Arrestin Specificity for G Protein-coupled Receptors in HumanAirway Smooth Muscle*□S

Received for publication, May 8, 2001Published, JBC Papers in Press, June 20, 2001, DOI 10.1074/jbc.M104143200

Raymond B. Penn‡§, Rodolfo M. Pascual¶�, You-Me Kim‡**, Stuart J. Mundell‡,Vera P. Krymskaya‡‡, Reynold A. Panettieri, Jr.‡‡, and Jeffrey L. Benovic‡

From the ‡Department of Microbiology and Immunology, Kimmel Cancer Institute, Thomas Jefferson University,Philadelphia, Pennsylvania 19107, the ¶Division of Critical Care, Pulmonary, Allergic and Immunological Diseases,Department of Medicine, Jefferson Medical College, Philadelphia, Pennsylvania 19107, and the ‡‡Division ofPulmonary and Critical Care, Department of Medicine, University of Pennsylvania School of Medicine,Philadelphia, Pennsylvania 19104

Despite a widely accepted role of arrestins as “uncou-plers” of G protein-coupled receptor (GPCR) signaling,few studies have demonstrated the ability of arrestins toaffect second messenger generation by endogenouslyexpressed receptors in intact cells. In this study we dem-onstrate arrestin specificity for endogenous GPCRs inprimary cultures of human airway smooth muscle(HASM). Expression of arrestin-green fluorescent pro-tein (ARR2-GFP or ARR3-GFP) chimeras in HASMsignificantly attenuated isoproterenol (�2-adrenergicreceptor (�2AR)-mediated)- and 5�-(N-ethylcarboxam-ido)adenosine (A2b adenosine receptor-mediated)-stimulated cAMP production, with fluorescent micros-copy demonstrating agonist-promoted redistribution ofcellular ARR2-GFP into a punctate formation. Con-versely, prostaglandin E2 (PGE2)-mediated cAMP pro-duction was unaffected by arrestin-GFP, and PGE2 hadlittle effect on arrestin-GFP distribution. The pharma-cological profile of various selective EP receptor ligandssuggested a predominantly EP2 receptor population inHASM. Further analysis in COS-1 cells revealed thatARR2-GFP expression increased agonist-promoted in-ternalization of wild type �2AR and EP4 receptors,whereas EP2 receptors remained resistant to internal-ization. However, expression of an arrestin whose bind-ing to GPCRs is largely independent of receptor phos-phorylation (ARR2(R169E)-GFP) enabled substantialagonist-promoted EP2 receptor internalization, in-creased �2AR internalization to a greater extent thandid ARR2-GFP, yet promoted EP4 receptor internaliza-tion to the same degree as did ARR2-GFP. Signalingvia endogenous EP4 receptors in CHO-K1 cells wasattenuated by ARR2-GFP expression, whereasARR2(R169E)-GFP expression in HASM inhibited EP2receptor-mediated cAMP production. These findingsdemonstrate differential effects of arrestins in alteringendogenous GPCR signaling in a physiologically rele-

vant cell type and reveal a variable dependence onreceptor phosphorylation in dictating arrestin-recep-tor interaction.

Signaling by G protein-coupled receptors (GPCRs)1 is regu-lated by multiple, diverse mechanisms. Among these processesis the well defined phosphorylation of GPCRs by the family ofserine-threonine kinases known as G protein-coupled receptorkinases (GRKs), originally defined by their capacity to specifi-cally phosphorylate agonist-occupied GPCRs and promote re-ceptor desensitization (1). GRK-mediated GPCR phosphoryla-tion induces receptor binding to arrestin molecules, whichserves to sterically inhibit GPCR-heterotrimeric G protein cou-pling and terminate G protein activation. Four members of thearrestin family have been identified: arrrestin-1 (also termedvisual arrestin) and arrestin-4 (cone arrestin) are specificallyexpressed in the visual system and serve to regulate photore-ceptors; arrestin-2 (�-arrestin1) and arrestin-3 (�-arrestin2)are more widely expressed and are involved in the regulation ofnonvisual GPCRs (2). In addition to their role in GPCR desen-sitization, additional functions of arrestins involving GPCRinternalization (3, 4) and resensitization (5), as well as roles intransducing mitogenic signals from GPCRs (6, 7), have beenrecently described.

Numerous studies have examined the role of arrestins inregulating heterologously expressed GPCRs in various celllines. In such model systems, a regulatory role for arrestins hasbeen ascribed in the agonist-dependent internalization or de-sensitization of numerous GPCRs, including the �2-adrenergic(�2AR) (3, 4), �1 adrenergic (8), A2b adenosine (A2bAR) (9), �1b

adrenergic (10), follicle-stimulating hormone (11, 12), neuroki-nin-1 (13, 14), � and � opioid (15, 16), PAR-2 (17), and lutein-izing hormone (18) receptor, and various chemokine receptors(19–22). Conversely, internalization or desensitization of theIP prostacyclin (23), � opioid (15, 24), AT1 angiotensin (25, 26),and m2 muscarinic receptors (27) has been shown to be arres-tin-independent, although such resistance may be cell type-specific (28, 29) or depend on the nature of the stimulatingagonist (24–26).

* This work was supported by Grants HL58506 and GM47417 fromthe National Institutes of Health. The costs of publication of this articlewere defrayed in part by the payment of page charges. This article musttherefore be hereby marked “advertisement” in accordance with 18U.S.C. Section 1734 solely to indicate this fact.

□S The on-line version of this article (available at http://www.jbc.org)contains supplemental material and three videos.

§ To whom correspondence should be addressed: Thomas JeffersonUniversity, Kimmel Cancer Institute, Rm. 930 B.L.S.B., 233 S. 10th St.,Philadelphia, PA 19107. E-mail: rpenn@lac.jci.tju.edu.

� Recipient of a Glaxo Wellcome Pulmonary Fellowship, a MerckYoung Investigator Award, and a Parker B. Francis Fellowship.

** Recipient of an American Heart Association PredoctoralFellowship.

1 The abbreviations used are: GPCR, G protein-coupled receptor;A2bAR, A2b adenosine receptor; ARR2, arrestin-2; ARR3, arrestin-3;�2AR, �2-adrenergic receptor; FACS, fluorescence-activated cell sort-ing; GRK, G protein-coupled receptor kinase; GFP, green fluorescentprotein; HASM, human airway smooth muscle; ISO, isoproterenol;NECA, (N-ethylcarboxamido)adenosine; PGE2, prostaglandin E2; HA,hemagglutinin; CON, control; ELISA, enzyme-linked immunosorbentassay.

THE JOURNAL OF BIOLOGICAL CHEMISTRY Vol. 276, No. 35, Issue of August 31, pp. 32648–32656, 2001© 2001 by The American Society for Biochemistry and Molecular Biology, Inc. Printed in U.S.A.

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Interestingly, the capacity of arrestins to regulate propertiesof endogenously expressed GPCRs in intact cells is relativelyunexplored. Initially, Iacovelli et al. (30) demonstrated thatexpression of wild type arrestin-2 in FRTL5 cells could atten-uate cAMP production mediated via endogenous thyrotropinreceptors. Two recent studies by Mundell et al. (31, 32) haveprovided the only analyses of arrestin specificity for endog-enously expressed GPCRs to date. Expression of antisensemRNA targeting arrestin-2 and arrestin-3 in HEK293 cellsresulted in an �50% decrease in cellular arrestin levels and anincrease in signaling through endogenously expressed �2AR,A2bAR, m1 muscarinic, somatostatin, and prostaglandin E2

(PGE2) receptors, whereas signaling through P2y (1), P2y (2),and AT1 angiotensin receptors was unaffected. By demonstrat-ing that arrestins and arrestin expression levels can be impor-tant determinants of endogenous GPCR signaling, these stud-ies represent an important step toward establishing therelevance of arrestins in regulating receptor-mediated func-tions in the in vivo condition.

In this study we examined arrestin specificity for endog-enously expressed GPCRs in a differentiated, physiologicallyrelevant cell type-human airway smooth muscle (HASM), inwhich GPCRs regulate numerous cellular functions (33). Het-erologous expression of arrestin-2- or arrestin-3-green fluores-cent protein (GFP) chimeras in HASM significantly attenuatedcAMP production mediated by endogenous �2ARs and A2bARs,and agonist-dependent subcellular redistribution of arrestinwas consistent with a role for arrestins in mediating the inter-nalization of activated �2ARs and A2bARs into clathrin-coatedpits. Alternatively, PGE2-stimulated second messenger accu-mulation was independent of arrestin expression, and PGE2

failed to elicit arrestin redistribution in HASM cells. Furtheranalysis using various cell lines expressing recombinant EP2or EP4 PGE2 receptors revealed that internalization of the EP2(but not of the EP4) subtype of PGE2 receptors was resistant toregulation by arrestins. This resistance could be attributed inpart to EP2 receptor phosphorylation state, as a phosphoryla-tion-independent arrestin-2 mutant was capable of promotingboth EP2 internalization and desensitization. Interestingly,arrestin effects on GPCR signaling could not be observed inexperimental models employing receptor overexpression, butwere consistently observed in analyses of endogenous receptorsignaling. These studies demonstrate the specificity of ar-restins for endogenous GPCRs in a physiologically relevant celltype and reveal the disparate regulation of EP2 and EP4 re-ceptors by arrestins that potentially contributes to the differ-ences in receptor function.

EXPERIMENTAL PROCEDURES

Materials—125I-adenosine 3�,5�-cyclicphosphoric acid (2,200 Ci/mmol) was purchased from PerkinElmer Life Sciences. cAMP antibodywas a gift from Mario Ascoli (University of Iowa). pEGFPN1 was pur-chased from CLONTECH (Palo Alto, CA). PGE2, butaprost, 11-deoxy-PGE1, 15-keto-PGE2, and SC-19220 were purchased from CaymanChemicals (Ann Arbor, MI). Anti-HA monoclonal antibody 101R waspurchased from Covance (Richmond, CA). All other reagents were pur-chased from Sigma or from sources described previously (34, 35).

Cell Culture—HASM cultures were established as described byPanettieri et al. (36) from human tracheae obtained from lung trans-plant donors, in accordance with procedures approved by the Universityof Pennsylvania Committee on Studies Involving Human Beings. Char-acterization of these cell lines with regard to immunofluorescence ofsmooth muscle actin and agonist-induced changes in cytosolic calciumhas been reported previously (36, 37). Third to sixth passage cells wereplated at a density of 104 cells/cm2 in either 24 or 48 well (for cAMPaccumulation assays in intact cells) and maintained in Ham’s F-12medium supplemented with 10% fetal bovine serum. Confluent cellswere growth-arrested by refeeding cells with Ham’s F-12 supplementedwith 5 �g/ml each insulin and transferrin (IT medium) for 24 h prior toassay.

Lines of HEK293-EBNA cells stably expressing either the humanEP2 receptor (HEK293EP2) or human EP4 receptor (HEK293EP4)were obtained from J. Regan (University of Arizona) and maintained in250 �g/ml G418 and 200 �g/ml hygromycin B. COS-1 cells were main-tained in Dulbecco’s modified Eagle’s medium supplemented with 10%fetal bovine serum. CHO-K1 cells were maintained in Ham’s F-12supplemented with 10% fetal bovine serum.

Plasmid Construction—Constructs encoding ARR2-GFP, ARR3-GFP, and ARR2(R169E)-GFP were generated by polymerase chain re-action amplification of the open reading frames of bovine ARR2, ARR3,and ARR2R169E (38) (all previously cloned into pcDNA3) and cloningin pEGFPN1 (CLONTECH, Palo Alto, CA) such that the C-terminalGFP sequence was in frame to generate the intended chimera. Plasmidsencoding the EP2 and EP4 receptors fused to an N-terminal 3-HA tagwere generated by polymerase chain reaction amplification of the hu-man EP2 and EP4 open reading frames from pCEP-EP2 and pCEP-EP4(provided by B. Ashby, Temple University) and ligation of the resultantEcoRI/XbaI digests into a pcDNA3 vector containing a 3-HA cassetteimmediately upstream of the EcoRI site (provided by T. Som, ThomasJefferson University). For all constructs, orientation, in frame align-ment, and sequence were confirmed by dideoxynucleotide sequencing.

Transfection Procedures—HASM cells seeded onto 15-cm plates weretransfected as described previously (39) by addition of a HEPES-basedCaPO4 mixture containing 10 �g of carrier DNA, and 30 �g of eitherpEGFP, pARR2EGFP, pARR3EGFP, or pARR2(R169E)EGFP. HASMcells expressing GFP were subsequently sorted to �99% purity byfluorescence-activated cell sorting (FACS) using a Coulter Epics EliteESP Flow Cytometer as described previously (39). Following sorting,cells were plated at a density of 3 � 104 cells/cm2 in 48-well plates andgrown in Ham’s F-12, 10% FBS. 24 h later cells were refed IT mediumfor 24 h, then subsequently stimulated as described below. Determina-tion of protein density per well was assessed in parallel wells using theBradford assay (40).

HEK293EP2, HEK293EP4, and COS-1 cells were transfected in60-mm plates with Fugene (Roche Molecular Biochemicals) as de-scribed previously (34). 24 h later, cells were passaged into 24-wellplates (for subsequent ELISA, cAMP assay), six-well plates (immuno-blot analysis of EP receptor, arrestin expression), 60-mm dishes(immunoprecipitation studies), or onto poly-L-lysine-coated coverslips(immunocytochemical localization of EP receptors, arrestin-GFPchimeras).

Stable Selection—HASM cells transfected as described above wereselected with 250 �g/ml G418 starting 48 h after transfection. Subcul-tures were screened for GFP or arrestin-2 expression. For one culturestably expressing ARR2-GFP (ARR2-GFP(A)), untransfected cells fromthe same culture were used for control cells in studies of cAMP accu-mulation. In a second culture stably expressing ARR2-GFP (ARR2-GFP(B)), cells stably expressing GFP (a subpopulation of the parentculture transfected with pEGPN1, grown and passaged in parallel withARR2-GFP(B)) served as control cells.

CHO-K1 cells were transfected with either pEGFP, pARR2EGFP, orpARR2(R169E)EGFP using Fugene as described above for COS-1 cellsand subsequently selected with 250 �g/ml G418. Ten days later cellssurviving selection were sorted by FACS for GFP expression and sub-sequently maintained in G418.

cAMP Assay—Except where noted, cells grown in 24-well plates weregrowth-arrested for 24 h, washed with cold phosphate-buffered saline,and subsequently stimulated with 500 �l of phosphate-buffered salinecontaining 300 �M ascorbic acid, 1 mM RO-20-1724 (phosphodiesteraseinhibitor), and either vehicle (basal), (�)-isoproterenol (ISO), PGE2,butaprost, 11-deoxy-PGE1, 15-keto-PGE2, 5�-(N-ethylcarboxamido)ad-enosine (NECA), or forskolin at the indicated concentrations for 10 minat 37 °C. cAMP was isolated and quantified by radioimmunoassay asdescribed previously (35). For CHO-K1 cells, to minimize promiscuousactivation of receptors (other than the EP4) by PGE2, a slightly lowerconcentration (100 nM) of PGE2 was used, and cells were stimulated inthe presence of 10 �M SC-19220, a specific EP1 receptor antagonist(41). Effects of arrestins on PGE2-mediated cAMP production in eitherHASM or CHO-K1 cells were similar when cells were pretreated for 8 hwith 100 ng/ml pertussis toxin (data not shown). For all cAMP data,values represent cAMP generated per well in response to agonist minusbasal (vehicle-stimulated) values, normalized to the maximal controlvalue. Protein concentration per well was similar between CON andexperimental conditions, and conclusions based on statistical analysiswere not affected by normalization.

Studies Involving Fluorescent Microscopy—Visualization of the ago-nist-induced translocation of arrestin-GFP chimeras in cells was per-formed in real-time using a Nikon Eclipse E800 fluorescence micro-

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scope. Cells transfected with the arrestin-GFP chimeras were passagedonto poly-L-lysine-coated coverslips, growth-arrested, then mounted ona temperature-regulated imaging chamber (Warner Instrument Corp.)equipped with an inlet port for introduction of media/agents. Cells wereobserved using a Plan-Apo 60 �1.40 NA oil immersion objective. Imageswere captured using QED Camera software. Agonist-induced redistri-bution of antibody-labeled (HA-tagged) receptors and ARR-GFP chime-ras was observed in fixed COS-1 cells as described previously (9).

Assay of Receptor Internalization—Effects of ARR2-GFP orARR2(R169E)-GFP expression on agonist-promoted internalization ofHA-tagged (�2AR, EP2, and EP4) receptors were assessed in COS-1cells by ELISA as described previously (9). Preliminary experimentsexamining the kinetics of �2AR, EP2, and EP4 receptor sequestrationdemonstrated that sequestration observed in all groups began to pla-teau at �15 min after agonist addition. Subsequent experiments there-fore focused on the effects of 15-min agonist treatment.

Immunoprecipitation Studies—COS-1 cells transiently transfectedto express GFP, ARR2-GFP, or ARR2(R169E)-GFP, and either HA-tagged EP2 or EP4 receptor, were stimulated for 5 min with vehicle or1 �M PGE2. Cells were then scraped in lysis buffer (50 mM Tris, pH 8.0,100 mM NaCl, 2 mM EDTA, 1% Triton X-100, 10% glycerol, 1 mM

phenylmethylsulfonyl fluoride, 10 �g/ml aprotinin, 10 �g/ml leupeptin)and transferred to microcentrifuge tubes, centrifuged at 30,000 rpmsfor 30 min in a TLA45 rotor, and the resultant supernatant was incu-bated overnight at 4 °C with purified anti-arrestin-2 antibody 178.Samples were then incubated with protein A-agarose for 2.5 h at 4 °C,washed in lysis buffer, and resuspended in SDS-sample buffer forimmunoblot analysis of co-precipitated receptors using anti-HA anti-body 101R.

Data Presentation and Statistical Analysis—Data points from indi-vidual assays represent the mean values from duplicate or triplicatemeasurements. Data are presented as mean � S.E. Statistically signif-icant differences among groups were assessed by either analysis ofvariance with Fisher’s post-hoc analysis (Statview 4.5, Abacus Con-cepts, Berkeley, CA) or by t test for paired samples, with p values � 0.05sufficient to reject the null hypothesis.

RESULTS

To assess the role of arrestins in GPCR signaling in HASMcells, cultures were transiently transfected with plasmids en-coding GFP chimeras of arrestin-2 (ARR2-GFP) or arrestin-3(ARR3-GFP) or with pEGFP (GFP). Because transfection effi-ciency in HASM cultures is low (presently optimized to 20–30%) (39), GFP-expressing cells were subsequently isolated byFACS, resulting in homogeneous populations of cells express-

ing the construct of interest (Fig. 1A). Immunoblot analysis ofsorted populations demonstrated high ARR2-GFP expressionrelative to the (low) endogenous arrestin-2 levels (Fig. 1B).Similarly, ARR3-GFP expression was high, but no endogenousarrestin-3 could be detected in HASM cells. In addition, twoseparate cultures were established under G418 selection toexpress ARR2-GFP with repeated passage. One culture exhib-ited a low level of ARR2-GFP expression (�3–4-fold that ofendogenous arrestin-2), whereas the other exhibited highARR2-GFP expression (Fig. 1C).

As shown in Fig. 2, A and C, expression of ARR2-GFP orARR3-GFP significantly inhibited cAMP accumulation elicitedby either ISO or NECA, implicating both arrestin-2 and arres-tin-3 as effective uncouplers of �2AR and A2bAR signaling inHASM cells. However, neither ARR2-GFP nor ARR3-GFP in-hibited PGE2-stimulated cAMP accumulation (Fig. 2B). A sig-nificantly lower level of ISO- and NECA-stimulated cAMP gen-eration was also observed in two separate lines of HASM stablyexpressing ARR2-GFP when compared with matched controlvalues (Fig. 2, D and F), whereas levels elicited by PGE2 re-mained unaffected (Fig. 2E). Interestingly, the reductions (rel-ative to matched control levels) in cAMP accumulation exhib-ited in the two stable ARR2-GFP lines were similar, despite thedifference in ARR2-GFP expression (Fig. 1C). Arrestin-GFPexpression did not significantly alter cAMP accumulation stim-ulated by forskolin (a receptor-independent activator of adeny-lyl cyclase; data not shown).

Subsequent experiments were performed to analyze ARR2-GFP translocation in transiently transfected HASM cells. Cellsexpressing ARR2-GFP were plated onto poly-L-lysine-coatedcoverslips and mounted in a temperature-regulated imagingchamber for observation of ARR2-GFP localization in real-time.Upon addition of ISO, ARR2-GFP underwent rapid redistribu-tion into punctate vesicles (Fig. 3A), suggesting a rapid inter-nalization of ASM �2ARs with arrestin into clathrin-coated pitsor early endosomes (42). A less profound but still clear redis-tribution of ARR2-GFP was observed upon addition of NECA(Fig. 3B), suggesting an arrestin-mediated internalization ofA2bARs. However, PGE2 failed to promote any redistribution of

FIG. 1. Expression of arrestin-GFP chimeras in transient and stably transfected HASM cells. A, HASM cells were transfected withpcDNA3 vector (Mock), pEGFPN1 (GFP), ARR2-GFP, or ARR3-GFP and analyzed for fluorescence using a Coulter Epics Elite ESP FlowCytometer. The population of cells exhibiting fluorescence greater than that established in mock-transfected cells (autofluorescence) was sortedand subsequently plated for analysis of arrestin expression, receptor-mediated cAMP production, or agonist-dependent ARR2-GFP localization asdescribed under “Experimental Procedures.” B, populations of ARR2-GFP- or ARR3-GFP- expressing cells isolated by FACS were plated onto12-well plates. Four days later cells were harvested, and ARR2-GFP and ARR3-GFP expressions were assessed by immunoblotting using thepolyclonal antibodies 178 (specific for arrestin-2) and 182 (specific for arrestin-3). C, two separate lines expressing ARR2-GFP were also establishedby selection with G418, with one line expressing a low level of ARR2-GFP (ARR2-GFP(A); �3–4-fold of endogenous arrestin-2 observed inuntranslated cells (UNT)) and another line expressing a high level of ARR2-GFP (ARR2-GFP(B); �100-fold of endogenous arrestin-2). Differentlengths of autoradiograph exposure account for differences in intensity of bands representing endogenous arrestin-2.

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ARR2-GFP (Fig. 3C); subcellular ARR2-GFP localization re-mained unchanged in cells observed up to 2 h following PGE2introduction. Cells stimulated with PGE2 remained responsiveto subsequent stimulation with ISO (Fig. 3C, far right panel).

Two different receptor subtypes of the PGE2 receptor family,

EP2 and EP4, are known to couple to Gs and stimulate adeny-lyl cyclase (43). These subtypes are distinguished by theirstructural, pharmacological, and functional features. The hu-man EP2 receptor possesses a short third intracellular loop anda short C-terminal tail, is responsive to the agonist butaprost,and is resistant to agonist-induced short term desensitization(44, 45). The EP4 receptor possesses a long third intracellularloop and a long C-terminal tail, is unresponsive to the agonistbutaprost, and undergoes rapid agonist-induced desensitiza-tion (45, 46). We characterized EP subtype expression inHASM cultures by examining cAMP accumulation stimulatedby PGE2 (equally selective for EP2 and EP4), butaprost (selec-tive for EP2), 11-deoxy-PGE1 (EP2/EP4-selective), and 15-ke-to-PGE2 (EP2-selective with slight activity at the EP4 receptor)(43) (Fig. 4). The dose-dependent response of HASM cells tothese compounds is consistent with a predominately EP2 pop-ulation of receptors, with the response to butaprost and 11-deoxy-PGE1 relative to that to PGE2 similar to that observed inHEK293 cells expressing recombinant human EP2 receptors(44). However, the relative efficacy of 15-keto-PGE2 is less thanthat reported for EP2 receptors (45), suggesting that a low levelof other PGE2-responsive receptors may be expressed inHASM. PGE2 did not stimulate phosphoinositide production ora calcium transient in HASM (data not shown), suggesting alack of EP1 receptors. Forskolin-stimulated cAMP generationwas virtually unaffected by sulprostone (an EP3 receptor ago-nist) (data not shown). However, PGE2-stimulated cAMP gen-eration in HASM is significantly increased by pertussis toxinpretreatment, and chronic sulprostone treatment caused asmall Gi-dependent sensitization of adenylyl cyclase,2 suggest-ing that EP3 receptors or possibly other Gi-coupled receptorsresponsive to PGE2 are expressed in HASM.

These data suggest that the EP2 receptor is largely respon-sible for PGE2-mediated signaling in HASM, and a lack ofagonist-promoted arrestin binding explains in part the resist-ance of this receptor to rapid homologous desensitization. To

2 R. M. Pascual and R. B. Penn, unpublished observations.

FIG. 2. Specificity of arrestin-GFP chimeras in Gs-coupled receptor signaling. A–C, HASM cultures were transiently transfected withplasmids encoding ARR2-GFP (ARR2), ARR3-GFP (ARR3), or with pEGFP (CON), and GFP-positive cells were subsequently isolated by FACS andplated at high density onto 48-well plates. Following growth arrest, cells were stimulated with 1 �M ISO, 100 �M NECA, or 1 �M PGE2 for 0–10min. cAMP was isolated and quantified by radioimmunoassay as described under “Experimental Procedures.” Maximal values in GFP-expressing(CON) cells (pmol cAMP/well, mean � S.E.): ISO, 11.5 � 1.8; PGE2, 30.2 � 5.5; NECA, 6.6 � 1.5. D–F, two separate HASM cultures stablyexpressing ARR2-GFP as described in the legend Fig. 1 were plated onto 48-well plates for analysis of cAMP as in A. CON represents the matched,untransfected culture for ARR2-GFP(A) or the matched culture expressing GFP for ARR2-GFP(B). Maximal values in CON cells (pmol cAMP/well,mean � S.E.): ISO, 14.6 � 3.6; PGE2, 38.4 � 7.9; NECA, 8.3 � 2.5. Values for 100 �M forskolin-stimulated cAMP production were not differentamong groups. Data represent mean � S.E. of four paired observations for A, B, and D—F and three paired observations for C. *, p � 0.05,ARR2-GFP or ARR3-GFP group versus matched control group.

FIG. 3. Agonist-dependent redistribution of ARR2-GFP inHASM cells. Cells transiently transfected to express ARR2-GFP wereplated at low density onto poly-L-lysine-coated coverslips and growth-arrested in serum-free medium. Coverslips were mounted in a 37 °Cchamber into which various agonists could be delivered, and real-timeanalysis of ARR2-GFP distribution could be observed. A, images ofARR2-GFP-expressing HASM cells before (left panel) and 30 s (middlepanel) or 10 min (right panel) after the addition of 1 �M ISO. B, imagesobtained before (left panel) and 10 (middle panel) or 30 min (right panel)after the addition of 100 �M NECA. C, images obtained before (leftpanel) and 30 min after the addition of 1 �M PGE2 (middle panel). After30 min of stimulation with 1 �M PGE2, the chamber was flushed, andfresh medium containing 1 �M ISO was added. Twenty min later, theimage represented in the right panel was captured. Supplemental vid-eos (showing real-time redistribution of ARR2-GFP in response to ISO,NECA, and PGE2 in HASM) are available at http://www.jbc.org.

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further establish the relationship between EP receptor signal-ing/internalization and arrestin-2, we analyzed heterologouslyexpressed EP2 and EP4 receptors in two separate cell lines. InHEK293 cells stably expressing the human EP2 (HEK293EP2),PGE2 failed to promote punctate formation of expressed ARR2-GFP (Fig. 5A). Conversely, in HEK293 cells expressing the EP4receptor (HEK293EP4) PGE2 was observed to induce ARR2-GFP redistribution, although the kinetics varied among cells(Fig. 5B). To further explore the selectivity of arrestins for EP2and EP4 receptors, we generated constructs encoding the hu-man EP2 or EP4 receptor, each containing an N-terminal 3-HAtag. These constructs were transiently expressed in COS-1 cellswith either ARR2-GFP or the arrestin mutant ARR2(R169E)-GFP. The R169E mutant of arrestin-2 has been previouslycharacterized to bind to agonist-activated �2ARs in a phospho-rylation-independent manner (38). Prior to stimulation withPGE2, both the EP2 and EP4 receptors were primarily localizedto the plasma membrane, whereas ARR2-GFP and ARR2(R169E)-GFP were more diffusely distributed with a tendencytoward nuclear/perinuclear localization (Fig. 6). Followingstimulation with PGE2, the EP2 receptor co-expressed withARR2-GFP tended to remain localized at the plasma mem-brane, although some clustering was observed. This clusteringwas more frequently observed in cells expressing ARR2(R169E)-GFP. Distribution of ARR2-GFP was largely unaf-fected by PGE2 addition. In some cells, a clear redistribution ofARR2(R169E)-GFP coinciding with clustered EP2 receptorswas observed, but in most cells ARR2(R169E)-GFP remaineddiffuse with only a small degree of redistribution that co-local-ized with the receptor. Conversely, PGE2 addition caused aclear redistribution of EP4 receptors into punctate vesicles thatco-localized with redistributed ARR2-GFP and ARR2(R169E)-GFP.

In parallel experiments, ARR2-GFP and ARR2(R169E)-GFPeffects on �2AR, EP2, and EP4 receptor internalization inCOS-1 cells were assessed by ELISA. Both the �2AR and EP4receptor exhibited significant agonist-mediated internalizationin the absence of heterologously expressed arrestins (i.e. incontrol cells expressing GFP), whereas the EP2 receptor did notinternalize (Fig. 7). ARR2-GFP significantly increased inter-nalization of both the �2AR and the EP4 receptor, but did littleto promote EP2 receptor internalization. Expression ofARR2(R169E)-GFP increased �2AR internalization to an evengreater extent than did ARR2-GFP, but had essentially thesame effect as ARR2-GFP on EP4 receptor internalization.Interestingly, ARR2(R169E)-GFP dramatically promoted EP2receptor internalization, suggesting that (a lack of) EP2 recep-tor phosphorylation limits the capacity of the EP2 receptor tointeract with and be regulated by arrestins.

To explore the relative effects of ARR2-GFP and

ARR2(R169E)-GFP on signaling via the EP2 and EP4 recep-tors, we examined agonist-mediated cAMP production in: 1)HEK293EP2 and HEK293EP4 cell lines transfected with ei-ther of the arrestin constructs at up to 70% efficiency or 2)COS-1 cells co-transfected with HA-tagged �2AR, EP2, or EP4receptor, and ARR2-GFP or ARR2169EGFP. Despite demon-strated effects of ARR2-GFP on �2AR signaling in HASM (Fig.2), of ARR2-GFP on �2AR and EP4 internalization (Fig. 7), andof ARR2(R169E)-GFP on internalization of the �2AR, EP2, orEP4 receptors (Fig. 7), we observed no effect of either ARR2-GFP or ARR2(R169E)-GFP on cAMP production mediated byany of the overexpressed receptors (data not shown). We there-fore explored alternative models to examine signaling regula-tion of the EP2 and EP4 receptor. PGE2-mediated cAMP pro-duction in CHO cells has been previously attributed toendogenously expressed EP4 receptors (47). Moreover, CHOcells are known to express relatively low levels of arrestins (48),rendering them a suitable system for examining the effects ofheterologously expressed arrestins. In CHO-K1 cells we ob-served a significant cAMP response to 100 nM PGE2 (�3-foldbasal levels at 10 min), yet no response to butaprost. In thesecells, cAMP production mediated via endogenous EP4 receptorswas significantly decreased by expression of ARR2-GFP (22 �7%, p � 0.05) or ARR2169EGFP (18 � 6%, p � 0.05) (Fig. 8A),and PGE2 promoted a punctate formation of ARR2-GFP (Fig.8B) as well as of ARR2(R169E)-GFP (not shown).

To assess the ability of ARR2(R169E)-GFP to regulate EP2receptor signaling, we revisited our model of EP2 receptorsignaling in HASM cells. Expression of ARR2(R169E)-GFP, butnot of ARR2-GFP, caused a small (21 � 5%, p � 0.05) butsignificant decrease in PGE2-mediated cAMP production (Fig.9A). ARR2(R169E)-GFP caused a slightly greater decrease inISO-mediated cAMP production than did ARR2-GFP. Of note,PGE2-induced punctate formation of ARR2(R169E)-GFP wasmore readily observed in HASM cells (Fig. 9B) than in COS-1cells expressing wild type EP2 receptor, perhaps reflectinglower levels of endogenous arrestins in HASM cells.

Given the large disparity in the effects of ARR2(R169E)-GFPon the EP receptors, we examined the interaction of ARR2-GFPand ARR2(R169E)-GFP with EP2 or EP4 receptor in intactcells. After 5-min stimulation with vehicle or 1 �M PGE2,ARR2-GFP or ARR2(R169E)-GFP was immunoprecipitatedfrom COS-1 cell lysates in which HA-tagged EP2 or EP4 recep-tor had been co-expressed (Fig. 10). Interestingly, for EP2receptor we failed to observe a significant effect of activation onthe amount of receptor that co-precipitated with arrestins,whereas only a small effect in ARR2-GFP expressing cells(�2-fold greater amount with activation) was observed with

FIG. 4. Pharmacological profile of EP receptor-selective ago-nists in HASM cells. Growth-arrested, confluent HASM were stimu-lated with PGE2, butaprost, 11-deoxy-PGE1, or 15-keto-PGE2, each atconcentrations ranging from 1 nM to 10 �M in the presence of 1 mM

RO-20-1724 for 10 min at 37 °C. Intracellular cAMP was subsequentlyisolated and quantified by radioimmunoassay as described under “Ex-perimental Procedures.”

FIG. 5. ARR2-GFP redistribution in HEK293 cells lines stablyexpressing EP2 or EP4 receptors. HEK293EP2 (top panels) orHEK293EP4 (bottom panels) cells transfected to express ARR2-GFPwere passaged onto poly-L-lysine-coated coverslips and stimulated forthe indicated duration with 1 �M PGE2. Images were captured at theindicated times.

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EP4 receptor activation (discussed below). However, clear dif-ferences between the EP2 and EP4 receptor were observed withrespect to the relative effects of ARR2-GFP and ARR2(R169E)-GFP in stimulated cells. Although co-precipitation of EP2 re-ceptor was observed with immunoprecipitation of ARR2-GFP, a5–8-fold greater (densitometry analysis of duplicate experi-ments) amount of EP2 receptor co-precipitated withARR2(R169E)-GFP. In contrast, the difference in the amount ofEP4 receptor co-precipitating with ARR2-GFP versusARR2(R169E)-GFP was minimal. Whereas a greater amount ofthe EP4 receptor migrating at �55 kDa was observed to co-precipitate with ARR2(R169E)-GFP, aggregates of the receptor(migrating at �100–150-kDa oligomers (49)) that co-precipi-tated with ARR2-GFP were more abundant, such that thedifference in total co-precipitating EP4 receptor was insignifi-cant (less than 20% in duplicate experiments). Thus, the rela-tive degree of interaction between EP receptors and the arres-tin chimeras suggested by co-precipitation parallels the effectsof the two arrestins on agonist-promoted internalization anddesensitization.

DISCUSSION

The present study demonstrates that alterations in arrestinlevels can differentially affect GPCR signaling in a physiolog-ically relevant cell type. By emphasizing cellular models ena-bling analyses of endogenously expressed GPCRs, we discerned

a capacity of arrestins to inhibit signaling of the �2AR and EP4receptor. Additional studies examining trafficking of epitope-tagged receptors determined that arrestins similarly promoteinternalization of the EP4 receptor. Conversely, our data sug-gest that arrestins are not involved in regulating either EP2receptor signaling or internalization. This failure of arrestinsto regulate the EP2 receptor appears to be related to a lack ofEP receptor phosphorylation, given that a phosphorylation-independent mutant of arrestin-2 is capable of promoting sig-nificant EP2 internalization and desensitization. Moreover, dif-ferences in the ability of phosphorylation-independentarrestin-2 to affect internalization of the �2AR, EP2, and EP4receptor suggest that receptor phosphorylation is of variedimportance among GPCRs in determining arrestin-receptorinteractions.

Numerous studies employing cell-free assays or cellularmodels of receptor overexpression have established a broadspecificity for arrestins in regulating GPCR internalization andsignaling. Although these studies have revealed much aboutthe capacity of arrestins to regulate GPCRs, the relative im-portance of arrestins among the numerous elements of controlsystems that regulate GPCR signaling under true physiologicalconditions remains unknown. Under such conditions it is un-clear whether arrestins are required, facilitating, or redundantin the process of GPCR desensitization. Moreover, it is alsouncertain whether cellular arrestin levels can limit the rateand magnitude of desensitization (and thus signaling) of agiven GPCR, as has been proposed for cellular GRK levels (50).

We have recently begun to address this issue by examiningarrestin specificity for endogenously expressed GPCRs in agiven cell type. In two previous studies (31, 32) we character-ized arrestin specificity among various Gs-, Gi-, and Gq-coupledreceptors in HEK293 cells utilizing an antisense approach toreduce cellular arrestin levels by �50%. In the present studywe were able to demonstrate that increasing cellular levels ofarrestins by as little as �3–4-fold significantly attenuatedcAMP production elicited by either ISO or NECA, suggestingthat in HASM cells, the level of arrestin expression is animportant determinant of agonist-specific desensitization ofthe �2AR and A2bAR. However, our finding that PGE2-medi-ated signaling was not inhibited by increased arrestin expres-sion seemed to contradict the recent findings of Mundell et al.(31) in which PGE2-mediated cAMP production was increasedby arrestin antisense expression in HEK293 cells. We thereforeexamined potential differences in arrestin effects on PGE2 re-

FIG. 6. Visualization of recombi-nant EP2, EP4 receptors, ARR2-GFP,and ARR2(R169E)-GFP in COS-1 cellsupon stimulation with PGE2. Imagesof COS-1 cells expressing either HA-tagged EP2 (upper panels) or EP4 (lowerspanels) receptors and either ARR2-GFP(first and third row panels) or ARR2(R169E)-GFP (second and fourth row pan-els) were captured for cells stimulatedwith vehicle (left panels) or 1 �M PGE2 for15 min (right panels). Prior to stimula-tion, cells were treated with a monoclonalanti-HA antibody. After stimulation, cellswere fixed and permeablized and incu-bated with a rhodamine-conjugated sec-ondary antibody. Receptor distribution wasvisualized with the appropriate rhodaminefilter and arrestin-GFP distribution with afluorescein isocyanate filter set.

FIG. 7. Effects of ARR2-GFP and ARR2(R169E)-GFP on �2AR,EP2, and EP4 receptor internalization. COS-1 cells were trans-fected with constructs encoding GFP, ARR2-GFP, or ARR2(R169E)-GFP, and either HA-tagged �2AR, EP2, and EP4 receptors. A, cells werepassaged onto 24-well plates and stimulated with 1 �M ISO or 1 �MPGE2 for 15 min, and receptor internalization was subsequently as-sessed by ELISA as described under “Experimental Procedures.” Datarepresent mean � S.E. from 8–10 paired observations. *, p � 0.05,ARR2-GFP group versus GFP group; **, p � 0.05, ARR2(R169)-GFPversus ARR2-GFP group.

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ceptor subtypes by analyzing trafficking of heterologously ex-pressed EP2 and EP4 receptors. Co-expression of ARR2-GFPwas shown to increase agonist-promoted internalization of theEP4 receptor, but had little effect on the EP2 receptor, whichfailed to sequester after exposure to agonist. In addition, ago-nist treatment caused a rapid redistribution of the EP4 recep-tor into punctate vesicles that co-localized with ARR2-GFP, butwas largely ineffective in promoting the association of the EP2receptor with ARR2-GFP.

To explore potential mechanisms underlying the observedarrestin specificity, we examined the effect of expressingARR2(R169E)-GFP. The ARR2(R169E) mutant was generatedbased on a previously characterized mutation in visual arrestin(51) to test the validity of the model which proposes two pri-mary binding sites of arrestins: an activation-recognition sitethat recognizes the agonist-activated conformation of the re-ceptor and the phosphorylation-recognition site that interactswith GRK-phosphorylated residues of the receptor (52, 53).Generation of the R169E mutation in arrestin-2 reverses thecharge of the phosphorylation-sensitive trigger in arrestin-2,resulting in the ability of ARR2(R169E) to bind and desensitizeactivated �2AR regardless of its phosphorylation status (38).Because the EP2 receptor has a short C-tail, one potentialexplanation for its desensitization- and internalization-resis-

FIG. 10. Association of arrestins with EP receptors in immu-noprecipitates. Cells were stimulated for 5 min with vehicle or 1 �MPGE2, and ARR2-GFP (ARR2) or ARR2(R169E)-GFP (ARR2(R169E)was immunoprecipitated from cell lysates incubated with anti-arres-tin-2 polyclonal antibody 178 as described under “Experimental Proce-dures.” Blots of immunoprecipitated proteins were probed with ananti-HA antibody to assess cellular interaction of arrestin chimeraswith each of the receptors.

FIG. 8. Effects of arrestin expression on endogenous EP4 receptor-mediated signaling in CHO-K1 cells. A, stable cell lines of CHO-K1expressing GFP, ARR2-GFP, or ARR2(R169E)-GFP were passaged onto 24-well plates and subsequently stimulated with 100 nM PGE2 in thepresence of 10 �M SC-19220 and 1 mM RO-20-1724 for 10 min at 37 °C. cAMP was isolated and quantified as described under “ExperimentalProcedures.” Data represent mean � S.E. from seven to eight paired observations. *, p � 0.05, ARR2-GFP group versus GFP group; **, p � 0.05,ARR2(R169)-GFP versus ARR2-GFP group. B, stable cell lines of CHO-K1 cells expressing ARR2-GFP were grown on poly-L-lysine-coatedcoverslips and stimulated with 100 nM PGE2 for 15 min, resulting in redistribution of ARR2-GFP into a punctate formation. Similar effects of PGE2were observed in CHO-K1 cells expressing ARR2(R169E)-GFP (not shown).

FIG. 9. Effects of ARR2(R169E)-GFP expression on agonist-stimulated cAMP production in HASM cells. A, HASM cells expressingGFP, ARR2-GFP, or ARR2(R169E)-GFP (transfected then enriched by FACS as described under “Experimental Procedures”) were stimulated in48-well plates with vehicle, 1 �M ISO, or 1 �M PGE2 in the presence of 1 mM RO-20-1724 for 10 min at 37 °C. Intracellular cAMP was isolatedand quantified by radioimmunoassay as described under “Experimental Procedures.” Data represent mean � S.E. from 6–7 paired observations.*, p � 0.05, ARR2-GFP group versus GFP group. B, HASM cells expressing ARR2(R169E)-GFP seeded onto poly-L-lysine-coated coverslips werestimulated with 1 �M PGE2 for 15 min, resulting in redistribution of ARR2(R169E)-GFP into a punctate formation.

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tant nature could be its inability to be phosphorylated byGRKs, which may in turn limit its affinity for arrestins. Basedon the demonstrated ability of ARR2(R169E) to rescue thehomologous desensitization of a truncated � opioid receptorlacking GRK phosphorylation sites (38), we hypothesized thatARR2(R169E)-GFP would be able to associate with the EP2receptor and promote its agonist-dependent internalization.Indeed, this was the case as ARR2(R169E)-GFP could bindactivated EP2 receptor (Fig. 10), promote its internalization(Fig. 7), and attenuate EP2 receptor signaling (Fig. 9). Therelative inability of ARR2-GFP to promote any of these effectssuggests that the lack of phosphorylation recognition mini-mizes arrestin-2-EP2 receptor interaction and thus the role ofarrestin in EP2 receptor regulation.

Interestingly, we also observed disparate effects ofARR2(R169E)-GFP on the �2AR and EP4 receptor. For the�2AR, ARR2(R169E)-GFP promoted a greater degree of agonist-dependent internalization than did ARR2-GFP. This finding isconsistent with previous findings demonstrating that in thepresence of GRK3, phosphorylation-independent arrestin-2 in-duced a more rapid desensitization of the �2AR than did wildtype arrestin-2, suggesting that ARR2(R169E) actually bindsmore readily to the phosphorylated �2AR (38). However, thisproperty of ARR2(R169E) does not appear to extend to the EP4receptor, as we found ARR2(R169E)-GFP was essentially equalto ARR2-GFP in promoting its internalization. Moreover,ARR2(R169E)-GFP was only marginally better than ARR2-GFP in associating with EP4 receptor based on co-precipitationanalysis. Previous studies have demonstrated agonist-pro-moted phosphorylation of the EP4 receptor (49) and a require-ment of C-tail phosphorylation sites in EP4 receptor homolo-gous desensitization (54). However, a recent study by Desai etal. (55) demonstrated that mutation of numerous potentialGRK phosphorylation sites in the EP4 receptor C-tail did notalter agonist-induced internalization, suggesting that phospho-rylation may be unimportant in EP4 receptor internalization.In light of this finding, our data suggest that arrestins cannotmake use of or do not require the phosphorylation-sensitivetrigger in its interaction with the EP4 receptor and that otherdeterminants dictate arrestin-EP4 interaction. Alternatively,changes in arrestin conformation that occur as a consequenceof phosphorylation recognition and are important in sequentialmultisite arrestin binding (52, 53) may nevertheless occur witharrestin-EP4 interaction regardless of receptor phosphoryla-tion state.

A somewhat curious finding of the present study was ourinability to deduce any effects of arrestin expression on signal-ing in cells overexpressing the �2AR or EP4 receptor. However,we did observe cAMP production via endogenously expressed�2AR (in HASM) and EP4 receptors (in CHO-K1) to be signif-icantly decreased by increased arrestin expression. This latterfinding suggests that the effects of reduced arrestin expressionon PGE2-mediated signaling in arrestin antisense-expressingHEK293 cells (31) reflected altered EP4 receptor signaling.Our inability to detect arrestin effects on signaling via overex-pressed GPCRs suggests that possibly spare receptors mayoverwhelm any effects of desensitization mechanisms onGPCR-Gs-adenylyl cyclase signaling, or perhaps some criticalcompartmentalization effect may be obscured in receptor over-expression models.

Of additional interest is the largely agonist-independent co-precipitation of the arrestin-GFP chimeras observed with theEP2 and EP4 receptors (Fig. 10). This contrasts with the ar-restin-promoted co-localization (Figs. 5 and 6) and internaliza-tion (Fig. 7) of EP2 and EP4 receptors that appeared to belargely agonist-dependent. These differences suggest that ar-

restins might weakly interact with EP2 and EP4 receptors inan agonist-independent manner and that this interactionmight be stabilized by the co-immunoprecipitation conditions.Agonist-independent interaction of arrestins with the EP re-ceptors may reflect an ability of arrestins to stabilize the acti-vated conformation as shown for the �2AR and M2AchR (56) orthe ability of constitutively active arrestins to interact in anagonist-independent manner as observed recently with theM2AChR (57).

In summary, the differing susceptibilities of the �2AR, EP2,and EP4 receptor to arrestin and a phosphorylation-independ-ent arrestin mutant suggest that arrestin-receptor interactionis determined by multiple factors that are of varied importanceamong GPCRs. With respect to the EP2 receptor, resistance toarrestin-promoted desensitization and internalization appearsattributed in part to a lack of EP2 receptor phosphorylation.Last, the demonstrated capacity to assess arrestin-GPCR spec-ificity in a differentiated cell type offers the opportunity toexplore the role of arrestins in regulating discrete cellularfunctions modulated by GPCRs.

Acknowledgments—We acknowledge Andrew Eszterhas and KristinBrodbeck for technical support and Vsevolod Gurevich for helpful dis-cussion in preparation of the manuscript.

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Krymskaya, Reynold A. Panettieri, Jr. and Jeffrey L. BenovicRaymond B. Penn, Rodolfo M. Pascual, You-Me Kim, Stuart J. Mundell, Vera P.

MuscleArrestin Specificity for G Protein-coupled Receptors in Human Airway Smooth

doi: 10.1074/jbc.M104143200 originally published online June 20, 20012001, 276:32648-32656.J. Biol. Chem. 

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