the protein kinase catalytic domain to an arginine. Ex- pressed and purified PARK-K220R was able to inhibit wild type PARK phosphorylation of the P&R in vitro.
Communication
THEJOURNAL OF BIOUJCICAL CHEMISTRY Vol. 269, No. 18,Issue of May 6, pp. 13084-13087,1994 0 1994 by The American Society for Biochemistry and Molecular Biology, Inc. Printed in U S A .
A P-Adrenergic Receptor Kinase Dominant Negative Mutant Attenuates Desensitizationof the P,-Adrenergic Receptor*
protein G,, and long term mechanisms that down-regulate &AR number by affecting the rates of receptor degradationand synthesis (2, 3). The p-adrenergic receptor kinase (PARK) has been implicatedas the major kinase involved in the activationdependent phosphorylationof t h e P f l (4). PARK is a member of a growing family of G protein-coupled receptor kinases, enzymes with the unique ability to recognize and phosphorylate (Received for publication, February 22, 1994, and in revised form, only the activated conformationof their receptor substrates (5, March 18, 1994) 6). Phosphorylation of the P&R by PARK then promotes the association of another protein, p-arrestin, whicheffectively unGuanghui Kong, Raymond Penn, and Jeffrey L. BenovicS couples the &AFt from G, by specifically binding t o the phosphorylated and activated form of the receptor (7). PARK is a From the Department of Pharmacology, Jefferson ubiquitous cytoplasmic enzyme that not only phosphorylates Cancer Institute, Thomas Jefferson University, Philadelphia, Pennsylvania 19107 the P f l to a high stoichiometry (8),but can also phosphorylate additional GPRs in vitro (9-11). Moreover, multiplestudThe @-adrenergic receptor kinase (PARK) specifically ies have demonstrated translocation of PARK activity fromthe phosphorylates the activated form of the &-adrenergic cytosol to the plasma membrane following activation of various receptor ( P a ) and related G protein-coupled receptors. To further elucidate the roleof PARK in receptor GPRs in intact cells(12-141, thus suggesting a role forPARK in desensitization, we generated a PARK dominant nega- regulating GPR-mediated signal transduction in vivo. tive mutant by converting an invariant lysine residue inThe structural featuresof PARK include a centrally localized the protein kinase catalytic domain to an arginine. Ex- catalytic domainthat contains all of the conserved amino acid residues shared by the extended family of protein kinases (15, pressed and purified PARK-K220R was able to inhibit in subdomain I1 of the catawild type PARK phosphorylation of the P&R in vitro. 16).A universally conserved lysine lytic domain (Lyszzoin PARK)has been shown to be directly When stably transfected into human bronchial epithelial BEAS-2B cells, PARK-K220R promoted a >2-fold in- involved in the phosphotransfer reaction(17).Amino acid subcrease in P-agonist-stimulated CAMPproduction with- stitutions for this lysine have been demonstrated to signifioutaffecting P&.R sequestration. In contrast, @IRK- cantly diminish or abolish activityin both serinelthreonine and K220R had no effect on thedesensitization ofthe tyrosine protein kinases (18-20). Based on these findings, we prostaglandin E, receptor response in BEASdB cells. generated a PARK m u t a n t that lacked kinase activity by subThese findings directly demonstrate a role for PARK in stituting the invariant lysine in PARK with an arginine. In an desensitization of the P&.R in intact cells and establish effort to elucidate the role of PARK in intact cells, we subsethe potential utility of using dominant negative mutants quently overexpressed this PARK dominant negative mutant of G protein- and characterized its effect on desensitization of the &-adretoelucidatethesubstratespecificity coupled receptor kinases. nergic and prostaglandinE, (PGEJ receptors in BEAS-2B epithelial cells. EXPERIMENTAL PROCEDURES Desensitization of transmembrane signaling systems has Materials-Restriction endonucleases and other molecular biology been extensively characterized for cell surface receptors that transduce extracellular signals through guanine nucleotide- reagents were purchased from New England Biolabs and Boehringer binding proteins (G proteins)' (1). A prototypic model for the Mannheim. Sequenase was from U. S. Biochemicals, while [y-32PlATP, [12611iodopindolol, and [(x-~'S]~ATP were purchased from DuPont NEN. study of G protein-coupled receptor (GPR) desensitization has Isoproterenol, PGE,, forskolin, and soybean phosphatidylcholine were been the &-adrenergic receptor (P&R)-coupled adenylyl cy- from Sigma. Cell culture reagents were obtained from Life Technoloclase system. Binding of extracellular catecholamines to t h e gies, Inc. or Sigma. All other materials were from sources described &AR leads to the formation of the intracellular second mes- previously (21, 22). Construction and Expression of a Dominant Negative @RK senger CAMP, which in turn mediates a host of cell-specific (15) was used to synthesize singlephysiological responses. Prolonged agonist activation of t h e Mutant-The bovine PARK cDNA stranded DNA that served as a template for site-directed mutagenesis P&R often leads to a loss of responsiveness or desensitization (Amersham Corp.). The oligonucleotide 5"GTCCAGACAGCGCATGof the &AR. Desensitization can occur via multiple pathways, GCGTA-3' was used to change residue 220 in PARK from lysine to including rapid mechanisms whereby receptor phosphorylation arginine, and the mutation was confirmed byDNA sequencing. The promotes the uncoupling of the P f l from the stimulatory G pARK-K220R cDNA was then excised with HindIII, blunted with Klenow, isolated on a low melting agarose gel, and subcloned into the blunted NheI site of the baculovirus vector pJVPlOE (23). A recombi*This research was supported in part by National Institutes of nant baculovirus was obtained by cotransfecting Sf9 cells with the Health Grants GM44944 and HL45964. The costs of publication of this pARK-K220R containing vector and wild type baculovirus DNA using article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance the calcium phosphate precipitation technique and then was purified through several rounds of plaque purification. To isolate purified PARKwith 18 U.S.C. Section 1734 solely to indicate this fact. $ To whom correspondence should be addressed: Thomas Jefferson K220R, Sf9 ce11s infected with recombinant pARK-K220R virus were University, 233 S. 10th St., Philadelphia, PA 19107. Tel.: 215-955-4607; harvested after 48 h by centrifugation and washed with phosphatebuffered saline. The cells were lysedusing a Polytron disrupter, and a Fax: 215-923-1098. high speed supernatant fraction was purified by successive chromatogI The abbreviations used are: G protein, guanine nucleotide-binding protein; P f l , P,-adrenergic receptor; PARK, P-adrenergic receptor ki- raphy on SP-Sepharose and heparin-Sepharose columns as described nase; G,, stimulatory G protein; GPR, G protein-coupledreceptor; PGE,, previously for wild type PARK (21). The PARK-K220R preparations prostaglandin E,. used in these studies were >95%pure as assessed by SDS-gel electro-
13084
A Dominant Negative pARK Mutant Reduces &#
Desensitization
13085
phoresis and Coomassie Blue staining. In Vitro Phosphorylation of &AR-The hamsterwas overexpressed in ,319 cells, purified on an alprenolol-Sepharose column, and reconstituted into phosphatidylcholine vesicles a s described previously w t BARK (21).G protein By subunits were purifiedfrom bovine brain as described BARK-KZZOR . previously (22). Phosphorylation reactions contained 20 mM Tris-HCI, PY pH 7.5, 2 mM EDTA. 100 p~ [y-:"P]ATP (-1 cpdfrnol), 10 mM M S I , , 1 mM sodium phosphate, 20p~ (-)-isoproterenol, and0.25 pmol of&AR in a total volume of 100 PI. Reactions included 10 nw wild type BARK a n d o r 150 nM PARK-K220R in the presence or absence of 100 nM G protein IJy subunits.Competitionstudiesincluded10 nM wild type BARK and 15-150 nw PARK-K220R in the presence or absence of 100 nM G protein B y subunits. Reactions were incubatedfor 20 min a t 30 "C and quenched by addition of 1 mlof cold 0.1 w sodium phosphate. pH 7.2, 10 mM EDTA buffer. The receptors were pelleted by centrifugation a t 100.000 rpm for 20 min, resuspended in SDS sample buffer. and then resolved by SDS-polyacrylamide gel electrophoresis and visualized by autoradiography. Following autoradiography, the phosphorylated &AR hands were cut and counted. Cell Culture-Human airway epithelial BEAS-2B cells were obtained from Dr. Curtis H a m s (National Institutesof Health). Asubcloneof this cell line (R1) reported to he resistant to serum-induced squamous differentiation (24) was used throughout these studies. Cells were cultured in Ham'sF-12 medium supplemented with50 fetal bovine serum, 200 unitdml penicillin, and200 pg/ml streptomycin a t 37 "C in 95%air 0 5 IO I5 20 and 5 8 CO,. rnulant profem Io wl BARK rafro IIFansfection of REAS-2B Cells-An EcoRV-PuuII fragment of the FIG.1. PARK-K!220Rinhibits BARK-mediatedphosphorylation PARK-K22OR cDNA was excised, isolated on a low melting agarose gel, ofthe &4Rin v i t r o . A , BARK and pARK-K220R phosphovlation ofthr ligated with BarnHI linkers, and then inserted into the BamHI site of &AR in the presence or absence of ljARK-W'LOR. Wild typr PARK, pCW-neo to yield the vector pCMV-neo-&4RK-K220R.REAS-2B cells PARK-K220R. and G protrin B y subunits wcrr purifird a s drscrihrd weretransfectedwithHindIII-linearized pCMV-neo or pCMV-neo- under "Experimental Procedures." Hamster &AR were expressrd. pupARK-K220R using DOTAPTn following the manufacturer's instrucrified, and reconstituted into phosphatidylcholinevesicles as drscrihed tions (Roehringer Mannheim). Stably transfected clones were selected previously 121). Phosphorylation reactions contained 20 mw Tris-HCI. with 200 pg/ml G418 and then isolated using cloning rings. Expression pH 7.5. 2 mw EDTA, 100 pw ly-:"PIATP ("1 cpdfmol). 10 mw MgCI,. 1 mw sodium phosphate,20 pw (-J-isopmterenol, and 0.25pmol of IjBR in of PARK-K220R in the various clonal lines was assessed by Western blot a total volume of 100 pl. In addition, 10 nw wild type PARK. 150 ny analysis using polyclonal anti-PARK antibodies generated in rabbits PARK-K220R, and 100 nM G protein B y subunits wrre presrnt where against purified recombinant bovine BARK. CAMP Assays-Wild type,pCMVneo-transfected,and pCMV-neo- noted. Reactions were incubated for 20 min a t 30 -C and qurnchrd hy addition of 1 mlof cold 0.1 w sodium phosphate. pH 7.2. 10 m y EDTA PARK-K220R-transfectedBEAS-2R cells were grown to 9045% conflubuffer. The receptors were pelleted by centrifugation a t 100.000 rprn for encein 24-well plates,washedwithphosphate-bufferedsaline,and 20 min, resuspended in SDS sample buffrr. and then rrsolvrd by SDSsubsequently incubated in 0.5 mlof serum-free Ham's F-12 medium polyacrylamide gel electrophoresis and visualizrd hy autoradiography. containing 0.3 mM ascorbic acid, 1 mM isobutylmethylxanthine, 4 mM Imnes 1 3 were exposed for 18 h. while lanes4 4 were rxposrd fnr 10 h. MSI,, and eitherno agent. 1 p~ (-)-isoproterenol, 1 pw PGE,. or 100 p~ B. phosphorylation a t various ratios ofwild typr and mutant BARK forskolin. Reactions were stopped following 0-60 min of incubation a t in the presence or absence of B y subunits, Assay conditions wrrr idrn37 "C by addition of 0.25 ml of 1.2% perchloric acid, and the samples ) or presencr ( 0 ) tical to those described ahove either in the ahsrnce were then neutralized with 0.5 ml of 7.50 sodium bicarbonate. CAMP of 100 nM B y subunits. Wild t-ype BARK was kept at 10 nM, whilr PARKproduction was measured by radioimmunoassay (DuPont NEN). K220R was vaned from 15 to 150 nw. Following gel electmphorrsls and autoradiography. the phosphorylated P.fiR hands were cut and counted. The data represent the average of four indrpendent experirnrnk RESULTS AND DISCUSSION
- .- .- .. .. .. 1
.
-
.
.
.
f3.m
In vitro mutagenesis of the bovine PARK cDNA was used to p f l . We generate a pARK-K220R mutant lacking the invariant lysine tive mutant that competitively interacts with the therefore hypothesized that PARK-K220R might be effective in residuecritical to phosphotransfer.PARK-K220Rwasthen Sf9 insect cells using the bacu-attenuating rapid desensitizationof the P&R-mediated s i p a l overexpressed and purified from lovirus system.In vitro phosphorylation studies using purified transduction in vivo. Overexpression of PARK-K220R in the P&R confirmed the absenceof kinase activity inPARK-K220R human airway epithelialcell line BEAS-2R provided us with a suitablemodelforexaminingthisquestion. BEAS-2R cells (Fig. lA). Lack of kinase activity in PARK-K220R was independent of the presence of G protein p y subunits, which have have been shown to undergo rapid, agonist-specific desensitiby P-agonists or PGE,.' been shown previouslyto activate PARK (22,25). WhilePARK- zationinresponsetostimulation BEAS-2B cells express -8500 &ARs/cell and PARK levels sufto K220Rdidnotphosphorylatethedirectly,itwasfound be an effective inhibitor of wild type PARK phosphorylation of ficiently low to render competition by constitutively overexA typicaltime t h e P&R (Fig. lA ). A 10-fold excess of PARK-K220R inhibited pressed dominant negative mutants feasible. -90% of wild typePARK phosphorylation of t h e &AR (Fig. 1B). course of CAMP production in wild type REAS-2R cells is deHowever, a significant but less pronounced effect of PARK- picted inFig. 2. Forskolin, a direct activatorof adenylyl cyclase. K220R was observed in the presenceof G protein Py subunits stimulated CAMP production ina near-linear manner over the rates of CAMP pro(Fig. 1).In an effortto find a mutant that could more potently examined 60-min time range. However, the inhibit receptor phosphorylationby PARK, an aspartate 317 to duction in response to isoproterenol and PGI?, diminished siaalanine mutant PARK (equivalent to AspLM in the CAMP-de- nificantly after -20 min of stimulation, indicative of desensiand PGE, receptors. Sf9in tization of the pendent protein kinase) was also constructed, expressed The in vitro studies shown in Fig. 1 suggest that ifPARK cells, and purified. While this mutant was also defective in kinase activity, it was found to be less effective than PARK- interacts with G protein Py subunits in the cell, a >IO-fold K220R in inhibiting PARK phosphorylation of the P f l (data - . not shown). R. €3. Penn. S. G . Kelsen. and .J. L. Rrnovic. suhmittrd for puhlicaThese data suggest that PARK-K220R is a dominant nega- tion.
A Dominant Negative @ARKMutant Reduces p#IR Desensitization
13086
0
10
20
30
40
50
60
70
Time (min)
FIG.2. Time c o m e of c A ” production in BEAS-2B cells. Cells grown to 90-95% confluence in 24-well plates were incubated in 0.5 ml of serum-free Ham’s F-12medium containing 0.3m ascorbic acid,1m isobutylmethylxanthine, 4 m MgCl,, and either no addition (O),1 p (-)-isoproterenol (O), 1w PGE, (V), or 100 p forskolin (V).Reactions were stopped at the indicated time by addition of 0.25 ml of 1.2% perchloric acid, and the samples were then neutralized with 0.5 ml of 7.5% sodium bicarbonate. CAMPproduction wasmeasured by radioimmunoassay. The values reported are the means from two independent experiments performed in duplicate.
overexpression of PARK-K220R might be required to significantlyinhibit phosphorylation by endogenous PARK. Indeed, in initial studies using the vector pBC12BI (271, a 3-4fold overexpression of PARK-K220R in BEAS-2B cells had no significant effect on the ability of isoproterenol or PGE, to stimulate CAMP production (datanot shown). To obtain a higher level of PARK-K220R expression, the cDNA was subcloned into pCMV-neo, a mammalian expression vector with a powerful promoter and enhancer(28). Transfection of this construct inBEAS-2B cells generated a number of G418-resistant clones expressing a n -15-20-fold excess of PARK-K220R over endogenous PARK as assessed by Western blotting. Northern blot analysisusing a 3”untranslatedhuman PARKcDNA probe revealed that thelevel of endogenous wild type PARK in these clones was unchanged. Two cell lines, pCMV-neo-KR7 and pCMV-neo-KR8, were selected for further studiesexamining receptor-mediated CAMP production. Fig. 3 depicts the time course of CAMPproduction in response to 1l.m (-)-isoproterenol stimulation of wild type cells, cells transfected with the pCMVneo vector only, and the two clonal pCMV-neo-KR cell lines. Basal levels of CAMPaccumulation were similar ineach of the four lines examined. Stimulation of either wild type or pCMVneo cells resulted ina predicted attenuation of responsiveness to isoproterenol with peak levels of CAMP accumulation achieved in 20-30 min. In contrast, CAMPaccumulation in the two PARK-K220R-containing lines continued throughout the first 45 min of stimulation, attainingpeak levels-2-fold higher than those observed for the wild type and pCMV-neo cells. To ascertain whether thesefindings reflect a true inhibition of desensitization per se as opposed to an inherentvariability among different clones, wild type BEAS-2B cells and multiple lines of pCMV-neo and pCMV-neo-KR were stimulated with 1 p~ (-)-isoproterenol for 45 min and CAMP accumulation was measured (Fig. 4). In agreement with results from the time course experiments, production of CAMPin pCMV-neo-KR lines was consistently 2-2.5-fold greater than that observed for either wild type or pCMV-neo lines. Interestingly, stimulation of clonal lines with 1 PGE, for 45 min resulted in no differences in CAMPaccumulation between the wild type, pCMV-neo, and pCMV-neo-KR lines tested (Fig. 4). Thus, the effect of PARK-K220R on receptor desensitization does not appear to extend to all GPRs. Moreover, this finding also suggests that the effect of PARK-K220R in intact cells is not mediated by alterations in inherentG,, adenylyl cyclase, or CAMPphospho-
0
0
10
20
30
40
50
60
70
Time (min)
FIG.3. Time c o m e of f3-agonist-stimulatedCAMP production in wild type, pCMV-neo-transfected, and pCMV-neo-fL4R.KK22ORtransfected BEAS-2Bcells. BEAS-2B cells were transfected with the vector pCMV-ne0 or the construct pCMV-neo-PARK-IC22ORas described under “Experimental Procedures.” The cell lines pCMV-neoKR7 and pCMV-neo-KR8 were determined to express an -15-20-fold ratio of the mutantpARK-K22OR compared to the endogenous wildtype PARK. Wild type (O),pCMV-neo (O), pCMV-neo-KR7 (V), and pCMVneo-KR8 (V)BEAS-2B cells werestimulated with 1p (-)-isoproterenol for various periods of time, and CAMPproduction wasthen determined as described in the legend to Fig. 2. To correct for modestexperiment to experiment variability, all CAMP values are reported as the amount of CAMP produced comparedto a 20-min incubation with 100 p forskolin. The values are presented as the means * S.E. from three independent experiments performed in duplicate.
n
n 5
20
0
wl cells
pCMV-neo pCMV-neo-KR
FIG.4. Isoproterenol and PGE, stimulation of C A M P productioninwildtype, pCMV-neo, and pCMV-neo-@RK-K220R BEAS2B cells. Cells wereincubated with either 11.1~(-)-isoproterenol or 1 p PGE, for 45 min before the reactions were quenched as described in the legend to Fig. 2. All values are reported as the mean * S.E. of CAMP produced compared to a 20-min incubation with 100 w forskolin. The values reported are either from wild type BEAS-2B cells (n= 6), five different pCMV-neo-transfectedBEAS-2B cell lines (n = 18 for isoproterenol; n = 12 for PGE,), or six different pCMV-neo-K220Rtransfected BEAS-2B cell lines (n = 19 for isoproterenol; n = 11 for PGE,). The basal levels of CAMP production (-3% of the forskolin level) were subtracted from all values.
diesterase activity. No significant differences were observed among the three rate or magnitude groups of cells in eitherP $ R density or in the of p $ R sequestration thataccompanies rapid desensitization in BEAS-2B cells (data notshown). We have demonstratedpreviously in BEAS-2B cells that agonist-nonspecific mechanisms of
A Dominant Negative @IRK Mutant Reduces desensitization invoked during the first 30 min of treatment produce only small changes in P&R sensitivity without influencing maximal production of CAMP by isoproterenol.' These findings strongly suggest that the PARK-K220R effect on desensitization is mediated by inhibition of rapid, agonist-specific receptor modification, possibly by inhibition of receptor phosphorylation by PARK. Although the success of the dominant negative approach implicates PARK as theprincipal kinase mediating short termdesensitization, the possibility remains that other protein kinase/P&R interactions are also involved in the desensitization process in BEAS-2B cells and that these interactions might also be affected by overexpression of PARKK220R. While this possibility cannot be completelyruled out, we have demonstrated that GRK5 and GRK6, two other members of the G protein-coupled receptor kinase family that arepresent in BEAS-2B cells (data not shown),are probably not involved in P & R desensitization given their reduced ability to phosphorylate the P&R when compared to PARK (29-31). The effect of the PARK-K220R mutant appears restricted to the first -90 min, since more prolonged stimulation (2-4 h) results in minimal differences in CAMPaccumulation between wild type and transfected cells (data not shown). The ultimate desensitization in mutant lines is likely a result of less rapid mechanisms (which mayinclude alterations in any of the signal transduction pathway components)and/or delayed PARK phosphorylation of P-s. In vitro data suggest that the effect of PARK-K220R might be limited by the activation of PARK by endogenous Py subunits in vivo (Fig. 1). Thus, a slower but progressive phosphorylation of P e s by active endogenous PARK might therefore contribute to the ultimate desensitization of the receptor. A more precise elucidation of the mechanism by which PARK-K220R inhibits P&R desensitization awaits adirect assessment of P&R phosphorylation in vivo.We are presently investigating the effect of PARK-K220R expression in other cell lines that express a sufficiently high density of P&R suitable for in vivo receptor phosphorylation studies. Previous in vitroand intactcell studies have definitivelydemonstrated the importantrole of phosphorylation in desensitization of the P$R (32-34). However, elucidation of the role of PARK in P & R desensitization has been limited to in vitroanalysis utilizing either purified proteins or permeabilized cells (26, 35-37). Thus, we report here the first intactcell study directly demonstrating a role for PARK in desensitization of the p&R. Moreover, these results demonstratethe potential utility of using dominant negative mutants to further elucidate the role of G protein-coupled receptor kinases inreceptor desensitization. Acknowledgments-We thank Dr. James Onorato for providing the purified hamster &-adrenergic receptors, Dr. Curtis Hams for providing BEAS-2B cells, Dr. Christopher Richardson for providing the vector pJVPlOE, Chong Kim for supplying purified G protein P-y subunits, and Drs. Rachel Sterne-Marr and VsevolodGurevich for helpful suggestions.
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