-139 © 1993 Elsevier Science Publishers B.V. All fights reserved 0167-0115/93/$06.00
131
REGPEP 01277
Binding and signal transduction of the cloned vascular angiotensin II (ATla) receptor cDNA stably expressed in Chinese hamster ovary cells Mafia L. W e b b a, Hossain Monshizadegan a, Kenneth E.J. Dickinson a, Randy Serafino b, Suzanne Moreland b, Inge Michel ~, Steven M. Seller ~ and T.J. Murphy c Departments of aBiochemistry and bpharmacology, Bristol-Myers Squibb Pharmaceutical Research Institute, Princeton, NJ (USA) and CDivision of Cardiology, Emory University, Atlanta, GA (USA) (Received 13 November 1992; accepted 11 December 1992)
Key words." SarcosineL[ 125I]tyrosine4-isoleucine8-AII; Intracellular calcium; Phosphoinositide metabolism; Adenylate cyclase; Losartan, PD 123,177
Summary The vascular angiotensin (A) II receptor cDNA (ATla) was transfected into Chinese hamster ovary (CHO) cells to generate the stable cell line CHO-ATIa. This cell line was used to investigate the binding and signal transduction properties of the cloned vascular AT 1 receptor. Specific binding of sarcosineX-[125I]tyrosine4isoleucineS-AII ([125I]SI-AII) to CHO-ATla membranes reached equilibrium after 1 h at 25°C and was consistently greater than 95 ~o of total binding. Saturation binding analyses demonstrated [125I]SI-AII bound to a saturable population of sites on membranes with an equilibrium dissociation constant (KD) of 0.7 nM and a binding site maximum of 1.2 pmol/mg protein. [125I]SI-AII binding to CHO cells was inhibited by the following compounds with a rank order of potency of SI-AII > All > losartan > AI > > PD 123,177. AII (1 #M) treatment of CHO-ATla cells caused an increase in inositol phosphates and intracellular calcium relative to basal levels. These responses were blocked by losartan but not by PD 123,177. AII (1 #M) did not effect adenylate cyclase activity in CHO-ATt~ cells, whereas the agonist inhibited adenylate cyclase activity in rat liver cell membranes. These effects were blocked by 10/~M losartan. These results indicate that CHO-ATla cells express functional ATla receptors which stimulate phospholipase C activity but not adenylate cyclase activity. CHO-ATt~ cells should provide a useful model for studies of ATla receptor domains which are critical to signaling pathways.
Correspondence to." M.L. Webb, Department of Biochemistry, Bristol-Myers Squibb Pharmaceutical Research Institute, P.O. Box 4000, Princeton, NJ 08543-4000, USA.
132
Introduction
Angiotensin (A) II is a vasoactive peptide hormone that has significant effects on cardiovascular, renal, endocrine and neuroendocrine function [27]. All exerts its effects by binding to specific cell-surface receptors in numerous cells and tissues including the adrenal, vascular smooth muscle, kidney, uterus and brain [37]. Two types of All receptors have been identified based on their biochemical and pharmacological properties [40]. Type I (AT1) receptors bind the nonpeptidic antagonist losartan (DuP 753) with high affinity and PD 123,177 with low affinity [10,12,16]. The AT 1 receptor mediates the Allinduced alterations in pressor responses, catecholamine and aldosterone secretion and drinking behavior [41] and therefore has been the focus of much study. By contrast, AT 2 receptors bind DuP 753 with low affinity and PD 123,177 with high affinity [12,13,35]. Bottari et al. [6] have reported that AT 2 receptors mediate an All-induced decrease in cGMP and tyrosine phosphorylation, however, others have not observed these effects [ 17,39]. Thus, the physiological relevance of this receptor is not presently known. In addition, while other receptors for angiotensins may exist [5,9,21,30,31], their identity and role is not clear. While the All receptor family may be more widely heterogeneous than originally thought, the AT 1 receptor remains the primary focus for therapeutic intervention. Several second messenger systems are involved in All activation of AT 1 receptors. In vascular smooth muscle and renal mesangial cells, All stimulates phospholipase C (PLC) catalyzed phosphoinositide turnover and subsequent release of calcium from intracellular stores [1,19] as well as phosphatidylcholine hydrolysis by activation of phospholipase D (PLD) [24,28]. However, in the liver, AT 1 receptors are coupled via G i to adenylate cyclase as well as via Gq to phospholipase C (PLC) [4,29]. Both the Allinduced increase in phosphoinositide metabolism and [Ca 2+ ]i, and the decrease in adenylate cyclase are attenuated by DuP 753 [4]. However, the poten-
tial existence of multiple subtypes of the AT~ receptor [22,31 ] leaves the possibility open that activation of multiple signal transduction systems occurs through distinct subtypes of the AT 1 receptor. The cloning of the rat vascular AT 1 receptor [25] and the development of AT 1 and AT 2 selective nonpeptidic agents [13,16,41] are powerful tools for the investigation of the mechanism of AT 1 receptor action. The purpose of this study was to express the vascular AT 1 receptor cDNA in CHO cells in order to investigate the coupling of this receptor to intracellular signaling pathways. We demonstrate here that transfected CHO cells expressing the ATI receptor provide a useful model for studies of selective AT 1 receptor antagonists and All-mediated biochemical responses.
Materials and Methods
Cell culture. CHO cells were co-transfected with the vascular AT 1 receptor cDNA (pCDM8/Ba23) [25] and the pSV2 vector which carries the gene for hygromycin resistance by calcium phosphate precipitation. Transfected CHO cells (CHO-ATIa) were grown in Dulbecco's modified Eagle's medium (DMEM) (Gibco, Grand Island, NY) supplemented with 10~o fetal calf serum (FCS) (Gibco), 200 #g/ml hygromycin B (Boehringer-Mannheim, Indianapolis, IN) and 200 #g/ml L-proline (Gibco) at 37°C in a humidified atmosphere containing 5~o CO2. Clonal populations of cells expressing the AT 1receptor were obtained by selection in hygromycin B (200 #g/ml). CHO-ATIa cells were passaged by detachment from the flasks with a solution of 0.25 ~o trypsin containing 1 mM EDTA every 7 days and the medium changed every 3 to 4 days. Rat liver cells (Clone 9 cells) were obtained from ATCC (RockviUe, MD) and cultured in Ham's F-12 medium containing 10 ~o FCS. Medium was renewed every 3-4 days and cells were maintained at 37 oC in a humidified atmosphere containing 5 ~o CO2. Membrane preparation. CHO-ATla cells were
133 harvested by centrifugation at 1000 rpm for 15 min at 4°C. The cell pellet was resuspended in cold phosphate-buffered saline (PB S), re-centrifuged, and resuspended in hypotonic buffer (5 mM Tris-HC1, 5 mM EDTA, pH 7.4). The cells were homogenized for 5-10 s with a Brinkman polytron at setting # 8 and the homogenate centrifuged at 48,000g for 30 min at 4°C. This step was repeated and the final pellet resuspended in 50 mM Tris-HC1 buffer (pH 7.4), containing 100 mM NaC1 and 1 mM MgC12. The membranes were aliquoted and stored at -80 ° C. Rat liver was minced and homogenized in 50 mM Tris-HC1, 1 mM EGTA, 10mM MgCI2, 1 mM PMSF, 0.24 TI units/ml aprotinin, 0.1 mg/ml 1,10phenanthroline with a Brinkman Polytron homogenizer (setting # 7 , 3 x 6 s). The homogenate was passed through two layers of cheesecloth, and centrifuged at 40,000g for 20 min at 4°C. The supernatant was discarded, and the membranes resuspended in buffer and washed three times. The pellet was resuspended in this buffer at a concentration of 10 mg protein/ml. The cell homogenate was stored in 1 ml aliquots at - 8 0 ° C until use. Radioligand binding assays. Membrane proteins (5-10 #g) were incubated with 0.2 nM [125I]SI-AII (2200 Ci/mmol; NEN, Boston, MA) for 1 to 2 h at 25 or 37°C in 5 0 m M Tris-HC1 (pH 7.4), 5 m M MgCI2, 0.24 unit/ml aprotinin, 10#g/ml, 1,10phenanthroline, 1 mM EDTA and 1 mg/ml BSA. Saturation binding was conducted in the absence or presence of 1 # M SI-AII, over increasing concentrations of radioligand. The receptor-ligand complex was filtered through Filtermat B T M (Pharmacia LKB, Gaitherburg, MD) pretreated with 0.3 ~o polyethyleneimine in 50 mM Tris-HC1 (pH 7.4), using a Tomtec T M Multiwell cell harvester (Orange, CT). Saturation binding data was analyzed using nonlinear regression least-square curve fitting to the nontransformed data. Linear transformation of the data was conducted as described [32]. Competition binding data were analyzed by iterative curve fitting to a one site model and inhibition constants (Ki) calculated from ICs0 values [ 11 ].
Phosphoinositide metabolism. CHO-ATla cells were incubated in DMEM/10Yo FCS overnight, the medium removed and replaced with 2 ml of inositol free D M E M , containing 10~o FCS and 4 #Ci/ml of [3H]myoinositol (Amersham, 94Ci/mmol) for 2 days. The supernatant was removed, the cell monolayer washed three times in 2 ml D M E M containing 0.1~o BSA, and the cells incubated in 2 ml of this medium containing 10 mM LiC1 for 10 min. The stimuli were added to the medium in 20 #1 volumes, and the cells incubated at 37°C for 15 min. Supernatants were removed and 2.5 ml of boiling 2 mM EDTA (pH 5-6) was added to the cells. Following cooling, 2 ml of the supernatant was added to a Dowex columns AG-1X8 anion exchange column, and the [3H]inositol, glycerophosphoinositol, and inositol 1-, 2- and 3-phosphates (IP) fractionated [39]. Aliquots were counted in a Packard Cobra liquid scintillation counter using Packard Ultima Gold XR scintillant (Packard Instruments, Meriden, CT). Measurement of intracellular calcium. Intracellular free calcium (Ca 2 ÷ ]i) estimation was performed with the fluorescent calcium indicator dye, fura-2. Cell concentration was adjusted to 1.106 cells/ml for determination of autofluorescence in a SPEX spectrofluorometer. Excitation wavelengths were set at 340 and 380 nm, while emission was monitored at 505 nm. Excitation slits were set at 1.0 mm and emission slits were set at 1.0 and 0.5 mm. Cells were incubated with 2 # M fura-2 acetoxymethyl ester (Molecular Probes, Eugene, OR) for 30 min at 37 ° C. The fura-2 loaded cells were washed once by centrifugation, resuspended in Hepes-buffered saline (HBS) plus 1 mg/ml BSA at 37°C and incubated in HBS for an additional 30 min. The suspension was centrifuged and the cell pellet was suspended in fresh HB S at room temperature. Fluorescence experiments were carried out in the SPEX spectrofluorometer at 37°C in a quartz cuvette. Fura-2 fluorescence was measured at the settings used for autofluorescence determination. [Ca2+]i was calculated using data analysis software developed by SPEX industries,
134 based on the following formula: [ Ca2 + ]i = g d ( R - Rrnin/Rmax - R ) (Sf,2/Sb2) where R is the ratio of the fluorescence of the sample at 340 nm and 380 nm; Rmax and Rmin represent the ratios for fura-2 free acid at the same wavelengths in the presence of saturating calcium and zero calcium, respectively; Sf2/Sb2 is the ratio of fura-2 at 380 nm in zero and saturating calcium; and K d is the dissociation constant of fura-2 for calcium, which was assumed to be 224 nM at 37°C [20]. Measurement of adenylate cyclase activity. Adenylate cyclase activity was assayed in a reaction media (200 #1 total volume) containing 30 mM Tris acetate (pH 7.6), 200 mM LiC1, 10 mM MgC12, 5 mM phosphocreatine, 50 U/ml creatine phosphokinase, 1 mM 3-isobutyl-l-methylxanthine, 0.2 mM adenosine triphosphate (50 cpm/pmol of [~-32p]ATP), with 10 # M guanosine triphosphate. The reaction was initiated with the addition of cell membrane protein (15-40 #g/assay) to temperatureequilibrated reaction tubes. The samples were incubated for 1 h at 37 ° C, and the reactions stopped with 100 #1 of a solution containing 2~o SDS, 45 mM ATP, 1.3 mM cAMP and 2.105 cpm/ml [3H]cAMP (to correct for column recovery). 1 ml of deionized H 2 0 was added, and the entire sample was subjected to chromatography on Dowex A G 50W-X4 and alumina columns [34]. Statistical analysis. Data are expressed as means+ S.E.M. or S.D. Statistical comparisons were made using the Student's t-test. The null hypothesis was rejected at P < 0.05.
sistent with a single class of receptor binding sites (Fig. 1). Specific [125I]SI-AII binding was saturable (Bmax = 1246 + 252 fmol/mg protein, n = 2) and was generally 65-95 Fo of the total binding over the range of [125I]SI-AII concentrations (0.1 to 5 nM). The equilibrium dissociation constant (Kn) for [1=5I]SIAII was 0.7 + 0.1 (n = 2). Specific [125I]SI-AII binding to CHO-ATla membranes was inhibited by angiotensin peptides and the nonpeptidic AT] receptor selective antagonist losartan in a concentration-dependent manner (Fig. 2). The AT= receptor selective agent PD 123,177 was a weak inhibitor of binding. The rank order of potency was S I - A I I > A I I > l o s a r t a n > A I > > P D 123,177. K i values and slope factors are given in Table I. The possibility that vascular-type AT~ receptors could couple to several signal transduction pathways
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was investigated. All (I/aM) stimulated phosphoinositide metabolism as seen by the 3 to 4-fold increase in IP-1 and 2-fold increase in IP-2/IP-3 levd s (Fig. 3). Pre-treatment of CHO-ATI~ cells with 10/aM of losartan blocked the All-induced increase in IP-1 and IP-2/IP-3. P D 123,177 did not affect the All stimulated IP response (data not shown).
TABLE I Inhibition constants (Ki) and slope factors of [ 125I]SI-AII binding to membranes from CHO-AT~ cells
Competitor
Ki (nM)
Slope factor
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0.4 _+0.05* 5.2 + 0.6 38.1 + 9.5 355 +_48.5 > 100,000
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Competition binding of [12sl]SI-AII (0.2nM) to CHO-ATI~ membranes. Membranes, competing compound and radioligand were incubated 2 hours at 25°C as described in Methods. Mean Ki values + S.E.M. or S.D.*, were derived from IC~o values according to Ref. 1I.
Fig. 3. Effect of AII on phosphoinositide hydrolysis. [3H]MyoinositoMabeled CHO'ATla cells were stimulated with 1/~M AII in the absence and presence of 10 #M losartan for 30 min at 37°C. Total inositol phosphates were extracted and quantitated as described in Methods, Results show the mean _+S.E.M. of three similar experiments.
The effects of All on [Ca 2+ ]i in fura-2-1oaded cells were studied. Basal [Ca2+]i averaged 161 + 9 nM (n = 13) in CHO-ATIa cells. Exposure of CHO-ATla cells to All resulted in an increase in [ Ca2 + ]i which was prevented by losartan (1/aM) but not by PD 123,177 (1 #M) (Fig. 4). AII (30 nM) stimulated calcium mobilization to an average 7.8 _+0.5 (n = 5) fold increase above basal levels. In the presence of losartan (1 #M), the All-induced increase in [Ca2÷]i was reduced to 1.6_+0.1 ( n = 3 ) fold over basal levels. The effect of losartan on the All stimulated calcium mobilization was concentration-dependent (data not shown). P D 123,177 (1/aM) did not affect the All-induced calcium response (8.6 _+0.6 fold above basal levels, n = 3). In contrast to the stimulatory effect of All on phospholipase C activity and calcium mobilization, the agonist failed to effect adenylate cyclase activity in the CHO-AT~, cells. Hepatic membranes consistently' demonstrated a 10-30~/o decrease in adenylate cyclase activity in response to 1/aM AII which was completely inhibited by the AT~ receptor antagonist losartan (10/~M) (Fig. 5). Conversely, All did
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Fig. 4. Effect of AII on intracellular calcium concentration ([ Ca2+ ]i). CHO-ATla cells were treated with 30 nM AII in the absence and presence of 1 # M losartan (broken line, upper panel) or PD 123,177 (broken line, lower panel). Results show a representative tracing of three separate experiments.
not inhibit adenylate cyclase activity in CHO-ATla cells.
Discussion
Stable expression of recombinant proteins in CHO cells is a powerful method for determination of the functional properties of the protein of interest. The aim of this study was to examine the binding and signal transduction characteristics of the cloned vascular ATla receptor. To this end, the cloned ATta receptor cDNA was expressed in CHO ceils and the stable cell line, CHO-ATla, was derived.
Fig. 5. Effects AII on adenylate cyclase activity in membranes from CHO-AT~a and rat liver cells. Membranes were incubated with 1 #M All with and without 10 # M losartan (DuP753). All the values were normalized to their respective control (basal) levels which were 11.0 + 1.0 pmol cAMP/mg/min for the CHO-AT:, cells membranes and 12.9 + 0.5 pmol cAMP/mg/min for the rat liver membranes. A statistically significant difference (P<0.01) from control values is denoted by the asterisk. This figure show the means _+S.D. of a single representative experiment and is representative of 3-4 determinations with similar results.
Saturation binding data indicates that the vascular ATla receptor cDNA is highly expressed. Comparison of the Bmax for [125I]SI-AII in CHO-ATIa cells (1.2pmol/mg protein) to that in rat aortic smooth muscle cells (0.9-2.9 pmol/mg protein) demonstrates that the level of expression for the recombinant receptor is similar (Dr. K. Dickinson, unpublished data). Competition binding demonstrates that SI-AII, AII, and losartan bind to the recombinant receptor with high affinity while PD 123,177, the AT 2 receptor-selective ligand, binds only weakly. The K i values and rank order of potency reported here are consistent with ATta receptor pharmacology in rat aortic smooth muscle cells [ 14] and for the vascular clone expressed transiently in Cos7 cells [25]. Thus,
137 the stably expressed recombinant ATla receptor exhibits similar binding characteristics to those previously described for endogenous AT 1 receptors in numerous cell and tissue types. Given the existence of two different subtypes of the AT 1 receptor, termed ATla and ATlb [22], the possibility existed that multiple signal transduction pathways were activated by different subtypes of the receptor rather than multiple coupling of the same receptor. Such has been shown to be the case for the ~-adrenergic receptor where activation of the 0qadrenergic receptor results in an increase in IP and [ Ca2+ ]i while activation of ~2-adrenergic receptor inhibits adenylate cyclase [7,33]. However, several reports indicate that a single receptor can in fact activate multiple effector pathways. The cloned M 2 muscarinic and ~2-adrenergic receptors can mediate stimulation of phosphoinositide metabolism and inhibition of adenylate cyclase activity [ 3,15 ]. In contrast, the cloned thyrotropin receptor stimulates both phosphoinositide metabolism and adenylate cyclase activity when expressed in Cos7 cells [38]. More recently, the recombinant calcitonin receptor was also shown to increase IP and cAMP accumulation in response to calcitonin treatment of HEK-293 cells [8]. The finding that fl?, subunits of Gi can activate adenylate cyclase [ 18] raises the possibility that costimulation of adenylate cyclase may be indirect. The data presented here demonstrate that agonist stimulation of CHO-ATI~ cells expressing the cloned vascular AT~ receptor cDNA results in phosphoinositide hydrolysis and increased intracellular calcium. Both responses were inhibited by losartan, consistent with signal transduction via the AT~ receptor. However, All did not effect CHO-ATla adenylate cyclase activity. All also failed to alter forskolin-stimulated cAMP production in CHOA T ~ cells or influence the conversion of ATP to cAMP in [3H]adenosine-labeled CHO-ATla or rat aortic smooth muscle cells (data not shown). Initially, several conditions were tried to maximize the amount of inhibition of adenylate cyclase by AII. We chose conditions similar to those described by Bauer
and colleagues [4], which produced consistent All inhibition of adenylate cyclase activity in rat liver membranes. We were somewhat surprised to find no inhibition of adenylate cyclase in CHO-ATla cells, especially given that others have observed inhibition of adenylate cyclase in rat heptic tissue [4,29], adrenal glomerulosa cells [42] and rat aorta [2]. More recently, Ohnishi et al. [26] reported that CHO cells stably transfected with the bovine adrenal ATIa cDNA responded to 10/~M AII with a 2 9 ~ decrease in forskolin-stimulated cAMP levels. It is interesting to note that the AII effect on cAMP had not reached a plateau by 10/~M nor was it shown to be losartan sensitive [26]. The high concentrations of AII used raise the possibility that the 1 #M concentration of AII used here is insufficient to effect adenylate cyclase. However, this concentration is greater than 100-fold the K i of AII for the CHO-ATla cell receptor and thus receptor occupancy was greater than 99~o. The Bmax in the C H O - A T l a cells used here (1.2 pmol/mg protein) is consistent with AT 1 receptor density in other cells and tissues so it is unlikely that the lack of AII effect on adenylate cyclase is due to limiting receptor concentration. However, it is possible that the AII effect in liver is mediated by the recently identified ATlb receptor [22] and not by the AT1a receptor or that co-stimulation of adenylate cyclase by the fl~ subunits of Gq has counter-balanced an AII-mediated decrease in cAMP levels. In addition, while the rat vascular and bovine adrenal cDNAs are 9 4 ~ homologous, it has been previously shown that minor differences in amino acid sequence can significantly effect receptor function [36]. The mechanism underlying the information encoded in the molecular structure of the AT 1 receptor which enables it to activate signaling pathways is presently unclear. Evidence from studies with chimeric and mutant seven transmembrane receptors indicates that regions in the second and third intracellular loops as welt as the carboxy-terminal tail are involved in the specificity of coupling to distinct G-proteins [23]. The regions of the AT 1 receptor
138
involved in G-protein coupling remain to be determined. In summary, the evidence provided here demonstrates that AII activation of CHO-ATla receptor stimulates phosphoinositide hydrolysis and increased intracellular calcium. The absence of an effect on adenylate cyclase activity in CHO-AT~a cells suggests that these cells may be useful in identifying critical elements in the receptor required for effecting adenylate cyclase.
Acknowledgement The authors thank Stephen Skwish for technical assistance in preparing rat liver membranes.
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30
31
ation of Ca 2÷ indicators with greatly improved fluorescence properties, J. Biol. Chem., 260 (1985) 3440-3450. Hanesworth, J. and Harding, J., Investigation of the AIV receptor in rabbit heart membranes, Neuroscience, (1991) Abstract # 3709. Kakar, S. S., Sellers, J.C., Devor, D.C., Musgrove, L.C. and Neill, J.D., Angiotensin II type-1 receptor subtype cDNAs: Differential tissue expression and hormonal regulation, Biochem. Biophys. Res. Commun. 183 (1992) 1090-1096. Kobilka, B. K., Kobilka, T. S., Daniel, K., Regan, J. W., Caron, M.G. and Lefkowitz, R.J., Chimeric ~,fl2-adrenergic receptors: delineation of domains involved in effector coupling and ligand binding specificity, Science, 240 (1988) 1310-1316. Lassegue, B., Alexander, R.W., Clark, M. and Griendling, K.K., Angiotensin II-induced phosphatidylcholine hydrolysis in cultured vascular smooth muscle cells. Regulation and localization, Biochem. J., 276 (1991) 19-24. Murphy, T.J., Alexander, R.W., Griendling, K.K., Runge, M. S. and Bernstein, K. E., Isolation of a cDNA encoding the vascular type-1 angiotensin II receptor, Nature, 351 (1991) 223-236. Ohnishi, J., Ishido, M., Shibata, T., Inagami, T., Murakami, K. and Miyazaki, H., The rat angiotensin II AT1A receptor couples with three different signal transduction pathways, Biochem. Biophys. Res. Commun., 186 (1992) 1094-1101. Peach, M.J., Renin-angiotensin system: Biochemistry and mechanism of action, Physiol. Rev., 57 (1977) 313-370. Pfeilschifter, J., Huwiler, A., Merriweather, C. and Briner, V.A., Angiotensin II stimulation of phospholipase D in rat renal mesangial cells is mediated by the AT~ receptor subtype, Eur. J. Pharmacol., 225 (1992) 57-62. Pobiner, B.F., Hewlett, E.L. and Garrison, J.C., Role of N i in coupling angiotensin receptors to inhibition of adenylate cyclase in hepatocytes, J. Biol. Chem., 260 (1985) 1620016209. Sandberg, K., Millan, M.A. and Catt, K.J., Amphibian myocardial angiotensin II receptors are distinct from mammalian AT 1 and AT 2 receptor subtypes, FEBS Lett., 284 (1991) 281284. Sandberg, K., Ji, H., Clark, A.J.L., Shapira, H. and Cart, K.J., Cloning and expression of a novel angiotensin II receptor subtype, J. Biol. Chem., 267 (1992) 9455-9458.
32 Scatchard, G., The attractions of proteins for small molecules and ions, Ann. NY Acad. Sci., 51 (1949) 660-672. 33 Simonds, W.F., Goldsmith, P.K., Codina, J., Unson, C.G. and Spiegel, A.M., Gi2 mediates ~-adrenergic inhibition of adenylate cyclase in platelet membranes: In situ identification with G~ C-terminal antibodies, Proc. Natl. Acad. Sci. USA, 86 (1989) 7809-7813. 34 Salomon, Y., Londos, C. and Rodbell, M., A highly sensitive adenylate cyclase assay, Anal. Biochem., 58 (1974) 541548. 35 Speth, R.C. and Kim, K.H., Discrimination of two angiotensin II receptor subtypes with a selective agonist analog of angiotensin II, p-aminophenylalanine 6 angiotensin II, Biochem. Biophys. Res. Commun., 169 (1990) 997-1006. 36 Strader, C. D., Candelore, M. R., Hill, W. S., Dixon, R. S. and Sigal, I. S., A single amino acid substitution in the fl-adrenergic receptor promotes partial agonist activity from antagonists, J. Biol. Chem., 264 (1989) 16470-16477. 37 Vallotton, M.B., The renin-angiotensin system, Trends Pharmacol. Sci., 8 (1987) 69-74. 38 VanSande, J., Raspe, E., Perret, J., Lejeune, C., Maenhaut, C., Vassart, G. and Dumont, J. E., Thyrotropin activates both the cyclic AMP and the PIP 2 cascades in CHO cells expressing the human cDNA of the TSH receptor, Mol. Cell. Endocrinol., 74 (1990) R1-R6. 39 Webb, M.L., Liu, E.C.K., Cohen, R.B., Hedberg, A., Bogosian, E.A., Monshizadegan, H., Molloy, C., Serafino, R., Moreland, S., Murphy, T.J. and Dickinson, K. E.J., Molecular characterization of angiotensin II type II receptors in rat pheochromocytoma cells, Peptides, 13 (1992) 499-508. 40 Whitebread, S., Mele, M., Kamber, B. and De Gasparo, M., Preliminary biochemical characterization of two angiotensin II receptor subtypes, Biochem. Biophys. Res. Commun., 163 (1989) 284-291. 41 Wong, P.C., Hart, S. D., Zaspel, A. M., Chiu, A.T., Ardecky, R.J., Smith, R.D. and Timmermans, P. B. M.W., Functional studies of nonpeptide angiotensin II receptor subtype-specific ligands: DuP753 (All-l) and PD 123,177 (AII-2), J. Pharmacol. Exp. Ther., 255 (1990) 584-592. 42 Woodcock, E.A. and Johnston, C.I., Inhibition of adenylate cyclase in rat adrenal glomerulosa cells by angiotensin II, Endocrinology, 115 (1984) 337-341.
131
REGPEP 01277
Binding and signal transduction of the cloned vascular angiotensin II (ATla) receptor cDNA stably expressed in Chinese hamster ovary cells Mafia L. W e b b a, Hossain Monshizadegan a, Kenneth E.J. Dickinson a, Randy Serafino b, Suzanne Moreland b, Inge Michel ~, Steven M. Seller ~ and T.J. Murphy c Departments of aBiochemistry and bpharmacology, Bristol-Myers Squibb Pharmaceutical Research Institute, Princeton, NJ (USA) and CDivision of Cardiology, Emory University, Atlanta, GA (USA) (Received 13 November 1992; accepted 11 December 1992)
Key words." SarcosineL[ 125I]tyrosine4-isoleucine8-AII; Intracellular calcium; Phosphoinositide metabolism; Adenylate cyclase; Losartan, PD 123,177
Summary The vascular angiotensin (A) II receptor cDNA (ATla) was transfected into Chinese hamster ovary (CHO) cells to generate the stable cell line CHO-ATIa. This cell line was used to investigate the binding and signal transduction properties of the cloned vascular AT 1 receptor. Specific binding of sarcosineX-[125I]tyrosine4isoleucineS-AII ([125I]SI-AII) to CHO-ATla membranes reached equilibrium after 1 h at 25°C and was consistently greater than 95 ~o of total binding. Saturation binding analyses demonstrated [125I]SI-AII bound to a saturable population of sites on membranes with an equilibrium dissociation constant (KD) of 0.7 nM and a binding site maximum of 1.2 pmol/mg protein. [125I]SI-AII binding to CHO cells was inhibited by the following compounds with a rank order of potency of SI-AII > All > losartan > AI > > PD 123,177. AII (1 #M) treatment of CHO-ATla cells caused an increase in inositol phosphates and intracellular calcium relative to basal levels. These responses were blocked by losartan but not by PD 123,177. AII (1 #M) did not effect adenylate cyclase activity in CHO-ATt~ cells, whereas the agonist inhibited adenylate cyclase activity in rat liver cell membranes. These effects were blocked by 10/~M losartan. These results indicate that CHO-ATla cells express functional ATla receptors which stimulate phospholipase C activity but not adenylate cyclase activity. CHO-ATt~ cells should provide a useful model for studies of ATla receptor domains which are critical to signaling pathways.
Correspondence to." M.L. Webb, Department of Biochemistry, Bristol-Myers Squibb Pharmaceutical Research Institute, P.O. Box 4000, Princeton, NJ 08543-4000, USA.
132
Introduction
Angiotensin (A) II is a vasoactive peptide hormone that has significant effects on cardiovascular, renal, endocrine and neuroendocrine function [27]. All exerts its effects by binding to specific cell-surface receptors in numerous cells and tissues including the adrenal, vascular smooth muscle, kidney, uterus and brain [37]. Two types of All receptors have been identified based on their biochemical and pharmacological properties [40]. Type I (AT1) receptors bind the nonpeptidic antagonist losartan (DuP 753) with high affinity and PD 123,177 with low affinity [10,12,16]. The AT 1 receptor mediates the Allinduced alterations in pressor responses, catecholamine and aldosterone secretion and drinking behavior [41] and therefore has been the focus of much study. By contrast, AT 2 receptors bind DuP 753 with low affinity and PD 123,177 with high affinity [12,13,35]. Bottari et al. [6] have reported that AT 2 receptors mediate an All-induced decrease in cGMP and tyrosine phosphorylation, however, others have not observed these effects [ 17,39]. Thus, the physiological relevance of this receptor is not presently known. In addition, while other receptors for angiotensins may exist [5,9,21,30,31], their identity and role is not clear. While the All receptor family may be more widely heterogeneous than originally thought, the AT 1 receptor remains the primary focus for therapeutic intervention. Several second messenger systems are involved in All activation of AT 1 receptors. In vascular smooth muscle and renal mesangial cells, All stimulates phospholipase C (PLC) catalyzed phosphoinositide turnover and subsequent release of calcium from intracellular stores [1,19] as well as phosphatidylcholine hydrolysis by activation of phospholipase D (PLD) [24,28]. However, in the liver, AT 1 receptors are coupled via G i to adenylate cyclase as well as via Gq to phospholipase C (PLC) [4,29]. Both the Allinduced increase in phosphoinositide metabolism and [Ca 2+ ]i, and the decrease in adenylate cyclase are attenuated by DuP 753 [4]. However, the poten-
tial existence of multiple subtypes of the AT~ receptor [22,31 ] leaves the possibility open that activation of multiple signal transduction systems occurs through distinct subtypes of the AT 1 receptor. The cloning of the rat vascular AT 1 receptor [25] and the development of AT 1 and AT 2 selective nonpeptidic agents [13,16,41] are powerful tools for the investigation of the mechanism of AT 1 receptor action. The purpose of this study was to express the vascular AT 1 receptor cDNA in CHO cells in order to investigate the coupling of this receptor to intracellular signaling pathways. We demonstrate here that transfected CHO cells expressing the ATI receptor provide a useful model for studies of selective AT 1 receptor antagonists and All-mediated biochemical responses.
Materials and Methods
Cell culture. CHO cells were co-transfected with the vascular AT 1 receptor cDNA (pCDM8/Ba23) [25] and the pSV2 vector which carries the gene for hygromycin resistance by calcium phosphate precipitation. Transfected CHO cells (CHO-ATIa) were grown in Dulbecco's modified Eagle's medium (DMEM) (Gibco, Grand Island, NY) supplemented with 10~o fetal calf serum (FCS) (Gibco), 200 #g/ml hygromycin B (Boehringer-Mannheim, Indianapolis, IN) and 200 #g/ml L-proline (Gibco) at 37°C in a humidified atmosphere containing 5~o CO2. Clonal populations of cells expressing the AT 1receptor were obtained by selection in hygromycin B (200 #g/ml). CHO-ATIa cells were passaged by detachment from the flasks with a solution of 0.25 ~o trypsin containing 1 mM EDTA every 7 days and the medium changed every 3 to 4 days. Rat liver cells (Clone 9 cells) were obtained from ATCC (RockviUe, MD) and cultured in Ham's F-12 medium containing 10 ~o FCS. Medium was renewed every 3-4 days and cells were maintained at 37 oC in a humidified atmosphere containing 5 ~o CO2. Membrane preparation. CHO-ATla cells were
133 harvested by centrifugation at 1000 rpm for 15 min at 4°C. The cell pellet was resuspended in cold phosphate-buffered saline (PB S), re-centrifuged, and resuspended in hypotonic buffer (5 mM Tris-HC1, 5 mM EDTA, pH 7.4). The cells were homogenized for 5-10 s with a Brinkman polytron at setting # 8 and the homogenate centrifuged at 48,000g for 30 min at 4°C. This step was repeated and the final pellet resuspended in 50 mM Tris-HC1 buffer (pH 7.4), containing 100 mM NaC1 and 1 mM MgC12. The membranes were aliquoted and stored at -80 ° C. Rat liver was minced and homogenized in 50 mM Tris-HC1, 1 mM EGTA, 10mM MgCI2, 1 mM PMSF, 0.24 TI units/ml aprotinin, 0.1 mg/ml 1,10phenanthroline with a Brinkman Polytron homogenizer (setting # 7 , 3 x 6 s). The homogenate was passed through two layers of cheesecloth, and centrifuged at 40,000g for 20 min at 4°C. The supernatant was discarded, and the membranes resuspended in buffer and washed three times. The pellet was resuspended in this buffer at a concentration of 10 mg protein/ml. The cell homogenate was stored in 1 ml aliquots at - 8 0 ° C until use. Radioligand binding assays. Membrane proteins (5-10 #g) were incubated with 0.2 nM [125I]SI-AII (2200 Ci/mmol; NEN, Boston, MA) for 1 to 2 h at 25 or 37°C in 5 0 m M Tris-HC1 (pH 7.4), 5 m M MgCI2, 0.24 unit/ml aprotinin, 10#g/ml, 1,10phenanthroline, 1 mM EDTA and 1 mg/ml BSA. Saturation binding was conducted in the absence or presence of 1 # M SI-AII, over increasing concentrations of radioligand. The receptor-ligand complex was filtered through Filtermat B T M (Pharmacia LKB, Gaitherburg, MD) pretreated with 0.3 ~o polyethyleneimine in 50 mM Tris-HC1 (pH 7.4), using a Tomtec T M Multiwell cell harvester (Orange, CT). Saturation binding data was analyzed using nonlinear regression least-square curve fitting to the nontransformed data. Linear transformation of the data was conducted as described [32]. Competition binding data were analyzed by iterative curve fitting to a one site model and inhibition constants (Ki) calculated from ICs0 values [ 11 ].
Phosphoinositide metabolism. CHO-ATla cells were incubated in DMEM/10Yo FCS overnight, the medium removed and replaced with 2 ml of inositol free D M E M , containing 10~o FCS and 4 #Ci/ml of [3H]myoinositol (Amersham, 94Ci/mmol) for 2 days. The supernatant was removed, the cell monolayer washed three times in 2 ml D M E M containing 0.1~o BSA, and the cells incubated in 2 ml of this medium containing 10 mM LiC1 for 10 min. The stimuli were added to the medium in 20 #1 volumes, and the cells incubated at 37°C for 15 min. Supernatants were removed and 2.5 ml of boiling 2 mM EDTA (pH 5-6) was added to the cells. Following cooling, 2 ml of the supernatant was added to a Dowex columns AG-1X8 anion exchange column, and the [3H]inositol, glycerophosphoinositol, and inositol 1-, 2- and 3-phosphates (IP) fractionated [39]. Aliquots were counted in a Packard Cobra liquid scintillation counter using Packard Ultima Gold XR scintillant (Packard Instruments, Meriden, CT). Measurement of intracellular calcium. Intracellular free calcium (Ca 2 ÷ ]i) estimation was performed with the fluorescent calcium indicator dye, fura-2. Cell concentration was adjusted to 1.106 cells/ml for determination of autofluorescence in a SPEX spectrofluorometer. Excitation wavelengths were set at 340 and 380 nm, while emission was monitored at 505 nm. Excitation slits were set at 1.0 mm and emission slits were set at 1.0 and 0.5 mm. Cells were incubated with 2 # M fura-2 acetoxymethyl ester (Molecular Probes, Eugene, OR) for 30 min at 37 ° C. The fura-2 loaded cells were washed once by centrifugation, resuspended in Hepes-buffered saline (HBS) plus 1 mg/ml BSA at 37°C and incubated in HBS for an additional 30 min. The suspension was centrifuged and the cell pellet was suspended in fresh HB S at room temperature. Fluorescence experiments were carried out in the SPEX spectrofluorometer at 37°C in a quartz cuvette. Fura-2 fluorescence was measured at the settings used for autofluorescence determination. [Ca2+]i was calculated using data analysis software developed by SPEX industries,
134 based on the following formula: [ Ca2 + ]i = g d ( R - Rrnin/Rmax - R ) (Sf,2/Sb2) where R is the ratio of the fluorescence of the sample at 340 nm and 380 nm; Rmax and Rmin represent the ratios for fura-2 free acid at the same wavelengths in the presence of saturating calcium and zero calcium, respectively; Sf2/Sb2 is the ratio of fura-2 at 380 nm in zero and saturating calcium; and K d is the dissociation constant of fura-2 for calcium, which was assumed to be 224 nM at 37°C [20]. Measurement of adenylate cyclase activity. Adenylate cyclase activity was assayed in a reaction media (200 #1 total volume) containing 30 mM Tris acetate (pH 7.6), 200 mM LiC1, 10 mM MgC12, 5 mM phosphocreatine, 50 U/ml creatine phosphokinase, 1 mM 3-isobutyl-l-methylxanthine, 0.2 mM adenosine triphosphate (50 cpm/pmol of [~-32p]ATP), with 10 # M guanosine triphosphate. The reaction was initiated with the addition of cell membrane protein (15-40 #g/assay) to temperatureequilibrated reaction tubes. The samples were incubated for 1 h at 37 ° C, and the reactions stopped with 100 #1 of a solution containing 2~o SDS, 45 mM ATP, 1.3 mM cAMP and 2.105 cpm/ml [3H]cAMP (to correct for column recovery). 1 ml of deionized H 2 0 was added, and the entire sample was subjected to chromatography on Dowex A G 50W-X4 and alumina columns [34]. Statistical analysis. Data are expressed as means+ S.E.M. or S.D. Statistical comparisons were made using the Student's t-test. The null hypothesis was rejected at P < 0.05.
sistent with a single class of receptor binding sites (Fig. 1). Specific [125I]SI-AII binding was saturable (Bmax = 1246 + 252 fmol/mg protein, n = 2) and was generally 65-95 Fo of the total binding over the range of [125I]SI-AII concentrations (0.1 to 5 nM). The equilibrium dissociation constant (Kn) for [1=5I]SIAII was 0.7 + 0.1 (n = 2). Specific [125I]SI-AII binding to CHO-ATla membranes was inhibited by angiotensin peptides and the nonpeptidic AT] receptor selective antagonist losartan in a concentration-dependent manner (Fig. 2). The AT= receptor selective agent PD 123,177 was a weak inhibitor of binding. The rank order of potency was S I - A I I > A I I > l o s a r t a n > A I > > P D 123,177. K i values and slope factors are given in Table I. The possibility that vascular-type AT~ receptors could couple to several signal transduction pathways
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was investigated. All (I/aM) stimulated phosphoinositide metabolism as seen by the 3 to 4-fold increase in IP-1 and 2-fold increase in IP-2/IP-3 levd s (Fig. 3). Pre-treatment of CHO-ATI~ cells with 10/aM of losartan blocked the All-induced increase in IP-1 and IP-2/IP-3. P D 123,177 did not affect the All stimulated IP response (data not shown).
TABLE I Inhibition constants (Ki) and slope factors of [ 125I]SI-AII binding to membranes from CHO-AT~ cells
Competitor
Ki (nM)
Slope factor
n
SI-AII AII Losartan AI PD 123,177
0.4 _+0.05* 5.2 + 0.6 38.1 + 9.5 355 +_48.5 > 100,000
1.0 + 0.06* 0.8 + 0.03 0.9 + 0.06 1.0 + 0.08 -
2 8 4 3 2
Competition binding of [12sl]SI-AII (0.2nM) to CHO-ATI~ membranes. Membranes, competing compound and radioligand were incubated 2 hours at 25°C as described in Methods. Mean Ki values + S.E.M. or S.D.*, were derived from IC~o values according to Ref. 1I.
Fig. 3. Effect of AII on phosphoinositide hydrolysis. [3H]MyoinositoMabeled CHO'ATla cells were stimulated with 1/~M AII in the absence and presence of 10 #M losartan for 30 min at 37°C. Total inositol phosphates were extracted and quantitated as described in Methods, Results show the mean _+S.E.M. of three similar experiments.
The effects of All on [Ca 2+ ]i in fura-2-1oaded cells were studied. Basal [Ca2+]i averaged 161 + 9 nM (n = 13) in CHO-ATIa cells. Exposure of CHO-ATla cells to All resulted in an increase in [ Ca2 + ]i which was prevented by losartan (1/aM) but not by PD 123,177 (1 #M) (Fig. 4). AII (30 nM) stimulated calcium mobilization to an average 7.8 _+0.5 (n = 5) fold increase above basal levels. In the presence of losartan (1 #M), the All-induced increase in [Ca2÷]i was reduced to 1.6_+0.1 ( n = 3 ) fold over basal levels. The effect of losartan on the All stimulated calcium mobilization was concentration-dependent (data not shown). P D 123,177 (1/aM) did not affect the All-induced calcium response (8.6 _+0.6 fold above basal levels, n = 3). In contrast to the stimulatory effect of All on phospholipase C activity and calcium mobilization, the agonist failed to effect adenylate cyclase activity in the CHO-AT~, cells. Hepatic membranes consistently' demonstrated a 10-30~/o decrease in adenylate cyclase activity in response to 1/aM AII which was completely inhibited by the AT~ receptor antagonist losartan (10/~M) (Fig. 5). Conversely, All did
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Fig. 4. Effect of AII on intracellular calcium concentration ([ Ca2+ ]i). CHO-ATla cells were treated with 30 nM AII in the absence and presence of 1 # M losartan (broken line, upper panel) or PD 123,177 (broken line, lower panel). Results show a representative tracing of three separate experiments.
not inhibit adenylate cyclase activity in CHO-ATla cells.
Discussion
Stable expression of recombinant proteins in CHO cells is a powerful method for determination of the functional properties of the protein of interest. The aim of this study was to examine the binding and signal transduction characteristics of the cloned vascular ATla receptor. To this end, the cloned ATta receptor cDNA was expressed in CHO ceils and the stable cell line, CHO-ATla, was derived.
Fig. 5. Effects AII on adenylate cyclase activity in membranes from CHO-AT~a and rat liver cells. Membranes were incubated with 1 #M All with and without 10 # M losartan (DuP753). All the values were normalized to their respective control (basal) levels which were 11.0 + 1.0 pmol cAMP/mg/min for the CHO-AT:, cells membranes and 12.9 + 0.5 pmol cAMP/mg/min for the rat liver membranes. A statistically significant difference (P<0.01) from control values is denoted by the asterisk. This figure show the means _+S.D. of a single representative experiment and is representative of 3-4 determinations with similar results.
Saturation binding data indicates that the vascular ATla receptor cDNA is highly expressed. Comparison of the Bmax for [125I]SI-AII in CHO-ATIa cells (1.2pmol/mg protein) to that in rat aortic smooth muscle cells (0.9-2.9 pmol/mg protein) demonstrates that the level of expression for the recombinant receptor is similar (Dr. K. Dickinson, unpublished data). Competition binding demonstrates that SI-AII, AII, and losartan bind to the recombinant receptor with high affinity while PD 123,177, the AT 2 receptor-selective ligand, binds only weakly. The K i values and rank order of potency reported here are consistent with ATta receptor pharmacology in rat aortic smooth muscle cells [ 14] and for the vascular clone expressed transiently in Cos7 cells [25]. Thus,
137 the stably expressed recombinant ATla receptor exhibits similar binding characteristics to those previously described for endogenous AT 1 receptors in numerous cell and tissue types. Given the existence of two different subtypes of the AT 1 receptor, termed ATla and ATlb [22], the possibility existed that multiple signal transduction pathways were activated by different subtypes of the receptor rather than multiple coupling of the same receptor. Such has been shown to be the case for the ~-adrenergic receptor where activation of the 0qadrenergic receptor results in an increase in IP and [ Ca2+ ]i while activation of ~2-adrenergic receptor inhibits adenylate cyclase [7,33]. However, several reports indicate that a single receptor can in fact activate multiple effector pathways. The cloned M 2 muscarinic and ~2-adrenergic receptors can mediate stimulation of phosphoinositide metabolism and inhibition of adenylate cyclase activity [ 3,15 ]. In contrast, the cloned thyrotropin receptor stimulates both phosphoinositide metabolism and adenylate cyclase activity when expressed in Cos7 cells [38]. More recently, the recombinant calcitonin receptor was also shown to increase IP and cAMP accumulation in response to calcitonin treatment of HEK-293 cells [8]. The finding that fl?, subunits of Gi can activate adenylate cyclase [ 18] raises the possibility that costimulation of adenylate cyclase may be indirect. The data presented here demonstrate that agonist stimulation of CHO-ATI~ cells expressing the cloned vascular AT~ receptor cDNA results in phosphoinositide hydrolysis and increased intracellular calcium. Both responses were inhibited by losartan, consistent with signal transduction via the AT~ receptor. However, All did not effect CHO-ATla adenylate cyclase activity. All also failed to alter forskolin-stimulated cAMP production in CHOA T ~ cells or influence the conversion of ATP to cAMP in [3H]adenosine-labeled CHO-ATla or rat aortic smooth muscle cells (data not shown). Initially, several conditions were tried to maximize the amount of inhibition of adenylate cyclase by AII. We chose conditions similar to those described by Bauer
and colleagues [4], which produced consistent All inhibition of adenylate cyclase activity in rat liver membranes. We were somewhat surprised to find no inhibition of adenylate cyclase in CHO-ATla cells, especially given that others have observed inhibition of adenylate cyclase in rat heptic tissue [4,29], adrenal glomerulosa cells [42] and rat aorta [2]. More recently, Ohnishi et al. [26] reported that CHO cells stably transfected with the bovine adrenal ATIa cDNA responded to 10/~M AII with a 2 9 ~ decrease in forskolin-stimulated cAMP levels. It is interesting to note that the AII effect on cAMP had not reached a plateau by 10/~M nor was it shown to be losartan sensitive [26]. The high concentrations of AII used raise the possibility that the 1 #M concentration of AII used here is insufficient to effect adenylate cyclase. However, this concentration is greater than 100-fold the K i of AII for the CHO-ATla cell receptor and thus receptor occupancy was greater than 99~o. The Bmax in the C H O - A T l a cells used here (1.2 pmol/mg protein) is consistent with AT 1 receptor density in other cells and tissues so it is unlikely that the lack of AII effect on adenylate cyclase is due to limiting receptor concentration. However, it is possible that the AII effect in liver is mediated by the recently identified ATlb receptor [22] and not by the AT1a receptor or that co-stimulation of adenylate cyclase by the fl~ subunits of Gq has counter-balanced an AII-mediated decrease in cAMP levels. In addition, while the rat vascular and bovine adrenal cDNAs are 9 4 ~ homologous, it has been previously shown that minor differences in amino acid sequence can significantly effect receptor function [36]. The mechanism underlying the information encoded in the molecular structure of the AT 1 receptor which enables it to activate signaling pathways is presently unclear. Evidence from studies with chimeric and mutant seven transmembrane receptors indicates that regions in the second and third intracellular loops as welt as the carboxy-terminal tail are involved in the specificity of coupling to distinct G-proteins [23]. The regions of the AT 1 receptor
138
involved in G-protein coupling remain to be determined. In summary, the evidence provided here demonstrates that AII activation of CHO-ATla receptor stimulates phosphoinositide hydrolysis and increased intracellular calcium. The absence of an effect on adenylate cyclase activity in CHO-AT~a cells suggests that these cells may be useful in identifying critical elements in the receptor required for effecting adenylate cyclase.
Acknowledgement The authors thank Stephen Skwish for technical assistance in preparing rat liver membranes.
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