GRK Phosphorylation and βarrestin Recruitment Regulate the Constitutive Signaling Activity of the Human

Cytomegalovirus US28 GPCR William E. Miller‡†¶, Daniel A. Houtz‡, Christopher D. Nelson‡, P.E. Kolattukudy¦ , and Robert J. Lefkowitz‡

1The abbreviations used are: GPCR G Protein Coupled Receptor, GRK G protein coupled receptor kinase, β2AR β2 Adrenergic Receptor, IL8R2 Interleukin 8 Receptor Type 2, RGS Regulator of G protein signaling, RANTES regulated on activation normal T cell expressed and secreted. Addresses: Howard Hughes Medical Institute, Departments of Medicine and Biochemistry, Duke University Medical Center, Box 3821, Durham, NC 27710, USA‡, Department of Molecular Genetics, Biochemistry, and Microbiology, University of Cincinnati College of Medicine, Cincinnati, OH 45267†, and Department of Biochemistry, The Ohio State University, Columbus, OH 43210, USA¦ ¶To whom correspondence should be addressed E-mail:william.miller@uc.edu Tel: (513) 558-0866 FAX: (513) 558-8474 Department of Molecular Genetics, Biochemistry, and Microbiology, University of Cincinnati College of Medicine, Cincinnati, Ohio 45267-0524.

Copyright 2003 by The American Society for Biochemistry and Molecular Biology, Inc.

JBC Papers in Press. Published on March 31, 2003 as Manuscript M303219200 by guest on June 16, 2020

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Summary

Phosphorylation of G protein coupled receptors (GPCRs) by GRKs and subsequent recruitment

of βarrestins to agonist occupied receptors serves to terminate or attenuate signaling by blocking G-

proteins from further interaction with the receptors. Human Cytomegalovirus encodes a GPCR termed

US28 that is homologous to the human chemokine family of GPCRs, but differs from the cellular

receptors in that it maintains high constitutive activity in the absence of agonist. Although US28 is

constitutively active, mechanisms that regulate this activity are unknown. We provide evidence that

US28 is constitutively phosphorylated by GRKs in cells and that in consequence, βarrestin2 is

localized to the plasma membrane. Deletion of the carboxy terminal 40 amino acids in US28 generates

a receptor that is severely impaired in its ability to become phosphorylated and recruit βarrestin and

accordingly demonstrates increased inositol phosphate signaling. This result indicates that the carboxy

terminus of US28 contains an important signaling regulatory region and mutational analysis deleting

carboxy terminal serines identified Serine-323 as a critical residue within this region. In addition,

overexpression of wildtype GRK5 leads to hyperphosphorylation of US28 that results in a decrease of

inositol phosphate accumulation. These results are consistent with the hypothesis that GRK

phosphorylation and recruitment of βarrestin to the US28 viral GPCR attenuates signaling to the

traditional Gαq stimulated inositol phosphate pathway. Finally, in contrast to the results with inositol

phosphate signaling, we provide evidence that the US28 carboxy terminal phosphorylation sites and

βarrestin interacting domain is required for maximal activation of the p38 MAP kinase. Taken

together, these results indicate that US28 interacts with these important regulatory proteins to control

multiple aspects of signal transmission. Understanding the regulation of viral GPCRs by GRKs and

βarrestins will likely provide important new insights into not only aspects of viral pathogenesis but also

basic mechanisms of receptor signaling.

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Introduction

G-protein coupled receptors (GPCRs)1 constitute a family of seven-transmembrane domain

proteins that transmit signals to the interior of the cell by activating a variety of signaling pathways (1-

3). Stimulation of GPCRs with agonist leads to increased GTPase activity of heterotrimeric G-proteins

that in turn modulate downstream effectors such as the second messenger generating adenylyl cyclases

and phospholipases. Phosphorylation of the receptors by GPCR kinases (GRKs) and recruitment of

βarrestins to the agonist occupied receptors attenuates signaling by blocking G-proteins from further

interaction with the receptors, a process also known as desensitization (4-7).

Many of the herpesviruses encode seven-transmembrane domain proteins that are homologous

to the cellular chemokine family of GPCRs (8-13). The function of these virally encoded “receptors”

is unknown although speculation has been that they might enable the host virus to access additional

tissue types in vivo and to evade immune surveillance (14-18). This is a particularly attractive

hypothesis as one of the hallmarks of the herpesviruses is their ability to set up a lifelong latent or

persistent infection in the host. The genomes of human cytomegalovirus (HCMV), human herpesvirus

6, human herpesvirus 7, and human herpesvirus 8 all contain one or more open reading frames with

homology to the chemokine receptors. Interestingly, several of these virally encoded GPCRs exhibit

high levels of constitutive activity in the absence of agonist (19-23).

HCMV encodes four GPCR homologues in its genome, US27, US28, UL33, and UL78, while

mouse cytomegalovirus (MCMV) and rat cytomegalovirus (RCMV) encode genes that correspond to

UL33 and UL78 (12,14,16,24). Studies using rodent cytomegaloviruses have indicated that the UL33

counterparts termed M33 and R33 are required for viral dissemination and replication in the salivary

gland and are therefore important for virulence in vivo (14,16). Recent biochemical studies indicate

that the HCMV US28 and MCMV M33 proteins share many common signaling activities indicating

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that US28 may also play an important role in viral replication in vivo (23). Expression of HCMV

US28 on the surface of leukocytes promotes cell rolling and adhesion in in vitro models, while US28

expression in smooth muscle cells stimulates cell migration (25,26). These studies suggest that US28

is important in promoting virus trafficking from the circulation to inflammatory sites and may have

significant effects on HCMV induced diseases such as atherosclerosis.

US28 is closely related to the receptors for β-chemokines such as CCR1 and accordingly US28

binds to several chemokines including RANTES, MCP-1, and Fractalkine (13,24,27,28). Although

US28 binds to these chemokines, the receptor has high constitutive activity even in the absence of

chemokine (19,23). Some reports indicate that binding of RANTES or MCP-1 can increase US28

activity, however this activity seems to vary with cell type as well as specific signaling pathway being

activated (24,29). Similar to other viral encoded GPCRs, US28 constitutively activates Gαq dependent

signaling pathways that stimulate phospholipases leading to elevation of inositol phosphate levels.

US28 signaling also activates mitogen activated protein kinase pathways as well as the CREB and NF-

κB transcription factors although the mechanisms by which these pathways are regulated by US28 is

less clear (19,23,29). US28 appears to be functionally similar to its cellular counterparts in that the

receptor undergoes internalization via the cellular endocytic machinery, however, unlike the cellular

GPCRs that are internalized in response to agonist stimulation, US28 appears to undergo constitutive

internalization (30,31). The data concerning constitutive signaling and constitutive internalization are

consistent with the idea that US28 mimics the agonist occupied form of the receptor. In this respect

US28 appears to function quite similar to cellular GPCRs except that it does so in the absence of

agonist.

Desensitization is a key regulatory mechanism required to control the magnitude and duration

of GPCR signaling (1). The important factors in controlling desensitization are the GRKs and

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βarrestins. Currently there are seven known GRKs (GRK1-GRK7) and four arrestins (βarrestin1,

βarrestin2, rod arrestin, and cone arrestin). The specificity of GRKs and arrestin family members for

different receptors is just beginning to be ascertained but it is clear that the limited number of GRK and

arrestin proteins are responsible for regulating the much larger number of receptor proteins (32).

Cellular GPCRs have differing numbers of GRK phosphorylation sites and this appears to be important

in the strength as well as duration of βarrestin binding to receptor (33,34). The strength of βarrestin

binding will likely prove to be important in determining the relative sensitivity of different receptors to

desensitization. This paradigm of GRK phosphorylation followed by βarrestin recruitment leading to

desensitization has been experimentally defined in biochemical experiments and in cultured cells and

has recently been confirmed by studies using knockout and transgenic mice (35-37). Deletion of GRK

phosphorylation sites results in receptors that have stronger signaling responses to agonist stimulation

in transfection studies (38-40). Recent experiments in GRK and arrestin knockout mice demonstrate

hypersensitive responses to several agonists (36,37).

US28 appears to mimic agonist occupied receptors in that it uses traditional GPCR pathways to

transmit signals and also undergo internalization via classic endocytic pathways. It is unknown if

traditional regulatory proteins such as GRKs and βarrestins modulate US28 signaling. It has recently

been demonstrated that US28 is constitutively phosphorylated in cells, however it remains unknown if

this phosphorylation leads to βarrestin binding or directly affects signaling to downstream effectors

such as inositol phosphate (31). Accordingly, in this study we examine the effects of GRK

phosphorylation and βarrestin binding on several aspects of US28 signaling. The data indicate that

US28 interacts with GRK and βarrestin proteins and that these interactions have multiple effects on

US28 directed signal transmission.

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Experimental Procedures

Cell Culture and Transfection- All cell lines were obtained from the American Type Culture

Collection (ATCC). HEK-293 cells were maintained in minimal essential media (MEM)

supplemented with 10% fetal bovine serum (FBS) and Penn/Strep (Gibco-BRL). Cells were routinely

sub-cultured 1:5 two times weekly. COS-7 cells were maintained in Dulbecco’s modified essential

media (D-MEM) supplemented as above. Cells were routinely sub-cultured 1:10 three times weekly.

HEK-293 cells were transfected in 100-mm-dishes with 5µg of total plasmid DNA using a modified

calcium phosphate method. COS-7 cells were transfected in 100-mm-dishes with 5µg total plasmid

DNA and 20µl lipofectamine according to the manufacturer’s instructions. Cells were routinely

harvested forty-eight hours post transfection.

Plasmids- The mammalian expression vector for US28 was derived from the HCMV strain

VHL/E. The US28 cDNA is cloned into the pcDNA3 mammalian expression vector and contains an

amino terminal FLAG tag and prolactin signal peptide followed by the US28 open reading frame. The

US28(1-314), US28(1-322), US28(1-324), and US28(1-332) mutants were constructed by PCR

incorporating a stop codon and Xba1 restriction enzyme site into the antisense primer. All sequences

were confirmed using an automated ABI DNA sequencer (Howard Hughes Nucleic Acid Facility,

Duke University). βarrestin2 tagged with GFP was a kind gift of Marc Caron and Larry Barak (Duke

University). pcDNA3 based expression plasmids for the β2AR and IL8R2 and pRK5 based expression

plasmids for GRK2 and GRK5 have been described previously (41,42). pRK5 based expression

constructs containing the kinase inactivating K220R (GRK2) and K215R (GRK5) have also been

described (43). The p38α expression plasmid was a kind gift of Roger J. Davis.

Inositol Phosphate Accumulation- Twenty fours hours post transfection cells were

transferred to 12-well tissue culture plates and allowed to adhere. Cells were incubated for 18 hours in

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complete media containing 3µCi/ml of [3H]-inositol (New England Nuclear), washed 1X with PBS for

30 minutes, and incubated in PBS containing 20mM LiCl (with or without 10nM Rantes) for 3 hours.

Reactions were stopped by aspirating medium, adding 1ml 0.4M perchloric acid, and cooling

undisturbed at 4°C for 5 minutes. 800µl of supernatant was neutralized with 400µl of 0.72M

KOH/0.6M KHCO3 and subjected to centrifugation. 1ml of supernatant was diluted with 3ml of dH20

and applied to freshly prepared dowex columns (AG1-X8, Bio-Rad laboratories). Columns were

washed 2X with dH20, total inositol phosphates were eluted with 4.0ml of 0.1M formic acid/1M

ammonium formate, and eluates were counted in a liquid scintillation counter.

[32P]-Orthophosphate Labeling and Receptor Immunoprecipitations- Forty-eight hours post

transfection cells were starved in phosphate-free DMEM supplemented with 2% dialyzed fetal bovine

serum and 20mM HEPES for 30 minutes. Cells were labeled with 250µCi/ml [32P]-orthophosphate for

120 minutes and either left unstimulated or stimulated with the appropriate ligand for 5 minutes. Intact

cells were washed 1X with ice cold PBS and whole cell extracts were prepared by lysing cells in 1ml

of lysis buffer (LB) (20mM HEPES, 0.5% NP-40, 250mM NaCl, 10% glycerol, 2mM EDTA, 1mM

PMSF, 2.5µg/ml Aprotinin, 25µg/ml Leupeptin, 100µM sodium orthovanadate and 50mM sodium

fluoride) for 20 minutes at 4°C. The supernatant was clarified by centrifugation and stored at -80°C

until use. FLAG tagged receptors were immunopurified from the supernatant with 20µl of M2 anti-

FLAG affinity matrix (Sigma) for > 3 hours with constant rocking at 4°C. Immunocomplexes were

washed 3-4X in lysis buffer and diluted in 50µl 3X laemmli sample buffer. Immunocomplexes (20µl)

were resolved by SDS-PAGE, and dried gels were subjected to autoradiography.

Immunoblotting and Detection of Phosphorylated p38α- Forty-eight hour post-transfection,

cells were harvested directly in 3X laemmli sample buffer, briefly sonicated and the cell extracts were

subjected to SDS-PAGE. Resolved proteins were transferred to nitrocellulose filters (Schleicher and

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Schuell) and non-specific reactivity was blocked by with Tris-buffered saline containing 0.1% Tween

20 (TBST) and 5% non-fat dried milk. Antisera directed against the phosphorylated form of p38α was

used at a 1:1500 in TBST to detect activated p38 (Cell Signaling Technology, Beverly, MA). Reactive

proteins were detected using the appropriate secondary antibodies in the enhanced chemiluminescence

system (ECL, Amersham).

FACS Analysis of Cell Surface US28 Expression and Confocal Microscopy- Transfected

cells were transferred into six-well dishes twenty four hours after transfection. Forty eight hours after

transfection, media was aspirated, cells were incubated with anti-FLAG primary antibody diluted

1:500 in PBS on ice for two hours, washed 1X in PBS and incubated with FITC conjugated anti-mouse

antibody diluted 1:250 in PBS on ice for one hour. Cells were fixed in phosphate buffered saline

containing 4% formaldehyde and the presence of cell-surface receptors was determined by flow

cytometry (Duke University Flow Cytometry Facility). For confocal microscopic localization of

βarrestin, HEK-293 cells were transfected with the appropriate combination of βarrestin2-GFP, US28,

and GRK plasmids. Twenty- four hours after transfection cells were transferred into collagen coated

glass bottom dishes. Forty- eight hours after transfection confocal microscopy was performed on a

Zeiss LSM510 laser-scanning microscope using a 100x/1.4 numerical aperature oil immersion lens.

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Results

HCMV US28 is constitutively phosphorylated in the absence of agonist- Stimulation of

cellular GPCRs with agonist leads to a rapid and transient increase in signaling activity. The transient

nature of this increase in signaling activity is due to the fact that activated receptors are strong targets

for phosphorylation by GRK proteins and subsequent interaction with βarrestin proteins. The

interaction of GPCRs with these regulatory proteins leads to the dissociation of receptor from its

cognate G-protein and effectively terminates signal transduction. Although the HCMV encoded US28

GPCR has been shown to maintain strong constitutive signaling capacity in the absence of agonist, it

remains unknown if phosphorylation and βarrestin binding regulate this signaling.

To determine if US28 signaling is regulated by phosphorylation and βarrestin binding, we first

analyzed its constitutive signaling activity. Since US28 appears to be predominantly coupled to Gαq

proteins, we analyzed the ability of US28 to induce accumulation of inositol phosphates, a classical

readout for Gαq signaling. US28 induces a 9.3-fold increase in the level of inositol phosphates when

compared with mock-transfected HEK-293 cells (Figure 1A, left panel). Stimulation with the CC

chemokine RANTES has only a weak effect on the ability of US28 to induce inositol phosphate

accumulation in HEK-293 cells. To analyze US28 phosphorylation, HEK-293 cells expressing US28,

IL8R2, or β2AR were labeled with [32P]-orthophosphate, stimulated with the appropriate ligand, and

receptors were immunoprecipitated. As expected, both the IL8R and the β2AR exhibited weak

receptor phosphorylation that is strongly stimulated by agonist (Figure 1A, right panel). Interestingly,

US28 is strongly phosphorylated in the absence of exogenous ligand and this phosphorylation appears

to be unaffected by the β-chemokine RANTES.

We also analyzed signaling and phosphorylation in COS-7 cells to determine if constitutive

phosphorylation of US28 was a general phenomenon or limited to HEK-293 cells. Similar to HEK-

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293 cells, US28 alone resulted in a 5.6-fold increase in inositol phosphate accumulation when

compared to mock transfected COS-7 cells. Stimulation with RANTES had a stronger effect on US28

signaling in COS-7 than in HEK-293 cells (Figure 1B, left panel). Analysis of US28 phosphorylation

in COS-7 cells (Figure 1B, right panel) demonstrated results similar to those obtained in HEK-293

cells indicating that unlike its cellular counterparts, US28 is constitutively active and constitutively

phosphorylated in the absence of agonist. Interestingly, while RANTES had a more dramatic effect on

increasing the signaling capacity of US28 in COS-7 cells, we observed no discernable effect of

RANTES on receptor phosphorylation. This result suggests that while RANTES can induce a

conformation more suitable for signaling, this ligand-induced conformation is not a better substrate for

phosphorylation.

Overexpression of GRK2 and GRK5 increases constitutive phosphorylation of US28- Since

a variety of cellular GPCRs are phosphorylated by GRKs we set out to determine if different members

of the GRK family were capable of phosphorylating US28. GRK2 and GRK5 represent two types of

GRKs that are present in mammalian cells. GRK2 contains several functional domains in addition to

its catalytic core (Figure 2A). The amino terminus contains an RGS (Regulator of G-protein

Signaling) homology domain and has been demonstrated to have Gαq binding capabilities and the

carboxy terminus contains a PH (pleckstrin homology) domain that has been shown to be important in

recruitment of GRK2 to the membrane (44-46). GRK5 also contains an RGS homology domain but

the domain appears to be inactive as it does not interact with Gαq. Kinase inactive versions of both

enzymes have been identified that contain Lysine à Arginine mutations in the ATP binding domain.

We hypothesized that if GRKs were capable of phosphorylating US28, overexpression of

GRK2 or GRK5 would lead to an increase in observable US28 phosphorylation. US28 was expressed

in COS-7 cells either alone or concomitant with either GRK2 or GRK5, cells were metabolically

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labeled with [32P]-orthophosphate and the level of US28 phosphorylation was analyzed in US28

immunoprecipitates (Figure 2B). GRK2 is a very potent stimulator of US28 phosphorylation while

GRK5 is less potent but still strongly increases US28 phosphorylation. These experiments indicate

that GRKs are capable of phosphorylating US28 and suggest that at least some of the basal US28

phosphorylation observed in cells is due to the activity of endogenous GRKs.

βarrestin2 is constitutively recruited to US28 in the absence of agonist- Agonist induced

phosphorylation of cellular GPCRs typically results in the rapid recruitment of βarrestins to the

membrane associated receptor complex. Although US28 appears to be localized to several cellular

organelles, previous reports have indicated that approximately 20% of US28 is localized to the plasma

membrane (30). To determine if βarrestins are also recruited to US28, we analyzed the subcellular

localization of a GFP-tagged βarrestin protein using confocal microscopy (Figure 3). βarrestin2-GFP

is distributed uniformly in the cytoplasm of MOCK transfected cells that do not contain US28 (Figure

3, left panel). Expression of US28 induces translocation of βarrestin2-GFP to the plasma membrane

(Figure 3, middle panel). This translocation can be visualized as punctate localization of βarrestin2 at

the cell periphery and is indicated by arrows. Expression of GRK2 leads to hyperphosphorylation of

US28 and more robust recruitment of βarrestin (Figure 3, right panel). These results, taken together

with the phosphorylation studies, indicate that although US28 is a constitutively active receptor,

cellular regulators of GPCR activity such as GRKs and βarrestins retain the ability to interact with this

viral encoded GPCR.

Construction of US28 deletion mutants and localization of US28 phosphorylation sites- A

large number of seven membrane spanning GPCRs contain ser/thr phosphorylation sites in the carboxy

terminal tail. The carboxy terminal tail of US28 is 58 amino acids long of which 14 are potential sites

of ser/thr phosphorylation. To analyze the effects of carboxy terminal ser/thr residues on US28

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phosphorylation and signaling we constructed four mutants, US28(1-314), US28(1-322), US28(1-324)

and US28(1-332) (Figure 5). The (1-314) mutant is deleted for all the carboxy terminal ser/thr

residues, while the other mutants contain 2 (1-322), 3 (1-324), and 7 (1-332) carboxy terminal ser/thr

residues.

To determine the phosphorylation status of mutant receptors we expressed US28(WT) as well

as US28(1-332) and US28(1-314) in COS-7 cells and immunoprecipitated receptors from [32P]-

orthophosphate labeled cells. As expected from earlier experiments US28(WT) is highly

phosphorylated when expressed in COS-7 cells (Figure 5). The majority of US28 phosphorylation is

located between amino acids 333 and 354 as US28(1-332) is largely defective in its ability to undergo

phosphorylation. US28(1-314) exhibits a phosphorylation status slightly weaker that US28(1-332)

indicating that although the majority of US28 phosphorylation is between amino acids 333 and 354,

residues between amino acids 315 and 332 are also weakly phosphorylated. Cell surface expression of

US28(WT), US28(1-332), and US28(1-314) was essentially equivalent as monitored by FACS analysis

of viable cells stained with FLAG antibody that recognizes the tagged US28 proteins (data not shown).

The US28(1-314) mutant does not recruit βarrestin2- Since phosphorylation of the US28

carboxy terminal mutants was severely impaired we sought to determine if βarrestin2 is recruited to

these two mutants. In HEK-293 cells expressing US28(WT) or US28(1-332), βarrestin2-GFP is

localized to the membrane as observed previously for wildtype US28 (Figure 6). In contrast, cells

expressing the US28(1-314) mutant did not exhibit membrane localization of βarrestin2-GFP (Figure

6). This result indicates that the US28(1-314) mutant is severely impaired in not only the ability to

undergo phosphorylation but also to recruit βarrestin. This finding is similar to those obtained for

carboxy terminal mutants of other receptors and prompted us to analyze the signaling properties of the

carboxy terminal US28 mutants.

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The US28(1-314) mutant exhibits increased signaling to the inositol phosphate pathway-

The duration and magnitude of signaling mediated by agonist activated GPCRs is regulated largely by

phosphorylation of activated receptors by GRKs and subsequent interaction with βarrestins. Our data

indicate that US28 is constitutively phosphorylated and bound by βarrestin2. We therefore

hypothesized that the US28(1-314) mutant that is unable to be phosphorylated or recruit βarrestin

might have significantly altered signaling properties. Wildtype and mutant US28 proteins were

expressed in COS-7 cells and analyzed for their abilities to induce accumulation of inositol phosphates

(Figure 7). US28(WT) induced inositol phosphate accumulation 5.7-fold over control cells. The

US28(1-332) mutant that demonstrated an intermediate level of phosphorylation and retained the

ability to interact with βarrestin2 induced inositol phosphate accumulation slightly weaker than

US28(WT). Interestingly the US28(1-314) mutant that exhibits low level phosphorylation and is

unable to interact with βarrestin induced levels of inositol phosphate accumulation 8.7-fold over

control cells. This activity exhibited by US28(1-314) is roughly 50% greater than that of US28(WT)

and supports the hypothesis that this mutant is unable to undergo regulation by GRK and βarrestin

proteins.

These data indicate that residues within amino acids 315 and 332 contain important

determinants for regulation of US28 signaling to inositol phosphates so we constructed several

additional mutants that systematically delete the serine residues between amino acids 315 and 332

(Figure 4). US28(1-322) and US28(1-324) delineate the important residues required for this regulation

as US28(1-322) induces accumulation of inositol phosphates 8.3-fold over control cells while the

US28(1-324) mutant induced accumulation of inositol phosphates only 3.6-fold over control cells

(Figure 7). These experiments indicate that Serine-323 is important for attenuation and regulation of

US28 signaling as the US28(1-322) mutant exhibits the increased signaling phenotype similar to

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US28(1-314) while the US28(1-324) mutant exhibits signaling equivalent to or less that US28(WT).

Effect of GRKs on US28(1-314) signaling to the inositol phosphate pathway- Previous

studies have indicated that overexpression of wildtype or kinase inactive GRK2 blocks signaling from

US28 (19). Based on these studies it is unclear if GRK phosphorylation plays a significant role in

US28 signaling since both wildtype and kinase inactive GRK2 blocked US28 signaling to the same

degree. We analyzed the ability of US28(WT) and US28(1-314) to induce inositol phosphate

accumulation in the presence of either GRK2 or GRK5 (Table 1). In agreement with previous studies

US28(WT) signaling is strongly inhibited by either wildtype GRK2 (52-62% inhibition) or kinase

inactive GRK2 (40-65% inhibition). This effect is dose dependent as increasing amounts of GRK

more strongly inhibited US28 signaling. We next analyzed the US28(1-314) mutant that is only

minimally phosphorylated in cells to determine if GRK2 overexpression would affect signaling of this

mutant. Since the effect of GRK2 is likely attributed to the RGS domain we expected wildtype and

kinase inactive GRK2 to potently inhibit signaling of US28(1-314). US28(1-314) signaling is

inhibited similar to US28(WT) by either wildtype GRK2 (47-61% inhibition) or kinase inactive GRK2

(40-60% inhibition). These data support our hypothesis that the effects of overexpressed GRK2 in this

system are largely independent of its activity as a GPCR kinase.

GRK5 on the other hand is unable to function as a Gαq sequestering protein since it contains an

inactive RGS domain in its amino terminus (Figure 2) and is therefore a useful tool to analyze the

specific effects of GRK kinase activity on US28 signaling. Overexpression of wildtype GRK5

potently inhibited US28(WT) signaling (34-66% inhibition) but kinase inactive GRK5 had little effect

and actually slightly enhanced signaling at a low concentration of transfected DNA (Table 1).

Equivalent expression of wildtype and kinase inactive GRK5 was confirmed by immunoblot (data not

shown). This result confirmed the notion that GRK5 would be useful for analyzing the effects of the

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kinase activity of GRKs on US28 signaling. US28(1-314) was only weakly inhibited by wildtype

GRK5 (6-26%) likely due to the fact that US28(1-314) does not contain the phosphorylation sites in

the carboxy terminus. Similar to the results with GRK5, the kinase inactive GRK5 had little effect on

US28(1-314) signaling and actually slightly enhanced signaling at a low concentration of transfected

DNA (9% increase). These results indicate that GRK phosphorylation of US28 is capable of

regulating US28 signaling and that the generation of the minimally phosphorylated US28(1-314)

mutant is a useful reagent to study US28 signaling in the absence of regulation by phosphorylation.

The US28(1-314) mutant exhibits decreased signaling to the p38α MAP kinase- We next

sought to determine if activation of the MAP kinase p38α pathway is induced by US28 and to examine

the effect of deletion of the carboxy terminus on this process. Using chemical inhibitors of p38 it has

been reported that US28 mediated activation of the p38 pathway is partially involved in stimulation of

the CREB transcription factor (23). These experiments do not specifically assess p38 activation so it

remains unknown if US28 can induce phosphorylation and hence activation of p38. We transfected

increasing amounts of the US28 expression plasmid (0.03µg to 1.0µg) along with p38α and used a

phospho-specific antibody to explore the ability of US28 to activate p38α (Figure 8A). US28 potently

induces phosphorylation of p38α indicating that this viral GPCR constitutively initiates a signaling

event that leads to activation of the p38 pathway. Interestingly, although US28(1-314) is capable of

activating p38α, the magnitude of this activation is decreased in comparison to US28(WT). This

decreased ability of US28(1-314) to activate p38 is particularly evident using 1µg of transfected DNA

and quantitation of phospho-p38 levels indicates that US28(1-314) exhibits a 34% defect compared to

US28(WT) in its ability to activate this signaling pathway (Figure 8B). The analysis of p38 signaling

is in sharp contrast to inositol phosphate signaling as the US28(1-314) mutant demonstrates increased

ability to induce inositol phosphate accumulation. These data indicate that US28 activation of these

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two signaling pathways is controlled by distinct mechanisms. While the GRKs and βarrestins are

critical negative regulators of the traditional Gαq stimulated inositol phosphate pathway it appears that

the GRKs and/or βarrestins play positive roles in activation of the p38 pathway.

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Discussion

We present evidence that the virally encoded GPCR US28 is constitutively phosphorylated and

recruits βarrestin in the absence of agonist. The data indicate that the GRKs are capable of inducing

strong phosphorylation of ser/thr residues in the carboxy terminal tail of US28 subsequently leading to

βarrestin recruitment. Deletion of the phosphorylation sites in the US28 carboxy terminal tail results in

a mutant, US28(1-314), that demonstrates weak overall phosphorylation, fails to recruit βarrestin and

accordingly exhibits enhanced signaling capacity to the second messenger inositol phosphate.

Additional mutational analysis identifies Serine-323 as a critical residues involved in US28 signaling.

Moreover, the US28(1-314) mutant exhibits decreased signaling to the MAP kinase p38α suggesting

that the US28 directed p38 pathway is regulated differently than the inositol phosphate pathway. This

regulation of signal transduction by GRK and βarrestin proteins is typical of a variety of cellular

GPCRs and can now be extended to include the viral family of GPCRs (1,39).

The data presented here are largely in agreement with a very recent study that examines the

effects of carboxy terminal deletion mutants on cell surface expression of US28 (47). This study

demonstrates that the US28 carboxy terminus contains determinants for constitutive internalization

thereby affecting the magnitude of signaling to inositol phosphates. However, the mutants we

generated and studied in this report exhibit cell surface expression patterns very similar to wildtype

US28 and indicate that the altered signaling we observe is due specifically to the fact that the US28(1-

314) mutant is unable to engage GRK and βarrestin proteins.

Our data indicate that in cells expressing constitutively active US28, the receptor is constantly

undergoing phosphorylation and regulation by the cellular machinery. This is somewhat surprising

since the constitutive nature of US28 might suggest that these viral receptors have evolved a

mechanism to avoid being regulated by cellular proteins such as GRKs and arrestins. One speculation

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is that the viral proteins need to be regulated to prevent chronic hyperstimulation of signaling pathways

that are not beneficial for viral replication and infection. Mammalian cells have evolved numerous

mechanisms to prevent aberrant chronic activation of signaling pathways and by US28 engaging cell

regulatory machinery it may able to signal only to moderate levels thus potentially avoiding

recognition by apoptotic or other cell defense machinery.

US28 appears to maintain an active conformation so that immediately upon synthesis and

trafficking to the plasma membrane US28 is capable of engaging G-proteins and activating signal

transduction pathways. Since the active conformation of US28 is also a substrate for phosphorylation,

the receptor becomes GRK phosphorylated and bound by βarrestin. The βarrestin bound receptor

complex is therefore sterically inhibited from interaction with G-proteins and traditional signaling

through these G-proteins is terminated. In direct support of this model is the data obtained with

overexpression of GRKs. As the level of GRK proteins in the cell is raised (in this case by gene

transfection experiments) the ratio of phosphorylated to unphosphorylated receptor increases and the

magnitude of signaling activity decreases. This may also be important with regards to the total

constitutive activity observed with these receptors in different cell types. The magnitude of US28

signaling in cells in vivo may be directly correlated with the relative amount of GRKs expressed, so

that cells expressing low levels of GRK may have high levels of constitutive inositol phosphate

signaling and vice-versa. Interestingly, data obtained with the HHV8 encoded ORF74 protein

indicates that this viral receptor constitutively induces inositol phosphate production in COS-7 and

HEK-293 cells but only induces inositol phosphate production in an agonist dependent manner in lung

endothelial cells (21,22,48). It will be interesting to determine if GRK phosphorylation and βarrestin

binding also regulate the ORF74 viral GPCR and to analyze the relative effects of GRKs on US28

signaling in additional cell types.

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It is interesting that the GRKs have evolved multiple mechanisms to shut off GPCR signaling

through traditional G-protein signaling pathways. The presence of multiple mechanisms of GRK

regulation is especially evident when studying the constitutive viral receptor US28 in comparison with

the carboxy terminal mutants that are impaired in phosphorylation. The discovery of the RGS

homology domain in the amino terminus of GRK2 first indicated that the mechanisms of GRK

mediated inhibition of GPCR signaling might extend beyond the ability to phosphorylate the receptor

(44,45). We and others have shown that kinase inactive GRK2 (K220R) inhibits US28 signaling to the

same degree as wildtype GRK2 (19). This kinase independent mechanism can likely be attributed to

the RGS homology domain in the amino terminus of GRK2, which potently binds Gαq proteins

preventing receptors from interacting with and activating the G-protein. The RGS homology domain

in GRK2 only inhibits signaling involving Gαq proteins, as it does not interact to any measurable

degree with other G-proteins. This effect of GRK2 is therefore specific to Gαq signaling. GRK5

mediated inhibition of US28 signaling utilizes the more traditional mode of signaling inhibition by

GRKs. While wildtype GRK5 potently inhibits US28 signaling, the kinase inactive mutant of GRK5

(K215R) is unable to inhibit US28 signaling. In this case, the mechanism of inhibition by GRK5 can

be attributed to its ability to phosphorylate US28. This traditional mechanism of inhibition is more

widespread and applies to GPCR signaling in general, as it is not specific for Gαq mediated signaling

pathways. US28 has been reported to also signal through Gαi and Gα16 proteins so the different GRKs

could certainly have different effects on US28 signaling depending on the specific pathway and G-

protein being used.

Accumulating evidence indicates that in addition to their roles in the termination of G-protein

signaling, the GRKs and βarrestins also function as adapter proteins to couple GPCRs to additional

types of signaling pathways (49,50). GRKs have been shown to interact with or phosphorylate several

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non-receptor proteins including tubulin, synuclein, and GIT-1(51-53). The βarrestins appear to be

important adapter proteins involved in coupling receptors to a variety of signaling pathways including

most notably the Src tyrosine kinases and several MAP kinases (54). GRKs and βarrestins have also

been shown to be important in interacting with members of the endocytic machinery and are important

in promoting receptor internalization (55).

We have shown in this study that the viral US28 GPCR is engaged and regulated by the GRKs

and βarrestins. Moreover, deletion of the carboxy terminal phosphorylation and βarrestin binding sites

generates a US28 protein with enhanced inositol phosphate signaling capacity due to an inability to be

regulated by GRKs and βarrestins. Interestingly, this same mutant demonstrates a defect in its ability

to induce high levels of the p38 MAP kinase in comparison with wildtype US28. This result is

consistent with the hypothesis that US28 uses a βarrestin scaffold to induce maximal levels of the p38

MAP kinase similar to that observed with ERK and JNK activation by cellular GPCRs such as the

Angiotensin AT1a receptor (56,57).

Several cellular chemokines including RANTES, MCP-1, and fractalkine interact with US28

(24,27,28). We have shown that RANTES modestly increases US28 induced signaling to inositol

phosphates but has no detectable effect on US28 phosphorylation. US28 has demonstrable capacity to

internalize these chemokines so it will be interesting to see how the binding of chemokines in addition

to RANTES affects receptor phosphorylation. In particular, fractalkine partially inhibits US28

signaling and it would be interesting if this chemokine induced additional phosphorylation leading to

decreased signaling capacity.

The phosphorylation and regulation of US28 by GRKs is unlikely to be unique to US28 within

the viral GPCR subfamily. The other herpesviral GPCRs also contain a significant number of serine

and threonine residues in their carboxy terminal domains. The HHV8 encoded ORF74 protein has 6

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potential sites in the carboxy terminal region while the HHV6 encoded U12 protein contains 9 sites

and the HCMV encoded UL33 contains 20 sites. Since these other viral proteins signal in a similar

manner to US28, it seems reasonable to speculate that the other viral GPCRs might be regulated by

cellular GRKs and βarrestins. Particularly interesting is the ORF74 protein that has demonstrable

oncogenic activities in vitro, interacts with c-Src, and activates several MAP kinase signaling pathways

(58,59). Recent evidence linking GRKs and βarrestins to c-Src and MAP kinase signaling pathways

suggests that these cell regulatory proteins may play an important role in ORF74 signaling.

Taken together, these findings indicate that phosphorylation of US28 and regulation of

signaling by GRK and βarrestin proteins play an important role in US28 mediated signal transduction.

We have identified a US28 mutant that demonstrates altered signaling through inositol phosphate and

p38 MAP kinase pathways due to deletion of the key determinants required for engagement of cellular

GRK and βarrestin proteins and have defined several mechanisms used by GRKs to regulate US28

signaling. Future studies on the role of GRK and βarrestin mediated regulation should provide

considerable new insight into not only the mechanism(s) by which US28 activates cellular signaling

pathways, but also into how cellular proteins such as GRKs and βarrestins function in mediating GPCR

mediated signal transduction in general.

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Acknowledgements

We thank Donna Addison and Julie Turnbough for excellent secretarial assistance, Katrina H.

Wilson and Mouhamadou Niang for technical assistance. This work was supported in part by National

Institutes of Health Grant HL16037. RJL is an Investigator of the Howard Hughes Medical Institute.

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Figure Legends

Figure 1. The HCMV US28 vGPCR maintains constitutive signaling and is phosphorylated in

the absence of agonist. A) HEK-293 cells were transfected with empty vector (MOCK) or FLAG-

US28 and identical populations of cells were either left untreated or stimulated with 10nM RANTES.

Inositol phosphates were isolated using anion exchange chromatography and the results represent the

mean induction ± S.E. of three independent experiments performed in duplicate (left panel). HEK-293

cells were transfected with empty vector (MOCK), FLAG-US28, FLAG-IL8R2, or FLAG-β2AR,

labeled with 250 µCi/ml [32P]-orthophosphate and either left untreated or stimulated for 5 minutes with

the appropriate ligand (10nM RANTES, 10nM GROα, or 10µM ISO respectively). Receptors were

immunoprecipitated with anti-FLAG affinity gel and phosphorylated receptors were visualized by

autoradiography (right panel). The autoradiograph shown is representative of two to three independent

experiments. B) COS-7 cells were transfected with empty vector (MOCK) or FLAG-US28 and

identical populations of cells were either left untreated or stimulated with 10nM RANTES. Inositol

phosphates were isolated using anion exchange chromatography and the results represent the mean

induction ± S.E. of 10 independent experiments performed in duplicate (left panel). COS-7 cells were

transfected with empty vector (MOCK), FLAG-US28, FLAG-IL8R2, or FLAG-β2AR, labeled with

250 µCi/ml [32P]-orthophosphate and either left untreated or stimulated for 5 minutes with the

appropriate ligand (10nM RANTES, 10nM GROα, or 10µM ISO respectively). Receptors were

immunoprecipitated with anti-FLAG affinity gel and phosphorylated receptors were visualized by

autoradiography (right panel). The autoradiograph shown is representative of three to four

independent experiments.

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Figure 2. The constitutive phosphorylation of US28 is strongly enhanced by GRK expression.

A) Structure of the GRK2 and GRK5 proteins. The GRKs are multi-functional proteins that have

several structural domains in addition to the catalytic kinase domain. GRK2 contains an active amino

terminal RGS domain, a central catalytic domain, and a carboxy terminal PH domain. GRK5 contains

an inactive amino terminal RGS domain and a central catalytic domain. The amino acid sunstitutions

that lead to the inactivation of the catalytic domains are indicated. B) COS-7 cells were transiently

transfected with empty vector (MOCK), FLAG-US28, or FLAG-US28 along with GRK2 or GRK5 and

labeled with 250 µCi/ml [32P]-orthophosphate. Receptors were immunoprecipitated with anti-FLAG

affinity gel and phosphorylated receptors were visualized by autoradiography. The autoradiograph

shown is representative of at least 5 independent experiments

Figure 3. βarrestin2 is constitutively recruited to phosphorylated US28. HEK-293 cells were

transiently transfected with empty vector and βarrestin2-GFP (Mock, left panel), FLAG-US28 and

βarrestin2-GFP (US28, middle panel), or FLAG-US28, GRK2, and βarrestin2-GFP (US28+GRK2,

right panel). Subcellular localization of βarrestin2-GFP was localized using a Zeiss Axiomat Confocal

Microscope. The confocal images shown are representative of three to five independent experiments.

Figure 4. Structure of the US28 carboxy terminal domain. The carboxy terminal domain of US28

contains 12 serines and 2 threonines that could be potential phosphorylation sites. Four mutants were

generated for this study; US28(1-332), US28(1-324), US28(1-322), and US28(1-314). These mutants

delete 22, 30, 32, and 40 amino acids respectively from the carboxy terminus of US28. The locations

of potential sites of serine and threonine phosphorylation are marked by arrows.

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Figure 5. Localization of US28 phosphorylation sites to the carboxy terminal domain. COS-7

cells were transiently transfected with empty vector (MOCK), FLAG-US28(WT), FLAG-US28(1-

314), and FLAG-US28(1-332) and labeled with 250 µCi/ml [32P]-orthophosphate. Receptors were

immunoprecipitated with anti-FLAG affinity gel, and phosphorylated receptors were visualized by

autoradiography. The autoradiograph shown is representative of at least four independent experiments.

Figure 6. βarrestin2 is not recruited to the US28(1-314) Mutant. HEK-293 cells were transiently

transfected with empty vector and βarrestin2-GFP (Mock, upper left panel), FLAG-US28 and

βarrestin2-GFP (US28(WT), upper right panel), FLAG-US28(1-314) and βarrestin2-GFP (US28(1-

314), lower left panel), or FLAG-US28(1-332) and βarrestin2-GFP (US28(1-332), lower right panel).

Subcellular localization of βarrestin2-GFP was localized using a Zeiss Axiomat Confocal Microscope.

The confocal images shown are representative of two to three independent experiments.

Figure 7. US28(1-314) has elevated constitutive signaling capacity in comparison with wildtype

US28. COS-7 cells were transiently transfected with 1.3µg or 1.5µg of FLAG-tagged wildtype and

mutant US28 proteins and replated into 12-well plates twenty-four hours after transfection. Forty-eight

hours after transfection, inositol phosphates were isolated using anion exchange chromatography. The

results are presented graphically and represent the mean ± S.E. of three to seven independent

experiments performed in duplicate. *, P < 0.05, comparing cells expressing US28(1-322) or US28(1-

314) with US28(WT).

Figure 8. US28(1-314) mutant exhibits decreased signaling to p38α MAP kinase. COS-7 cells

were transiently transfected with increasing amounts (0.03µg to 1.0µg) of FLAG-tagged US28(WT)

and US28(1-314) proteins. A) Forty-eight hours after transfection, cell extracts were analyzed by

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immunoblot using a phospho-specific p38 antibody. B) The results are presented graphically and

represent the mean +/- S.E. of four to five independent experiments. *, P < 0.05, comparing cells

expressing 1µg of US28(1-314) with cells expressing 1µg of US28(WT).

Table Legends

Table 1. Inhibition of US28 signaling by GRK overexpression. COS-7 cells were transiently

transfected with FLAG-US28(WT) or FLAG-US28(1-314) along with increasing amounts of wildtype

and kinase inactive GRKs. Forty-eight hours after transfection, inositol phosphates were allowed to

accumulate for 3 hours in the presence of 20 mM LiCl and total inositol phosphates were isolated

using anion exchange chromatography. The results are presented in table form and represent the effect

of GRK proteins relative to US28(WT) or US28(1-314) alone. The numbers reported are the mean

decrease in signaling ± S.E. of three to four independent experiments performed in duplicate.

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MO

CK

US28 IL8R2 B2AR+ + + Agonist (5 min)

Phospho-B2ARPhospho-IL8R2Phospho-US28

Inos

itol P

hosp

hate

Acc

umul

atio

n(F

old

Indu

ctio

n)

Mock US28+ +- - RANTES

(10nM)

A)

B)

IP:FLAG

Agonist (5 min)

Phospho-B2AR

Phospho-IL8R2Phospho-US28

IP:FLAG

MO

CK

US28 IL8R2 B2AR+ + +

Inos

itol P

hosp

hate

Acc

umul

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n(F

old

Indu

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Mock US28+ +- - RANTES

(10nM)

10

7.5

5

2.5

0

15

10

5

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HEK-293

COS-7

Miller et. al. Figure 1

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US

28

US

28+G

RK

2

US

28+G

RK

5

IP:FLAG

Phospho-US28

GRK2 N

CGRK5 N

C

K220R (kinase inactivating mutation)

K215R (kinase inactivating mutation)

ACTIVE RGSDOMAIN

INACTIVE RGSDOMAIN

KINASE DOMAIN PH DOMAIN

KINASE DOMAIN

A)

B)

Miller et. al. Figure 2

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US2

8U

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KFRQELHCLLAEFRQRLFSRDVSWYHSMSFSRRSSPSRRETSSDTLSDEACRVSQIIP

KFRQELHCLLAEFRQRLFSRDVSWYHSMSFSRRSSP

KFRQELHCLLAEFRQRLF

US28(WT)US28(1-332)

US28outside

inside

US28(1-314)

Miller et. al. Figure 4

KFRQELHCLLAEFRQRLFSRDVSWYHSM

KFRQELHCLLAEFRQRLFSRDVSWYHUS28(1-322)US28(1-324)

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Miller et. al. Figure 5

MO

CK

US

28(W

T)U

S28

(1-3

14)

US

28(1

-332

)

Phospho-US28

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Miller et. al. Figure 6

MOCK US28(WT)

US28(1-314) US28(1-332)

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Miller et.al. Figure 7

Con

trol WT

1-33

2

1-32

4

1-32

2

1-31

4

US28

* *

Inos

itol P

hosp

hate

Acc

umul

atio

n(F

old

Indu

ctio

n)

2.5

5.0

7.5

10.0

0

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Miller et.al. Figure 8

A)

phospho-p38α

Con

trol US28

(WT)US28

(1-314)

B)

*

Con

trol

Phos

pho-

p38α

(Arb

itrar

y U

nits

)

25000

50000

75000

US28(WT)

US28(1-314)

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2+/-11

US28 (WT) US28(1-314)

0.5 ug GRK2 (wildtype)

2.5 ug GRK2 (kinase inactive)

2.5 ug GRK2 (wildtype)0.5 ug GRK2 (kinase inactive)

52+/-11**62+/-340+/-865+/-2

47+/-961+/-340+/-660+/-4

US28 (WT) US28(1-314)0.5 ug GRK5 (wildtype)

2.5 ug GRK5 (kinase inactive)

2.5 ug GRK5 (wildtype)0.5 ug GRK5 (kinase inactive)

34+/-666+/-319+/-16

6+/-826+/-69+/-113+/-7

(-)(-)(-)(-)

(-)(-)(-)(-)

(-)(-)(+)(-)

(-)(-)(+)(-)

**numbers are percent inhibition relative to US28 or US28(1-314) alone

Miller et. al. Table 1

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LefkowitzWilliam E. Miller, Daniel A. Houtz, Christopher D. Nelson, P. E. Kolattukudy and Robert J.

activity of the human cytomegalovirus US28 GPCR arrestin recruitment regulate the constitutive signalingβGRK phosphorylation and

published online March 31, 2003 originally published online March 31, 2003J. Biol. Chem. 

  10.1074/jbc.M303219200Access the most updated version of this article at doi:

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