Brian K. Kobilka, Carolyn MacGregor, Grace P. Irons, Marc G. Caron, and Robert J. Lefkowitz ...... Chirgwin, J. M., Przybyla, A. E., MacDonald, R. J., and Rutter,.
THEJOURNALOF BIOLOGICAL CHEMISTRY
Vol. 264, No. 28,Iame of October 5, pp. 1678616792, I989 Printed in U.S.A.
0 1989by The American Society for Biochemistry and Molecular Biology, Inc
Two Distinct Pathways for CAMP-mediated Down-regulation of the &-Adrenergic Receptor PHOSPHORYLATION OF THE RECEPTOR AND REGULATION OF ITS mRNA LEVEL* (Received for publication, April 7, 1989)
Michel Bouvier$#, Sheila Collinsq, Brian F. O’Dowd, Paul T. Campbell, Antonio de Blasi, Brian K. Kobilka, Carolyn MacGregor, Grace P. Irons, Marc G. Caron, and Robert J. Lefkowitz From the Departments of Medicine, Biochemistry,and Cell Biology, Howard Hughes Medical institute, Duke University Medical Center, Durham, North Carolina 27710 and the $Department of Biochemistry, University of Montreal, Montreal H3C-3J7, Canada
We have studied cyclic AMP-mediatedregulation of the &-adrenergicreceptor (&AR). The effects of cAMP were assessed in Chinese hamster fibroblast (CHW) cells expressing eitherthe wild type human B2AR receptor (CH-B2)or mutated forms of the receptor lacking theconsensus sequences for phosphorylation by the CAMP-dependentprotein kinase. Treatment of the CHBz cells with the cAMP analogue dibutyryl cAMP (BtZcAMP) induces a time-dependent “down-regulation” of the number of B2AR. This down-regulation of the receptors is accompanied bya decline in the steady state level ofBzARmRNA. Moreover, the treatment with Bt2cAMP induces an increasein the phosphorylation level of the membrane-associated &AR. Both the reduction in B2AR mRNA andthe enhanced phosphorylation of the receptor are rapid andprecede the loss of receptor. The down-regulation of &AR induced by BtzcAMPis concentration-dependent and mimicked by the other biologically active cyclic nucleotide analogue, 8-Br-cAMP, by forskolin, and by the phosphodiesterase inhibitor,isobutylmethylxanthine. In the CHW cell lines expressing receptors lacking the putative protein kinase A phosphorylation sites, the BtzcAMP-induced phosphorylation of BzAR is completely abolished. In these cells the down-regulation of bzAR receptor number produced by cAMP is significantly slowed, whereas the reduction in BzARmRNA level is equivalent to that observed in CH-j32 cells. These data indicate that there are at least two pathways by which cAMPmay decrease the number of P2ARs in cells:one involves phosphorylation of the receptor by the CAMP-dependent protein kinase and the other leads to a reduction in steady state bzAR mRNA levels.
The processes by which cellular sensitivity to hormone and neurotransmitter stimulation become attenuated over time are collectively referred to asdesensitization. The underlying mechanisms, which are complex and only incompletely understood, have been extensively investigated for the P-adrenergic stimulation of the adenylyl cyclase system. Desensitization may result from alterations in the properties of the receptor,
* The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Recipient of a scholarship from the Medical Research Council of Canada. 11 To whom correspondence should be addressed.
the guanine nucleotide regulatory protein, or the adenylyl cyclase itself (1). At least three distinct types of regulatory phenomena involving the receptor have been described 1) functional uncoupling of the receptor from the adenylyl cyclase complex, 2) reversible physical sequestration of the receptors away from the cell surface, and 3) “down-regulation” of the totalnumber of receptors. Both CAMP-dependent and -independent pathways have been shown to contribute to the overall cellular mechanisms of desensitization (2). Phosphorylation of the &adrenergic receptor (P,AR)’ by the CAMP-dependent protein kinase, as well as by a CAMP-independent protein kinaseknown as PAR kinase, has been proposed to contribute to theagonist-induced functional uncoupling of the receptor from adenylyl cyclase stimulation (3-6). Two consensus sequences for PKA phosphorylation located on cytoplasmic domains of the PAR are likely candidates for receptor phosphorylation by PKA, whereas phosphorylation byBAR kinase seems to involve primarily the carboxyl-terminal domain (6, 7). Most studies addressingthe molecular basis of PAR desensitization have focused on rapid agonist-promoted changes leading to receptor uncoupling. From this work it has been shown that phosphorylation of the receptor by PKA and PAR kinase plays a crucial role in such regulation (3-6). In contrast, the molecular mechanisms operating over more prolonged time (hours), which serve to ultimately diminish cellular sensitivity by decreasing the total number of cellular receptors, have not been extensively explored. In this regard, the role of cAMP in mediating down-regulation of the PzAR is of particular interest (8-10). In thepresent studies we have directly addressed the role of the second messenger CAMP in mediating feedback control of receptor number. Two distinct pathways for such regulation, involving the phosphorylation of the receptor and regulation of mRNA levels, have been delineated. EXPERIMENTALPROCEDURES
Materiuk-Carrier-free 32Pi,‘251-cyanopindolol(CYP), [1251]iodopindolol, [3H]cAMP, [cY-~’P]ATP, and [w3’P]dCTP were obtained from Du Pont-New England Nuclear. Isoproterenol, alprenolol, ATP, GTP, CAMP, dibutyryl CAMP, 8-bromo-cAMP, forskolin, sodium The abbreviations used are: &AR, &adrenergic receptor; PKA, CAMP-dependentprotein kinase; CYP, cyanopindolol; DMEM, Dulbecco’s modified Eagle’s medium; BtZcAMP, dibutyryl cyclic AMP; BIM, bromoacetylindolyloxyhydroxypropyldiaminomethane; PBS, phosphate-buffered saline; Hepes, 4-(2-hydroxyethyl)-l-piperazineethanesulfonic acid; G., stimulatory guanine nucleotide-binding regulatory protein of adenylyl cyclase.
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CAMP-mediated Down-regulation of the &-Adrenergic Receptor
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Adenylyl Cycluse Assay-Adenylyl cyclase activities were measured fluoride, Sepharose CL-4B, phosphoenolpyruvate, myokinase, and collagenase were purchased from Sigma. Pyruvate kinase and isobu- by the method of Salomon et al. (16). The assay mixture contained tylmethylxanthine were from Calbiochem. Digitonin was purchased 0.02 ml of membrane suspension (-25 pg of protein), 30 mM Tris, 10 from Gaillard Schlessinger. DMEM, fetal calf serum, geniticin mM MgC12,0.8 mM EDTA, 0.12 mM ATP, 1 pCi of [w3'P]ATP, 0.1 mM CAMP, 0.06 mM GTP, 2.8 mM phosphoenolpyruvate, 5.2 pg/ml (G418),penicillin, streptomycin, and amphotericin B were purchased from GIBCO. Bromoacetylindolyloxyhydroxypropyldiaminomethane pyruvate kinase, and 10 pg of myokinase in a final volume of 0.05 ml. (BIM) was a generous gift from Dr. Joseph Pitha (National Institutes Enzyme activities were determined in the presence of (-)-isoproterenol (0-100 p ~ )Reactions . were terminated by the addition of 1 ml of Health). CGP-12177 was generously provided by Ciba Geigy. Site-directed Mutagenesis-Two mutants of the human &AR of 0.4 mM ATP, 0.3 mM CAMP, and [3H]cAMP (-20,000 cpm), and cDNA were designed to generate receptors lacking either one or both CAMP was isolated by sequential chromatography on Dowex cation consensus sequences for phosphorylation by PKA. In thefirst mutant, exchange resin and aluminum oxide. Determinations were performed a sequence of four amino acids (Ser-Lys-Leu-Glu) was inserted be- in triplicate anddata analysis carried out using nonlinear least tween Arg-260 and Ser-261 of the native &AR, disrupting the con- squares regression as described previously (17). Whole CellRadioligand Binding Assay-To measure the total numsensus sequence for phosplnorylation by PKA located in the third cytoplasmic loop. This mutant is termed (PKA-),. In the second ber of cellular &AR, 50 pl of the cell suspension in PBS (2 X lo6 in the presence or mutant, consensus sequences in both the third cytoplasmic loop and cells/ml) were incubated with 200 p~ 1251-pindolol the carboxyl terminus were obliterated by selectively substituting absence of 1.0 p~ (-)-propranolol to define total @-adrenergicrecepserines 261, 262, 345, and 346 with alanines (PKA-12. To generate tor binding. Binding assays were performed in triplicate in DMEM these mutants, the &AR coding sequence was cloned into theEcoRI- buffered with 25 mM Hepes (pH 7.4) in a final volume of 1 ml at Hind111 site of the vector pTZ (Pharmacia LKB Biotechnology Inc.). 13 "C for 3 h. The binding was terminated by rapid filtration over Oligodeoxynucleotideswere annealed and served as primers for syn- Whatman GF/C glass fiber filters. To assess the level of sequestered binding sites which thesis of double-stranded plasmid DNA using a standardmutagenesis receptors, the percentage of specific 1251-pindolol kit (Amersham Corp.). These mutant constructs were verified by could not be displaced by the hydrophilic antagonist CGP-12177 (0.3 p ~ was ) measured before and after treatment with Bt2cAMPor DNA sequencing. Expression of WildType and Mutant Human PAR in Chinese isoproterenol. Whole Cell Phosphorylation Experirnents-Nearly confluent cells Hamster Fibroblasts (CHW-1102)"Wild type human &AR and (PKA-), cDNAs, containing 45 base pairs of 5"untranslated se- were detached from the flasks by treatment with collagenase (1.5 mg/ quence, were cloned into the eukaryotic expression vector pKSVlO ml) containing 0.05 mg/ml soybean trypsin inhibitor for 30 min at (Pharmacia LKB Biotechnology Inc.) as described previously (11). 37 'C. The cells were then washed twice with fresh phosphate-free For (PKA-), cells, the vector pBC12MI was used (12). The resulting DMEM and preincubated with carrier-free 32P1(30 mCi) in 60 ml of constructs were cotransfected with pSVneo into Chinese hamster phosphate-free DMEM a t 37 "C for 60 min. The Bt2cAMP (1 mM) CHW-1102 cells by calcium phosphate precipitation (13), and neo- was then added to the cells and incubated at 37 "C for 10 min, mycin-resistant cells were selected in DMEM 10% fetalcalf serum followed by centrifugation at 200 X g to terminate the reaction, and containing 150 pg/ml G418. Individual clones were then screened for the cells were washed twice with ice-cold PBS. Cells were lysed by &AR expression by radioligand binding assay in membranes using sonication (three bursts for 10 s) in ice-cold 20 mM Tris-HC1, 5 mM 7.4) and Centrifuged at 40,000 X g. The lZ5I-CYPas the ligand. Untransfected CHW cells contain very low EDTA, 10 mM Na2HP04 (pH resulting pellets were washed twice with the same buffer (30 ml). The levels of P2AR (11). Cell Culture-The CHW-1102 (14) and clonal derivative cells were crude membrane preparations obtained were then solubilized in 100 grown as monolayers in 75-cm2 flasks containing DMEM supple- mM NaCl, 10 mM Tris-HC1, 5 mM EDTA, 2% digitonin (pH 7.4) a t mented with 10% fetalcalf serum, penicillin (100 units/ml), strepto- 4 "C for 2 h and the &AR purified by alprenolol-Sepharose affinity mycin (100 pglml), and amphotericin B (0.02 pg/ml) until apparent chromatography as described (18). SDS-Polyacrylamide Gel Electrophoresis-Gel electrophoresis was confluency in an atmosphlere of 75% air, 5% CO, at 37 "C. Cell monolayers at 75% confluency were washed twice with 5 ml of fresh performed according to the method of Laemmli (19) with 10% slab medium. DMEM containing Bt2cAMP (1 mM) or other drugs as gels. Sample buffer consisted of 8%dodecyl sulfate, 10% glycerol, 5% indicated in the figure legends were added (10 ml) to the cells. After 0-mercaptoethanol, 25 mM Tris-HC1, 0.003% bromphenol blue (pH incubation at 37 "C for the indicated periods of time, the treatments 6.5). Equal amounts of receptor were applied to each lane of a gel for were terminated by removal of the media and threerapid washes with a single experiment as monitored by '251-CYP radioligand binding. 10 ml of PBS. The cells were then quickly detached and prepared for After electrophoresis, the gels were dried and autoradiographed at radioligand binding to whole cells or membranes and adenylyl cyclase -90 "C with Kodak XAR-5 film. activity assays. In some experiments a fraction of the cells was used RNA Analysis-Total cellular RNA wasprepared from cells at the to determine the level of &AR mRNA. indicated times following Bt,cAMP treatment by the cesium chloride Membrane Radioligand Binding Assays-Washed cells were resus- gradient method (20). RNA levels for &AR and actin transcriptswere pended in ice-cold 5 mM Tris, 2 mM EDTA (pH 7.4) and homogenized quantitated by slot blot analysis. Blots were prepared by denaturing with a Polytron homogenizer (4 X 5-s bursts at maximum setting). RNA (25 pg for PzAR or 5 pg for actin) in 6.15 M formaldehyde, 10 X The lysates were centrifuged at 200 X g for 10 min at 4 "C. The saline sodium citrate (1X = 0.15 M NaCl, 0.015 M sodium citrate (pH resulting supernatants were centrifuged at 40,000 X g for 30 min at 7.0)) at 65 "C for 15 min and applied to nitrocellulose membranes 4 "C. The pelleted membranes were washed once and resuspended in using aslot blot apparatusas described by the manufacturer 75 mM Tris-HC1, 12.5 mM !klgC12,2 mM EDTA (pH 7.4). Protein was (Schleicher & Schuell). To confirm the specificity of hybridization in measured, following NaOl3 treatment of the membranes, by the slot blots, Northern blots were prepared. Following denaturation in method of Bradford (15) using bovine serum albumin as standard. 1 M glyoxal, 50% (v/v) dimethyl sulfoxide, 10 mM sodium phosphate Radioligand binding assayls were conducted essentially as described (pH 6.8) at 50 "C for 1 h and fractionation by electrophoresis (21), (11)using 0.01 ml of membrane suspension (-12 pg of protein) in a the RNA was immobilized on nylon membranes. Blots were hybridtotal volume of 0.05 ml. Triplicate assay tubes contained 400 PM lZ5I- ized to PAR and actin cDNA probes as previously detailed (22, 23). CYP in the presence and absence of 10 p~ (-)-alprenolo1 to determine To quantitate BzAR and actin mRNA levels, blots were scanned by the specific binding. The binding studies were terminated by rapid laser densitometry. filtration over Whatman GF/C glass fiber filters. Alklylation of the PAR by BZM-Irreversible alkylation of the RESULTS 13,AR was performed by incubating CH-& cells with BIM at concenEffects of Dibutyryl CAMP Treatment on &Adrenergic trations varying from 5 to !jO mM in DMEM supplemented with 10% fetal calf serum or with media alone for 30 min at 37 "C. The Receptor Levels and Adenylyl Cyclase Stimulation-To evaltreatment was terminated by removal of the media and three rapid uate the direct effect of cAMP on PzAR levels, CHW-1102 washes with 10 mlof PBS. Membranes were then prepared as cells expressing the human P2AR (CH-P,) (11) were treated described above, and the number of intact &4R remaining were with the membrane-permeable analogueof CAMP,Bt2cAMP. measured by radioligand binding assay using 1251-cyanopindolol(see above). The isoproterenoLstimulated adenylyl cyclase activity was As shown in Fig. 1, following a 24-h treatment the number of measured (see below) in the same membrane preparation. The extent P2AR measured in membranes derived from CH-6, cells was ofP2AR alkylation and effects on the isoproterenol-stimulated d e - reduced by-50%. Treatment of t h e CH-P2cells with another nylyl cyclase activity could therefore be directly calculated. permeable cAMP analogue 8-Br-CAMP or with agents which
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FIG.1. Effect of treatment with cAMP analogues, isobutylmethylxanthine (ZBMX), and forskolin on membrane &AR number in CH-flZ cells. The number of receptors is expressed as percent of receptor number in untreated cells studied in parallel. The cells were treated with the various drugs or vehicle for 24 h at 37 “C. Drug doses were as follows: Bt2cAMP, 1 mM; 8-Br-cAMP, 1 mM; isobutylmethylxanthine, 0.1 mM; and forskolin, 0.1 mM. The membrane &AR number was determined by radioligand binding using T - C Y P as theligand (see “Experimental Procedures”) and was 1.12 pmol/mg protein in the control conditions. Thedata shown are representative of three separate experiments, and the amplitude of the down-regulation ranged from 50 to 80%.
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FIG.3. Effects of BtzcAMP pretreatment of CH-& cells on membrane isoproterenol-stimulated adenylyl cyclase. CH-& cells were incubated with Bt2cAMP (1 mM) (A) or with the vehicle alone (0)for 24 h at 37 “C; membranes were prepared, and the isoproterenol-stimulated adenylyl cyclase activity was measured as described under “Experimental Procedures.” The adenylyl cyclase activity was expressed as percentof maximal isoproterenol-stimulated activity incontrol cells. Thedata represent the mean of seven experiments. The basal level of adenylyl cyclase activity was 8.6 2 1.0 pmol/min/mg protein, and themaximal isoproterenol-stimulated activity was 47.2 k 11.3 pmol/min/mg protein.
of isoproterenol to stimulate adenylyl cyclase was reflected by a rightward shift of the ECeo following the Bt,cAMP treatment (control: 202 +- 37 nM; n = 7, Bt2cAMP-treated: 974 f 11nM; n = 7). The observation that maximal isoproterenol-stimulated adenylyl cyclase activity was not reduced in spite of a 50-6075 reduction in thenumber of PZARinitially appeared paradoxical. However, we reasoned that the totalpopulation of &AR may not be required for maximal adenylyl cyclase activation by isoproterenol due to thelarge number of receptors in these cells (-1.2 pmol/mg membrane protein). To test thishypothesis CH-B2cells were exposed to various concentrations of the alkylating /3-adrenergic antagonist BIM, to irreversibly alkylate graded proportions of the &AR population. As shown in Fig. 4 no reduction in the maximal isoproterenol stimulat5 -4 1 I -3 tion was observed until -60% of the receptors were alkylated. In fact, when only a small fractionof receptors was alkylated log [dibutyryl - CAMP] M a modest increase in the maximal cyclase activity was obFIG.2. Dose-response curve of the BtzcAMP-induceddownregulation of the BzAR number. CH-& cells were exposed to served, analogous to that seen when receptor reduction was various concentrations of BtZcAMP for 24 h at 37 “C. The membrane caused by Bt2cAMP.However, similar to thedown-regulation &.AR number was determined by radioligand binding using ‘251-CYP induced by BtzcAMP, a rightward shift in the ECso values of as the ligand (see “Experimental Procedures”) and was 1.8 pmol/mg isoproterenol to stimulate adenylyl cyclase was induced by protein in untreatedcontrol cells. The datashown are representative the alkylating agent. of two separate experiments. Role of the PKA-dependent Phosphorylation of BAR in CAMP-induced Down-regulation-To address the potential elevate intracellular cAMP levels such as forskolin and iso- role of CAMP-dependent phosphorylation of the &AR in butylmethylxanthinealso induces down-regulation of the producing receptor down-regulation, mutated forms of the &AR number to the same extent asBtzcAMP (Fig. 1). The receptor were constructed where the putative sites of phoseffect of BtZcAMP on the number of pzAR is concentration- phorylation by PKA were disrupted by site-directed mutagendependent (Fig. 2). To exclude the possibility that thebutyryl esis. These receptors were then expressed in CHW-1102 cells. groups of Bt2cAMP could be responsible for the reduction in In the first approach a sequence of four amino acids (Serreceptor number induced by BtsAMP, cells were incubated Lys-Leu-Glu) was inserted between Arg-260 and Ser-261 of with butyric acid (2 mM) for 6 h. In sharp contrast with the the PzAR (PKA-)I, disrupting the consensus sequence for effects of BtZcAMP, the butyric acid treatment induced a 2- PKA (Arg-Arg-Ser-Ser) found in the third cytoplasmic loop. fold increase, as previously reported (24), and not a reduction This sitewas selected initially since preliminary studies with synthetic peptides indicated that thissequence from the BzAR in receptor number. Somewhat surprisingly the decrease in the &AR number was the most effective substrate for PKA. Indeed, whereas was not associated with a reduction in the maximal isoproter- both peptides Glu-249-Lys-270 and Gln-337-Asn-357, which enol-stimulated adenylyl cyclase (Fig. 3). Rather, a modest encompass the two consensus sequences for PKA phosphorylation found in the&AR, serve as substratesin vitro for increase of the maximally stimulated adenylyl cyclasewas observed. Nevertheless, a significant reduction in the potency PKA, the peptide from the third cytoplasmic loop (Glu-249-
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that observed in CH-b, (Fig. 5). Essentially identical results were obtained in CHW cells expressing a receptor construct in which both putative PKA phosphorylation sites were eliminated by substitution of alanines for serines 261, 262, 344, and 345 (PKA-):! (data notshown). To establish whetherthe CAMP-induced decrease in receptor number is due to true receptor down-regulation or to reversible receptor sequestration, '251-pindolol binding to whole cells was also monitored. Equivalent down-regulation of the total bZARnumber by BtzcAMP was revealed in these whole cell binding experiments (Fig. 5, inset). Itis noteworthy 85 % that when compared with agonist-induced down-regulation 66% the decrease in PzAR number induced by Bt2cAMPwas found 100% to be slower and of lesser magnitude. As seen in Fig. 5 (inset), incubation of cells expressing the wild type b2AR with isopro4 I Yo terenol leads to a faster decrease in whole cell '251-pindolol binding sites than incubation with BtZcAMP. Moreover, following a 24-h treatment, isoproterenol leads to a reduction of -80% of the receptor number compared with 50% for BtzcAMP. An additional difference between BtzcAMP and isoproterenol-induced down-regulation is that the former oc18 % curs without a concomitant sequestration of the receptor away from the cell surface. Treatment with isoproterenol induces a I rapid sequestration of -30% of the total cellular PzAR as measured by the proportion of '251-pindololbinding sites that LOG [ISOPROTERENOL] M cannot be displaced by the hydrophilic antagonist CGP-12177 FIG. 4. Effects of irreversible BzAR alkylation in CH-B2 (see "Experimental Procedures"). In contrast the proportion cells by various concentrations of BIM on isoproterenol-stimulated adenylyl cyclase ;activity. The percentages that appear on of receptors inaccessible to CGP-12177 ( 4 0 % ) remained the right side of the curves indicate the proportion of nonalkylated unchanged throughout the treatment with BtzcAMP (data (intact) PAR present in cells treated with concentrations of BIM not shown). This indicates that receptor sequestration did not varying from 5 to 50 nM. CH-pz cells were exposed to the various accompany the CAMP-mediated down-regulation of the b2AR. concentrations of BIM at 3'7 "C for 30 min; membranes were prepared, As previously observed in several other cell types (26, 27), and the isoproterenol-stimulated adenylyl cyclase was measured as described under "Experimental Procedures." The number of nonal- BtzcAMP induced a 2-fold increase in 32P04incorporation kylated receptors was determined by radioligand binding in mem- into the &AR of CH-b, cells (Fig. 6). In contrast, following branes using "T-CYP as the ligand. The adenylyl cyclase activity is BtzcAMP treatment phosphorylation was not increased over expressed as percent of maximal isoproterenol-stimulated activity in basal levels in either the bzAR receptor (PKA-), (Fig. 6 A ) or untreated cells (0).Thedata shown are representative of three the receptor in which both putative PKA phosphorylation experiments. The total number of receptors in this experiment was 0.7 pmol/mg protein, and the basal level of adenylyl cyclase activity sites were eliminated by substitution of the serines (PKA-)z was 11.8 pmol/min/mg protein, while the maximal isoproterenol- (Fig. 6B). stimulated adenylyl cyclase activity was 38.1 pmol/min/mg protein. PAR mRNA Levels-Down-regulation of the &AR number
Lys-270) has an -100-fold lower K,,, value (13 -+ 5 p M ) for PKA than the peptide from the proximal segment of the cytoplasmic tail (Gln-337-Asn-357) (K,= 947 f 238 mM). These findings are consistent with a previous report (25). The (PKA-), pzARdislplayed a classical &adrenergic pharmacology and mediated normal isoproterenol stimulation of adenylyl cyclase (data not shown). However, the rateof downregulation of the mutated P,AR in response to Bt,cAMP was significantly delayed and reduced in amplitudecompared with
FIG. 5. Effects of BtZcAMP treatment on B2AR number in CH-B2 (0) cells and in cells expressing themutated B2AR (PKA-)l ( 0 ) . The cells were incubated with BtZcAMP (1 mM) for various periods of time, membranes prepared, and receptor number in membranes measured by radioligand binding using lZ5I-CYP.The number of CYP binding sites is expressed as percent of the receptor number in the absence of Bt2cAMP. The datapoints represent the mean f S.E. of 5-15 independent experiments. The absolute number of receptors for control CH-8, cells was 1.48 t 0.19 pmol/mg protein. In the (PKA-), cells total receptor number was 1.33 f 0.15 pmol/mg protein. Inset, effects of BtZcAMP (dbcAMP) (0)or isoproterenol (ZSO) (A)on p2AR number in CHp 2 cells. The number of receptors was determined bywhole cell radioligand binding assay using '251-p~~ndolol as described (see "Experimental Procedures") following incubation for various periods of time with either BtZcAEdP (1 mM) or isoproterenol (2 p ~ ) .
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FIG. 6. Effects of Bt2cAMP treatment on phosphorylation of the B2AR in CH-B2 cells, in cells expressing the mutated receptor (PKA-), ( A ) , and in cellsexpressingthe mutated receptor that lacks both PKA consensus sequences (PKA-)2 ( B ) .Cells pretreated with :’*Pi were exposed to Bt2cAMP (1 mM) or the vehicle alone for 10 min; the receptorwas solubilized and purified by affinity chromatography as described under “Experimental Procedures.” Identicalamounts of receptors as assessed by ’*‘I-CYP binding were applied in each lane of a given panel ( A and B ) . A, autoradiograph of partially purified &AR solubilized from CH-P2wild type ( W T ) and(PKA-), cells treatedornot with Bt2cAMP. J?, autoradiograph of partially purified P2AR solubilized from CH-& and (PKA-), cells treated or not with Bt2cAMP. The data shown are representative of two experiments in eachcase.
induced by Bt2cAMP treatment was also accompanied by a very rapid reduction in the steady state level of the P2AR mRNA (Fig. 7). This effect of Bt2cAMP on theP2AR mRNA levels can be observed as early as 0.5 h after the addition of Bt,cAMP and reached a maximum by -6 h. Thus, the decrease in mRNA level clearly preceded the reduction in the P2AR number and may be at least in part responsible for it. While the rateof CAMP-induced down-regulation of the mutant receptor in (PKA-)Icells was delayedrelative to thewild type, P2AR mRNA levels declined in the same fashion as wild type cells (Fig. 7). Since transcription of the &AR gene in these cell lines is under the control of the SV40 promoter (11) which is relatively insensitive to cAMP (see “Discussion”), rather than the P2ARpromoter, these changes in mRNA levels are presumably duetopost-transcriptionalevents. Fig. 8 demonstrates that when cellular cAMP levels were raised by incubation with forskolin, reductions in both &AR number and mRNA levels were comparable with thoseobserved with Bt2cAMP.
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FIG. 7. Effects of BtzcAMP treatment of CH-B2 cells (0,W) and cells expressingthe mutated B2AR (PKA-), (0,O) on 82AR numberand B2ARmRNA levels. Cells were incubated with BbcAMP (1 mM) for various periods of time a t 37 “C; membranes prepared for &AR number determination and &AR mRNA levels were determined as described under “Experimental Procedures.” The mRNA levels and receptor numberwere determined in the samecells in parallel and expressed as percent of values assessed in untreated cells. The data presented are representative of five different experiments.
gating the regulationof receptor number in various mutants of the S49 lymphoma series and in HC-1 hepatomacells (810). The results of these studies suggested that receptor-(;. coupling, rather than the generationof cAMP and activation DISCUSSION of PKA, was the key determinant of down-regulation. For In the present studywe have shown that cAMP analogues, example, in S49 kin- cells that lack detectable PKA as well as well as agents that elevate intracellular levels of CAMP, as in HC-1 cells that are deficient in the catalytic subunit of can directly induce down-regulation of the P2AR in mammathe adenylyl cyclase, agonist-induced down-regulationof the lian cells. Occupancy of the receptor by an agonist is not P,AR was observed. In the S49 cyc- and UNC cell lines in requiredfor the down-regulation mediated by this second which R-G. coupling cannot occur,down-regulation was messenger. Both an increase in the level of phosphorylation of the receptor anda decrease in its mRNA level precede the blunted. Finally, in H21a cells (a variant of S49) in which RG, coupling is normal butG,-cyclase coupling is not, the rate reduction inreceptornumber. AlthoughCAMP-mediated receptor phosphorylation appears to be involved in thedown- of receptor down-regulation was faster than wild type. Here we present direct evidence that cAMP can function regulationprocess, it is notan obligatoryevent. Thisis alone to induce down-regulation of the B2AR. However, the suggested by results with mutants of the P2AR in which one or both consensus sequencesfor phosphorylation by PKA time course of this down-regulation is much slower than that were disrupted. Incells expressing these altered receptors, theinduced by @-agonists in thesesame cells (Fig. 5, inset). CAMP-mediated increase in phosphorylation of the receptor Therefore, it appears that there are both CAMP-dependent was completely blocked,whereas down-regulationwas slowed and -independent mechanisms that canlead to down-regulation of the &AR. Since the CAMP-dependent mechanism(s) but not abolished. Earlier studies have addressed therole of cAMP and PKA seem to proceed more slowly, the rapid CAMP-independent in agonist-induced down-regulation of the &AR by investi- down-regulation of the receptor that occursuponagonist
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24 0 24 FORSKOLIN INCUBATION TIME (HOURS)
FIG. 8. Effect of forskolin treatment of CH+2 cells on ,&AR and &AR mRNA levels. Cells were incubated with forskolin (0.1 mM) or media alone for 24 h and harvested in parallel for radioligand binding studies using ‘”I-CYP or for determination of &AR mRNA levels by blot hybridization. The data are expressed as percent of values in untreated cells and represent the mean -C S.D. of either three experiments (receptor number) or two experiments (mRNA levels).
occupancy may have previously obscured the role of cAMP in regulating steps involved in receptor biosynthesis (e.g. via changes in PzAR mRNA level). In view of the reported long half-life of the PzAR (>12 h) in the plasma membrane (28, 29), the slower CAMP-dependent mechanism may play an important role in the long term regulation of receptor density. Consistent with this notion is the recent report that isoproterenol treatment induces both down-regulation of PzARnumber and a reduction of steady state pzAR mRNA (30). It should, however,be pointed out that the isoproterenol-induced reduction in receptor number preceded the decrease in PzAR mRNA level. It therefore appears that the early phase of agonist-induced down-regulation is independent of the reduction in mRNA levels. It is of interest to attempt to dissect the roles of CAMPinduced receptor phosphorylation and reduction in receptor mRNA in the down-regulation process. Treatment of CH-& cells with Bt2cAMP promoted a rapid increase in &AR phosphorylation (Fig. 6) which persisted for several hours (data not shown). This phosphorylation induced by BtzcAMP was completely abolished in receptors that were mutated to disrupt one or both PKA phosphorylation consensus sequences. These receptors also displayed a delay in theprocess of CAMPinduced down-regulation. These results would suggest that phosphorylation of the receptor by PKA increases the rateof &AR down-regulation, especially over the first several hours after cAMP exposure. Interestingly, the mutation of the PKA consensus sequence in the third cytoplasmic loop (Ser-261, Ser-262) alone completely abolished BtzcAMP-induced phosphorylation. Indeed, when the two consensus sequences for PKA phosphorylation were mutated by specific substitution of alanines for the serines (261,262,344, and 345), no further effects on receptor phosphorylation or on the rate of downregulation were observed. It therefore appears that ‘‘in UI’UO” the consensus sequence located in the third cytoplasmic loop represents the most favored substrate for PKA. Whether occupancy of the receptor by an agonist, which is also known to increase the rate of phosphorylation of the PzAR in vitro by PKA (3, 4), would favor the phosphorylation of the two serines (344 and 345) in the carboxyl terminus of the receptor remains an open question. It is unlikely that the two mutations constructed confer a nonspecific effect on the down-
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regulation process, since these two mutants arealso effective at eliminating BtzcAMP-induced receptor phosphorylation. One possible way that phosphorylation of the &AR might enhance therate of down-regulation is by enhancing the susceptibility to degradation and thusdecreasing the half-life of the receptor in the membrane. In the case of the photoreceptor system, phosphorylation of rhodopsin has been proposed to induce certain conformational changes that facilitate the action of several proteases (31, 32). After a delay of several hours, the mutant &ARs were down-regulated at a rate which closely paralleled that of the wild type receptors. This finding argues that while receptor phosphorylation is animportant component of the early events of desensitization (l),it cannot be the major factor responsible for mediating CAMP-induced receptor down-regulation at later times. Rather, theCAMP-dependent decrease in steady state levels of pzAR mRNA and, hence, the synthesis of PzAR may be largely responsible for the down-regulation of the receptor observed at these later times. Support for this proposal of long term down-regulation of the &AR induced by agonist is provided by the observations of Neve and Molinoff (28). They found that in L6 myoblasts the major effect of long term (8 h) treatmentwith isoproterenol was a persistent depression in the rate of appearance of the membraneassociated PAR. Regulation of the steady state levels of pzAR density by controlling its rate of synthesis is consistent with the observation that, in many systems, recovery from agonist-induced down-regulation requires new proteinsynthesis (33-35). Moreover, consistentwith our proposal that regulation of PzAR mRNA levels represents a prominent mechanism of BtzcAMP-induced down-regulation at later times, we have found that treatment of CH-& cells with actinomycin D, an RNA polymerase inhibitor, leads to a decrease of’261-CYP binding withkinetics very similar tothat produced by BtzcAMP (data not shown). For another G-protein coupled receptor, the TRH receptor, down-regulation was also shown to be accompanied by a rapid decrease in the level of its mRNA (36). In thiscase, Oron et al. (36) suggested that posttranscriptional regulation of the RNA was responsible in part for the decrease in steady state mRNA levels. For the &AR, the mechanism by which cAMP decreases the P2AR mRNA level is presently unknown. However, an effect on gene transcription is most unlikely because the receptor gene constructs used in thesestudies are under the control of the SV40 promoter and not the original PzAR promoter. Thus, while the SV40 early promoter has been shown in some cells to be modestly up-regulated by cAMP as a consequence of the activation and binding of transcription factor AP-2 (37), we observe a rapid decrease in PzAR mRNA levels. Moreover, in related work with cells expressing PzAR under the control of its own promoter, we have recently discovered that, following short termexposure to agonists and agents that elevate intracellular levels of CAMP, the transcription rate of the pzAR gene is stimulated (38). Elements in thePzAR promoter region that correspond to thecAMP enhancer elementsdescribed in several other genes (39) appear to be responsible for this activity. Comparable cAMP enhancer element sequences are not found in the SV40 promoter. Thus, aneffect of CAMPon post-transcriptional eventssuch as &AR mRNA stabilization must be considered. This idea is supported by the finding that, for a number of other cellular genes, cAMP has effects on both transcription rate and mRNA stability (40-42). We were initially surprised to find that the decrease in PzAR number in CH-PZ cells, which generally reached 50% following a 24-h treatment with BtZcAMP,was not associated
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with a proportionate decrease in maximal isoproterenol stimulation of adenylyl cyclase activity. Rather, the reduction in PzAR density was reflected in decreased potency of isoproterenol to stimulate adenylyl cyclase. Similarly, irreversible alkylation of up to 60% of the BAR did not decrease the maximal isoproterenol-stimulated adenylyl cyclase activity, suggesting that in theCH42 cell type the full complement of P2AR is not necessary to achieve maximal adenylyl cyclase stimulation. Paradoxically, the down-regulation of the receptor number was even associated with a slight increase in the maximal isoproterenol-stimulated adenylyl cyclase activity. Wedo not know the reason for such a phenomenon but suspect that it is related to the high number of receptors presentin the cells used in this study, since in cell lines expressing lower receptor number, Bt2cAMP-induced downregulation affects both thepotency and efficacy of a ,&agonist to stimulate adenylyl cyclase (data notshown). In summary we conclude that two distinct CAMP-dependent mechanisms contribute to the down-regulation of the P2AR. First, CAMP-dependent phosphorylation of the PzAR by PKA increases the rate of down-regulation, especially in the first few hours, presumably by shortening the half-life of the receptors inthe cell membrane. Second, a regulatory action of CAMP, the mechanism of which is not currently understood, leads to a reduction in the steady state level of BzAR mRNA and thuslowers receptor density. Acknowledgments-We are grateful to Oded Herbsman, Mark Leader, and Chantal Persechino for their technical support and to Mary Holben for typing the manuscript. The BIM was kindly provided by Dr. J. Pitha andCGP-12177 was a gift from Ciba Geigy. REFERENCES 1. Benovic, J. L., Bouvier, M., Caron, M. G., and Lefkowitz, R. J. (1988) Annu. Reu. Cell Biol. 4 , 405-428 2. Clark, R. B. (1986) Adv. Cyclic Nucleotide Protein Phosphorylation Res. 2 0 , 155-209 3. Benovic, J. L., Pike, L. J., Cerione, R. A., Staniszewski, C., Yashimasa, T., Codina, J., Caron, M. G., and Lefkowitz, R. J. (1985) J. Biol. Chem. 260,7094-7101 4. Bouvier, M., Leeb-Lundberg, L. M., Benovic, J. L., Caron, M. G., and Lefkowitz, R. J. (1987) J. Biol. Chem. 262,3106-3113 5. Benovic, J. L., Kuhn, H., Weyland, I., Codina, J., Caron, M. G., and Lefkowitz, R. J. (1987) Proc. Natl. Acad. Sci. U. S. A. 8 4 , 8879-8882 6. Bouvier, M., Hausdorff, W. P., DeBlasi, A., O’Down,B. F., Kobilka, B. K., Caron, M. G., and Lefkowitz, R. J. (1988) Nature 333,370-373 7. Dohlman, H. G., Bouvier, M., Benovic, J. L., Caron, M. G., and Lefkowitz, R. J. (1987) J. Biol. Chem. 262,14282-14288 8. Mahan, L.C., Koachman, A. M., and Insel, P. A. (1985) Proc. Natl. Acad. Sci. U. S. A. 8 2 , 129-123 9. Su, Y-F., Harden, T. K., and Perkins, J. P. (1980) J. Biol. Chem. 266.7410-7419 Insel, P. A., Melmon, K.L., and Coffino, P. (1976) J. 10. Shear,”., Biol. Chem. 251,7572-7576
of &-Adrenergic the Receptor 11. Bouvier, M., Hnatowich, M., Collins, S., Kobilka, B. K., DeBlasi, A., Lefkowitz, R. J., and Caron, M. G. (1988). Mol. Phurmacol. 33,133-139 12. Cullen, B. R. (1987) Methods Enzymol. 1 5 2 , 684-704 13. Mellon, P. L., Parker, V., Gluzman, T., and Maniatis, T. (1981) Cell 27,279-288 14. Sheppard, J. R., Wehner, J. M., McSwigan, J. D., and Shous, T. B. (1983) Proc. Natl. Acad. Sci. U. S. A. 80, 233-236 15. Bradford, M. M. (1976) Anal. Biochem. 7 2 , 248-254 16. Salomon, Y.,Londos, C., and Rodbell, M. (1974) Anal. Biochem. 58,541-548 17. De Lean, A., Stadel, J. M., and Lefkowitz, R. J. (1980) J. Bwl. Chem. 255,7108-7117 18. Shorr, R. G. L., Lefkowitz, R. J., and Caron, M. G. (1981) J . Bwl. Chem. 256,5820-5826 19. Laemmli, U. K. (1970) Nature 227, 680-685 20. Chirgwin, J. M., Przybyla, A. E., MacDonald, R. J., and Rutter, W. J. (1979) Biochemistry 1 8 , 5294-5299 21. McMaster, G. K., and Carmichael, G. C. (1977) Proc. Natl. Acad. Sci. U. S. A. 7 4 , 4835-4838 22. Thomas, P. S. (1980) Proc. Natl. Acad. Sci. U. S. A. 77,52015205 23. Collins, S., Caron, M. G., and Lefkowitz, R. J. (1988) J. Biol. Chem. 263,9067-9070 24. Tallman, J. F., Smith, C. C., and Henneberry, R. C. (1977) Proc. Natl. Acad. Sei. U. S. A. 74,873-877 25. Blake, A. D., Mumford, R. A., Stroud, H. U., Slater, E. E., and Strader, C.D. (1987) Biochem. Biophys. Res. Commun. 1 4 7 , 168-173 26. Sibley, D. R., Peters, J. R., Nambi, P., Caron, M. G., and Lefkowitz, R. J. (1984) J. Bwl. Chem. 2 5 9 , 9742-9749 27. Sibley, D. R., Daniel, K. D., Strader, C. D., and Lefkowitz, R. J. (1987) Arch. Biochem. Bwphys. 2 5 0 , 24-27 28. Neve, K. A., and Molinoff, P. B. (1986) Mol. Phurmacol. 30,104111 29. Mahan, L. C., and Insel, P. A. (1986) Mol. Phurmacol. 2 9 , 7-15 30. Hadcock, J. R., and Malbon, C. C. (1988) Proc. Natl. Acad. Sci. U. S. A . 85,8415-8419 31. Pellicone, C., Nullans, G., Cook, N. J., and Virmax, N. (1985) Biochem. Biophys. Res. Commun. 127,816-821 32. Kuhn, H., Mommertz, O., and Hargrave, P. A. (1982) Bwchem. Biophys. Acta 6 7 9 , 9 5 1 0 0 33. Marshema, I., Thompson, W. J., Robinson, G. A., and Strada, J. J. (1980) Mol. Pharmacol. 18,370-378 34. Doss, R.C., Perkins, J. P., and Harden, T.K. (1981) J. Biol. Chem. 256,12281-12286 35. Frederich, R. C., Jr., Waldo, G . L., Harden, T. K., and Perkins, J. P. (1983) J. Cyclic Nucleotide Protein Phosphorylation Res. 9,103-118 36. Oron, Y., Straub, R. E., Traktman, P., and Gershengorn, M. D. (1987) Science 2 3 8 , 1406-1408 37. Inagawa, M., Chiu, R., and Karin, M. (1987) Cell 51,251-260 38. Collins, S., Bouvier, M., Bolanowski, M. A., Caron, M. G., and U. S. A. 86,4853Lefkowitz, R. J. (1989) Proc. Natl. Acad. Sci. 4857 39. Roesler, W. J., Vandenbark, G. R., and Hanson, R. W. (1988) J. Biol. Chem. 263,9063-9066 40. Jungmann, R. A., Kelley, D. C., Miles, M. F., and Milkowski, D. M. (1983) J. Bwl. Chem. 258,5312-5318 41. Hod, Y., and Hanson, R. W. (1988) J. Biol. Chem. 2 6 3 , 77477752 42. Smith, J. D., and Liu, A. Y-C. (1988) EMBO J. 7,3711-3716
