Vol. 268, No. 5, Issue of February 15, pp. 3201-3208, 1993 Printed in U.S.A.

THEJOURNALOF BIOLOGICAL CHEMISTRY 0 1993 by The American Society for Biochemistry and Molecular Biology, Inc.

Overexpression of ,&Arrestin and ,&Adrenergic Receptor Kinase Augment Desensitization of P2-Adrenergic Receptors* (Received for publication, May 5, 1992)

Susanne Pippigs, SabineAndexingerS, Kiefer Daniel§, Mechthild PuzichaS, MarcG . CaronB, Robert J. LefkowitzQ, and Martin J. LohseS From the $Laboratory of Molecular Biology, University of Munich, Max-Planck-Institute of Biochemistry, 8033 Martinsried, Federal Republic of Germany and the §Howard Hughes Medical Institute, Duke University Medical Center, Durham, NorthCarolina 27710

Receptor-specific or homologous desensitization of whereasheterologous desensitization is amoregeneralized &-adrenergic receptors is thought to be effected via process affecting a variety of receptors. The alterations of phosphorylation of the receptor by the &adrenergic receptor responsiveness appear tobe mediated by a whole set receptor kinase (BARK), followed by binding of B-ar- of mechanisms occurring over time scales ranging from secrestin. We have generated stably transfected Chinese onds to days (reviewed by Hausdorff et al. (1990) and Lohse hamster ovary cell lines overexpressing either of the (1992a)). Slow mechanisms involve alterations of receptor two regulatory proteins and also expressing low or synthesis and probably also degradation of the receptor prohigh levels of &-adrenergic receptors (-80 and -600 tein (reviewed by Hadcock and Malbon (1991) and Collins et fmol/mg of membrane protein). In these cells, we stud-al. (1991)). Rapid mechanismsinclude sequestration of recepied the process of desensitization induced by the Badrenergic receptor agonist isoproterenol. Incells ex- tors away from the cellsurface and functional uncoupling pressing high levelsof &adrenergic receptors, desen- between receptors andG, triggered by phosphorylation of the sitization to high concentrations of isoproterenol (pre- receptors. Such receptor phosphorylation can be mediated either by viously shown to be mediated by bothBARK and prothe CAMP-dependent protein kinase, protein kinase A (Betein kinaseA) amounted to-50% in controlcells, -80% in BARK-overexpressing cells,and -90% in B-arrestin- novic et d . , 1985; Clark et al., 1988; Hausdorff et d., 1989; overexpressing cells. In cells expressing low levels of Lohse et al., 1990b), or by the specific P-adrenergic receptor &adrenergic receptors, these values were -50, -60, kinase, PARK (Benovic et al., 1989; Hausdorff et al., 1989; and -60%,respectively. Desensitization to low concen- Lohse et al., 1990b). It is thought that protein kinase Atrations of isoproterenol (previously shown to be essenmediated phosphorylation leads to heterologous desensitizatially protein kinaseA-mediated and not receptor-spe- tion, whereas homologous desensitizationis triggered by cific, i.e. heterologous) was not affected by overexPARK (Clark et al., 1988; Hausdorff et al., 1989; Lohse et al., pression of either BARK or B-arrestin.These data 1990b). PARK is a member of the family of receptor-specific suggest that in cells expressing high levelsof &-adre- kinases that also includes rhodopsin kinase (Lorenz et al., nergic receptors, B-arrestin and BARK become limiting 1991) and a second receptor kinase, termedPARK-2, of as yet for homologous receptor desensitization. They provide unclear receptorspecificity(Benovic et al., 1991). These further support for the involvementof these two prokinases are unique in their substrate specificity as they recteins in the regulation of &adrenergic receptor funcognize only the active conformations of Gprotein-coupled tion. receptors. This dependence on receptor activation may be the basis of the receptor-specific pattern of homologous desensitization. While the kinases are apparently not specific for a single type of receptor, PARK clearly prefers P-adrenergic When receptors in intact cells or tissues are exposed to receptors over rhodopsin, and rhodopsin is the preferential agonists, there is often a rapid decline in responsiveness. This substrate for rhodopsin kinase (Benovic et al., 1986a, 1989; process is called receptor desensitization. It has been particLorenz et al., 1991). ularly well studied for the @-adrenergicreceptor/(;, I/adenyThese kinases are thought to act in concert with another lylcyclase system, a receptor system that responds to P-recepset of cytosolic proteins, the “arresting” proteins. Two such or isoproterenol with the tor agonists such as epinephrine generation of CAMP (reviewed by Levitzki (1988) and Dohl- proteins have been identified the rhodopsin-specific retinal protein arrestin (Wilden et al., 1986; Shinohara et al., 1987) man et al. (1991)). Two forms of desensitization are often distinguished on a descriptive basis: homologous desensitiza- and p-arrestin, aubiquitous protein that, just likePARK, prefers 0-adrenergic receptors over rhodopsin (Lohse et al., tion involves only those receptors that had been activated, 1990a, 1992). Phosphorylation of the receptors by their specific kinases seems to enhance the ability of the arresting *This study was supported by grants from theDeutscheForreceptors, the which schungsgemeinschaft, Bundesministerium fur Forschung und Tech- proteins to bind to and thereby inactivate nologie, Fonds der Chemischen Industrie, and National Institutes of causes the desensitized state of the system. Health. The costs of publication of this articlewere defrayed in part These hypotheses have been developed essentially on the by the paymentof page charges. This article must therefore be hereby basis of experiments utilizing reconstituted systems of the marked “advertisement” in accordance with 18 U.S.C. Section 1734 purified components. While such experiments demonstrate solely to indicate this fact. that these proteinscan interact in thisway, they do not prove The abbreviations used are: G,, stimulatory guanine nucleotidebinding protein; BARK, @-adrenergicreceptor kinase; GTPyS, guan- that such interactionsdo indeed take place in intact cells and osine 5’-O-(thiotriphosphate). that they do in fact provide the basis of homologous desensi-

3201

3202

Overexpression of P-Arrestin 0-Adrenergic and Receptor Kinase

tization. To address this question, we have previously inhib- limiting dilution to obtain individual clones. These clones were screened for the levels of expression of &-adrenergic receptors and ited the phosphorylations either by mutating the presumed PARK or 0-arrestin, respectively. Cells used for further study were phosphorylation sites i n the &adrenergic receptor sequence grown in selection medium (150 pg/ml G418 plus 0.1 p~ methotrex(Bouvier et al., 1988;Hausdorff et al., 1989)orbyusing ate) forup to 8 weeks. Expression of the various proteins was inhibitors of PARK a n d protein kinase A (Lohse et al., 1989, monitored during these times andfound to be stable. Determination of &Adrenergic Receptor Expression-To screen 1990b; Roth et al., 1991). While these experiments support the modelsof desensitization described above, there are none- clones for the expression of the various proteins, cells were harvested by scraping in a small volume (2 m1/107 cells) of lysis buffer (5 mM that may be theless caveats concerning potential artifacts Tris-HCI, 2 mM EDTA, pH 7.4, containing as protease inhibitors 10 caused by conformational changesof receptor mutants or by pg/ml soybean trypsin inhibitor, 15 pg/ml benzamidine, and 100 p~ insufficient specificity of kinase inhibitors. phenylmethylsulfonyl fluoride). Cells were disrupted with a Polytron In this study, we have therefore chosen another approach device, and debris and nuclei were removed bycentrifugation at 1000 to examine the functional role of PARK and p-arrestin. We X g for 10 min. The supernatant was centrifuged at 450,000 X g for have overexpressed both proteins in combination with differ- 30 min to give membrane and cytosolic preparations. These preparations were normalized to their protein content as measured with e n t levels of &receptors in stably transfected cell lines, and the protein assay of Bradford (1976). we have investigated the effects of their overexpression on Expression of &-adrenergic receptors was determined in binding the patternof receptor desensitization. experiments with the membrane preparations using saturating concentrations (250PM) of (-)-['251]iodocyanopindolol.Assays contained EXPERIMENTALPROCEDURES 10-50 pg of membrane protein and were conducted for 2 h at 25 "C in 50 mM Tris-HC1, pH 7.4, and terminated by filtration through Construction of Overexpression Vectorsfor PARK and 0-ArrestinThe vector for the overexpression of 0-arrestin was constructed on Whatman GF/C filters. Determination of PARK Expression-PARK activity in the cytosol the basis of the vector pBC-SK-dhfr (Lohse, 199213).The lattervector preparations was determined essentially as described by Benovic et is basedon the plasmid pBC/CMV/IL2 containing a cytomegalovirus promoter (Cullen, 1987); a polylinker had been added downstream al. (1989). In brief, cytosolic preparations (5 pg of protein/tube) were incubated with bovine rod outer segments (-200 pgof protein) in from the cytomegalovirusimmediate early promoter, and thegene for buffer containing 100 p~ [Y-~'P]ATP(0.5 pCi/tube), 2 mM EDTA, 5 mouse dihydrofolate reductase (dhfr) under the control of the SV40 mM MgCl,, 10 mM NaCl, and 20 mM Tris-HC1, pH 7.5. Incubations early promoter had been inserted. The coding region of the cDNA for were done at 30 "C for 10 min or for the times indicated on the p-arrestin was excised from pBC-Parrestin (Lohse et al., 1990a) with figures. They were terminated by addition of SDS sample buffer, and Not1 and ApaI and inserted intothe corresponding sitesinthe the samples wereloaded on 12% Laemmli (1970) gels with 4% polylinker of pBC-SK-dhfr to give pBC-Parrestin-dhfr. stacking gels. Urea-treated rod outer segments, which consisted of A similar vector was constructed (see Lohse (1992b)) for the >95% rhodopsin, the substrate for PARK, were prepared for these overexpression ofPARK. The vector pBC-PARK (Benovic et al., experiments as described by Wilden and Kuhn (1982). Phosphoryl1989) was cut with SfiI in the SV40 origin of replication, and blunt ated rhodopsin was visualized by autoradiography of the dried gels. ends were generated with T4 DNA polymerase. Into this site, the For quantitative assays, these bands were excised, and radioactivity PvuII-BamHI fragment of pSV2-dhfr (Subramani et al., 1981), con- was determined by Cerenkov counting. taining an intact SV40 origin of replication and early promoter and Generation of Anti-P-arrestin Antibodies and Western Blots-The the dhfr cDNA, was inserted after filling the BamHI site with the peptide VDTNLIELDTNDDDI, corresponding to amino acids 372Klenow fragment, such that the orientation was the same as for the 386 in bovine 0-arrestin (Lohse et al., 1990a), a region that is absent rest of the vector. in retinal arrestin, was synthesized and coupled via glutaraldehyde The correctness of the two constructs was confirmed by restriction (Harlow and Lane, 1988) to keyhole limpet hemocyanin (5 and 10 endonuclease mapping and by sequencing of the ligation sites. The mg, respectively). 150 pg of the coupled peptide/injection was injected functionality of the two expression vectors was verifiedby transiently into rabbits, initially with Freund's complete adjuvant, and for transfecting COS-7 cells and assaying for overexpression of the booster injections at 4-week intervals with Freund's incomplete adrespective proteins as described below. This resulted in a >30-fold juvant. Antibodies were purified by affinity chromatography using increase of PARK activity or in the appearance of p-arrestin immu- the peptide coupled to activated CH-Sepharose and elution by 100 noreactivity, respectively, in the COS-7 cell cytosol. COS-7 cells used mM glycine, pH 2.5. The eluates were neutralized, dialyzed against for these experiments weregrown in Dulbecco'smodified Eagle's phosphate-buffered saline, and stored at -80 "C. medium supplemented with 10% fetal calf serum, 100 units/ml peniFor Western blots, proteins were transferred from 10% Laemmli cillin, and 0.1 mg/ml streptomycin. The cells were transfected a t SDS-polyacrylamide gels onto polyvinylidene difluoride membranes -50% confluency with the vectors by the DEAE-dextran method by semidry blotting. The blots were blocked with 2% nonfat dried (Cullen, 1987). Two days later, the cells were screened for overex- milk, 1% ovalbumin in phosphate-buffered saline. P-Arrestin was pression of the respective proteins as described below. detected with the anti-0-arrestin antibodies plus peroxidase-coupled Generation of Stable Cell Lines-Stable cell lines expressing differ- goat anti-rabbit second antibodies and chemoluminescent substrate ent amounts of &adrenergic receptors and overexpressing either (ECL, Amersham Corp.). PARK or (3-arrestinwere generated as follows. Chinese hamster ovary Functional Determination of @-Arrestin Expression-The funccells deficient in dihydrofolate reductase (CHO-dhfr- cells) were tional activity of the overexpressed p-arrestin was assessed by its grown in Dulbecco's modified Eagle's medium, 10% fetal calf serum ability to inhibit the function of PARK-phosphorylated &receptors. supplemented with hypoxanthine (13.6 mg/liter), thymidine (3.88 This was determined by measuring the (-)-isoproterenol-stimulated mg/liter), penicillin (100 units/ml), and streptomycin (0.1 mg/ml). GTPase activity of purified G. co-reconstituted with purified PARKThey were transfected by the calcium phosphate method (Cullen, phosphorylated (>3 mol of phosphate/mol of receptor) &-receptors. 1987)with 20 pg of the respective overexpression vector plus 10 pg of Cytosolic preparations (in 5 mM Tris-HCI, pH 7.4, 2 mM EDTA) pBC12BI-P2,an expression vector for human &adrenergic receptors were preincubated for 30 min on ice with 10 p M GTPrS inthe MgC1, to reduce the activity of endogenous under the control of the Rous sarcoma virus promoter (Kobilka et al., presence of10 mM 1987) plus 3 pgof pSV2-neo. All three vectors were linearized with GTPases. GTPyS and MgCI, were then removed by gel filtration on PD-10 columns (Pharmacia LKB Biotechnology Inc.), followed by PvuI before transfection. Three different transfections were done: pBC12BI-P2 alone, pBC12BI-P2 plus pBC-parrestin-dhfr, and washing on Centricon 30 devices (Amicon Corp.) in 100 mM NaCI, 10 mM Tris-HCI, pH 7.4. Different amounts of this preparation were pBC12BI-f12plus pBC-PARK-dhfr (all plus pSV2-neo). Two days after the transfection, a first round of selection was then added to assays measuring the &receptor-stimulated GTPase using PARK-phosphorylated receptors as described started by addition of 150 mg/liter G418 (GIBCO) to the medium. activity ofG. The resulting resistant cell population was selected for high expres- earlier (Lohse et al., 1992; Bauer et al., 1992). Desensitization Experiments-Cells were grown to -90% conflusors in a second selection step by omitting hypoxanthine and thymidine from the cell culture medium. The resistant mixed cell popula- ence as described above. They were held in serum-free Dulbecco's tion emerging after "2 weekswas subjected to a third round of modified Eagle's mediumovernight. Fresh medium containing 10 nM selection by adding 0.1 p~ methotrexate to themedium. Cells growing or 10 p~ (-)-isoproterenol (or none for controls) was added, and the after this step (emerging again after -2 weeks) were subcloned by cells were held at 37 "C for 10 min to effect desensitization. Subse-

Overexpression of 0-Arrestin@-Adrenergic and Receptor Kinase

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The adenylylcyclase data in Table I demonstrate that the expression of higher receptor levels results in a much higher signal transduction efficacy: the maximum stimulation by isoproterenol increasesfrom 6-fold. Fig. 1 shows that the PARK activities in the two PARKoverexpressing cell lines (BB9 and BB19) were about equal and -10 times higher than in control cell lines not transfected with PARK. Transfection of the cells with p-arrestin had no effect on PARK activity (data not shown). Overexpression of p-arrestin was monitored on Western blots (Fig. 2 A ) . Endogenous levels of p-arrestin were too low to be detected by simple Western blots (but were detectable after immunoprecipitation with another anti-p-arrestin antibody (data not shown)). In Fig. 2 A , /3-arrestin was just detectable in lanes containing 1.5 pg of cytosolic protein from the P-arrestin-overexpressing cells (BA16 and BA48), but not in the lane containing 50 pgof cytosolic protein from the control cells. Upon overexposure (not shown), @-arrestin was clearly visualized in the lanes containing 1.5 pg of cytosolic protein from the P-arrestin-overexpressing cells and was still not visible in control cell cytosol. Thus, the extent of overexpression as determined by Western blotting was >30-fold. The functional activityof the overexpressed p-arrestin was determined by its ability to inhibit the stimulation ofG,/ GTPase activity by PARK-phosphorylated &-receptors in a reconstituted system (Fig. 2B). The inhibitory activityin the cytosol of the P-arrestin-overexpressing cell lines was -100fold higher than in the control cells. Full inhibition was seen with 1 pg of cytosolic protein from P-arrestin-overexpressing cells, but not even half-maximal inhibitionwas observed with 10 pg of cytosolic protein from the controlcells (higher levels of cytosolic protein could not be used because of endogenous GTPase activities). Thus, the functional (-100-fold data overexpression) agree well with the data obtained from Western blots (>30-foldoverexpression) and document the marked (although not totally equal) overexpression of p-arrestin in the two cell lines BA16 and BA48. Comparison of the PARK and p-arrestinlevels in these cell lines with the signals generated by purified PARK (Benovic et al., 1987) and purified P-arrestin (Lohse et al., 1992) allows an estimate of the absolute levels of the two proteins: the (3arrestin Western blot signal obtained per milligram of cytosolic protein from overexpressing cells is equivalent to -10 pmol of p-arrestin, which correspondsto -5 pmol of 0arrestin/mg of total cell protein. For the control cell lines, this value would be 30-100-fold lower, i.e. in the range of 0.1 pmol of P-arrestinlmg of total cell protein. In the case of PARK, control andoverexpressing cell lines have activities of -20 and -200 pmol of phosphate/mg of cytosolic protein/ min, respectively, whereas pure PARK under the same conRESULTS ditions has an activity of up to 80 nmol of phosphate/mg of The transfection of CHO-dhfr- cells resulted in the gener- protein/min. Per milligram of total cell protein, this corresponds to -1.5 and -15 pmol of PARK in the control and ation of a large number of clones expressing &adrenergic receptors and overexpressing either PARK or P-arrestin or overexpressing cell lines, respectively. Forcomparison, the neither of the two. About 50 clones were screened from each receptor levels per milligram of total cell protein in the low of the transfections, and six cell lines were used for further and high expressing cell lines are -0.015 and -0.12 pmol, studies. Thesewere divided into two groups expressing recep- respectively. In these matched groups of cell lines, we then studied the tors either in the low or high physiological range (-80 and -600 fmol/mg of membrane protein, respectively). In each pattern of desensitization to the @-receptor agonist isoprotergroup, there was one cell line each that overexpressed either enol. The resultsfor desensitization tohigh concentrations of PARK or P-arrestin or neither of the two (control). Cell lines agonists, required to elicit homologous desensitization (Lohse overexpressing PARK or p-arrestin in thelow and high recep- et al., 1990b), are given in Figs. 3 and 4; and their quantitative tor groups were matched as far as possible for comparable analysis is summarized in Fig. 5. In these experiments, adenoverexpression (see below). Table I gives the details of the ylylcyclase activity was measured a t a freeMg2+concentration determination of &adrenergic receptor number as well as of of 4 mM, conditionsthat allow a preferentialanalysis of P2-receptor-induced stimulation of adenylylcyclaseactivity. homologous desensitization versus the protein kinase A-me-

quently, the cells were washed three times with ice-cold phosphatebuffered saline and then scraped in 30 ml of ice-cold 50 mM TrisHC1, pH 7.4, per 150-mm Petri dish. They were disrupted with a Polytron device, and crude membranes were prepared as described above. Adenylylcyclase activity was determined in these membranes as described earlier (Lohse et al., 1990b), but using the termination of the assays described by Jakobs et al. (1976). Incubations contained =20 pg of membrane protein, 50 mM Tris-HC1, pH 7.4, 1 mM EDTA, T P pCi/tube), 100 pM CAMP, 50 p M GTP, 5 mM 100 p~ [ C I - ~ ~ P I A(0.2 creatine phosphate, 0.4 mg/ml creatine kinase, and 1 mg/ml bovine serum albumin. MgCIZwas added to give a free Mg*+ concentration in these assays of 4 mM to facilitate the detection of homologous uersus heterologous desensitization; only for assays determining heterologous desensitization (seeFig. 6) was the free Mg2+ concentration 0.8 mM (see Clark et al. (1987) and Lohse et al. (1990b)). Incubations were done at 37 "C for 30 min. Accumulation of CAMP was linear under these conditions. Data Analysis-Quantitative data are presented as means of a t least three independent experiments. Concentration-response curves were normalized to the basal activity (which is given in the figure legends). Desensitization was quantitated as the loss of signal transduction efficacy (T) as detailed earlier (Lohse, 1990). In brief, the concentration-response curvesof control anddesensitized membranes were fitted and compared using the following algorithm (Black et al., 1985): E = E,,,[T"A"/((KA + A)" + T"A")], with E denoting the effect, E , the maximum possible effect, Athe agonist concentration, KA the dissociation constant of the agonist-receptor complex, and n a slope factor, which was never significantly different from 1 in all experiments presented here. T is a parameter describing the signal transduction efficacy of the system and is estimatedindividually for each curve, whereas all the other parameters are shared. T~ denotes the T is thevalue for the desensitized value under control conditions, and a measure of desensitization curve. (1- ( T / T ~ ) ) X 100 is then taken as (in percent). Intuitively, this parameter can best be described as the percentage of receptors that need to be destroyed to give an equivalent loss of receptor-stimulated adenylylcyclaseactivity. The details of this quantitationprocedure have been describedearlier (Lohse,1990). In addition,we also evaluated desensitizationby the more conventional approach of determining the decrease of the maximum effect (Emax) of isoproterenol: the E,,, values for the control (E,,,,) and curves were calculated by curve-fitting (Lohse et desensitized (Emard) al., 1986) to the Hill equation, and desensitization (in percent) was calculated as (1 - (Emaxd/EmaXc)) X 100. Materiak-[1251]Iodocyanopindolol was obtained from Amersham Corp. [cY-~*P]ATP, [Y-~*P]ATP, and [Y-~'P]GTP were purchased from ICN. [w3*P]ATP and [Y-~'P]GTP were purified by anion-exchange chromatography on Dowex 1-X2 C1- (Walseth and Johnson, 1979). Cell culture media, fetal calf serum, and antibiotics were from GIBCO. Enzymes formolecular biology were from BoehringerMannheim. Electrophoresis chemicals were from Bio-Rad, with theexception of agarose, which was purchased from Bethesda Research Laboratories. Activated CH-Sepharose was obtained from Pharmacia LKB BiotechnologyInc.Polyvinylidenedifluoride membranes for Western blotting were from Millipore, and peroxidase-coupled goat anti-rabbit antibodies were from Bio-Rad. Chemoluminescence kits (ECL) were from Amersham Corp. and were used in conjunction with Kodak XAR films.

Overexpression of P-Arrestin and P-Adrenergic Receptor Kinase

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TABLE I Levels of &adrenergic receptors and stimulation of adenylylcyclase activity by the B-receptor agonist (-)-isoproterenol in membranes from Chinese hamster ovary cell lines Receptor numbers were determined by radioligand binding with saturating concentrations of (-)-[‘251]iodocyanopindolol.Adenylylcyclase activity was measured under basal conditions and in the presence of 10 g~ (-)-isoproterenol. Basalactivities were not significantly different in the cell lines and were 18.2 k 2.6 pmol of cAMP/min/mg of membrane protein. Data are means f S.E. of three (binding) andat least five

(adenylylcyclase) independent determinations. Cell line

Overexpressed

protein

&Receptors pmollmg membrane -fold protein

Isoproterenol-stimulated adenylylcyclase activity stimulation

Low receptor levels B-36

BA48 BB19 High receptor levels

Control P-Arrestin PARK

B-17

Control

BA16

@-Arrestin

BB9

BARK

92 f 13 76 f 15 82 f 16

2.5 & 0.2 2.4 f 0.2 2.4 & 0.1

650 & 90 650 -t 50 6.4 560 f 60

7.3 f 0.5 8.0 f 0.5 &

0.4

hancement reaches statistical significance for the overexpression of p-arrestin ( p < 0.05), but not of PARK ( p > 0.05). In cells with high levels of receptors (Figs. 4 and 5 , lower panel), the extent of desensitization increases significantly with the overexpression of PARK ( p < 0.01), but even more so with that of p-arrestin ( p < 0.001). Whereas in control cells desensitization was about the samefor cells expressing high or low levels of receptors, desensitization was enhanced in cells with high receptor densities when either p-arrestin or BARK was overexpressed. Emax The results obtained by analyzing the drop in the values (Table 11) generally agree with those obtained by the algorithm-based method. Using this method to evaluate de0 2 4 6 8 10 12 sensitization, the effects of PARK or P-arrestin overexpresTIME (rnin) sion in the cells expressing low levels of receptors are more FIG. 1. BARK activity in cytosol of BARK-overexpressing visible and reach statistical significance ( p < 0.05) in both and control cells. The activity of BARK was determined by meas- cases. uring the ability of crude cytosolic fractions to catalyze light-dependThese datashow that overexpression of p-arrestin or PARK ent phosphorylation of rhodopsin in rod outer segments. Data are does indeed enhance homologous desensitization effected by means f S.E. of three independent experiments. high concentrations of isoproterenol. T o test whether overexpression of these proteins also affects heterologous desendiated heterologous form of desensitization (Clark etal., 1987; sitization, we modified the experimental conditions: the conLohse et al., 1990b). centration of isoproterenol used to desensitize the systemwas Fig. 3 shows the desensitization to 10 PM isoproterenol in lowered to 10 nM, and the free Mg2+ concentration in the cells expressing low levels of receptors. Fig. 4 shows the same adenylylcyclase assays was 0.8 mM. Theseareconditions for cellswith a high receptor complement. In allcases, desen- under which heterologous, protein kinase A-mediated desensitization caused a rightward shift of the concentration-re- sitization isobserved (Clark etal., 1987; Hausdorff et al., 1989; sponse curve and a decrease of maximum stimulation. Partic- Lohse et al.,1990b). Sincethealterations of homologous ularly in cells with high receptordensities (Fig. 4), both desensitization caused by overexpression of PARK or P-arreschanges were more pronouncedin PARK- and p-arrestintin were much more pronounced in cells with high receptor overexpressing cells compared to controlcells. It is important densities, only these cells were tested for possible alterations to realize in this context that both parameters (the rightward of heterologous desensitization. The results of these experishift and the decrease of maximum stimulation) areindicative ments are given in Fig. 6. There was no significant effect of of a loss of signal transduction efficacy. These complex patthe overexpression of either of the two proteins on heteroloterns of the desensitization process make it mandatory to perform a quantitative analysis togauge the extent of desen- gous desensitization. Desensitization measured under these 40-50% regardless of whether Psitization. Accordingly, the curves shown in Figs. 3 and 4 were conditionsamountedto arrestin or PARK was overexpressed or not. fitted using the previously described algorithm (see “Experimental Procedures”) to quantitate the extentof desensitizaDISCUSSION tion. The resultsof this analysis are summarized in Fig. 5 . In Homologous or receptor-specific desensitization of &-adaddition, because the algorithm-based analysis appears tobe less sensitive if the signal transduction efficiency (ie. the T renergic receptors has been proposed to be effected via phosvalue) is low, the results of the more conventional analysis phorylation of agonist-occupied receptors by PARK, followed equating desensitizationwith a loss of maximum stimulation by binding of @-arrestin to the receptors. This proposal is based on a number of observations. 1) PARK is a kinase that are presented in Table11. a strictly agonist-dependent manIn cells expressing low levels of &receptors (Figs. 3 and 5 , phosphorylates receptors in upper panel), overexpression of either P-arrestin or PARK ner and is therefore a good candidate for a desensitization results in a minor enhancement of desensitization. This en- process that affects only those receptors that had been stim-

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Overexpression of @-Arrestinand @-AdrenergicReceptor Kinase

C

15 1.5 5

8-36 (control)

(parrestin)

0

50 1.5 5

50 15 1.5 5 EA16

.

0.1

45 31

15 50

BA48 (parrestin)

1

ISOPROTERENOL

(M)

ISOPROTERENOL

(MI

ISOPROTERENOL

(M)

10

cytosolic preparation (pg protein) FIG. 2. Overexpression of @-arrestin.A, Western blot of 8arrestin in the cytosol of 8-arrestin-overexpressing and control cells. Crude cytosolic preparations of the cells (1.5-50 pg of protein) were electrophoresed on a 12% Laemmli SDS-polyacrylamide gel, transferred onto polyvinylidene difluoride membranes, and developed with an anti-0-arrestin antibody as described under “Experimental Procedures.” B, the cytosolic preparations were measured for their ability to inhibit the stimulation of G./GTPase activity by PARK-phosphorylated P2-receptorsin a reconstituted system. Tubes contained 5 fmol of o2-receptors and 25 fmol ofG,. 100% values were 2.1 f 0.3 molof Pi/mol ofG./30 min of incubation ( i e . 55 fmol/tube); the background activity of endogenous GTPases remaining in the cytosolic preparations even after the GTPrS pretreatment (see “Experimental Procedures”) was -70 fmol/tube/30 min of incubation at the highest protein concentration (10 pgltube) and correspondingly less at the lower concentrations.

ulated with agonist (Benovic et aL, 1986b). 2) In reconstituted systems with partially purified (Lohse et al., 1990a) or purified (Lohse et al., 1992) components, PARK in concert with 8arrestin can impair coupling between P2-receptors and G, by up to 75%. 3) Inhibitors of BARK inhibit homologous desensitization of P2-receptors in permeabilized cells (Lohse et al., 1989, 199Ob; Roth et aL, 1991; Zhou and Fishman, 1991). 4) Mutations that delete or alter thepotential phosphorylation sites for PARK cause impaired homologous desensitization (Bouvier et al., 1988; Hausdorff et al., 1989). However, while others have confirmed our results (Lohse et al., 1989,1990b) that inhibition of PARK prevents homologous desensitization of P2-receptors in A431 cells (Zhou and Fishman, 1991) and ofcup-receptors in transfected Chinese hamster ovary cells (Liggett et al., 1992), Zhou and Fishman (1991) reported that similar effects were not observed for the desensitization of PIreceptors in SK-N-MC neurotumor cells. Furthermore, whereas some mutation studies support the hypothesis of a

FIG. 3. Desensitizationof &-receptors incells with lowlevels of &,-receptors.Upper panel,control cells (B-36); center panel, 8-arrestin-overexpressing cells (BA48); lower panel, PARK-overexpressing cells (BB19). Cells were exposed (W) or not (0)to 10 p M (-)-isoproterenol for 10 min at 37 “C. Adenylylcyclase( A C )activities were determined in the membranes prepared from these cells under basal conditions and in the presence of the indicated concentrations of (-)-isoproterenol. Dataare means f S.E.from 5 (BB19), 10 (BA48), and 15 (B-36) independent experiments.

major role for PARK-mediated phosphorylation in homologous desensitization ofB2-receptors (Bouvier et al., 1988, Hausdorff et al., 1989), one mutation study has questioned this role (Cheung et al., 1989). Accordingly, we wished to develop additional experimental approaches to confirm further theproposed mechanisms. In this study, we have therefore investigated the effects of

Overexpression of @-Arrestin @-Adrenergic and Receptor Kinase

3206

&,-recaplors:

i

auul

-80 Imollmg mombranbprotein

f-

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ISOPROTERENOL

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I

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ISOPROTERENOL

(MI

FIG. 5 . Quantitation of &-receptor desensitization in control cells and p-arrestin- and @ARK-overexpressingcells with low (upper panel)and high (lower panel)levels of &-receptors. The desensitization experiments shown in Figs. 3 and 4 were analyzed with the algorithm given under "Experimental Procedures," and the percent desensitization was calculated as the loss of signal transduction efficacy: (1- ( T / T O ) ) X 100. *, p < 0.05; **, p < 0.01;***, p < 0.001 by analysis of variance.

ISOPROTERENOL

(MI

FIG. 4. Desensitization of &-receptors in cells with high levels of Bz-receptors. Upper panel, control cells (B-17); center panel, 8-arrestin-overexpressing cells (BA16); lower panel, PARKoverexpressing cells (BB9). Cells were exposed).( or not (0)to 10 p~ (-)-isoproterenol for 10 min a t 37 "C. Adenylylcyclase ( A C ) activities were determined in the membranes prepared from these cells under basal conditions and in the presence of the indicated concentrations of (-)-isoproterenol. Data are means +. S.E. from six independent experiments.

overexpression of the two proteins proposed to mediate homologous desensitization (PARK and p-arrestin) on the extent of desensitization. The main resultof this study is that overexpression of either protein enhancedhomologous desensitization of &adrenergic receptors. These effects were more apparent in cells with high levels of &receptors, suggesting that in Chinese hamster ovary cells expressing high levels of

TABLE I1 Evaluation of &receptor desensitization i n control cells and p-arrestin- and PARK-overexpressing cells with low and high leu& of P2-receptors The desensitization experiments shown in Figs. 3 and 4 were values for analyzed for the drop in maximum stimulation (Emax). Emax the control (E,,,,) and desensitized (Emaxd) curves were calculated by curve-fitting to the Hill equation, and desensitization (in percent) X 100. was calculated as (1- (EmaxdEmaxc)) Cell line

Overexpressed protein

Desensitization %

Low receptor levels B-36 Control BA48 @-Arrestin BB19 PARK High receptor levels B-17 Control BA16 P-Arrestin BB9 PARK " p < 0.05 by analysis of variance. * p < 0.01 by analysis of variance.

23 f 2 32 f 5" 37 f 2b

18 f 2 40 f 5' 33 f 3b

receptors, PARK or p-arrestin more significantly limits the extent of homologous desensitization thanin those cells with low receptor levels, a finding consistent with the observed

Overexpression of &Arrestin p-Adrenergic and Receptor Kinase

3207

of the levels of PARK, 0-arrestin, and &receptors indicate that the P-arrestin levels are in the range of the &receptor x levels in thehigh receptor-expressing cell lines and that PARK levels are -10-fold higher. This suggests that at least for Parrestin, stoichiometric limitation may play a role and that consequently,thelimiting effects of P-arrestin expression should be more pronounced in cells with high receptor levels. The same reasoning applies to the effects of PARK expression, although because of the higher PARK levels compared to those of p-arrestin, stoichiometric limitation should be less pronounced. Indeed, in our experiments, overexpression of either (3-arrestin or PARK had more visible effects in cells expressing high levels of &receptors, and these effects were more pronounced for P-arrestin than for PARK. Oneobservation, however, is difficult to explainin this 6-17 EA16 669 context. In this study as well as in an earlier study on A431 (Wnlrol) (D-ArrerUn) (OARK) cells (Lohse, 1990), we have observed less or at most the same FIG. 6. Quantitation of heterologous Bz-receptor desensitization in control cells and B-arrestin- and BARK-overexpress- desensitization(as calculated by thealgorithm-basedaping cells withhigh levels of Bz-receptors.The data were derived proach) in cells with few receptors compared with those with in a manner analogous to those presented in Fig.5 from experiments high receptor levels. In this study, this was observed also for measuringalmostexclusivelyheterologousdesensitization to (-1pairs of cell linesoverexpressing P-arrestin (BA48 uersus isoproterenol: desensitization for 10 min with10 nM (-)-isoproterenol BA16) or PARK (BB19 uersus BB9). If stoichiometric limiand with adenylylcyclaseassays with low concentrations of free M$+ tation as mentioned above does indeed occur, cells with less (0.8 mM). receptors shoulddesensitize better than thosewith high receptor levels. If only or mainly concentration-dependent limitalevels of PARK and P-arrestin compared with those of the tion occurs, then desensitization should be the samein these receptors (see below). pairs of cell lines. This discrepancy may in part bedue to the Moreover, we observed no effects on heterologous desensi- low sensitivity of the algorithm-based analysis of desensititization. These results lend further support to our proposal zation incellswith low signal transduction efficiency (i.e. that PARK and P-arrestin areinvolved in homologous desen- little receptor-stimulatedadenylylcyclase activity) since it was sitization. On the other hand, there is no indication that theynot seen when the more conventional analysis of a decrease are involved in heterologous desensitization, which is thought in E,,, was used to evaluate desensitization (Table 11). Howto be protein kinase A-mediated. In a recent study employing ever, there may also beadditional, biological factors that purified reconstituted proteins (Lohse et al., 1992), we found permit better desensitization in those cells that express high that P-arrestin was essentially requiredfor theinhibitory levels of receptors. This question will require further studies. effects of PARK-mediated&receptorphosphorylation.In The observation that @ARKcan belimiting for homologous contrast, the impairmentof receptor function caused by prodesensitization may be of interest for attempts to interfere tein kinase A-mediated phosphorylation of the receptors was with homologous desensitization. Such attemptsmay provide not affected by 0-arrestin. Similar observations were made the means for maintaining the therapeuticeffects of receptor etal., 1992). Thus, two withrespect toarrestin(Pitcher agonists when these are lost due to homologous desensitizaindependentapproaches suggest that P-arrestin is not retion. We have shown previously that inhibitorsof PARK can quired for heterologous protein kinase A-mediated desensitilargely attenuate homologous desensitization (Lohse et al., zation. 1989). If PARK is indeed a limiting factor for homologous Even under optimized conditions, the desensitization induced by high concentrations of isoproterenol represents not desensitization, then it could become a target for the develonly homologous desensitization, but also contains a heterol- opment of inhibitors of this form of desensitization. In this context, we have recently observed that the levels ogous component.Thiscomponenthas been estimated at -25% (Lohse et al., 1990b). Thus, of the -50% total desen- of PARK enzymatic activity and @ARKmRNA are increased sitization seen in theB-17 control cells (Fig. 4, lower panel), severalfold in thefailing human heart, a clinical setting charof the@-receptor only -25% is indeed due to homologous desensitization. An acterized by pronounceddesensitization increase in total desensitization from ~ 5 0 % in control cells system (Ungereret al., 1993). This may indicate that in order to -80% (BB9 cells, PARK) or -90% (BA16 cells,0-arrestin) to effect maximum desensitization, the levels of PARK need can be therefore estimated to indicate anincrease in homolo- to be increased, i.e. that indeed the cytosolic concentrations of @ARKcan be limiting the extentof desensitization. gous desensitization from -25 to -55 and 65%, respectively. The levels of P-arrestin (or PARK) can limit desensitization The proposal of a role for PARK and P-arrestin in homolvia two possible mechanisms. Stoichiometric limitation will ogous desensitization has essentially been developed on the occur if the stoichiometry of P-arrestin:@-receptors islow. In basis of studies employing reconstituted systemswith purified proteins. Subsequently, experimentsemploying kinase inhibthis case, thereisnotenoughP-arrestintobindtoand desensitize all receptors. Alternatively, if the concentration of itors and receptor mutants have provided evidence in agreeP-arrestin is low compared to its affinity for the receptors, ment with this proposal. This studyis the first to demonstrate this may limit the extent of desensitization even if there is directly cellular effects of these two proteins by expression of more P-arrestin than P-receptors. It is clear that stoichiomet- their cDNAs. It supports our proposal that homologous deric limitation must bemore apparent incells with high recep- sensitization of &receptors (andpossibly also of other similar tor levels, whereas concentration-dependent limitation should receptors) is mediated by the concerted action of PARK and be the same irrespective of the receptor levels. We do not P-arrestin. It also indicates that neither protein plays a role know which of the two mechanisms ismore important because in the heterologous form of desensitization, indicating that the relevant affinities are unknown. However, our estimates these are two entirely separate processes. This further illus~-receplon:-600 fmollmg membrane protein

-

E js-

Overexpression of P-Arrestin&Adrenergic and Receptor

3208

trates the diversity of mechanisms that have evolved to regulate receptor function. Acknowledgments-We thank Grace Irons and Gabriele Bittner for help with the cell cultures. REFERENCES Bauer, P. H., Muller, S., Puzicha, M., Pippig, S., Obermaier, B., Helmreich, E. J. M., and Lohse, M. J. (1992) Nature 3 5 8 , 73-76 Benovic, J. L., Pike, L. J., Cerione, R. A., Staniszewski, C., Yoshimasa, T., Codina. J.. Caron. M. G.. and Lefkowitz. R. J. (1985) . . J. Biol. Chem. 260. 7094-7ioi Benovic, J. L., Mayor, F., Jr., Somers, R. L., Caron, M. G., and Lefkowitz, R. J. (1986a) Nature 322,869-872 Benovic, J. L., Strasser, R. H., Caron, M. G., and Lefkowitz, R. J. (1986b) Proc. Natl. Acad. Sci. U. S. A. 8 3 , 2797-2801 Benovic, J. L., Mayor, F., Staniszewski, C., Lefkowitz, R. J., and Caron, M. G. (1987) J.Biol. Chem. 262,9026-9032 Benovic, J. L., DeBlasi, A,, Stone, W. C., Caron, M. G., and Lefkowitz, R. J. (1989) Science 2 4 6 , 235-240 Benovic, J. L., Onorato, J. J., Arriza, J. L., Stone, W. C., Lohse, M., Jenkins, N. A,, Gilbert, D. J., Copeland, N. G., Caron, M. G., and Lefkowitz, R. J. (1991) J. Biol. Chem. 266,14939-14946 Black, J. W., Leff, P., and Sbankley, N. P. (1985) Br. J. Pharmacol. 8 4 , 561571

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~

I

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Kinase

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