THE JOURNAL OF BIOLOGKAL CHEMISTRY 0 1990 by The American Society for Biochemistry and Molecular Biology, Inc.

Vol. 265, No. 1, Issue of January 5, pp. 63%69,199O Printed in U.S. A.

Multiple Second Messenger Pathways of cu-Adrenergic Receptor Subtypes Expressed in Eukaryotic Cells*

(Received for publication, May 11, 1989)

Susanna Cotecchia, Brian K. Kobilka, Kiefer W. Daniel, Roger D. Nolan*, Eduardo Y. LapetinaS, Marc G. Caron, Robert J. Lefkowitz, and John W. Regang From the Howard Hughes Medical Institute, Departments of Medicine, Biochemistry and Cell Biology, Duke University Medical Center, Durham, North Carolina 27710 and SBurroughs Wellcome Company, Research Triangle Park, North Carolina 27709

The a-adrenergic receptors mediate the effects of epinephrine and norepinephrine on cellular signaling systems via guanine nucleotide binding regulatory pro- teins (G-proteins). Three a-adrenergic receptor sub- types have been cloned: the (Ye, the a2-ClO, and the az- C4 adrenergic receptors. To investigate functional dif- ferences between the different subtypes, we assessed the ability of each to interact with adenylyl cyclase and polyphosphoinositide metabolism by permanently and transiently expressing the DNAs encoding the al, the a&10, and the a&4 adrenergic receptors in cells lacking endogenous a-adrenergic receptors. Both a2- Cl0 and a2-C4 couple primarily to inhibition of ade- nylyl cyclase and to a lesser extent to stimulation of polyphosphoinositide hydrolysis. a2-Cl0 inhibits ade- nylyl cyclase more efficiently than a2-C4. Effects of the a2-adrenergic receptors on adenylyl cyclase inhi- bition and on polyphosphoinositide hydrolysis are both mediated by pertussis toxin-sensitive G-proteins.

The major coupling system of the aI-adrenergic receptor is activation of phospholipase C via a pertussis toxin-insensitive G-protein. aI-Adrenergic receptor stimulation can also increase intracellular CAMP by a mechanism that does not involve direct activation of adenylyl cyclase. As with the muscarinic cholinergic receptor family our results show that each of the a- adrenergic receptor subtypes can couple to multiple signal transduction pathways and suggest several gen- eralities about the effector coupling mechanisms of G- protein-coupled receptors.

The cY-adrenergic receptors are G-protein-coupled’ recep- tors, which together with the /3-adrenergic receptors mediate the physiological effects of epinephrine and norepinephrine.

* The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be-hereby marked “uduertisement” in accordance with 18 U.&C. Section 1734 solely to indicate this fact.

§ Present address: Dept. of Pharmacology and Toxicology, College of Pharmacy, University of Arizona, Tucson, Tucson, AZ 85721.

1 The abbreviations used are: G-protein, guanine nucleotide-bind- ing regulatory protein; az-ClO, the human platelet a*-adrenergic receptor whose gene is on chromosome 10; a2-C4, a human 01~. adrenergic receptor whose gene is on chromosome 4; PI, polyphos- phoinositide; IBMX, isobutylmethylxanthine; PTX, pertussis toxin; “‘I-HEAT, 2-~~-(4-hydroxy-3-[‘25I]iodo-phenyl)ethylaminomethyl~ tetralone; H7,1-(5-isoquinolinylsulfonyl)-2-methylpiperazine; HPLC, high performance liquid chromatography; DMEM, Dulbecco’s modified Eagle’s medium; GTPrS, guanosine 5’-O-(thiotriphos- phate); CHO, Chinese hamster ovary; HEPES, 4-(2-hydroxyethyl)- l-piperazineethanesulfonic acid; EGTA, [ethylenebis(oxyethylene- nitrilo)]tetraacetic acid.

At present, three subtypes of the a-adrenergic receptors are known. Two of these subtypes, the (Ye and the (~~-C10, have been purified from a hamster smooth muscle cell line (1) and from human platelets (2), respectively, and the DNAs encod- ing these receptors have been cloned (3, 4). A third subtype, the c~~-C4, was recently identified by cross-hybridization and was cloned from a human kidney cDNA library using a probe encoding the ~-Cl0 adrenergic receptor (5). Pharmacological characterization of the two recombinant a*-adrenergic recep- tor subtypes following de nouo expression suggests that they correspond to the anA and olzB subtypes as defined previously on the basis of ligand binding (6). Thus, ~u~-C10 and a2-C4 would be equivalent to the olzA and (Y~B, respectively. How- ever, since this is not conclusive and since the o(~-C~ and (Y~B subtypes may be different, we have used the designations of c-~-C10 and a*-C4 in this study.

The (~&lo adrenergic receptor is present in human plate- lets and from pharmacological studies it appears to have a wide distribution, being present in such tissues as brain, kidney, and adipose tissue. In these tissues cuZ-Cl0 receptor stimulation results in the inhibition of adenylyl cyclase (7). With respect to the (rZ-C4 adrenergic receptor subtype, vir- tually nothing is known about its tissue distribution and biochemical signaling mechanism(s). The a,-adrenergic receptor, on the other hand, is known to have a wide tissue distribution, and its activation results in the stimulation of polyphosphoinositide (PI) metabolism (8).

Other receptors whose signal transduction pathways utilize either the inhibition of adenylyl cyclase or stimulation of PI metabolism include the muscarinic cholinergic (9), the sero- tonin (lo), the angiotensin (11, 12), and vasopressin (11) receptors. Until recently it was generally assumed that a given receptor subtype was faithfully coupled either to inhibition of adenylyl cyclase or to PI metabolism activation. In cases where both responses were observed, for example with cholin- ergic stimulation, it was thought to be a consequence of receptor heterogeneity. The cloning and expression of mus- carinic receptor subtypes has shown, however, that a single receptor subtype can couple to both adenylyl cyclase inhibi- tion and PI metabolism activation, even though one of the responses is predominant (13). This finding is significant since it suggests that the ultimate cellular response may depend upon these bifurcating signaling pathways.

Given the growing diversity of the cY-adrenergic receptor family, the present study was performed to assess and com- pare the biochemical second messenger systems of the (Ye, the CQ-ClO, and the (u2-C4 receptors. In particular we investigated the ability of each a-adrenergic subtype to interact both with adenylyl cyclase and PI metabolism. This has been accom- plished by separately expressing recombinant DNA encoding the CQ, the ocZ-ClO, and the a*-C4 adrenergic receptor subtypes

63

by guest on July 12, 2018http://w

ww

.jbc.org/D

ownloaded from

64 a-Adrenergic Receptor Subtypes

in cells lacking endogenous a-adrenergic receptor activity. Using transient and stable expression systems, it was found that both the a*-C4 and ~-Cl0 adrenergic receptor subtypes could couple to the inhibition of adenylyl cyclase as well as to PI metabolism activation. The oll-adrenergic receptor on the other hand had a predominant effect on PI metabolism, but it could also increase intracellular CAMP concentrations.

EXPERIMENTAL PROCEDURES

Materials

Drugs and reagents were obtained from the following sources: epinephrine, norepinephrine, propranolol, IBMX, oxymetazoline, in- domethacin, nordihydroguaiaretic acid, 8-bromo-CAMP sphingosine (Sigma); phentolamine (CIBA-Geigy); prazosin (Pfizer); forskolin, H7 (Calbiochem); pertussis toxin (List); DMEM (Gibco); ‘H-rauwol- seine, “‘I-HEAT, and myo-]3H]inositol (DuPont-New England Nu- clear); restriction enzymes (Promega).

Methods

Expression of Recombinant DNAs Encoding a-Adrenergic Recep- tors-stable expression of a&10 and az-C4 was obtained using the inducible mammalian expression vector pMAM-neo (Clontech, Palo Alto, CA). pSPa+ZlO and pSPa&4 (5) were cut with EcoRI, were blunt-ended with Klenow, and were then cleaved with SalI. The EcoRI-Sal1 fragments were isolated and were ligated to pMAM-neo which had been cut previously with NheI, blunt-ended, and then cut with SalI. Chinese hamster lung fibroblasts (strain PS120, gift of J. Pouyssegur, Universite de Nice) (14) were transfected using Ca’+- phosphate precipitation (15), and G418-resistant clones were selected and examined for their ability to bind the a*-selective antagonist, [3H]rauwolscine. Two clones, PC10 and PC4, which expressed the tip- Cl0 and a&4 adrenergic receptor subtypes, respectively, were se- lected. Transient expression of olZ-Cl0 and 01~-C4 in COS-7 cells was accomplished as described previously (5).

Transient expression of the al-adrenergic receptor was obtained with a vector derived from pBC12-BI (15). pBC12-BI was cut with Hind111 and BamHI and was blunt-ended with Klenow. The 3.99- kilobase fragment was isolated and was ligated to a P-kilobase cDNA encoding the a,-adrenergic receptor (3) to yield pBC&AR.

Cell Culture-Cells were grown in DMEM supplemented with 10% fetal calf serum, penicillin (100 units/ml), and streptomycin (100 rg/ ml) in 5% COZ at 37 “C. PC cells were plated in 30-mm dishes and harvested when confluency was reached (1 x lo6 cells/dish). Since the pMAM-neo vector contains a glucocorticoid-inducible promoter, PC cells were treated with dexamethasone (lo-’ M) 15-18 h before harvesting. This treatment resulted in 4-5-fold increases in receptor expression. COS-7 cells were plated at 0.5 x lo6 cells in 30-mm dishes and were harvested 72 h after transfection.

Ligand Binding-Membrane preparation and ligand binding using “‘I-HEAT for orI-adrenergic receptors (3) and [3H]rauwolscine for a*-adrenergic receptors (5) were performed as described using 10-a M prazosin and 1O-5 M phentolamine to determine nonspecific binding, respectively. Binding data were analyzed by computer using nonlinear curve fitting procedures (16).

CAMP and Inositol Phosphate Determination-CAMP was meas- ured by radioimmunoassay according to the manufacturers instruc- tions (DuPont-New England Nuclear). Briefly, attached cells were incubated for 30 min at 37 “C in serum-free DMEM containing 20 mM HEPES, pH 7.5, and for an additional 10 min in the same media containing 250 PM IBMX. After a lo-min incubation with different drugs at 37 “C, the supernatant was aspirated and 100 mM HCl was added. Aliquots were taken and assayed for their CAMP content.

For inositol phosphate measurement, cells were labeled with [3H] inositol in DMEM supplemented with 5% fetal calf serum for 15-18 h before harvesting. After labeling, the cells were washed for 30 min in PBS (no calcium) and were preincubated for an additional 30 min in PBS containing 0.02 M LiCl. Propranolol (10m4 M) was included in the final incubation to block endogenous p-adrenergic receptors pres- ent in the cells. The agonists were dissolved in ascorbate which was present at lo-’ M in the final incubation. Inositol phosphates were extracted as described by Martin (17) and separated on Dowex AG 1-X8 columns (18) or by HPLC as previously described (19).

Adenylyl Cyckzse Assay-PC cells, grown in 75-cm* flasks, were rinsed with phosphate-buffered saline and were scraped into 5 ml of the same buffer. After centrifugation (200 X g for 10 min) they were

resuspended in 5 ml of 5 mM Tris (pH 7.4), 5 mM EDTA. After a lo- min incubation on ice, a homogenate was prepared with a Brinkman homogenizer (model PT 10/35; three times for 10 s; setting 4). After centrifuging (200 X g for 10 min) the pellet containing the nuclear fraction was discarded and the supernatant was centrifuged (40,000 X g for 10 min). The pellet was resuspended in 50 mM sodium-Hepes (pH 8), 2 mM EDTA and was washed twice in the same buffer. Membranes were resuspended at a concentration of 5-10 mg of protein/ml prior to storage at -80 “C. Adenylyl cyclase activity was measured as described by Katada et al. (20). Membranes (5-10 pg of protein) were incubated in 50-pl assays containing 20 mM sodium- Hepes (pH 8), 0.8 mM EDTA, 1.6 mM MgCl, 100 mM NaCl, 0.12 mM ATP, 0.05 mM GTP, 0.1 mM CAMP, 2.7 mM phosphoenolpyruvate, 0.05 III/ml myokinase, 0.01 III/ml pyruvate kinase, [w~*P]ATP (2.5 X lo6 cpm/tube), and other reagents as indicated in the text. In some experiments NaCl was omitted and MgCl was 1 mM in the incubation. Incubation was for 15 min at 37 “C.

COS-7 cell membranes were prepared as described above for the PC cells. Adenylyl cyclase activity was measured as described above in assays containing 20 mM Tris-HCl (pH 7.4), 0.4 mM EDTA, 4 mM MgCl, 0.12 mM ATP, 0.05 mM GTP, 0.1 mM CAMP, 2.7 mM phos- phoenolypyruvate, 0.05 IU/ml myokinase, 0.01 III/ml pyruvate ki- nase, [w~‘P]ATP (2.5 X lo6 cpm/tube), and other reagents as indi- cated in the text. Incubation was for 30 min at 37 “C. Adenylyl cyclase reactions were terminated by addition of 1 ml of 0.4 mM ATP, 0.3 mM CAMP, and [3H]cAMP (20,000 cpm) [w~*P]cAMP was isolated as described previously (21).

RESULTS

Stable Expression of cYz-Adrenergic Receptor Subtypes-Two clonal cell lines, PC10 and PC4, which stably expressed the 01~-C10 and (u&4 adrenergic receptors, respectively, were characterized by ligand binding. The original, wild type, Chinese hamster lung fibroblasts lacked endogenous a*-ad- renergic receptor binding activity. After transfection, how- ever, saturation analysis of the binding of the cup-adrenergic antagonist [3H]rauwolscine to PC10 and PC4 membranes gave receptor densities of 377 and 534 fmol/mg protein, respectively (data not shown). The affinity of [3H]rauwolscine was higher for az-C4 (& = 1.2 X lo-’ M) than for cuZ-Cl0 (KD = 4.2 X lo-’ M) and is consistent with previous results showing that rauwolscine has higher affinity for the a&4 subtypes (5). These cuz-receptor subtypes were also distinguished in competition studies using [3H]rauwolscine. Fig. lA shows that the al-selective antagonist, prazosin, had approximately 50- fold higher affinity for CQ-C4 as compared with oz-ClO. On

I (A)

-log [Prazosin] (Ml -log [Oxymstaroline] (Ml

FIG. 1. Competition for the binding of [3H]rauwolscine by prazosin (A) and by oxymetazoline (B) in membranes pre- pared from PC4 cells (circles) and PC10 cells (squares). The PC4 and PC10 cell lines were obtained following transfection of Chinese hamster tibroblasts (PS 120) with DNA encoding the (u2-C4 and a&10 adrenergic receptor subtypes, respectively. The final concentrations of [3H]rauwolscine were 7.7 and 2.1 nM for assays conducted with the PC10 and PC4 membranes, respectively. 100% equals 200 pM for PC10 membranes and 160 pM for PC4 membranes. K, values in PC10 and PC4 cells, respectively, were 4.6 and 0.1 NM for prazosin and 0.12 and 3.6 nM for oxymetazoline. The results are representative of three experiments.

by guest on July 12, 2018http://w

ww

.jbc.org/D

ownloaded from

a-Adrenergic Receptor Subtypes 65

the other hand, Fig. 1B shows that the a-adrenergic agonist oxymetazoline had approximately 30-fold lower affinity for a&4 as compared with an-ClO. Again these results are con- sistent with previous data obtained following transient expression of these receptors in COS-7 cells (5) and confirm the stable expression of the two a*-receptor subtypes in the PC cells.

Effects of aI- and a2-Adrenergic Receptor Subtypes on CAMP Metabolism-CAMP levels were measured following agonist stimulation of PC4 and PC10 cells to see if the a2-C4 and CY~- Cl0 adrenergic receptor subtypes could mediate a response through the inhibition of adenylyl cyclase activity. IBMX (250 I.IM) was present in the assay to minimize changes in CAMP levels due to possible effects on phosphodiesterase activity. Fig. 2 shows that in the presence of 50 PM forskolin, intracellular CAMP levels were increased approximately 40- fold in both the PC4 and PC10 cell lines. This increase in CAMP formation was near-maximal both with respect to time and to the concentration of forskolin (data not shown). In the presence of 10 ELM epinephrine, however, CAMP concentra- tions resulting from forskolin stimulation of adenylyl cyclase were decreased 65 and 56% in the PC4 and PClO, cells, respectively. This decrease in CAMP could be blocked com- pletely by 10 FM rauwolscine and did not occur in the wild type cell line PS120 (data not shown). Dose-response curves for the inhibition of forskolin-stimulated CAMP formation by epinephrine yielded similar ECso values (300 nM) for both the PC4 and PC10 cell lines (data not shown).

To check whether or not these decreases in CAMP levels were due to changes in the efflux of CAMP from the cell, intra- and extracellular CAMP was measured in the presence and absence of epinephrine (10 FM) either under basal con- ditions or in the presence of forskolin (50 PM). Under basal conditions and in the absence of epinephrine, the distribution of CAMP was 30% intracellular and 70% extracellular. Neither the concentration nor the distribution of CAMP changed in the presence of epinephrine. When forskolin alone was pres- ent, the proportion of the total CAMP content that was intracellular increased to 60%. In the presence of forskolin and epinephrine, CAMP concentrations decreased but the distribution of CAMP was still 60% intracellular and 40% extracellular.

The ability of epinephrine to inhibit prostaglandin E1- stimulated CAMP formation was tested to see if activation of

I200

T n BAS

I 000

PC10 PC4

+ EPI

FIG. 2. Epinephrine inhibition of CAMP formation in PC10 and PC4 cells. Cells (3 X lO”/dish) were incubated for 10 min at 37 “C with either vehicle @AS). 50 YM forskolin (RX). or 50 KM forskolin plus 10 fiM epinephrine iFSi( + EPI). The’figurb’represehts the mean f S.E. of eight experiments each performed in triplicate.

a&4 and an-C!10 could inhibit CAMP formation caused by an endogenous receptor as opposed to forskolin. In the pres- ence of prostaglandin E1 alone (10 pM for 10 min) CAMP concentrations increased 281 + 79 and 329 f 26% in PC10 and PC4 cells, respectively (mean f S.E. of three experi- ments). In the additional presence of epinephrine (10 wM), however, CAMP concentrations were decreased 40 + 3% in PC10 cells and 32 + 6% in PC4 cells, relative to prostaglandin E1 alone (mean + S.E. of three experiments).

Adenylyl cyclase activity was measured in PC10 and PC4 cell membranes to determine if decreases in CAMP caused by the activation of a&IO and as-C4 were due to inhibition of the enzyme. As shown in Fig. 3A, epinephrine inhibited basal adenylyl cyclase activity by a maximum of 68% in PC10 membranes but only by 27% in PC4 membranes. The EC60 of epinephrine was approximately 5 PM for both the PC10 and PC4 membranes. The differential effect on the maximal in- hibition appears to be due to differences in the coupling efficiency of the receptors rather than to differences in the sensitivity of the enzyme between the two clonal cell lines. Thus, GTPyS and thrombin inhibited basal adenylyl cyclase to the same extent in both cell membranes (Fig. 3B). Fur- thermore, Western blot analysis with antibodies specific for Giz and Gi3 (22) showed that the PC10 and PC4 cells has similar amounts of these G-proteins.’ The inhibition of ade- nylyl cyclase by epinephrine was not dependent on NaCl and was also obtained at low magnesium concentrations (Cl mM; data not shown). The greater inhibition of adenylyl cyclase by activation of CQ-C~O, as compared with az-C4, was also observed after stimulation of adenylyl cyclase by forskolin. Thus, in PC10 and PC4 cell membranes treated with forskolin (50 uM), epinephrine inhibited adenylyl cyclase activity by 35 f 1 and 13 + l%, respectively (mean f S.E. of three experi- ments).

Previous reports have indicated that al-adrenergic receptor stimulation can increase CAMP concentrations in some tis- sues (23-25). To examine this possibility, CAMP was meas- ured following stimulation of ai-adrenergic receptors by nor- epinephrine. Fig. 4 shows that following transient expression of al-adrenergic receptors in COS-7 cells, norepinephrine caused a significant increase in intracellular CAMP accumu- lation. This effect was blocked by 10 pM prazosin and was absent in the untransfected COS-7 cells. The EC& of norepi- nephrine for increasing CAMP in COS-7 cells was 500 nM

(data not shown). To assess whether or not these increases in CAMP were the

result of a direct effect on the enzyme, adenylyl cyclase activity was measured in COS-7 cell membranes expressing al-adrenergic receptors. As a control, stimulation of endoge- nous P-adrenergic receptors with isoproterenol (10 PM for 30 min) led to a 67 f 12% increase in cyclase activity (mean + S.E. of three experiments). However, no increase in adenylyl cyclase activity was observed following al-adrenergic receptor stimulation.

Previous studies with rat pinealocytes have shown that CQ- adrenergic receptor activation potentiates P-receptor stimu- lation of CAMP formation and that this effect is mediated by protein kinase C (25). H7 and sphingosine, which have been shown to inhibit protein kinase C in some cell systems (26), were used to examine the possible role of protein kinase C in the al-adrenergic receptor-mediated CAMP increase in COS- 7 cells. The presence of H7 (50 PM) or sphingosine (50 pM) decreased the a,-adrenergic receptor-mediated CAMP in- crease by 60 and 50%, respectively. The presence of these agents, however, did not modify the stimulation of adenylyl

’ S. E. Senogles, personal communication.

by guest on July 12, 2018http://w

ww

.jbc.org/D

ownloaded from

66

(A)

80

a-Adrenergic Receptor Subtypes

(0)

? q GTPyS q THR

-log [EPINEPHRINE] (MI

FIG. 3. Inhibition of adenylyl cyclase activity in PC10 and PC4 cell membranes. A, PC10 (triangles) and PC4 (circles) cell membranes were incubated with different concentrations of epinephrine for 15 min at 37 “C. The results are the mean + S.E. of three experiments performed in triplicate. Receptor density measured by the binding of [3H]rauwolscine (20 nM) was 630 + 100 and 950 + 150 fmol/mg protein in PC10 and PC4 cell membranes, respectively (mean f SE. of three experiments). B, PC10 and PC4 cell membranes were incubated for 15 min at 37 “C with vehicle (BAS), 100 pM epinephrine (EPZ), 1 FM GTPrS, and 5 NIH units/ml thrombin (Z’HR). The results are the mean + SE. of three experiments performed in triplicate. * p < 0.05; ** p < 0.01.

22 120

g 100 \ z 80

4 60

$ 40

- 20

COS7-a,

FIG. 4. a,-Adrenergic receptor-induced increase in CAMP concentrations in COS-7 cells. COS-7 (1 x lo6 cells/dish) cells were transfected with the plasmid pBCti,-AR as described under “Methods.” 2 pg of plasmid DNA were used/O.5 X lo6 cells. The (Ye- adrenergic receptor density was 3 pmol/mg protein as measured by the binding of iZ51-HEAT (500 PM). Membrane preparations from 5 x lo6 cells yielded 1 mg of protein. Cells were incubated with either vehicle (BAS), 10 PM norepinephrine (NE), or 10 PM norepinephrine plus 10 pM prazosin (NE + PRA) for 10 min at 37 “C. 100 pM propranolol was included in the final incubations to block native fi- adrenergic receptors which are present in small numbers in COS-7 cells. Results are the mean f S.E. of three experiments performed in triplicate.

cyelase by p-adrenergic receptors or the stimulation of PI hydrolysis by al-receptors (data not shown).

Indomethacin and nordihydroguaiaretic acid are potent in- hibitors of arachidonic acid metabolism and their effects on al-adrenergic receptor mediated CAMP formation were ex- amined. Treatment of COS-7 cells with either indomethacin (50 FM) or nordihydroguaiaretic acid (50 jtM) did not modify the increase in CAMP caused by a,-adrenergic receptor acti- vation. This is in agreement with previous studies (27) that exclude a role for arachidonic acid metabolites in the (Ye- adrenergic receptor effect on CAMP formation.

a-Adrenergic Receptor-induced Changes in Polyphosphoi- nositide Metabolism-Recent studies have shown that stimu- lation of M2 and M3 muscarinic receptors leads both to inhibition of adenylyl cyclase and activation of PI metabolism (13). To see if this might also occur with other classes of G- protein-coupled receptors, a*-Cl0 and a2-C4 were examined with respect to inositol phosphate release in PC10 and PC4 cells labeled with [3H]inositol. Stimulation of PC10 and PC4

(A)

DNA (micrograms/O.5 million cells) Receptors (pmol/mg protein)

FIG. 5. Expression of a-adrenergic receptor subtypes in COS-7 cells (A) and their stimulation of PI metabolism by epinephrine (B). COS-7 cells were transfected with increasing amounts of recombinant plasmid DNA encoding either the cq (O), cu&lO (W), or OI,-C4 (A) adrenergic receptors. Receptor concentra- tions were determined by ligand binding using final concentrations of 500 pM “‘I-HEAT for al-adrenergic receptor and 10 nM [3H] rauwolscine for the cuz-adrenergic receptors. For the inositol phos- phates determination, cells were prelabeled for 15-18 h with 5-10 &i/ml [3H]inositol and were stimulated with 100 pM epinephrine for 30 min. The results represent measurements obtained from two to four experiments performed in triplicate.

cells with 10m4 M epinephrine for 30 min resulted in a small but significant increase of inositol phosphates production over basal (PC10 mean f S.E.: 23.5 f 2.5%, n = 4, P C 0.01; PC4 mean + S.E.: 32 + 7.6%) n = 4, P < 0.05). This effect was not observed in untransfected cells and it was blocked completely by 10 PM rauwolscine in both cell lines (data not shown). The EC& of epinephrine for activation of PI metabolism was 5 x 10m6 M for both the PC16 and PC4 cells.

To further characterize the coupling of az-Cl0 and a&4 to PI metabolism, the effects of oc2-adrenergic receptor acti- vation on inositol phosphate release were compared with that elicited by the ai-adrenergic receptor. Intracellular variation was minimized by transient expression of a,-ClO, at-C4, and a,-adrenergic receptors in COS-7 cells. Fig. 5A shows that up to a point, the expression of cuZ-ClO, a2-C4, and ai-adrenergic receptors was increased as the amount of DNA used to trans- feet the cells was increased. The expression of all three

by guest on July 12, 2018http://w

ww

.jbc.org/D

ownloaded from

a-Adrenergic Receptor Subtypes 67

receptors peaked at plasmid DNA concentrations ranging from 2 to 4 pg/O.5 million cells and there were intrinsic differences in the maximal level of expression reached by each receptor subtype. With respect to inositol phosphate metab- olism, Fig. 5B shows that ai-adrenergic receptor stimulation by epinephrine gave a large increase of total inositol phos- phates which was highly dependent on the number of (pi- adrenergic receptors expressed. Epinephrine stimulation of an-C10 and c~-C4 also increased the release of inositol phos- phates, but it was much less dependent on receptor number.

The coupling of the cYz-adrenergic receptor subtypes to PI metabolism is less efficient as compared with the al-adrener- gic receptor. This is indicated by the fact that the ocr-receptor induced release of inositol phosphates was only a fraction of the cYi-receptor-mediated response at each comparable level of expressed receptors, as shown in Fig. 5B. At the maximal level of expression of the ai-adrenergic receptor there was a 295 + 54% (n = 4) increase of inositol phosphates over basal in response to epinephrine. By contrast, at maximal expres- sion of (~*-C10 and 01~-C4 the increase of inositol phosphates was 58 + 9 and 40 + 5%, respectively (n = 4). These epineph- rine-stimulated increases in inositol phosphates were com- pletely blocked by the appropriate antagonists and were not observed when COS-7 cells were transfected with DNA en- coding the ,0-adrenergic receptor (data not shown). The EC& of epinephrine for stimulating PI metabolism through CQ- adrenergic receptors was approximately 250 nM and was com- parable to the EC& for increasing CAMP levels.

Kinetic analysis of inositol phosphate isomers indicates that inositol phosphate release in response to a-receptor stimulation is due to the activation of phospholipase C. Fig. 6 shows that within 0.5 min inositol 1,4,5-phosphate which is the primary product of phospholipase C-catalyzed phospha- tidylinositol bisphosphate hydrolysis, increased as a result of epinephrine stimulation of (pi, az-ClO, and a*-C4. Inositol1,4- phosphate was also a major product after 0.5 min of epineph- rine stimulation. A small increase of inositol 1,3,4,5-phos- phate was also detected following both 0.5 and 5 min stimu- lation with epinephrine. After 5 min inositol 1,4,5phosphate decreased to basal levels while inositol 1,3,4-phosphate in-

creased, probably as a result of the metabolism of inositol 1,3,4,5-phosphate. The pattern of accumulation of inositol phosphate isomers after epinephrine stimulation is very sim- ilar among the three oc-adrenergic receptors, except for the larger accumulation of inositol 1,4-phosphate in the case of the cui-adrenergic receptor. cul-Adrenergic receptor induced inositol 1,4-phosphate formation might be due to direct hy- drolysis of phosphatidylinositol monophosphate via phospho- lipase C.

To exclude the possible interference of LiCl on the (Y- adrenergic receptor induced activation of PI metabolism, ino- sitol phosphates formation was measured in the presence and absence of LiCl. The three ol-adrenergic receptor subtypes were expressed transiently in COS-7 cells and, after a 0.5-min exposure to epinephrine, the levels of inositol phosphates were found to be unaffected by pretreatment with LiCl. After a 5-min exposure, however, the levels of inositol mono-, bis-, and trisphosphate were increased by 200, 300, and lOO%, respectively, in cells preincubated with LiCl.

The possibility that the stimulation of PI metabolism was secondary to other effects of ae-adrenergic receptor activation was assessed. To show that decreases in CAMP were not involved, epinephrine-stimulated PI release was examined in the presence of a CAMP analog in COS-7 cells transfected with az-Cl0 and a~-C4. Thus, 100 PM 8-bromo-CAMP did not modify the release of inositol phosphates induced by a 30-min stimulation with 100 pM epinephrine. Stimulation of PI re- lease may also occur indirectly as a consequence of raising intracellular Ca’+, either by increasing influx (28, 29), or by releasing intracellular stores via an arachidonate pathway (30). To exclude these mechanisms, the effects of the Ca*+ chelator, EGTA, and of the arachidonic acid metabolism inhibitors, indomethacin and nordihydroguaiaretic acid, were examined in COS-7 cells expressing either a&J4 or a*-ClO. The presence of either 2 mM EGTA, 50 pM indomethacin, or 50 PM nordihydroguaiaretic acid did not modify inositol phos- phates release resulting from a 30-min stimulation with epi- nephrine.

Pertussis Toxin Effects on Functional Responses Mediated by a-Adrenergic Receptors-Pertussis toxin was used to char-

aeCIO I

a$4

TIME (mid

FIG. 6. Time course of inositol phosphates accumulation stimulated by epinephrine via a-adrenergic receptor subtypes expressed in COS-7 cells. Transfections were done using 2 +g of recombinant plasmid DNA encoding either the oil, ol.&lO, or (~&4 adrenergic receptors per 0.5 X lo6 cells. The receptor density was 4, 12, and 3 pmol/mg protein for ~lr, ~y&lO, and (UZ-C4, respectively. The yield of membrane protein from 5 x lo6 cells was 1 mg. Cells (106) were labeled with 50 &i/ml of [3H]inositol for 15-18 h and were stimulated with 100 pM epinephrine for the indicated times. Inositol phosphates were isolated by HPLC as previously described (19). cpm represent the total radioactivity contained in each HPLC fraction after epinephrine stimulation and after subtraction of the radioactivity present in unstimulated cells for each condition. These results are representative of three experiments. ZP,,,, inositol1,4-phosphate; ZP.,, inositol4-phosphate; ZP 1,3.4, inositol 1,3,&phosphate; ZP1,4,5, inositol 1,4,5-phosphate; ZP1,3,4,5, inositol 1,3,4,5-phosphate.

by guest on July 12, 2018http://w

ww

.jbc.org/D

ownloaded from

68 a-Adrenergic Receptor Subtypes

acterize the possible G-protein(s) involved in the coupling of as-Cl0 and c&4 to adenylyl cyclase inhibition and activation of PI metabolism. Fig. 7A shows that PTX abolished, in a dose-dependent fashion, the inhibition of adenylyl cyclase by epinephrine activation of a&4 and (~~-C10. The effect of PTX was maximal after treatment with 1 rig/ml for 15 h. Treatment of PC10 cells with higher doses of PTX lead to a small but significant increase in CAMP formation following epinephrine stimulation in the presence of forskolin. This increase in CAMP concentration is similar to previous results showing an increase in CAMP formation by an az-adrenergic mechanism (31).

Fig. 7B shows that PTX also abolished the az-adrenergic receptor mediated stimulation of PI metabolism in cells stably expressing az-C4 and az-CIO. The concentrations of PTX needed to block PI metabolism were comparable to those needed to block the inhibition of adenylyl cyclase.

The effect of PTX on PI metabolism in COS-7 cells was studied to compare the coupling of the cu-adrenergic receptor subtypes to the PI pathway in the same cell system. In COS- 7 cells PTX (100 rig/ml for 15 h) was able to block ~y*-C10 and (r2-C4 stimulated PI metabolism. On the other hand, PTX doses as high as 1000 rig/ml did not modify al-adrener- gic-mediated inositol phosphate release (data not shown).

DISCUSSION

By utilizing transient and stable expression of recombinant DNAs encoding the cY-adrenergic receptor subtypes, we ex- amined their ability to interact with the major signal trans- duction pathways involving CAMP and phosphatidylinositol metabolism. For the recently discovered cYz-adrenergic recep- tor subtype, az-C4, it was determined that its primary effect was to decrease intracellular CAMP concentrations. The same finding was obtained for the (~&lo. In addition, we found unexpectedly that both a*-adrenergic receptor subtypes, (YZ- Cl0 and (~&4, had a modest effect to stimulate phosphati- dylinositol metabolism. Both effects were mediated by pertus- sis toxin-sensitive G-proteins. The major coupling of the LYE- adrenergic receptor as deduced from transient expression was to the phospholipase C pathway via a pertussis toxin-insen- sitive G-protein. al-Adrenergic receptor stimulation also in- creased intracellular CAMP.

Interestingly, while no quantitative difference was observed between a*-Cl0 and az-C4 with respect to decreasing CAMP in whole cells, in isolated membranes ocz-Cl0 showed a 2-fold greater inhibition of adenylyl cyclase than did CQ-C4. This difference was not the result of differences either in receptor density or in affinity for epinephrine between these receptors as expressed in the PC4 and PC10 cell lines. It appears, therefore, that the coupling of cuz-Cl0 to adenylyl cyclase is more efficient than (Y~-C~ which may reflect differences in the molecular interactions of these receptors with inhibitory G- proteins.

Reasons why the greater coupling efficiency of (YZ-C~O to the inhibition of adenylyl cyclase in membranes were not also observed in the whole cell CAMP measurements are not obvious, but similar observations have been made before with other hormone receptors. For example, platelet-activating factor and vasopressin do not decrease CAMP in intact human platelets; however, they do inhibit adenylyl cyclase in isolated membranes and adenylyl cyclase is PTX-sensitive (32). These results attest to the fact that the outcome of receptor G- protein interactions in whole cells are influenced by biochem- ical events other than those which occur in isolated mem- branes.

Previous studies have shown that activation of native 012-

receptors can potentiate forskolin-stimulated CAMP produc- tion in intact HT29 cells (31). Recently, it was shown in CHO cells, transfected with (~*-C10, that exposure to epinephrine concentrations up to 100 nM inhibits forskolin-stimulated CAMP accumulation, while concentrations greater than 1 PM potentiate the accumulation of CAMP (33). PTX blocked this inhibition of CAMP accumulation but it enhanced the poten- tiation of CAMP production. We did not observe such a dual effect on CAMP formation in intact cells by either cYz-receptor subtype, nor, did we observe such an effect on adenylyl cyclase activity in isolated membranes. However, after PTX treat- ment of PC10 cells, a significant 20% increase of forskolin- stimulated CAMP accumulation was observed (Fig. 7A) fol- lowing activation of the az-Cl0 receptors by epinephrine. This finding corresponds with the results obtained in the CHO cells (33); however, it is interesting that we did not observe a similar effect with the PC4 cells which express the CQ-C~ receptor subtype. This may be related to the number of receptors being expressed. In the CHO cells there was a clear correlation between the receptor-mediated potentiation of forskolin-stimulated CAMP accumulation and increased num- bers of receptors (33). In our studies, however, the expression of (Y*-C~ and (~&lo were similar, although somewhat lower than the expression of (~&lo in CHO cells. This further indicates that the interactions of at-C4 and az-Cl0 with G- proteins and adenylyl cyclase are different.

The effects of the Lu2-adrenergic receptors on the adenylyl cyclase and PI signaling pathways appear to be direct and are mediated by interactions with G-proteins. For example, the stimulation of PI metabolism is demonstrably independent of CAMP levels, arachidonic acid metabolism, and extracellular Ca2+ levels. Of note is a recent report demonstrating that CQ- receptor stimulation increases intracellular Ca*+ via a CAMP- independent mechanism in cultured human erythroleukemia cells (34). These workers, however, were unable to detect increases in PI turnover after as-adrenergic receptor stimu- lation. This may be related to receptor density in that the number of crz-adrenergic receptors in HEL cells is s-fold lower as compared with our transfected cells.

An important issue is whether the linkage of the cYz-adre-

"0 60 c

(A) (B>

- 0.1 I 10

xz- PC4 PC10 PC4

FIG. 7. Pertussis toxin (PTX) effects on the inhibition of adenslsl cvclase (A) and on the stimulation of PI metabolism (B) by-~&adrene$c receptor subtypes. PC10 and PC4 cells stably expressing the ocn-Cl0 and a&24 receptor subtypes were treated for 15 h with either 0.1, 1, or 10 ng of PTX/ml of media. For CAMP determinations, cells were incubated for 10 min at 37 “C in the presence of 50 NM forskolin and 10 jcM epinephrine. For inositol phosphate deter.minations, cells were labeled with 10 pCi/ml [3H] inositol for 15 h and were stimulated with 100 /.LM epinephrine for 30 min. The data are expressed as a percentage of the forskolin (FSK) stimulated CAMP or as a percentage of the basal inositol phosphates release which was determined separately for each concentration of PTX examined. The results are the mean _+ S.E. of three experiments performed in triplicate. *p < 0.01 by paired f-test as compared with untreated cells.

by guest on July 12, 2018http://w

ww

.jbc.org/D

ownloaded from

a-Adrenergic Receptor Subtypes 69

nergic receptors to the two signaling pathways is mediated by the same or different G-proteins. Unfortunately our data do not permit a definitive assessment. On the one hand a single member of the Gi family may be involved since the pertussis toxin sensitivity of the two processes is identical. Alterna- tively, two different G-proteins with similar sensitivity to PTX could be involved.

In contrast to the coupling of the cuz-adrenergic receptors with these second messenger systems, the coupling of al- receptors to increased CAMP production is indirect. The most likely mechanism would be secondary to activation of the phospholipase C system. Thus, increases in diacylglycerol and intracellular Ca*+ might lead to activation of adenylyl cyclase by protein kinase C (35) or by calmodulin-dependent mecha- nisms (36), respectively. This hypothesis is consistent with the evidence that the ECso of norepinephrine was the same for increasing PI release as it was for raising intracellular CAMP. Previous studies have suggested that protein kinase C plays a role in the ol,-adrenergic receptor-mediated stimula- tion of CAMP formation in rat pinealocytes (25). Our results showing that the potential protein kinase C inhibitors H7 and sphingosine, partially block increases in CAMP caused by the activation of al-receptors are consistent with this hypothesis.

The biochemical mechanisms involved in coupling the (Ye- and az-adrenergic receptors to phosphatidylinositol metabo- lism are different. Thus, stimulation of PI metabolism by the LYE- and a2-adrenergic receptors was clearly mediated by G- proteins which were respectively insensitive and sensitive to PTX. These results agree with the idea that several G-pro- teins, with varying sensitivities to PTX, can activate phos- pholipase C (37,38).

The pattern of biochemical events produced by the stimu- lation of a-receptor subtypes is similar to that reported for the muscarinic receptor subtypes (13,38). Both receptor fam- ilies can be divided in two groups. The a,-adrenergic, together with the Ml and M4 cholinergic receptors, are coupled pri- marily to phospholipase C by a G-protein that is resistant to PTX. The a2-C10 and (Y*-C~ adrenergic together with the M2 and M3 cholinergic receptors are coupled primarily to the inhibition of adenylyl cyclase and to a lesser extent to phos- pholipase C. These latter effects are both mediated by a PTX- sensitive G-protein. In addition, the recently characterized 5HT-1A receptor can also inhibit adenylyl cyclase and can stimulate PI metabolism (39). This heterogeneity of receptor- effector interactions could be explained by at least two mech- anisms. First, each receptor might couple to a unique G- protein which then interacts with multiple effector systems. Second, each receptor might couple to a variety of G-proteins which are then faithfully coupled to a single effector system. Moreover, the pattern of coupling of a single receptor molecule might be different from cell to cell depending on the tissue content of G-proteins and/or effector systems and on the regulatory mechanisms of their interactions.

Acknowledgments-We gratefully acknowledge Sabrina Taylor for her expert assistance in tissue culture and Donna Addison for her skillful help in the preparation of the manuscript. We wish to thank Drs. Toshi Kurose and Annick Fargin for helpful discussions and Dr. Mark R. Hnatowich for his advice in adenylyl cyclase experiments.

REFERENCES

1. Lomasney, J. W., Leeb-Lundberg, L. M. F., Cotecchia, S., Regan, J. W., DeBernadis, J. F., Caron, M. G., and Lefkowitz, R. J. (1986) J. Biol. Chem. 261, 7710-7716

2. Regan, J. W., Nakata, H., DeMarinis, R. M., Caron, M. G., and

Lefkowitz, R. J. (1986) J. Biol. Chem. 261, 3894-3900 3. Cotecchia, S., Schwinn, D. A., Randall, R. R., Lefkowitz, R. J.,

Caron, M. G., and Kobilka, B. K. (1988) Proc. Natl. Acad. Sci. U. S. A. 85.7159-7163

4. Kobilka, B. K., Matsui, H., Kobilka, T. S., Yang-Feng, T. L., Francke, II., Caron, M. G., Lefkowitz, R. J., and Regan, J. W. (1987) Science 238,650-656

5. Regan, J. W., Kobilka, T. S., Yang-Feng, T. L., Caron, M. G., Lefkowitz, R. J., and Kobilka, B. K. (1988) Proc. N&l. Acad. Sci. U. S. A. 85, 6301-6305

6. Bvlund, D. B. (1985) Pharmacol. Biochem. B&au. 22, 835-843 7. Limbird, L. E., and Sweat& J. D. (1986) in The Receptors (Conn,

M.. ed) Vol. II. DD. 281-305. Academic Press. Orlando. FL 8. Berridge; M. J. (1‘984) Biochek J. 220, 345-360 9. Nathanson, N. A. (1987) Reu. Neurosci. 10,195-236

10. Hover, D. (1989) in Peripheral Actions of 5-HZ’ (Fozard. J. R., ed) pp. 72-99, Oxford University Press,‘New York

11. Charest. R.. Prnic. V.. Exton. J. H.. and Blackmore. P. F. (1985) Biochkm.‘J. i2i, 76-90 ’ ’

12. Pobiner, B. F., Hewlett, E. L., and Garrison, J. C. (1985) J. Biol. Chmz. 260,16200-16209

13. Peralta, E. G., Ashkenazi, A., Winslow, J. W., Ramachandran, J., and Capon, D. J. (1988) Nature 334,434-437

14. Pouyssegur, J., Franchi, A., L’Allemain, G., Magnaldo, I., Paris, S.: and Sardet, C. (1987) Kidney Znt. 32;Suppl. 23, i44-149

15. Cullen. B. R. (1987) Methods Enzvmol. 152.684-704 16. DeLean, A., Hancock, A. A., and Lefkowitz, R. J. (1982) Mol.

Phurmacol. 2 1,5-16 17. Martin, T. F. J. (1983) J. Biol. Chem. 258, 14816-14822 18. Berridge, M. T., Dawson, R. M. C., Dowries, C. P., Hyslop, J. P.,

and Irvine, R. F. (1983) Biochem. J. 212.473-482 19. Vedia, L. M. Y., Nolan, R: D., and Lapetina,E. G. (1988) Biochem.

J. 256, 795-800 20. Katada, T., Bokoch, G. M., Northrup, J. K., Ui, M., and Gilman,

A. G. (1984) J. Biol. Chem. 259, 3568-3577 21. Salomon, Y., Londos, C., and Rodbell, M. (1974) Anal. Biochem.

58,541-548 22. Goldsmith, P., Rossiter, K., Carter, A., Simonds, W., Unson, C.

G., Vinitskv, R., and Spiegel. A. M. (19881 J. Biol. Chem. 264. 6476-6479-. - -

23. Davis, J. N., Arnett, C. D., Hoyler, E., Stalvey, L. P., Daly, J. W., and Skolnick, P. (1978) Brain Res. 159, 125-135

24. Garcia-Sainz, J. A., and Hernandez-Sotomayor, S. M. T. (1985) Proc. Natl. Acad. Sci. U. S. A. 82, 6727-6730

25. Sugden, D., Vanecek, J., Klein, D. C., Thomas, T. P., and Ander- son, W. B. (1985) Nature 314.359-361

26. Lambeth, J. D., Burnham, D. N.; and Tyagi, S. R. (1988) J. Biol. Chem. 263,3818-3822

27. Ho, A. K., and Klein, D. C. (1987) J. Biol. Chem. 262, 11764- 11770

28. Timmermans, P. B. M. W. M., Chiu, A. T., and Thoolen, J. J. M. C. (1987) Can. J. Physiol. Phurmucol. 65,1649-1657

29. Johnson, P. C., Ware, J. A., and Salzman, E. W. (1985) Z’hromb. Res. 40,435-443

30. Sweatt, J. D., Blair, I. A., Cragoe, E. J., and Limbird, L. E. (1986) J. Biol. Chem. 261,8660-8666

31. Jones, S. B., Toews, M. L., Turner, J. T., and Bylund, D. B. (1987) Proc. Natl. Acad. Sci. U. S. A. 84, 1294-1298

32. Brass, L. F., Woolkalis. M. J., and Manning. D. R. (1988) J. Biol. Chem. 283,5348-5$55

Yl

33. Fraser, C. M., Arakawa, S., McCombie, W. R., and Venter, J. C. (1989) J. Biol. Chem. 264, 11754-11761

34. Michel, M. C., Brass, L. F., Williams, A., Bokoch, G. M., LaMorte, V. J., and Motulsky, H. J. (1989) J. Biol. Chem. 264. 4986- 4991

35. Yoshimasa, T., Sibley, D. R., Bouvier, M., Lefkowitz, R. J., and Caron, M. G. (1987) 327, 67-70

36. Salter, R. S., Krinks, M. H., Klee, C. B., and Neer, E. J. (1981) J. Biol. Chem. 256,9830-9833

37. Casey, P. J., and Gilman, A. G. (1988) J. Biot. Chem. 263,2577- 2580

38. Ashkenazi, A., Peralta, E. G., Winslow, J. W., Ramachandran, J., and Capon, D. J. (1989) Cell 56, 487-493

39. Fargin, A., Raymond, J. R., Regan, J. W., Cotecchia, S., Lefkow- itz, R. J., and Caron, M. G. (1989) J. Biol. Chem. 264, 14848- 14852

by guest on July 12, 2018http://w

ww

.jbc.org/D

ownloaded from

Lefkowitz and J W ReganS Cotecchia, B K Kobilka, K W Daniel, R D Nolan, E Y Lapetina, M G Caron, R J

expressed in eukaryotic cells.Multiple second messenger pathways of alpha-adrenergic receptor subtypes

1990, 265:63-69.J. Biol. Chem. 

  http://www.jbc.org/content/265/1/63Access the most updated version of this article at

 Alerts:

  When a correction for this article is posted• 

When this article is cited• 

to choose from all of JBC's e-mail alertsClick here

  http://www.jbc.org/content/265/1/63.full.html#ref-list-1

This article cites 0 references, 0 of which can be accessed free at

by guest on July 12, 2018http://w

ww

.jbc.org/D

ownloaded from