Inhibition of Chemoattractant N-Formyl PeptideReceptor Trafficking by Active Arrestins

T. Alexander Key1,2,3, Charlotte M. Vines1,3,Brant M. Wagener1,3, Vsevolod V. Gurevich4,Larry A. Sklar2,3 and Eric R. Prossnitz1,3,*1Department of Cell Biology and Physiology,2Department of Pathology, and3Cancer Research and Treatment Center, University ofNew Mexico Health Sciences Center, Albuquerque, NM87131, USA4Department of Pharmacology, Vanderbilt UniversityMedical Center, Nashville, TN 37232, USA*Corresponding author: Eric R. Prossnitz,eprossnitz@salud.unm.edu

Recent studies have highlighted the emergence of a class ofG protein-coupled receptors that are internalized in anarrestin-independent manner. In addition to demonstratingthat the N-formyl peptide receptor belongs in this family,we have recently shown that recycling of the receptorrequires the presence of arrestins. To further elucidatemechanisms of arrestin-dependent regulation of G protein-coupled receptor processing, we examined the effects ofaltering the receptor–arrestin complex on ternary complexformation and cellular trafficking of the N-formyl peptidereceptor by studying two active arrestin-2 mutants (trun-cated arrestin-2 [1–382], and arrestin-2 I386A, V387A,F388A). Complexes between the N-formyl peptide receptorand active arrestins exhibited higher affinity in vitro thanthe complex between the N-formyl peptide receptor andwild-type arrestin and furthermore were observed in vivoby colocalization studies using confocal microscopy. Toassess the effects of these altered interactions on receptortrafficking, we demonstrated that active, but not wild-type,arrestin expression retards N-formyl peptide receptor inter-nalization. Furthermore, expression of arrestin-2 I386A/V387A/F388A but not arrestin-2 [1–382] inhibited recyclingof the N-formyl peptide receptor, reflecting an expandedrole for arrestins in G protein-coupled receptor processingand trafficking. Whereas the extent of N-formyl peptidereceptor phosphorylation had no effect on the inhibitionof internalization, N-formyl peptide receptor recycling wasrestored when the receptor was only partially phosphory-lated. These results indicate not only that a functionalinteraction between receptor and arrestin is requiredfor recycling of certain G protein-coupled receptors, suchas the N-formyl peptide receptor, but that the pattern ofreceptor phosphorylation further regulates this process.

Key words: arrestin, desensitization, endocytosis,formyl peptide receptor, G protein-coupled receptor,internalization, phosphorylation, recycling

Received 15 June 2004, revised and accepted forpublication 14 October 2004

The modulation of G protein-coupled receptor (GPCR)

activity, including that of the b2-adrenergic (b2AR), angio-tensin II type 1A, adenosine A2, D2 dopamine, CCR-5, and

vasopressin V2 receptors, is effected through the con-

certed actions of kinases, arrestins, and endocytic proteins

(reviewed in (1–3)). Agonist activation induces the rapid

phosphorylation of serines and threonines in the carboxyl-

terminus and/or intracellular loops of GPCRs by a G

protein-coupled receptor kinase (GRK). Arrestins then

physically prevent the signaling of activated, phospho-

rylated receptors by sterically precluding heterotrimeric G

protein binding. For many GPCRs, arrestin binding also

results in the simultaneous recruitment of endocytic pro-

teins to the plasma membrane followed by translocation of

the receptor–arrestin complex to clathrin-coated pits. As

receptors spatially segregate from both agonists and G

proteins upon internalization, they are further desensitized.

Internalized GPCRs can re-sensitize after ligand and

arrestin dissociation, phosphatase activity, and efferent

trafficking (4). Arrestins therefore likely play a broad

range of roles in the processing of GPCRs, with effects

on signaling, internalization, trafficking and recycling.

In recent years, however, GPCRs have been described that

internalize in an arrestin-independent manner. These include

the m2-muscarinic (5,6), protease-activated-1 (7), gonado-

tropin-releasing hormone (8), and N-formyl peptide recep-

tors (FPR (9–12)). Following agonist binding, these receptors

are phosphorylated by a GRK (13,14). However, unlike class-

ical GPCRs, phosphorylation in some cases may be suffi-

cient for partial desensitization (15,16). Thus, arrestin

binding may not be absolutely required to quench the ago-

nist-stimulated signaling of such receptors. Furthermore,

studies have revealed the presence of a novel endocytic

pathway in the internalization of some of these receptors,

since dominant-negative clathrin, dynamin and arrestin (or a

subset thereof) do not affect receptor kinetics of endocyto-

sis or recycling (5,9). Despite these dissimilarities, as with

classical GPCRs, arrestins do in fact translocate to themem-

brane following agonist stimulation, bind to these GPCRs

and can traffic with internalized receptors (15). Furthermore,

in vitro studies with liganded, phosphorylated receptors in

this class suggest complex interactions of the two proteins

(15–17). Nevertheless, a clear-cut role for arrestins in the

biology of certain receptors remains uncertain (18).

The formyl peptide receptor (FPR) is a chemoattractant

GPCR that couples to a pertussis toxin-sensitive G protein

in leukocytes, where it mediates superoxide formation,

degranulation, and chemotaxis (19). The FPR may be

Traffic 2005; 6: 87–99Copyright # Blackwell Munksgaard 2005

Blackwell Munksgaard doi: 10.1111/j.1600-0854.2004.00248.x

87

considered a nonclassical GPCR, since both desensiti-

zation (at least in vitro) and internalization can proceed

through arrestin-independent mechanisms (9,10,12,15,16).

Furthermore, MAPK signaling, granule exocytosis and

chemotaxis can function through arrestin-independent

processes (9,14,20). Despite these findings, recent stud-

ies with arrestins and phosphorylation-deficient receptors

have illustrated key hallmarks of arrestin-FPR assemblies,

as well as critical determinants underlying ternary complex

formation (15–17). In addition, the FPR binds arrestin-2 and

-3 with near equal affinity (16,21) and associates with

arrestins for prolonged time periods after agonist stimula-

tion and receptor internalization (15). Notwithstanding

these findings, the scope of functions served by arrestins

in the biology of the FPR has remained unclear until very

recently. A new finding from our lab, however, has

revealed that FPR recycling is absent in cells lacking arrest-

ins, providing the first clear evidence for a role for arrestins

in FPR trafficking and a novel role for arrestins in general

(12,18). Furthermore, FPR stimulation in the absence of

arrestins leads to the rapid induction of apoptosis, suggest-

ing additional roles for arrestins (22).

Previous investigations focusing on mechanisms of

arrestin activation have relied on the development and

characterization of activating mutations (23–25), given the

high basal inactivity of the protein (26). In general, these

studies have focused on in vitro receptor binding studies in

the context of various states of GPCR activation and phos-

phorylation, as well as on rates of receptor desensitization.

From that work, an appreciation of the significant intra-

molecular interactions involving the carboxy-terminus and

the phosphate-sensing polar core has emerged. Recently,

however, the study of active arrestin mutants and specifi-

cally their effects on GPCR function and trafficking has

begun to shed light on the mechanisms involved in

arrestin-mediated regulation of certain GPCRs (27–29). In

the case of the b2AR, receptor activation in the presence

of two active arrestin mutants had no effect on internaliza-

tion but accelerated the recycling of the receptor, and in so

doing decreased its long-term degradation (27).

In the current report, we sought to extend our recent

studies that demonstrate a requirement for arrestin in FPR

recycling (12). We initially employed a well-characterized

spectrofluorometric assay of ligand–receptor–arrestin inter-

actions to examine the differences between two activating

mutations in arrestins: arrestin-2–3A, which contains alter-

ations of three bulky hydrophobic residues in the tail of

arrestin, and a truncated form of arrestin (amino acids

1–382). We then utilized a flow cytometric assay to explore

the effects of activated arrestin expression on FPR internal-

ization and recycling. Active arrestins significantly delayed

the internalization of the FPR, including the wild-type and

phosphorylation-deficient receptors. Most surprisingly,

however, we show that arrestin-2–3A, but not truncated

arrestin-2 [1–382], inhibits the recycling of the FPR.

Results

High agonist affinity complexes of arrestin-2–3A with

phosphorylated forms of the FPR

Arrestin-2 contains a series of consecutive amino acids in the

carboxy-terminus with bulky, hydrophobic side chains (Ile386

Val387 Phe388). These residues are hypothesized to bind to the

core of arrestin in the resting state and have been demon-

strated to contribute significantly toward basal inactivity of

the protein (30). Removal of the bulky hydrophobic side

chains of these residues, as with the mutant arrestin-2–3A,

has been shown to lead to phosphorylation-independent

binding to the b2AR, as well as more rapid desensitization

of the b2AR and d-opioid receptor (23). As the FPR can

internalize in an arrestin-independent manner (12), as

opposed to the b2AR (31), we sought to determine the

effects of the activation state of arrestins in the context of

the FPR biology.

In order to determine the affinity of arrestin binding to the

FPR as well as the effect of arrestin binding on ligand

affinity, we employed a soluble spectrofluorometric recon-

stitution assay which assesses reconstitution between a

fluorescent ligand, the FPR and either G proteins or arrest-

ins (16). The system operates on the following bases.

First, the antifluorescein antibody specifically binds to and

quenches the fluorescence of unbound ligand, providing a

direct measurement of the ligand dissociation rate. Second,

ligand dissociation rates depend on the receptor assemblies

formed. Ternary complexes of G proteins and wild-type

arrestins with the nonphosphorylated and phosphorylated

FPR, respectively, display high affinity for agonist. Isolated

receptor, on the contrary, displays low affinity. Lastly, add-

ition of nonhydrolyzable guanine nucleotides induces the

rapid dissociation of G proteins from receptors, which

consequently results in the rapid dissociation of ligand,

since uncoupled receptors exhibit low affinity for agonist.

Conversely, guanine nucleotide addition has no effect on

the stability of FPR-arrestin complexes.

We initially examined the effects of the 3A mutation on

the receptor-ligand dynamics of the phosphorylated, wild-

type FPR. Incubation of the phosphorylated, wild-type

receptor with 10 mM arrestin-3A and fluorescent ligand

leads to the formation of a slowly dissociating complex

(Figure 1A), as previously described for arrestin-2 [1–382],

a truncated form of arrestin-2 that also displays constitu-

tive activation (17). Arrestin-2–3A reconstitution at an

equivalent concentration results in a similar fraction of

slowly dissociating species (Figure 1A). Titration studies

of arrestin-2–3A with the phosphorylated FPR demon-

strate an EC50 of 183� 32 nM (Figure 1C) that closely

resembles binding of arrestin-2 [1–382] (EC50�220nM

(17)) and wild-type arrestin-2 (EC50�600nM (16), see

table 1). Similar to results with truncated arrestins, where

the Bmax of the ligand affinity shift is significantly higher

than for wild-type arrestins, the arrestin-2–3A may bind to

Key et al.

88 Traffic 2005; 6: 87–99

a greater subset of a heterogeneously phosphorylated

receptor population, activating receptors to the same

degree. Alternatively, it may bind to the same subset of

phosphorylated receptors as the wild-type protein but acti-

vate them to greater degree. As with arrestin-2 [1–382],

there is no evidence of activation-independent reconstitu-

tion (i.e. FPR-arrestin complex formation in the absence of

ligand) in the case of the soluble phosphorylated FPR (data

not shown), in contrast to studies with other GPCRs (23).

We have previously demonstrated that two phosphor-

ylation-deficient mutants of the FPR are incapable of

binding to wild-type arrestins either in vivo or in vitro,

although they are fully capable of signaling and undergoing

internalization (15). These FPR mutants, termed DA and

DB, contain substitution serines and threonines in the

carboxy terminus of the FPR with nonphosphorylatable

residues (alanine and glycine). In a recent report, we char-

acterized the binding of arrestin-2 [1–382] to both the phos-

phorylated DA and DB receptors (17). In the current study,

we sought to determine whether the arrestin-2–3A protein

was similarly competent at binding to and activating these

phosphorylation-deficient receptors.

The DA receptor contains targeted substitution of a cluster

of four serines and threonines between amino acids

328–332 of the FPR. In response to prolonged agonist

exposure, the DA receptor exhibits minimal desensitiza-

tion, as determined by whole cell calcium mobilization

and GTPase assays, despite displaying full proficiency in

terms of signaling and internalization (11). Similar to results

with truncated arrestin-2, incubation of the phosphorylated

DA receptor with arrestin-2–3A results in the formation of

a high affinity complex (Figure 1B), in contrast to an equi-

valent concentration of wild-type arrestin-2 (data not

shown, shown in (17)). This result suggests at the very

least that the arrestin-2–3A, like the truncated protein,

likely acts on a greater subset of the receptor population

than wild-type arrestins, and not simply with greater effect

on the same receptors, since wild-type arrestins display no

detectable binding to this phosphorylation-deficient receptor

(16). Titration studies reveal that the affinity of the arrestin-

2–3A for the phosphorylated DA is approximately equal to

the truncated protein (Kd �2mM, data not shown). Like the

truncated arrestin, therefore, arrestin-2–3A is able to induce

a ligand affinity change in a submaximally phosphorylated

receptor population, despite having a weaker affinity.

Low agonist affinity complexes of arrestin-2–3A with

the FPR

Incubation of the nonphosphorylated, wild-type FPR with

arrestin-2–3A had no discernible effects on ligand affinity

(data not shown), in contrast to results with the phos-

phorylated FPR. Therefore, in order to discriminate

between a lack of binding and a lack of a ligand affinity

shift, we undertook competition studies between hetero-

trimeric G proteins and arrestin-2–3A. We have previously

shown that incubation of the nonphosphorylated FPR with

Figure 1: High agonist affinity complexes of the

phosphorylated wild-type and DA FPR with arrestin-2–3A.

Phosphorylated, wild-type FPR (A) and phosphorylated DAFPR (B) were incubated with 10nM fMLFK-FITC, a fluorescent

agonist, on ice for 90min with binding buffer alone (–), 10mMexogenous arrestin-2 [1–382] (*), or 10mM arrestin-2–3A (^). In

order to assess the relative affinity of the 3A protein for the

phosphorylated, wild-type receptor, dose–response curves were

generated by titration of the mutant arrestin protein from 35nM to

17mM, yielding an EC50 value of 183�36nM (C). In general,

following reconstitution on ice, samples were prepared

immediately prior to spectrofluorimetric analysis by dilution in

room temperature binding buffer and transfer to glass cuvettes.

At 10s, 60nM antifluorescein antibody was added through a

microinjection port, which quenches the fluorescence of unbound

ligand. At 40s in all experiments, excess GTPgS was added,

which disrupts coupling of receptor and G proteins. Block curves (–)

represent the quenching of free fluorescent ligand in the presence

of a large excess of fMLFF. Normalized means are representative

of at least three independent experiments conducted in duplicate.

Active Arrestins Inhibit FPR Trafficking

Traffic 2005; 6: 87–99 89

G proteins results in the formation of a high affinity,

nucleotide-sensitive complex (32), in contrast to ligand–

receptor–arrestin complexes and some ligand–phosphory-

lated receptor–arrestin complexes that exhibit low agonist

affinity (15–17). Since we have also demonstrated that G

protein binding can be blocked by truncated arrestin-2, we

incubated the FPR with arrestin-2–3A and G protein to

assess competition. As shown in Figure 2A, minimal

inhibition of high agonist affinity complex formation was

observed at 10 mM arrestin-2–3A, despite discernible

(� 50%) inhibition by truncated arrestin-2 at the same

concentration. This suggests that the 3A mutant can com-

pete for the nonphosphorylated FPR carboxyl-terminus,

though with lower affinity than the truncated protein.

The DB receptor contains targeted substitution of a cluster

of four serines and threonines between amino acids 334–

339 of the FPR. Despite normal signaling and internaliza-

tion, the DB receptor exhibits partial phosphorylation and

desensitization. Similar to recent results with truncated

arrestin-2 (17), incubation of the phosphorylated DB recep-

tor with arrestin-2–3A had no discernible effects on ago-

nist affinity (data not shown). Since truncated arrestin-2

can bind the carboxyl-terminus of the DB receptor, as

revealed by competition studies with exogenous G pro-

teins (17), we performed competition studies to examine

binding of arrestin-2–3A. Competition of G protein with

arrestin-2–3A did result in the conversion of the receptor

from a high affinity to low agonist affinity state (Figure 2B).

However, the level of competition at 10mM arrestin-2–3A

was somewhat less than with arrestin-2 [1–382] at an

equivalent concentration. Thus, the arrestin-2–3A in vitro

appears to display less phosphorylation-independence for

the FPR than the truncated arrestin protein, consistent with

the results above for the unphosphorylated wild-type recep-

tor. These results suggest that the interaction of the two

forms of activated arrestins with the FPR is distinct, leading

to the possibility that the effects of the two proteins on FPR

function may also be distinct. The affinities of wild-type and

mutant arrestins as well as ligand for the various states of

the FPR are summarized in Table1.

In vivo colocalization of arrestin–GFP with the FPR

To establish the binding characteristics of the arrestin-2–

3A with the FPR in vivo, we used U937 cells stably expres-

sing various forms of the FPR and transiently expressing

arrestin-2–GFP, arrestin-2 [1–382]–GFP, or arrestin-2–3A–

GFP. After a 24-h recovery period, cells expressed

arrestin–GFPs at 2–3 times the level of endogenous

arrestin, as determined by flow cytometric analysis of

anti-arrestin antibody stained cells (data not shown). Trans-

fected cells were stimulated with Alexa-546-labeled formyl

hexapeptide, fixed, and analyzed by confocal microscopy.

As shown in Figure 3, stimulation of the wild-type FPR for

3 and 10min at 37 �C resulted in the colocalization of

arrestin-2–3A and receptors into punctate, internal struc-

tures, consistent with prior results using wild-type

arrestin-2. Stimulation on ice produced neither receptor

clustering nor arrestin colocalization. In agreement with

in vitro binding results, both the DA and DB receptors

exhibited colocalization with the arrestin-2–3A, but not

with the wild-type arrestin, into punctate, endosomal

structures (Figure 4), similar to results with truncated

arrestin-2 (17). Interestingly, a significant fraction of the

arrestin-2–3A–GFP protein seemed to associate with the

plasma membrane in the wild-type, DA and DB FPR

expressing cells. This has not been observed to the

same extent with the wild-type arrestin–GFP (see Figure

3) or arrestin-2 [1–382]–GFP (17). These results confirm

the interaction of the arrestin-2–3A mutant with

Figure 2: Low agonist affinity complexes of the wild-type

and phosphorylated DB FPR with arrestin-2–3A. Given the lack

of discernible reconstitution between arrestins and low agonist

affinity states of putative receptor–arrestin complexes,

competition studies were undertaken between heterotrimeric G

proteins and arrestin-2–3A in order to assess coupling. Solubilized

samples of the nonphosphorylated, wild-type (A) and

phosphorylated DB FPR (B) were incubated with fluorescent

ligand (10nM), 1.0mM bovine brain G proteins, and either buffer

alone (&), 10mM truncated arrestin-2 (*), or 10 mM arrestin-2–3A

(^) for 120min prior to spectrofluorimetric analysis. Addition of

the 3A protein at high concentrations only minimally inhibited the

formation of the high agonist affinity, nucleotide-sensitive G

protein-coupled complexes, in comparison with the truncated

protein, suggesting a greater dependence on the phosphorylation

status of the FPR. Injections of both antifluorescein antibody and

GTPgS were made at 10 and 40 s, respectively. Normalized

means are representative of at least three independent

experiments conducted in duplicate.

Key et al.

90 Traffic 2005; 6: 87–99

phosphorylation-deficient mutants of the FPR but suggest

that the spatial and/or kinetic aspects of active arrestin

binding to the FPR may differ from that of the wild-type

arrestin.

Effects of active arrestins on FPR internalization

A previous study of the b2AR study has suggested that the

truncated ‘active’ form of arrestin translocates to liganded

receptors more quickly and stays associated for longer

than wild-type arrestins (33). To fully assess the effects

of active arrestin expression on FPR trafficking and proces-

sing, we employed a multiparameter flow cytometric

assay, with simultaneous gates for live cells, arrestin–

GFP expression, and a novel Alexa-labeled FPR agonist.

As shown in Figure 5A, overexpression of wild-type

arrestin-2–GFP had no significant effect on the rate of

wild-type FPR internalization in comparison with nontrans-

fected control cells, or non–GFP-expressing transfected

cells (data not shown). We therefore considered nontrans-

fected cells, which represented a significant fraction of our

electroporated samples, as valid, internal controls for the

expression of wild-type arrestins.

In contrast to wild-type arrestins, both arrestin-2–3A–GFP

and arrestin-2 [1–382]–GFP significantly inhibited the rate of

wild-type FPR internalization (Figure 5B). The most dramatic

inhibition was observed at early time points, with 94% and

80% percent inhibition at 3min for the truncated and 3A

mutant arrestin proteins, respectively. The observed inhibi-

tion was dose dependent, since gating on cell populations

with differential levels of arrestin–GFP expression yielded a

positive correlation with the extent of inhibition (Figure 5C).

Active arrestin–GFP chimerae, furthermore, also markedly

inhibited the rate of internalization of both phosphory-

lation-deficient, DA and DB, receptors, in comparison to

nontransfected controls (Figure 6A,B). Wild-type arrestin

overexpression, on the other hand, again had no effect

(data not shown). It therefore appears that in vitro binding

interactions of phosphorylation-deficient forms of the FPR

with activated arrestins can predict FPR internalization

dynamics, as in every case of demonstrable in vitro binding

interactions, inhibitory in vivo effects were observed.

Recent studies have suggested that active arrestins can

mediate the internalization of the b2AR in the absence of

significant receptor phosphorylation (27). In order to test

whether the reduced rate of internalization of the FPR

could be due to active arrestins mediating internalization

of the unphosphorylated FPR, we examined the effects of

active arrestin–GFP chimerae on the internalization of an

unphosphorylatable mutant form of the FPR (designated

DST, in which all the serine and threonine residues of the

carboxy terminus have been replaced with alanine and

glycine residues). This form of the FPR undergoes neither

ligand-mediated internalization nor desensitization in

the presence of endogenous arrestins (13,14). Our results

demonstrate that expression of active arrestin

mutants (arrestin-2–3A and arrestin-2 [1–382]) does not

induce internalization of this active unphosphorylated

form of the FPR (Figure 6C). These results are consistent

with previous conclusions that FPR internalization

Table 1: Arrestin and ligand affinities of FPR complexes a

FPR form Binding partner

G

proteinbWT

arrestin-2

arrestin affinityc

WT arrestin-2

ligand

affinityd

Arrestin-2 [1-382]

arrestin

affinity

Arrestin-2

[1-382]

ligand affinity

Arrestin-2–3A

arrestin

affinity

Arrestin-2 3A

ligand

affinity

WT FPR yes — NDe þ low þ low

Phosphorylated

WT FPR no þþþ med þþþ high þþþ high

(0.6 mM) (0.22 mM) (0.18 mM)DA yes — ND þ low þ low

Phosphorylated

DA yes — ND þþ high þþ high

(2.2 mM) (� 2 mM)DB yes — ND þ low þ low

Phosphorylated

DB yes — ND þþþ low þ/þþ low

(0.34 mM) (�10 mM)

aCompiled from this work and refs. (15–17).bRefers to the ability of the indicated FPR state to form a complex with G protein in vitro and initiate cell signaling in vivo.c— indicates an affinity >> 30 mM and not measurable; þ indicates an affinity >10 mM and weakly measurable; þþ indicates an affinity

between 1 and 10mM; þþþ indicates an affinity < 1mM. Where accurate values are known, they are shown in parentheses.dA qualitative determination inferred from both the direction and magnitude of the shift in the ligand dissociation curve of the indicated

complex. See also footnote a.eNot determinable due to the lack of observable complex formation. By default the receptors exist in a low affinity state for ligand.

Active Arrestins Inhibit FPR Trafficking

Traffic 2005; 6: 87–99 91

requires receptor phosphorylation yet is not mediated by

arrestins (12), in contrast to the b2AR.

Effects of active arrestins on FPR recycling

Given the prior results, one might hypothesize that the

apparent inhibition of internalization could be the result of

an enhanced recycling rate such that active arrestin

expression would lead to more rapid trafficking of internal-

ized receptors back to the cell surface, as has recently

been described for the b2AR (27). Additional support for

this hypothesis lies in the fact that the kinetics of arrestin

binding to the carboxyl-terminus of many GPCRs dictates

their kinetics of recycling (34). However, belying this

hypothesis, high affinity complexes are less likely to

dissociate than low affinity ones. In that sense, ‘active’

arrestins could stay associated with the FPR for longer

time periods, since they are of higher affinity, and there-

fore could retard, not enhance, recycling.

To assess directly the effects of active arrestin expression

on FPR recycling, we used the same flow cytometric

system as employed for internalization assays. However,

after agonist stimulation at 37 �C and washing on ice, cells

were rewarmed to 37 �C in the absence of ligand for vary-

ing times in order to allow trafficking from endosomes

back to the cell surface. As shown in Figure 7, overexpres-

sion of arrestin [1–382]–GFP had no observable effect on

the rate of wild-type FPR recycling in comparison with

nontransfected control cells. Surprisingly, however,

arrestin-2–3A–GFP transfected cells exhibited diminished

recycling rates with the wild-type receptor. Thus, the basis

of retarded internalization rates associated with active

arrestin expression is not accounted for in terms of

Figure 3: Co-localization of

arrestin-2–3A with Alexa546-

labeled wild-type FPR. Wild-type

FPR-expressing U937 cells were

electroporated with DNA for either

arrestin-2–GFP or arrestin-2–3A–

GFP. After 24–48h of recovery,

cells were treated with an Alexa-

568-labeled formyl hexapeptide

for 10min, fixed with parafor-

maldehyde, and observed by

confoca l mic roscopy . The

trafficking of arrestin-2–3A after

stimulation on ice (top row) or at

37 �C for 3min (2nd row from top)

and 10min (3rd row from top) and

wild-type arrestin for 10min at

37 �C (bottom row) was assessed

by imaging of ligand-receptor

complexes (red) and arrestins

(green). Co-localization (yellow) of

arrestins and receptors after

stimulation at 37 �C confirmed the

activation-dependent trafficking of

the proteins into punctate endo-

somes. Trafficking of arrestin-2–3A

to both the plasma membrane and

endosomes was observed for both

theDAandDB receptors, in contrast

to results with wild-type arrestins.

Images are representative of the

majority of transfected cells from

three separate experiments.

Key et al.

92 Traffic 2005; 6: 87–99

enhanced recycling rates. In fact, it is likely that the

diminished recycling rate mitigates the inhibitory inter-

nalization effect. To determine whether the extent of

FPR phosphorylation affected the inhibition of recycling

seen with the wild-type FPR, we tested both the DAand DB FPR mutants for their recycling kinetics upon

expression of activated arrestins. As with the wild-type

FPR, arrestin-2–3A–GFP markedly inhibited the rate of

recycling of the DA receptor, in contrast to nontrans-

fected controls (Figure 7B), unlike truncated arrestin

(Figure 7B) or wild-type arrestin (data not shown)

overexpression. Surprisingly, however, neither active

arrestin inhibited the recycling of the DB receptor

(Figure 7C).

Effects of active arrestins on intracellular localization

of the FPR

Given that the arrestin-2–3A inhibited recycling of the wild-

type and DA forms of the FPR, we sought to determine

whether the intracellular localization of the FPR was

altered in the presence of the arrestin-2–3A mutant. We

have recently demonstrated that arrestins are required for

FPR recycling (12). In addition, we observed that, in

arrestin-replete cells under equilibrium conditions repre-

senting continuous internalization and recycling, the FPR,

its ligand and arrestin display significant, though partial,

colocalization with recycling endosomes, as defined by

the marker Rab11. However, in the absence of arrestins,

the FPR and ligand accumulate in this compartment over

time, suggesting that exit from this compartment requires

arrestin. To examine the effects of active arrestins, we

cotransfected FPR-expressing U937 cells with Rab11–

GFP and monomeric red fluorescent protein chimeras of

wild-type and mutant arrestins and stimulated the cells

with an Alexa-633 derivative of the formylated hexapeptide

(Figure 8). Stimulation of cells expressing wild-type

arresin-2 and Rab11 for 30min yielded FPR in somewhat

enlarged endosomes compared to the 10min point (cf.

Figures 3 and 4). As we have observed before, a fraction

of these endosomes colocalize with the perinuclear recyc-

Figure 4: Co-localization ofGFP-

arrestins with Alexa546-labeled

FPR. DA (top two rows) and DB(bottom two rows) FPR-expressing

U937 cells were electroporated

with DNA for either arrestin-2–

GFP (rows 1 and 3) or arrestin-2–

3A–GFP (rows 2 and 4). After 24h

of recovery, cells were stimulated

with 10 nM Alexa-568-labeled

formyl hexapeptide for 10min,

fixed with paraformaldehyde, and

observed by confocal microscopy.

Colocalization of arrestins and the

phosphorylation-deficient forms of

the FPR was observed only with

the arrestin-2–3A mutant and not

thewild-type arrestin. Trafficking of

arrestin-2–3A to both the plasma

membrane and endosomes was

observed for both the DA and DBreceptors, in contrast to results

with wild-type arrestins. Images

are representative of the majority

of transfected cells from three

separate experiments.

Active Arrestins Inhibit FPR Trafficking

Traffic 2005; 6: 87–99 93

Figure 5: Active arrestins inhibit internalization of the wild-

type FPR. U937 cells stably expressing the wild-type FPR were

transiently transfected with arrestin–GFP constructs. Cells in

serum-free RPMI were stimulated with 10mM fMet-Leu-Phe for

up to 30min at 37 �C. Samples were repetitively washed in ice-cold

PBS and resuspended in PBS with 10nM fNle-Leu-Phe-Nle-Tyr-Lys-

Alexa633. A) Arrestin-2–GFP (*) expression had no discernible

effects on the internalization rate of the FPR, in comparison to

nontransfected control cells (&). We therefore used

nontransfected cells within the electroporated samples as

internal controls representative of the effects of wild-type

arrestin-2 for all subsequent experiments. B) Both arrestin-2 [1–

382]–GFP (~) and arrestin-2–3A–GFP ( .) expression significantly

delay internalization of the wild-type FPR. C) Plot of the extent of

internalization inhibition at the 3min point for the wild-type FPR as a

function of arrestin-2–3A–GFP fluorescence levels, based on gating

of FL1 events. A direct relationship between the level of arrestin-2–

3A–GFP expression and endocytic inhibition is evident. Data are

plotted as normalized means� standard error from at least three

experiments conducted in duplicate.

Figure 6: Active arrestins inhibit internalization of

phosphorylation deficient forms of the FPR. U937 cells

stably expressing the DA (A), the DB (B) or the DST (C) form of

the FPR were transiently transfected with arrestin-2–GFP,

arrestin-2–3 A–GFP, arrestin-2 [1–382]–GFP constructs or

vector only as indicated. Cells were stimulated with 10 mMfMet-Leu-Phe for up to 30min at 37 �C, repetitively washed in

ice-cold PBS and resuspended in PBS with 10 nM fNle-Leu-Phe-

Nle-Tyr-Lys-Alexa633 for analysis. Non-transfected cells

represent those in the transfected sample that did not

express green fluorescence above the level of control

untransfected cells. Data are plotted as normalized

means� standard error from at least three experiments.

Key et al.

94 Traffic 2005; 6: 87–99

ling compartment. A similar pattern was observed with

the arrestin-2 [1–382] mutant. However, although the FPR

was found predominantly in endosomes in cells expressing

the arrestin-2–3A mutant, very few of these endosomes

colocalized with the Rab11 marker. In separate experi-

ments, many of the endosomes were found to colocalize

with Rab5 containing early endosomes (data not shown).

These results suggest that the constitutively active arrestin-

2–3A may block receptor transit from early endosomes to

recycling endosomes.

Discussion

The N-formyl peptide receptor is a chemoattractant GPCR

that binds equally well to arrestin-2 and -3 (16), remaining

associated with arrestins after stimulation and stably traf-

ficking with them into endosomes (15,17). In addition, unlike

the well-characterized b2-adrenergic receptor, the FPR is

capable of undergoing internalization in an arrestin-indepen-

dent manner (9,12,15). We have previously demonstrated,

moreover, that agonist-promoted chemotaxis (14), MAPK

signaling (9) and granule exocytosis (20) can occur independ-

ently of receptor phosphorylation and therefore arrestin

binding, and furthermore, that in vitro desensitization

(uncoupling from G proteins) can occur in the absence of

arrestin binding through phosphorylation alone (15,16).

Therefore, despite numerous studies that detail complex

associations of the two proteins, the roles for arrestins in

the biology of the FPR remained unclear until recently, when

we demonstrated a requirement for arrestins in the recy-

cling of the FPR (12,18).

In the current report, we initially compared the in vitro and

in vivo interactions of an active arrestin point mutant with a

previously characterized truncated arrestin protein to the

FPR (17). Soluble reconstitution studies demonstrated that

the arrestin-2–3A mutant exhibited similar specificity and

affinity compared to the carboxy-terminal truncation

mutant (arrestin-2 [1–382]). Furthermore, experiments

examining in vivo association of the arrestin-2–3A with

both wild-type and phosphorylation-deficient forms of the

FPR were indistinguishable from that of the truncated

arrestin-2 [1–382]. Prior studies with the 3A protein have

demonstrated its capacity for phosphorylation-independent

binding in vitro, as well as for in vivo desensitization of

unphosphorylated forms of the b2AR and d-opioid receptor

(23). Our results therefore extend those findings from

receptors, such as the b2AR and d-opioid receptor, which

only undergo transient plasma membrane-localized

associations with wild-type arrestins (35,36), to GPCRs,

such as the FPR, that exhibit stable endosomal associations

with arrestins.

Subsequent characterization of the effects of activating

arrestin mutations on endocytic processing of the FPR

revealed that active, but not wild-type, arrestins signifi-

cantly inhibited agonist-dependent internalization of

wild-type and phosphorylation-deficient forms of the FPR

in a dose-dependent manner. Although it has recently

been established that the binding of arrestin can differen-

Figure 7: Arrestin-2–3A, but not arrestin-2 [1–382], inhibits

recycling of high agonist affinity receptors. Cells were

stimulated with 10 mM fMet-Leu-Phe for 20min at 37 �C to

internalize the majority of the FPR and then repetitively washed

in ice-cold PBS to remove ligand. Cells were then rapidly warmed

to 37 �C and incubated for up to 30min in order to assess efferent

trafficking of the receptor in the absence of external ligand. Cells

were subsequently pelleted and resuspended in ice-cold PBS with

10 nM fNle-Leu-Phe-Nle-Tyr-Lys-Alexa633 to quantitate cell

surface receptors. Acquisition was otherwise identical to that

for internalization assays. A) Non-transfected control cells (&)

expressing the wild-type receptor recycle at a rapid rate, with

nearly full recovery within 30min. Arrestin-2–3A–GFP expression

(.), but not arrestin-2 [1–382] (~), dramatically inhibited the rate of

recycling for the wild-type (A) and DA (B) receptor, but not for the

DB receptor (B). Normalized data� standard error are plotted from

at least three experiments conducted in duplicate.

Active Arrestins Inhibit FPR Trafficking

Traffic 2005; 6: 87–99 95

tially dictate the kinetics of GPCR recycling as a function of

the density of phosphorylation sites in the carboxy-termi-

nus of the receptor (34), this is the first study that links the

rate of GPCR internalization to the activation state of

arrestin. Furthermore, we demonstrate an inverse correla-

tion between the state of arrestin activity and the rate of

receptor internalization, since active arrestins actually

retarded FPR internalization.

Prior studies of a constitutively active arrestin point mutant,

arrestin-2-R169E, demonstrated that the active arrestin

could augment in an agonist-dependent, GRK-independent

manner, the rate of desensitization of the slowly desensitiz-

ing m-opioid receptor so that it was indistinguishable from

the rapidly desensitizing d-opioid receptor (37). This sug-

gested that it is the activation of arrestin, rather than its

binding, which is the rate limiting step in m-opioid receptor

desensitization. Since both the 3A and truncated arrestin-2

proteins used in our studies are preactivated, we can

hypothesize that the timing of arrestin association with acti-

vated states of the FPR is altered significantly in comparison

with wild-type arrestins. Furthermore, because the phos-

phorylation requirements for internalization of the FPR are

in general more relaxed than for desensitization and arrestin

binding (i.e. partially phosphorylated forms of the FPR, such

as DA and DB, internalize, although they do not bind, wild-

type arrestin), it follows that the process of FPR internaliza-

tion may be initiated temporally before the binding of endo-

genous arrestins. As we have recently shown, arrestin may

bind to the wild-type FPR only after initiation of the inter-

nalization process (45). It is therefore possible that the

activated forms of arrestin, which bind with high affinity to

the partially phosphorylated forms of the FPR, inhibit inter-

nalization by precluding the access of factors that must

interactwith the FPR prior to arrestin binding. In the absence

of specific knowledge regarding the mechanisms of FPR

internalization, however, it is difficult to propose what this

disrupted timing may be altering.

It is also possible that the activated arrestins are binding to

the ligand-bound, yet still unphosphorylated, FPR, thereby

inhibiting internalization. In the case of the b2AR, such an

interaction was demonstrated to permit phosphorylation-

independent internalization of this arrestin-dependent

receptor with unaltered kinetics (27). However, this possi-

bility seems unlikely for the FPR, as we have demon-

strated that an active unphosphorylated form of the FPR

can not be internalized by active arrestins (Figure 6C), that

the FPR internalizes in the absence of arrestins (12) and

furthermore that activated arrestins bind to fully and par-

tially phosphorylated forms of the FPR with 50–100-fold

higher affinity than to the unphosphorylated FPR (this

study and (17)). Although the b2AR preferentially interna-

lizes using arrestin-3 (31), it has been demonstrated to

associate with truncated arrestin-2 more quickly than

with wild-type arrestin and subsequently to traffic intra-

cellularly for prolonged times (33). However, in the case

of the b2AR, expression of active arrestins mediates much

more rapid recycling of the receptor (27). The likelihood

therefore that the inhibition of FPR internalization is a

consequence of decreased phosphorylation, despite an

inextricable link between these two processes, is greatly

diminished. Lastly, since internalization of the low agonist

affinity DB receptor was also significantly impaired, similar

to results with high agonist affinity receptors, we can

conclude that the resulting ligand affinity of the ternary

complex per se does not underlie the inhibitory internaliza-

tion effect. The same conclusion can be drawn with

Figure8: The FPR fails to traffic

t o the Rab11 r e cy c l i ng

compartment in the presence of

arrestin-2–3 A. U937 cel ls

express ing the FPR were

electroporated with Rab11–GFP

(green) and arrestin-2-mRFP1,

arrestin-2–3A-mRFP1 or arrestin-

2 [1–382]-mRFP1 constructs (red).

After 16–24h of recovery, cells

were treated with fNle-Leu-

Phe-Nle-Tyr-Lys-Alexa633 (blue)

f o r 3 0 m i n , f i x e d w i t h

paraformaldehyde, and examined

by confocal microscopy. Whereas

colocalization between a significant

fraction of the FPR with the Rab11

compartment is seenwithwild-type

arrestin-2 and arrestin-2 [1–382],

little colocalization is evident in

cells expressing the non-recycling

arrestin-2–3A mutant. In all cases,

arrestin is seen to colocalize with

the receptor. Images are repre-

sentative of transfected cells from

two separate experiments.

Key et al.

96 Traffic 2005; 6: 87–99

respect to the affinity of the mutant arrestin for the recep-

tor, since the DA mutant displays approximately 10-fold

lower affinity for both arrestin-2–3 A and arrestin-2 [1–382]

compared to the WT FPR. Finally, since the DA and DB

mutants are capable of recycling in the absence of WT

arrestin binding but the WT FPR requires arrestin for recy-

cling (12), we must presume that the extent of phospho-

rylation of the FPR dictates the requirement for arrestin in

recycling.

Perhaps the most puzzling result of our study is that

arrestin-2–3 A, but not wild-type arrestin-2 or arrestin-

2 [1–382], inhibits the recycling of the wild-type and DA,

but not DB, form of the FPR. It has been suggested that

the stability of a receptor–arrestin interaction dictates the

fate of an internalized GPCR (34). In that regard, the for-

mation of a transient receptor–arrestin complex on the

plasma membrane only favors rapid dephosphorylation

and return to the plasma membrane, whereas the forma-

tion of a stable receptor–arrestin complex (as defined

by colocalization on endosomes) retards resensitization

and may favor targeting of the receptor for eventual degra-

dation. Presumably, receptor resensitization requires

endosomal arrestin dissociation in addition to receptor

dephosphorylation. Since the principal difference between

the DA and DB receptors in terms of ternary complex

formation is agonist affinity, it is possible that differences

in complex stability (i.e. ligand affinity) account for the

differences in re-sensitization kinetics. That is, since the

DA receptor binds to arrestin-2–3 A and forms a high ago-

nist affinity complex, such assemblies are on average less

likely to dissociate in an acidic endosomal environment

than low agonist affinity assemblies composed of DB

receptors and are, concomitantly, less likely to recycle. It

should also be noted, however, that the somewhat lower

affinity of the arrestin-2–3 A mutant for the DB receptor

compared with the WT and DA FPR, could allow the for-

mer receptor but not the latter receptors to recycle.

A further explanation may lie in the loss of key regulatory

sites as a result of carboxy-terminal truncation of the

arrestin. The distal tail, which is intact in the 3 A, but not

the truncated arrestin-2 mutant, has been demonstrated to

contain several domains through which arrestins interact

with protein factors, as well as critical sites of regulation.

These protein factors include the b2-adaptin subunit of the

adaptor protein AP-2 (38), which has been shown to func-

tion in classical GPCR internalization (39), as well as

N-ethylmaleimide-sensitive factor, an ATPase which

binds to the distal tip of arrestin and functions in receptor

transport and sorting (40). Furthermore, it has been shown

that dephosphorylation of serine 412 in the tail of arrestin

is necessary for classical receptor–arrestin complexes to

engage the endocytic machinery (41). The truncated pro-

tein also lacks this important phosphorylation site. And

finally, there may be unknown conformational differences,

including sites of ubiquitination (42), between the two

proteins that would account for the differences in recy-

cling. The presence or lack of a critical domain may affect

not only the kinetics of trafficking, but also the endosomal

compartmentalization of internalized assemblies. Our

results suggest that the inhibition of recycling may in fact

result from a defect in the ability of the FPR to traffic

properly from early endosomes to recycling endosomes

in the presence of the arrestin-2–3 A mutant.

In summary, our recent results have demonstrated that

the presence of arrestin is required for FPR recycling (12).

The results presented here extend this observation to

reveal how the activation state of arrestin is a critical

determinant in regulating receptor recycling. Thus, an

arrestin mutant that is perhaps incapable of returning to

its basal inactive state (i.e. dissociating from the receptor)

is capable of blocking intracellular trafficking and thus pre-

vent the recycling of the FPR. Our results suggest that the

carboxy-terminus of arrestin as well as the ability of the

receptor to attain a conformation upon arrestin binding

displaying high affinity for the ligand are required for this

inhibition. These studies further suggest that dissociation

of the GPCR–arrestin complex is a critical determinant in

receptor recycling. It will be of interest to examine

whether disruptions in the timing and quality of arrestin

associations will similarly disrupt the endocytic profile of

other GPCRs that internalize in an arrestin-independent

manner. Lastly, our results underscore the fundamental

differences in the mechanisms involved in the internaliza-

tion and subsequent trafficking of the FPR as compared to

receptors such as classic b2AR.

Materials and Methods

ReagentsfNle-Leu-Phe-Nle-Tyr-Lys-Alexa546 and -Alexa633 were synthesized by

incubating Alexa Fluor-546 and -633 carboxylic acid succinimidyl esters

(Molecular Probes, Eugene, OR) in anhydrous DMSO (containing 100 mM

triethylamine as catalyst) with equimolar fNle-Leu-Phe-Nle-Tyr-Lys (Sigma,

St. Louis, MO) at room temperature overnight (17). GTPgS (guanosine 50-3-

O-(thio)triphosphate) was from Sigma. Frozen stocks were thawed imme-

diately prior to use. Monomeric RFP (mRFP1) was a generous gift from

Dr. Roger Tsien (43).

Cell stimulation and membrane preparationAs previously described, U937 cells stably expressing the FPR were cul-

tured in RPMI at 37 �C with 5% CO2 and passaged every 3–4 days by

reseeding at 2�105 cells/mL. For membrane harvest, cells were expanded

in sealed Pyrex spinner flasks, stimulated for 8 min at 37 �C with 10 mM

fMLF, which results in near maximal receptor phosphorylation, and trans-

ferred to ice (16).

Stimulated cells were collected by centrifugation, resuspended in ice-cold

cavitation buffer (10 mM HEPES, 100 mM KCl, 30 mM NaCl, 3.5 mM MgCl2,

600 mg/mL ATP, pH 7.3), and bombed by nitrogen cavitation for 20 min at

500 psi. Following centrifugation, the membrane fraction was resuspended

in HEPES sucrose buffer (200 mM sucrose, 25 mM HEPES, pH 7.0) with

protease inhibitor and phosphatase inhibitor cocktails (Calbiochem,

San Diego, CA) and flash frozen. Aliquots were stored until use at � 80 �C.

Detergent solubilizationThawed membranes were diluted in an intracellular binding buffer (BB:

30 mM HEPES, 100 mM KCl, 20 mM NaCl, 1 mM EGTA, 0.1% w/v BSA,

Active Arrestins Inhibit FPR Trafficking

Traffic 2005; 6: 87–99 97

0.5mM MgCl2), isolated by centrifugation, and resuspended in BB contain-

ing protease inhibitor cocktail set I, phosphatase inhibitor cocktail, and 1%

n-dodecyl b-D-maltoside (Calbiochem/EMD Biosciences, San Diego, CA).

Following solubilization for 90min at 4 �C, the soluble fraction was collected

by centrifugation for immediate experimentation.

Receptor reconstitutionSolubilized FPR was incubated with either bovine brain Gi/Go heterotrimer,

purified arrestins, or BB, as well as with fluorescent agonist, N-formyl-met-

leu-phe-lys-fluorescein 5-isothiocyanate (10 nM). The liganded samples

were gently mixed at 4 �C for up to 120min. Blocked samples received a

large excess of unlabeled peptide, N-formyl-met-leu-phe-phe (fMLFF), prior

to fluorescent ligand addition.

Spectrofluorometric analysisFluorescence was measured with an SLM 8000 spectrofluorometer (Spec-

tronics, Urbana IL) in time acquisition, photon-counting mode. Excitation

was fixed at 490 nm with a 490 nm band pass filter and the excitation

monochromater. Emission was monitored using a 520 nm band pass inter-

ference filter and a 500-nm-long pass filter. Antibody and nucleotide add-

itions were made through a microinjection port above the sample holder.

Following reconstitution at 4 �C, samples were diluted with room tem-

perature BB with 0.1% n-dodecyl b-D-maltoside and inhibitors, transferred

to glass cuvettes, and placed into the spectrofluorometer with gentle stirring.

For the first 10s, equilibrium levels were obtained. At 10s, excess antifluor-

escein antibodies, as previously described, were added to the sample. The

antibodies rapidly quench the fluorescence of the unbound, fluorescein-con-

jugated ligand. For some samples, excess GTPgS (guanosine 50-3-O-(thio)tri-

phosphate) was added at 40 s in order to disrupt G protein-receptor coupling.

ElectroporationsThe arrestin-2–GFP construct in pEGFPN1 was a generous gift from

Dr. Jeffrey Benovic (Thomas Jefferson University). Mutant arrestin

constructs were generated through PCR amplification using arrestin-specific

primers and verified in their entirety by dideoxy sequencing. For

transfections, DNA (25mg) was added to 8� 106 FPR-expressing U937

cells in serum-free RPMI. Cells were electroporated at 200V, 2000O, with

a 50ms pulse time on a BTX (Holliston, MA) electroporator and transferred to

T25 flasks with serum-containing RPMI. After 24–48h, cells were collected

by centrifugation, and resuspended in serum-free RPMI for assay.

Confocal microscopyTransfected cells were incubated with 10nM fNle-Leu-Phe-Nle-Tyr-Lys-

Alexa546 or 633 for the indicated time at either 0 �C or 37 �C. Stimulated

samples were immediately fixed with ice-cold, 2% paraformaldehyde for

30min. Cells werewashed, resuspended in Vectashield (Vector Laboratories,

Burlingame, CA), and mounted onto glass slides. Fluorescence images were

acquired on a Zeiss LSM510 confocal microscope (Carl Zeiss Inc., Thornwood,

NY, USA) to localize both the FPR (red) and arrestin (green) signals.

Internalization and recyclingTransfected cells in serum-free RPMI were stimulated with 10 mMfMet-Leu-Phe (FPR) or buffer for up to 30min at 37 �C. Samples were

repetitively washed in ice-cold PBS and resuspended in PBS with either

10 nM fNle-Leu-Phe-Nle-Tyr-Lys-Alexa633. Samples were kept on ice until

analysis. Fluorescence measurements were acquired on a Beckman Dick-

inson FACS Calibur. Live cells were initially gated by forward scatter vs.

side scatter plots. GFP-expressing cells were then gated by FL1 histogram

in comparison to untransfected control cells. Finally, the receptor-asso-

ciated fluorescence was resolved FL4 histogram analysis. The mean chan-

nel fluorescence for 50 000 events per sample was collected and analyzed.

For recycling assays, prior to fluorescent ligand/antibody addition, cells

were warmed for up to 30min at 37 �C to allow for receptor recycling,

quenched on ice, and collected by centrifugation. Acquisition was other-

wise identical to that for internalization assays.

Data analysisData were analyzed using FCSQuery (44) and Prism software (Graphpad,

San Diego, CA). In general, data were plotted as normalized fluorescent

intensity for receptor reconstitution or mean channel fluorescence for

internalization and recycling as a function of time. Non-linear regression

analyses were performed to generate dose–response curves and normal-

ized to Bmax values. For repetitive spectrofluorometric reconstitution

assays, sample data were pooled prior to analysis.

Acknowledgments

ThisworkwassupportedbyNIHgrantAI36357 toE.R.P.;NIHgrantsGM60799/

EB00265 and RR01315 to L.A.S., theNewMexico Cancer Research Fund; and

NIH grants EY11500 andGM63097 to V.V.G. Flow cytometry data and confocal

images in this paper were generated in the Flow Cytometry and Fluorescence

Microscopy Facilities, respectively, at the University of New Mexico Health

Sciences Center, which received support from NCRR 1S10 RR14668, NSF

MCB9982161, NCRR P20 RR11830, NCI R24 CA88339, the University of

NewMexicoHealthSciencesCenter, and theUniversity ofNewMexicoCancer

Center. C.M.V. received support from the University of New Mexico Cancer

Research and Treatment Center and is a recipient of an NIH post-doctoral

training fellowship (T32 AI007538). B.M.W. received support from an NIH pre-

doctoral training fellowship (T32 AI007538) and Department of Defense Breast

Cancer Research Program pre-doctoral award (BC030217).

Note added in Proofs

At the time of publication of this article, Dinh et al. (DinhDT, QianH, Seeber R,

Lim E, Pfleger K, Eidne KA, Thomas WG. Helix I of b-arrestin is involved in

post-endocytic trafficking, but is not required for membrane transloca-

tion, receptor binding and internalization. Mol Pharmacol 2004 (in press))

demonstrated that residues L100/L104/L108 in helix I of arrestin, which inter-

acts directly with I386/V387/F388 (the residues mutated in arrestin-2-3A), are

required for trafficking of type 1 Angiotensin II receptors to deep-core (likely

recycling) endosomes. This result is consistent with our conclusion that

disruption of the L100/L104/L108-I386/V387/F388 prevents the FPR from traf-

ficking to recycling endosomes and thus recycling.

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Active Arrestins Inhibit FPR Trafficking

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