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A novel mechanism of control of NFkB activation andinflammation involving A2B adenosine receptors

Ying Sun1, Yuanyuan Duan1, Anna S. Eisenstein3, Wenbao Hu1, Adrien Quintana1, Wai Kwan Lam1, Yan Wang1,Zhenguo Wu1,2, Katya Ravid3 and Pingbo Huang1,2,*1Division of Life Science, and 2State Key Laboratory of Molecular Neuroscience, Hong Kong University of Science and Technology, Hong Kong,People’s Republic of China3Department of Medicine and Biochemistry, and Whitaker Cardiovascular Institute, Boston University School of Medicine, Boston, MA 02118, USA

*Author for correspondence (bohuangp@ust.hk)

Accepted 28 May 2012Journal of Cell Science 125, 4507–4517� 2012. Published by The Company of Biologists Ltddoi: 10.1242/jcs.105023

SummaryThe nuclear factor kappa B (NFkB) pathway controls a variety of processes, including inflammation, and thus, the regulation of NFkB hasbeen a continued focus of study. Here, we report a newly identified regulation of this pathway, involving direct binding of the transcriptionfactor NFkB1 (the p105 subunit of NFkB) to the C-terminus of the A2B adenosine receptor (A2BAR), independent of ligand activation.

Intriguingly, binding of A2BAR to specific sites on p105 prevents polyubiquitylation and degradation of p105 protein. Ectopic expression ofthe A2BAR increases p105 levels and inhibits NFkB activation, whereas p105 protein levels are reduced in cells from A2BAR-knockoutmice. In accordance with the known regulation of expression of anti- and pro-inflammatory cytokines by p105, A2BAR-null mice generateless interleukin (IL)-10, and more IL-12 and tumor necrosis factor (TNF-a). Taken together, our results show that the A2BAR inhibits NFkB

activation by physically interacting with p105, thereby blocking its polyubiquitylation and degradation. Our findings unveil a surprisingfunction for the A2BAR, and provide a novel mechanistic insight into the control of the NFkB pathway and inflammation.

Key words: NFkB, A2B adenosine receptor, p105, Inflammation, GPCR

IntroductionExtracellular adenosine elicits a wide array of physiological and

pathological responses via binding to its four subtypes of cell

surface receptors A1, A2A, A2B and A3, each of which has a

unique pharmacological profile, tissue distribution and effector

coupling. Adenosine receptors are typical G protein coupled

receptors (GPCRs), which transmit signals through adenylyl

cyclase/cAMP and phospholipase C/Ca2+ pathways. It has been

well documented that these receptors play important parts in the

regulation of inflammation. Depending on the receptor subtype,

they may exert anti- or/and pro-inflammatory effects. The A2B

adenosine receptor (A2BAR) is a low-affinity adenosine receptor,

expressed in immune cells, endothelial cells, aortic vascular

smooth muscle, cecum, large intestine and urinary bladder (Yaar

et al., 2005). Considering its low affinity in some cases (Schulte

and Fredholm, 2000; Schulte and Fredholm, 2003), the A2BAR

was hypothesized to play important roles under inflammation and

cell stress/damage conditions, where extracellular adenosine is

drastically increased (Fredholm, 2007). Interestingly, A2BAR

protein level could be selectively upregulated as a result of

inflammation, injury, hypoxia and other types of cell stresses

(Fredholm et al., 2001; Hart et al., 2009; Kolachala et al., 2005;

Kong et al., 2006; Xaus et al., 1999). However, there seems to be

no consensus regarding the exact role of A2BAR in the regulation

of inflammation (Ryzhov et al., 2008a).

NFkB is a ubiquitously expressed transcription factor regulating

various biological functions, including inflammation. Mammalian

cells express five NFkB members, including NFkB1 (p50 and its

precursor p105), NFkB2 (p52 and its precursor p100), RelA (p65),

RelB and c-Rel. Five NFkB inhibitory proteins have been

described: IkBa, b and c as well as p100 and p105 (Hayden and

Ghosh, 2004). In resting cells, p105, like other IkB proteins,

sequesters NFkB dimers into the cytoplasm. In response to

stimulation, p105 is completely degraded by the proteasome,

releasing NFkB dimers to translocate into the nucleus and regulate

expression of target genes (Pereira and Oakley, 2008). Previous

work indicated that p105 plays a pivotal role in immune responses.

Deletion of p105 in mice caused severe inflammation and

increased susceptibility to opportunistic infections, indicating

p105 as a suppressor of inflammation (Ishikawa et al., 1998).

In the present study, we report that the A2BAR directly binds to

NFkB1/p105. Our in vitro and in vivo experiments demonstrated

that A2BAR binding stabilizes p105 protein by blocking its

polyubiquitylation, thereby inhibiting the NFkB signaling

pathway. Our study is the first to report that a GPCR directly

interacts with p105, thereby identifying a new, unexpected

regulator of the NFkB pathway.

ResultsNFkB1/p105 binds to A2BAR C-terminus domain in yeast

two-hybrid assays

In order to better understand A2BAR function, we searched for

novel protein binding partners of A2BAR by the yeast two-hybrid

(YTH) screening assay, using a human lung cDNA library with the

C-terminal 40 amino acids of the A2BAR (A2BAR-C) as a bait. Out

of three million screened transformants, 26 positive clones were

obtained, of which 20 positive clones encode p105. These results

were verified by retransforming yeast with p105 and A2BAR-C

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(Fig. 1A). Non-identical, overlapping sequences of the positiveclones suggested that the high frequency hit was not an artifact of

over-amplification of a particular p105 segment in the library, butrather reflected a possible strong interaction of A2BAR and p105.

To further determine the A2BAR binding site of p105, weconstructed various p105 deletion mutants (Fig. 1B). The

deletion of the N-terminal amino acids 1–542 or C-terminalPEST domain of p105 completely disrupted its interaction withA2BAR-C in the YTH assay. In contrast, p105 fragments

containing amino acids 497–542 and PEST domain bound toA2BAR-C as well as the full-length p105 (Fig. 1B). Thus, thePEST domain and residues 497–542 appear to be essential for the

interaction with A2BAR.

In addition, a series of A2BAR-C deletion mutants wereconstructed to assess the p105 binding site. A2BAR-CD292–302

and A2BAR-CD292–312 lost their ability to interact with p105

(Fig. 1C). In contrast, the C-terminal deletion mutants A2BAR-CD312–332 and A2BAR-CD322–332 were still associated with p105to an extent comparable to the intact A2BAR-C. These data

indicated that amino acids 292–302 of A2BAR are necessary, andamino acids 292–312 are sufficient for p105 binding.

Association of A2B adenosine receptor and p105 inmammalian cells

To further confirm A2BAR-p105 interaction, we tested theirphysical binding by pull-down assays. The full length A2BARand A2BAR-C were fused to glutathione S-transferase (GST) togenerate GST-A2B and GST-A2B-C, respectively. GST-A2B and

GST-A2B-C, but not GST alone, efficiently retained endogenousp105 (Fig. 2A, left panel). Reciprocal pull-down assays showedthat GST-p105 and GST-p105-C (p105 without p50 region, amino

acid residues 433–968), but not GST-p50 or GST alone, pulleddown the V5-tagged A2BAR (Fig. 2A, right panel). The results ofGST-p105-C are consistent with the yeast mapping data in Fig. 1B.

A2BAR-p105 interaction was further verified by co-

immunoprecipitation assays in HEK293T cells, using a tagged-A2BAR because of the lack of a reliable anti-A2BAR antibody(supplementary material Fig. S1). Cell extracts of HEK293T cells

transfected with V5-tagged A2BAR were immunoprecipitated withan anti-V5 antibody. Endogenous p105 coprecipitated withA2BAR, whereas no immunoreactive material was detected fromcells transfected with mock vector pcDNA4-V5 (Fig. 2B, left

panel). In a reciprocal experiment, V5-tagged p105 was expressedwith Flag-tagged A2BAR in HEK293T cells, followed byimmunoprecipitation. A2BAR was co-immunoprecipitated by

anti-V5 antibody but not by control mouse IgG (Fig. 2B, rightpanel). Taken together, these results indicated that the A2BAR andp105 are also capable of forming a complex in mammalian cells. In

addition, the co-localization of A2BAR and p105 was analyzed byconfocal microscopy, in living cells to avoid shrinkage of thecytoplasm during the cell fixation process and therefore betterassess the intracellular localization of A2BAR, and in fixed cells to

examine endogenous p105. A2BAR-CFP was localized in both theplasmalemma and cytoplasm (Fig. 2C), as has been observed forsome other GPCRs (Wang et al., 2005). Importantly, A2BAR-CFP

were co-localized with p105-GFP or endogenous p105 in both theplasmlemma and cytoplasm (Fig. 2C), consistent with the idea thatA2BAR and p105 physically interact in the cell.

Next, we asked whether A2BAR-p105 association is dependent

on A2BAR agonist stimulation. At 24 h after transfection ofV5-tagged A2BAR, cells were treated with 10 mM N-ethylcarboxamidoadenosine (NECA), an A2-type adenosine receptor

agonist. NECA, although eliciting a robust elevation of cAMPlevel (supplementary material Fig. S2), had little effect onA2BAR-p105 interaction, arguing for an agonist-independentA2BAR and p105 coupling (Fig. 2D). It was noted that p105

protein level markedly increased in the presence of A2BAR(Fig. 2B,D, also see Fig. 4).

Direct interaction of A2BAR with p105

Although GST pull-down, co-immunoprecipitation and co-

localization assays detected an association of A2BAR and p105,these assays are incapable of distinguishing between directprotein-protein interactions and those mediated by an additional

protein(s). Early studies suggested that b-arrestin physicallyinteracts with p105 (Parameswaran et al., 2006) or A2BAR(Matharu et al., 2001). To address whether p105-A2BAR

Fig. 1. Identification of p105 as an A2BAR-interacting protein in the YTH

system. (A) Yeast colonies co-transformed with pDBLeu-A2BAR-C and

pPC86-p105 vectors were first grown on synthetic dropout plates lacking

leucine and tryptophan (SD/Leu2 Trp2) to ensure successful co-

transformation of the two vectors pDBLeu and pPC86, and subsequently were

tested for the interaction of p105-A2BAR-C by growing on synthetic dropout

plates lacking leucine, tryptophan and histidine, supplemented with 50 mM 3-

aminotriazole (SD/Leu2 Trp2 His2 +50 mM 3-AT). pDBLeu-A2BAR-C +

pPC86, and pDBLeu + pPC86-p105 are negative controls. (B,C) Mapping the

A2BAR-binding region of p105 (B) and the p105-binding region of A2BAR

(C) with YTH assays. In B, yeasts were co-transformed with pDBLeu-A2B-C

(denoted as A2B-C) and different deletion mutants of p105, as indicated. In C,

pPC86-p105 and deletion mutants of A2BAR-C were co-transformed. +,

growth; 2, no growth. Data shown in A-C are representative of three

independent experiments. RHD, Rel homology domain; GRR, glycine-rich

region; ANK, ankyrin repeats; DD, death domain; PEST, proline-, glutamic-

acid-, serine- and threonine-rich sequence.

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association is mediated by b-arrestin or not, b-arrestin-null

murine embryonic fibroblasts (MEFs) were used (supplementary

material Fig. S3). Importantly, p105 interacted with the A2BAR

even in b-arrestin-null MEFs (Fig. 3A, right panel). This

observation demonstrated that b-arrestin is not necessary for

p105-A2BAR binding, although it does not rule out the

involvement of b-arrestin in p105-A2BAR interaction. To

further exclude the presence of additional protein mediators of

p105 and A2BAR interaction, GST-p105 or GST-p105-C and

maltose-binding protein (MBP)-tagged A2BAR-C or MBP alone

were purified from E. coli (supplementary material Fig. S4) and

then used for pair-wise GST pull-down assays. Results showed

that MBP-tagged A2BAR-C, but not MBP alone, binds to p105 or

p105-C (Fig. 3B; supplementary material Fig. S5), further

strengthening the conclusion that A2BAR-C-p105 interaction is

independent of accessory protein(s).

A2BAR stabilizes p105 by blocking its polyubiquitylation-

dependent degradation, resulting in suppression of NFkB

activation

Remarkably, the two A2BAR binding sites of p105 (Fig. 1B)

overlap with the potential ubiquitin ligase binding sites (Beinke

Fig. 2. The association of A2BAR with p105 in mammalian cells. (A) GST pull-down assays. Endogenous p105 in HEK293T cells was pulled down by GST-

A2B or GST-A2B-C fusion protein, but not by GST alone (left panel). Data are representative of three independent experiments. In a reciprocal experiment, V5-

tagged A2BAR in HEK293T cells was pulled down (denoted as A2B) by GST-p105 or GST-p105-C, but not by GST-p50 or GST alone (right panel). The amounts

of GST fusion proteins in each lane are shown at the bottom of each panel. Note that there is also GST-p50 when generating GST-p105, as expected (Beinke and

Ley, 2004). Data are representative of four independent experiments. (B) Co-immunoprecipitation assays. Cell lysates from HEK293T cells expressing (+) or not

expressing (2) pcDNA4V5-A2BAR (A2B-V5) were immunoprecipitated with anti-V5 antibody. The immunoprecipitates were immunoblotted with anti-p105 or

anti-V5 antibodies (left panel). Data are representative of five independent experiments. In a reciprocal experiment, cell lysates from HEK293T cells transfected

with Flag-tagged A2BAR and V5-tagged p105 were immunoprecipitated with either mouse anti-V5 or IgG antibodies, followed by immunoblotting with anti-Flag

or anti-V5 antibodies (right panel). Data are representative of three independent experiments. Of note, in this right panel, the two different immunoprecipitation

experiments involved exactly the same cell lysate (divided into two equal portions) but with two different antibodies (mouse IgG and anti-V5 antibody, and mouse

IgG immunoprecipitation was used as an irrelevant immunoprecipitation control). (C) The expression vectors, p105-GFP with CFP alone (upper panel) or A2BAR-

CFP (middle row) were transfected into HEK293T cells. After 36 h, the cells were examined by confocal microscopy. In the lower panel, HEK293T cells

expressing A2BAR-CFP were fixed and processed for double-staining of p105 and A2BAR with anti-p105 and anti-GFP antibodies, respectively. Scale bar: 5 mm.

Data shown are the representative of six independent experiments. ‘Zoom’ views show greater detail of the degree of colocalization of p105 and A2BAR.

(D) Co-precipitation of A2BAR and p105 (similar to as shown in B) was not affected by NECA. At 24 h after transfection of V5-tagged A2BAR, cells were treated

with A2BAR agonist 10 mM NECA or DMSO (vehicle) for 30 min. Cell lysates were then immunoprecipitated with anti-V5 antibody. Data are the representative

of three independent experiments. Note that the p105 level increases in the presence A2BAR in panels B and D.

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and Ley, 2004; Orian et al., 1999). We reasoned that A2BAR

binding may interfere with p105 ubiquitylation and therefore

regulate its stabilization. Indeed, ectopic expression of the

A2BAR dramatically increased endogenous p105 protein, while

it had little impact on p105 mRNA level (Fig. 4A, also

Fig. 2B,D), indicating a post-transcriptional regulation. A2BAR

expression increased p105 protein expression in a dose-

dependent manner (Fig. 4B). It was also noted that A2BAR

expression was accompanied by a reproducible increase in the

level of p50 (Fig. 4A–C), presumably secondary to augmented

levels of p105 protein, which is processed into p50 (Beinke and

Ley, 2004). It has been well known that the overexpression of

GPCRs without agonist stimulation mimics agonist-dependent

activation of the receptors (Milano et al., 1994). To exclude the

possibility that the stabilizing effect of A2BAR on p105 protein

expression results from A2BAR activation by A2BAR

overexpression, or by minimal adenosine present in the culture

media, we examined the effect of NECA on p105 expression.

NECA (10 mM) had no effect on p105 protein level (Fig. 4C;

supplementary material Fig. S6), indicating that A2BAR

expression stabilizes p105 in an agonist-independent manner. In

another set of experiments, we assessed p105 protein level after

knocking down endogenous A2BAR by siRNA. Knockdown of

endogenous A2BAR robustly reduced endogenous p105 protein

level in resting cells (Fig. 4D), but had no effect on p105 mRNA

level (supplementary material Fig. S7).

In the canonical NFkB pathway, IkB kinase b (IKKb) induces

quick degradation of p105 and activates NFkB signaling (Beinke

and Ley, 2004). We, thus, assessed A2BAR-mediated p105

stabilization after p105 degradation was accelerated by the

expression of IKKb-SS/EE, the constitutively active form of

IKKb. The level of p105 was reduced by IKKb-SS/EE

expression, as expected, and further decreased by A2BAR

knockdown (Fig. 4D). Because amino acids 292–302 of A2BAR

are necessary for binding to p105 in YTH assays (Fig. 1C), we

generated a binding deficient mutant (A2BARD292–302) of

A2BAR by deleting amino acids 292–302. As expected, the

mutation abolished the interaction of A2BAR and p105 in

HEK293T cells (Fig. 4E). More importantly, the mutant failed

to increase, even slightly decreased, p105 protein expression

(Fig. 4E), demonstrating that the physical association with p105

is critical for the stabilizing effect of A2BAR on p105.

In view of the fact that A2BAR and ubiquitin ligases

binding sites in p105 overlap (Fig. 1B), we tested whether

A2BAR binding obstructs p105 ubiquitylation and thereby

prevents ubiquitin-proteasome dependent degradation of p105.

Significantly, A2BAR expression markedly suppressed p105

polyubiquitylation (Fig. 5A). MG132, the proteasome inhibitor,

greatly elevated p105 polyubiquitylation, confirming that

polyubiquitylated p105 is mostly, if not all, directed to the

proteasome for degradation. In marked contrast, MG132 had little

effect on p105 polyubiquitylation in the presence of A2BAR

expression, further supporting that the A2BAR effectively blocks

the proteasome-targeted ubiquitylation of p105. As control, the

expression of another integral membrane protein cystic fibrosis

transmembrane conductance regulator (CFTR) slightly increased

p105 ubiquitylation, arguing against the possibility that the

decrease in p105 polyubiquitylation upon A2BAR expression is

an artifact resulting from protein overexpression. It is well known

that IKKb phosphorylates serines 927 and 933 in p105, triggering

the ubiquitylation and subsequent degradation of p105. To test

whether A2BAR binding also affects p105 phosphorylation, we

compared the phosphor-p105 (Serine 933) level in the presence

or the absence of A2BAR. A2BAR had no effect on the

phosphorylation level of p105 (Fig. 5B), suggesting that

A2BAR specifically blocks the binding of ubiquitin ligases, but

not IKKb, to p105.

p105 acts as a NFkB-inhibitory protein, retaining p50, p65 or

c-Rel in the cytoplasm and inhibiting NFkB signaling pathway.

Our data showed that A2BAR/p105 complex could interact with

Fig. 3. Direct interaction of p105 with

the A2BAR. (A) Association of A2BAR

with p105, both in b-arrestin1/2 wild-type

(WT) and knockout (KO) MEF cells. b-

arrestin1/2 WT or KO MEF cells were

transfected with V5-tagged A2BAR. After

36 h, cell lysates were incubated with

purified GST, GST-p50, GST-p105-C or

GST-p105 fusion protein (30 mg each).

A2BAR was pulled down by GST-p105 or

GST-p105-C, but not by GST-p50 or GST

alone in both b-arrestin1/2 wild-type (left

panel) and knockout MEF cells (right

panel). Data shown are the representative

of four independent experiments. (B) GST,

GST-p50, GST-p105 or GST-p105-C

fusion protein was immobilized on

glutathione beads and incubated with

purified MBP-tagged A2B-C protein.

Pulled-down proteins were then visualized

by immunoblotting with anti-MBP

antibodies. The loading of purified MBP-

A2B-C and GST fusion proteins is also

shown. Data shown are the representative

of three independent experiments.

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p65 and p50 (supplementary material Fig. S8). To examine the

effect of A2BAR-p105 interaction on NFkB activation, HEK293T

cells were first transiently co-transfected with the V5-tagged

A2BAR and an NFkB-dependent luciferase reporter gene. A2BAR

expression modestly suppressed basal NFkB transcription

activity, but this effect was much more profound in the

presence of IKKb-SS/EE stimulation (Fig. 6A, left panel, see

Discussion). CFTR, as a control, failed to alter NFkB activation.

Moreover, NFkB activation was significantly increased by

knocking down endogenous A2BAR with siRNA treatment

under both basal and IKKb-SS/EE induced conditions (Fig. 6A,

right panel), supporting that endogenous A2BAR expression

inhibits NFkB signaling. These observations are further

strengthened by the nuclear translocation assays using

TransAM NFkB ELISA kit. Ectopic expression of the A2BAR

inhibited p65 and p50 nuclear translocation, especially with

IKKb-SS/EE stimulation, while knocking down endogenous

A2BAR increased the nuclear translocation of p65 and p50 with

or without IKKb-SS/EE. As control, A2BAR did not change the

nuclear translocation level of AP1 transcription factor c-Fos

(Fig. 6B). It is noted that in the absence of IKKb-SS/EE

stimulation the inhibitory effect of ectopic A2BAR expression

on p65 and p50 nuclear translocation is small and not statistically

significant, presumably because of the limited sensitivity of the

assay.

p105 expression is reduced in A2BAR2/2 mice

Next, we examined the interrelation between A2BAR and p105

expression in mice. We previously reported that the A2BAR is

expressed primarily or only in the vasculature of tissues, such as

the pancreas, lung, and spleen. This was traced using b-

galactosidase (b-gal) staining, as our A2BAR knockout mice

include b-gal-encoding gene under the control of the A2BAR

gene promoter, instead of the deleted A2BAR gene (Yang et al.,

Fig. 4. A2BAR stabilized p105 and p50 protein expressions in vitro. (A) HEK293T cells were transfected with V5-tagged A2BAR (400 ng plasmid), and after

36 h, p105, p50, p65, A2BAR and b-actin protein levels were measured in whole cell lysates by immunoblotting (upper panel). p105-encoding transcripts were

analyzed by RT-PCR (middle panel). The lower panel shows the quantification of the relative levels of mRNA encoding p105 in four experiments that were

similar to that shown in the middle panel. (B) Immunoblotting, with the indicated antibodies, of the lysates of HEK293T cells transfected with increasing doses of

V5-tagged A2BAR (with a maximum of 400 ng plasmid). (C) At 24 h after transfection of V5-tagged A2BAR (100 ng plasmid), HEK293T cells were treated with

10 mM NECA or DMSO (vehicle) for 12 h, then cell lysates were collected for immunoblotting of p105, p50 and p65 protein expression. Note the relatively small

changes of p105 and p50 because there was less A2BAR expression (100 ng) in these experiments than those shown in panel A and B (400 ng A2BAR). Data

shown in panels A-C are the representative of 4–5 independent experiments. (D) At 6 h after transfection of A2BARSiRNA or NcSiRNA (control), HA-tagged

IKKb-SS/EE or mock vectors (pcDNA3.1-HA) were expressed in HEK293T cells. The expression of various proteins was examined with immunoblotting, and the

expression of endogenous A2BAR was examined by RT-PCR rather than immunoblotting because of lack of reliable A2BAR antibodies (upper panel). The lower

panel shows a summary of the relative protein amount of p105 normalized to b-actin in six experiments that were similar to those shown in the upper panel.

*significantly different from NcSiRNA, P50.010 for cell without IKKb-SS/EE, **P50.008 for cell with IKKb-SS/EE. b-actin was used as a loading control in

panels A-D. (E) V5-tagged wild-type A2BAR, mutant A2BAR (A2BARD292–302) or pcDNA4-V5 (Mock) expressed in HEK293T cells was immunoprecipitated

with anti-V5 antibody. The immunoprecipitates were immunoblotted with anti-p105 or anti-V5 antibodies. Whole cell lysates were analyzed with anti-p105 and b-

actin antibodies (upper panel). The lower panel shows quantification of the relative protein amount of p105 normalized to b-actin in five experiments similar to

those shown in the upper panel. **different from Mock cells, P50.0014.

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2006). High A2BAR expression was noted in arteries (Yang et al.,2006). In accordance, A2BAR deficiency is associated with

reduced levels of p105 in the mesenteric artery (Fig. 7A), but itdoes not induce a significant change in the basal level of p50 or

p65 (supplementary material Fig. S9A). Macrophages are also asignificant site of expression of this receptor (Yang et al., 2006).

In these cells too p105 level is reduced compared to wild type(Fig. 7B), with no significant changes in p50 or p65

(supplementary material Fig. S9B). Finally, A2BAR deficiencydoes not promote a change in the level of p105 in various tissues,

which typically do not express significant levels of the A2BAR(supplementary material Fig. S9C). Lipopolysaccharide (LPS)

upregulates the expression of the A2BAR in various tissues (StHilaire et al., 2008; and data not shown). Here, we show that

p105 is downregulated in different tissues following in vivo

administration of LPS into A2BAR knockout mice, relative to

wild-type mice (Fig. 7C). As control, p65, p50 and IkBaexpression levels did not change significantly between the

control and KO samples. Taken together, these results are inline with our above-described biochemical studies, which showed

that A2BAR expression stabilizes the level of p105 protein.

Cytokines production in A2BAR2/2 mice

The expression of target genes of NFkB signaling was examinedas additional functional evidence of reduced p105 expression in

A2BAR knockout mice. Numerous genes are modulated by NFkBsignaling, however, we primarily focused on cytokine gene

expression in order to better understand the role of A2BAR ininflammation. Previous studies indicated that NFkB signaling

regulates the production of numerous cytokines, including TNF-a, IL-2, IL-6, IL-8 and IL-12 (Blackwell and Christman, 1997).

In addition, it was suggested that following LPS treatment p105/p50 deficient mice produce more pro-inflammatory cytokines,

such as TNF-a and IL-12, and less anti-inflammatory cytokines,including IL-10 (Cao et al., 2006). Therefore, to define the

impact of A2BAR expression on p105-mediated NFkB signaling,we measured serum concentrations of TNF-a, IL-12 and IL-10 in

LPS-treated A2BAR knockout mice. A2BAR deficiency resultedin 31% suppression of IL-10 and 35% elevation of TNF-a at 1 h,

and 35% augmentation of IL-12 at 5 h after LPS stimulation(Fig. 8). These results are consistent with data reported by Yang

et al. (Yang et al., 2006) and support the notion that A2BARexpression, in association with elevated p105 (Fig. 7), regulates

cytokine production, thereby suppressing inflammatoryresponses. Since adenosine is present at some level in different

cellular milieus, we cannot conclude that some or all these effectsin vivo are agonist-independent.

DiscussionOur study has unveiled a novel mechanistic insight into the control

of the NFkB pathway and inflammation, and a surprising functionof A2BAR, a typical member of the GPCR family. This study is the

first to report that a GPCR directly interacts with p105. We haveshown that A2BAR binds to p105 in regions overlapping with the

ubiquitin ligase binding sites, and therefore sequesters p105from polyubiquitylation and proteosome-dependent degradation,

resulting in downregulation of NFkB signaling in an agonist-independent way. This agonist-independent A2BAR regulation of

Fig. 5. A2BAR expression suppressed p105 polyubiquitylation but not phosphorylation. (A) HEK293T cells expressing Flag-tagged ubiquitin and V5-tagged

p105 with or without CFP-tagged A2BAR or GFP-tagged CFTR. At 48 h after transfection, cells were treated with 10 mM MG132 or DMSO (vehicle) for 3 h,

followed by immunoprecipitation of p105 with anti-V5 antibodies. Immunoblotting of polyubiquitylated p105 (upper panel) and corresponding

immunoprecipitated p105 (second panel) are shown. Note that loading of p105 in the two lanes with A2B-CFP expression are only 40% of other lanes in order to

better view the differences in p105 ubiquitylation. Summary data of the relative ubiquitylation amount of p105 (normalized to the amount of immunoprecipitated

p105 protein) were shown in the lower panel. *significantly different from Mock cells (pEGFP-N1 transfected cells), P50.021 for DMSO, P50.026 for MG132

(n54). (B) HEK293T cells were transfected with V5-tagged A2BAR or GFP-tagged CFTR or pcDNA4-V5 (Mock) in the presence or absence (control) of HA-

tagged IKKb-SS/EE. After 36 h, the levels of total and phosphorylated p105, and other various proteins were subjected to immunoblot analysis (upper panel). The

summary data of the relative phosphorylation of p105 (normalized to the total amount of p105 protein) is shown in the lower panel (n54; P.0.05).

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NFkB signaling may have important biological implications, given

that inflammation and cell stress selectively upregulate A2BAR

expression by 3–4 folds (Blackburn et al., 2009). The elevation of

A2BAR expression induced by inflammation could downregulate

NFkB signaling and suppress inflammation, forming a negative

feedback loop (summarized in supplementary material Fig. S10).

Emerging evidence suggests that GPCRs can also signal

through G-protein independent pathways, although the classical

paradigm of GPCR signaling involves interaction of ligand with

the receptor, followed by G protein activation by the ligand-

bound receptor and modulation of intracellular signaling proteins,

or targets. The effectors of the G-protein independent pathways

include ion channels, and c-Src and Jak kinases (Liu et al., 2000).

Although the present study is the first report of a Family A

GPCR directly interacting with a transcriptional factor, two

independent studies have previously found that the metabotropic

GABAB receptor, a Family C GPCR, directly interacts with the

transcription factors ATF4 (CREB2) and ATFx to modulate their

cytosol-to-nucleus translocation, even though in an agonist- and

G-protein-dependent manner (Nehring et al., 2000; White et al.,

2000). In addition, GABAB receptor also interacts with the

transcription factor CCAAT/enhancer-binding protein (C/EBP)

homologous protein (CHOP), resulting in reduced surface

expression of the receptor (Sauter et al., 2005). In our study,

we addressed whether the physical and functional interaction

between A2BAR and p105 depends on A2BAR agonist, as the

interactions of GABAB and b2 adrenergic receptors with

transcriptional factors are agonist-dependent (Gao et al., 2004;

White et al., 2000). Our data support an agonist-independent

mechanism. Of note, however, in our experiments, we examined

the effect of A2BAR activation by NECA on the interaction of

A2BAR and p105, but not on NFkB activation per se. Our data

suggest that NECA did not affect NFkB by altering the

interaction of A2BAR and p105, but the current study did not

test whether or not the activation of A2BAR could stimulate

NFkB through other signaling pathways. We also examined

the effect of A2BAR-p105 interaction on A2BAR function,

considering that its interaction with the transcription factor

CCAAT/enhancer-binding protein (C/EBP) homologous protein

(CHOP) resulted in reduced surface expression of GABAB

receptor (Sauter et al., 2005). p105 expression did not seem to

have any impact on A2BAR-mediated cAMP generation (not

shown).

It is well documented that GPCRs regulate NFkB signaling via

activation of PKA, PKC and small GTPases (Diaz-Meco et al.,

1994; Zhong et al., 1998). A GPCR has recently been linked with

IkBa and NFkB/p105 through arrestin (Gao et al., 2004;

Parameswaran et al., 2006; Witherow et al., 2004), which binds

to ligand-bound GPCRs and mediates receptor endocytosis.

Among the four isoforms of the arrestin family, two are

expressed exclusively in the visual system, and the other two

(b-arrestin 1 and 2) are ubiquitously expressed. It was suggested

Fig. 6. A2BAR negatively regulates NFkB transcriptional activity. (A) A2BAR expression decreased basal and IKKb-SS/EE-induced NFkB transcriptional

activity. HEK293T cells were transfected with plasmids encoding the 3XkB-luciferase reporter, b-gal and V5-tagged A2BAR or GFP-tagged CFTR or pcDNA4-

V5 (Mock) in the presence or absence (control) of HA-tagged IKKb-SS/EE. Luciferase activity was normalized to b-gal activity to correct for variability in

transfection efficiency. **significantly different from mock cells, P50.004, n59 for control; ***P50.0001, n57 for IKKb-SS/EE (left panel). After knocking

down endogenous A2BAR by A2BSiRNA, basal and IKKb-SS/EE-induced NFkB transcriptional activities were determined in HEK293T cells transfected with

3XkB-luciferase reporter plasmid and b-gal vector. Non-targeting shRNAi (NcSiRNA) was used as a control. *significantly different from NcSiRNA, P50.044

for control; P50.030 for IKKb-SS/EE, n54 (right panel). (B) HEK293T cells were transfected with indicated vectors. After 36 h, p65, p50 and c-Fos nuclear

translocations were measured using the TransAMTM NFkB or AP1 c-Fos ELISA kit, following the manufacturer’s instructions (Active Motif). Left panel,

*significantly different from IKKb-SS/EE, P50.012 for p65, P50.028 for p50, n56. Right panel: *significantly different from NcSiRNA, P50.030 for p65,

**P50.008 for p50; **significantly different from NcSiRNA+IKKb-SS/EE, P50.005 for p65, P50.010 for p50, n56.

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that arrestin directly bind to the DD/PEST domains of IkBa (Gao

et al., 2004; Witherow et al., 2004) or p105 (Parameswaran et al.,

2006) and prevents their phosphorylation and degradation.

Interestingly, agonist stimulation of b2 adrenergic receptor

enhances the interaction of arrestin and IkBa (Gao et al.,

2004). However, this effect could be mimicked neither by the

stimulation of other GPCRs, such as d opioid receptor and

bradykinin receptor, nor by stimulation of adenylyl cyclase (Gao

et al., 2004). The mechanism underlying this receptor-specific

effect remains unknown. In view of these observations, we asked

whether A2BAR-p105 association is a direct protein-protein

interaction or is also bridged by b-arrestin. By using b-arrestin

null MEF cell lines, we demonstrated that the A2BAR is able to

bind to p105 directly.

The role of the A2BAR in inflammation has been seemingly

puzzling. Numerous pharmacological studies demonstrate that

the activation of A2BAR by adenosine triggers proinflammatory

effects by upregulating proinflammatory cytokines and growth

factors and downregulating anti-inflammatory cytokines in many

cell types (Donoso et al., 2005; Feoktistov and Biaggioni, 1995;

Fiebich et al., 1996; Rees et al., 2003; Ryzhov et al., 2004;

Ryzhov et al., 2008a; Zhong et al., 2004), while there is also

some contradictory evidence, depending on the systems and

conditions used (Eckle et al., 2008a; Eckle et al., 2008b). Yang

and colleagues showed that A2BAR knockout mice display mild

elevation of proinflammatory TNF-a and IL-6, and decreased

levels of anti-inflammatory IL-10 in the plasma under basal

conditions, and more so under LPS stimulation. This was

associated with upregulation of the NFkB pathway (Yang et al.,

2006). These findings implied that the A2BAR is constitutively

activated by the basal concentration of adenosine. This was rather

puzzling, considering that in some cellular systems the A2BAR is

only activated by high adenosine levels, occurring during cell/

tissue stress and damage (Fredholm, 2007). The observation of

Yang et al. (Yang et al., 2006), pointing to an anti-inflammatory

role of A2BAR under basal conditions, was supported by a

number of following studies in A2BAR knockout mice (Csoka

et al., 2010; Eckle et al., 2008a; Eckle et al., 2008b; Frick et al.,

Fig. 7. Reduced levels of p105 in macrophages and tissues from A2BAR

knockout mice. (A,B) Under basal conditions, total proteins isolated from the

mesenteric artery (a site of high A2BAR expression; panel A) and peritoneal

macrophages (non activated; see Materials and Methods; panel B) of wild-

type (WT) and A2BAR knockout (KO) mice were used. Representative

immunoblotting results with anti-p105 antibody are shown in the upper

panels, with b-actin used as loading control. The lower panels of show the

quantification of western blots. In the mesenteric artery, p105 values for KO

are significantly different from WT, ***P50.0006 (n56). Similarly, in

macrophages the differences between WT and KO are statistically significant;

**P50.0095 (n53, each run twice). (C) Wild-type and A2BAR KO mice

were injected intraperitoneally with LPS (10 mg/kg body weight). Proteins

from various tissues were collected after 5 h and immunoblotted (upper

panel). The lower panel shows the quantification of p105 expression in eight

experiments similar to that shown in the upper panel. ***significantly

different from WT A2BAR mice, P50.001 for thymus; *P50.038 for lung;

**P50.007 for spleen; *P50.020 for pancreas.

Fig. 8. Cytokine production in wild-type and A2BAR knockout mice.

Wild-type (WT) and A2BAR knockout KO mice (n514) were injected with

10 mg/kg LPS intraperitoneally. After 1, 5 or 10 h, serum levels of IL-10

(A), TNF-a (B) and IL-12 (C) were examined with ELISA. For IL-10,

*significantly different from wild-type mice, P0 h50.048; ***P1 h50.001;

**P10 h50.006. For TNF-a, *P1 h50.039. For IL-12, **P0 h50.01 and

P5 h50.002; ***P1 h50.0005; *P10 h50.044.

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2009; Grenz et al., 2008; Hart et al., 2009; Hua et al., 2007;Ryzhov et al., 2008b; Yang et al., 2008; Zhou et al., 2009).

However, most of these studies did not scrutinize whether thebasal phenotype in A2BAR knockout mice resulted from loss ofdirect agonist activation, or rather an unidentified function of theA2BAR. Ryzhov et al., (Ryzhov et al., 2008a) observed an

increase in inflammatory indices under basal conditions (nonLPS) in A2BAR knockout mice compared to the wild type in theabsence of adenosine stimulation, similar to the studies by Yang

et al. (Yang et al., 2006) and other groups. Similar to theconclusion of these authors, we deduced that the A2BARcan also exert an adenosine-independent downregulation of

pro-inflammatory cytokines by associating with a previouslyunrecognized signaling pathway(s), identified in our currentstudy. We also consider the report of direct regulation ofexpression of IL-10 by NFkB (Cao et al., 2006). Our data suggest

an alternative explanation of how the A2BAR could regulate IL-10 production through A2BAR-dependent NFkB signaling, inaddition to the A2BAR-p38-CREB pathway reported by Koscso

et al. (Koscso et al., 2012).

In our in vivo experiments, no significant change in the basalprotein level of p105 was observed in tissues from A2BAR

knockout mice in which the A2BAR is typically poorly expressed,compared with wild-type mice. It is also possible that in in vivo

resting cells p105 is ‘‘inactive’’ and sequesters NFkB dimer in thecytoplasm, and therefore, the protective effect of A2BAR is not

manifested. LPS effectively upregulates the expression of theA2BAR (St Hilaire et al., 2008). Upon LPS stimulation, mostp105 is degraded in A2BAR knockout mice, whereas in wild-type

mice p105 levels are high, which would be expected if theA2BAR prevents p105 polyubiquitylation and degradation. Ofnote, luciferase activity and nuclear translocation of p65 or p50,

indicative of NFkB signaling was modestly downregulated uponectopic expression of the A2BAR, although p105 level wasaugmented. This is probably due to already saturating levels of

p105 in the control cells prior to A2BAR overexpression.Alternatively, the overexpression of A2BAR activates thereceptors without agonists, as it is well known that theoverexpression of GPCRs without agonist stimulation mimics

agonist-dependent activation of the receptors (Milano et al.,1994). The activation of A2BAR could stimulate NFkB signalingvia PKA and PKC (Diaz-Meco et al., 1994; Zhong et al., 1998),

masking its agonist-independent protective effect on p105 inunstimulaled cells, and this masking effect is likely trivial whenNFkB signaling is substantially activated by IKKb.

We consistently observed elevation of p50 level with p105protein increase as a result of ectopic A2BAR expression(Fig. 4A,B). However, p105 decrease associated with eitherA2BAR knock down/knockout or IKKb stimulation was not

accompanied with a p50 decrease (Fig. 4D; Fig. 7C;supplementary material Fig. S9). It has been previouslyreported that decrease of p105 was not associated with any

changes in basal p50 level (Heissmeyer et al., 2001), suggestingthat basal p50 is primarily generated through de novo synthesis(with co-translation of p105) (Ciechanover et al., 2001) and p105

processing into p50 is largely inhibited with docking of p50under basal conditions (Cohen et al., 2001). However, when p105is upregulated post-translationally without accompanying p50

increase from co-translation, pre-existing level of p50 may not besufficient to block p105 processing into p50 and therefore anincrease of p50 will occur.

Finally, initial co-immunoprecipatation experiments indicatedthat p105 does not bind to the A2AAR (data not shown).

However, we cannot rule out the possibility that p105 interactswith other members of the adenosine receptor family. This couldconstitute a future line of investigations.

In sum, our study unveils novel mechanistic insights into thecontrol of the NFkB pathway and inflammation. It is the first

report to show that the A2BAR negatively regulates NFkBactivation by physically interacting with p105, thereby blockingits polyubiquitylation and degradation, with clear implications onNFkB-regulated pathways (supplementary material Fig. S10).

Materials and MethodsMice

A2BAR knockout mice were generated in an earlier study (Yang et al., 2006) andwere backcrossed to C57BL/6J mice to reach a pure C57BL/6 background (Yanget al., 2008). Wild-type and A2BAR knockout mice were strain-, sex-, and age-matched (6–8 weeks old, unless otherwise indicated). All animal procedures wereapproved by the University Committee on Research Practices at Hong KongUniversity of Science and Technology.

Reagents and antibodies

Anti-p105, anti-p65, anti-IkBa, anti-HA, anti-b-actin and anti-GFP antibodies werefrom Santa Cruz, anti-NFkB1/p105 for immunostaining and anti-phospho-NFkBp105 (Ser933) from Cell Signaling, anti-V5 antibody from Invitrogen, anti-CFTRantibody from Chemicon, and anti-GST antibody from GE Healthcare (UK).

Plasmids

Human p105 cDNA was PCR amplified and subcloned into pcDNA4-V5(Invitrogen). A2BAR-pcDNA4-V5/Flag and pCFP-N1-A2BAR vectors weregenerated by subcloning human A2BAR cDNA from pRK5-A2BAR (a gift fromDr S. V. Sitaraman). A2BARD292–302-pcDNA4-V5 was generated by deletingamino acids 292–302 from C-terminus of A2BAR using overlapping extensionPCR. Flag-tagged ubiquitin vector was provided by Drs. Gerd Walz and Yan Wangat University of Freiburg (Wang et al., 2007). NFkB-dependent luciferase reportervector and HA-tagged IKKb were from Z. W.’s lab. The constitutively active HA-tagged IKKb (HA-IKKb-SS/EE) vector was generated using the QuickChange(Stratagene). pIRES2-EGFP-CFTR vector was obtained from Dr Xiandi Gong atNanyang Technological University. For GST or Maltose-binding protein (MBP)-tagged recombinant plasmids, various proteins were subcloned into pGEX6p-1(Amersham Pharmacia Biotech) or into pMAL-c5X vector (New EnglandBiolabs). In yeast two-hybrid screens, various cDNA fragments of A2BAR andp105 were constructed into pDBLeu and pPC86 (gifts from Dr Jun Xia atHKUST). All constructs were verified by DNA sequencing.

Yeast two-hybrid screen

Yeast two-hybrid screen was performed with the Matchmaker GAL4 Two-hybridSystem (Clontech) and the human lung cDNA library (Invitrogen).

Cell culture and transfection

Wild-type and b-arrestin-null mouse embryonic fibroblast (MEF) cell lines (giftsfrom Dr R. J. Lefkowitz at Duke University) and HEK293T cells (ATCC CRL-11268TM) were maintained in Dulbecco’s modified Eagle’s medium (DMEM)supplemented with 10% fetal bovine serum (FBS), 100 units/ml penicillin/streptomycin in an atmosphere of 95% air-5% CO2 at 37 C. All transfection wereperformed with Lipofectamine 2000 reagent kit (Invitrogen).

RNA isolation and RT-PCR

Total RNA was isolated from HEK293T cells with TRIZOLH reagents (Invitrogen).RT-PCR was performed with OneStep RT-PCR kits (Qiagen). The primer pairs usedwere as follows: for A2BARs, 59-CTCTTCCTCGCCTGCTTCGT-39 (sense) and 59-GGGCAGAACACACCCAAAGAA-39 (antisense) (expecting a 345 bp fragment);for p105, 59-ATGGCAGAAGATGATCCA-39 (sense) and 59-AAATTTTGCC-TTCTAGAGG-39 (antisense) (expecting a 2.9 kb fragment). b-actin was used as aloading control. The identities of the PCR products were confirmed by DNAsequencing.

RNA interference

RNA oligonucleotides were synthesized by Dharmacon (Lafayette, CO). TheA2BAR siRNA 59-AACCGAGACUUCCGCUACA-39or non-targeting siRNA(Catalog no. D-001210-01-05) was inserted into pSuper vector (Oligoengine,Seattle, WA). HEK293T cells grown on plates at 70–80% confluency were pre-incubated for 6 h with A2BAR or non-targeting siRNA and Lipofectamine 2000

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(Invitrogen) in FBS-free DMEM. The cells were then transfected with othervarious vectors and grown for 36–48 h before collection.

Pull down assaysAfter bound with GST or GST fusion proteins generated and purified from E. colistrain BL21, the glutathione Sepharose beads were loaded with cell lysates at 4 Covernight, followed by 3 washes. The bound proteins were analyzed by westernblotting. MBP-A2B-C fusion protein was expressed in BL21 bacteria and purifiedby amylose resin kit (New England Biolabs).

Immunoprecipitation and western blottingApproximately 100–200 mg of whole cell extracts were incubated with ,25 ml bedvolume of protein A Sepharose beads (Santa Cruz) and 1 mg antibody. The totalvolume of the reaction was then adjusted to 400 ml with a CO-IP lysis buffer(HEPES 20 mM, NaCl 175 mM, NP-40 0.25%, glycerol 10%, EDTA 1 mM, DTT1 mM, PMSF 1 mM, pH 7.6 adjusted with NaOH), supplemented with 16Complete Protease Inhibitor cocktail (Roche). After incubation at 4 C overnight,the beads were washed 3 times with 700 ml CO-IP lysis buffer. Then, the immuno-complexes were collected for western blotting, as previously described (Sun et al.,2008).

ImmunostainingHEK293T cells grown on coverslips were transiently transfected with variousfluorescent vectors for 36 h. Cells were fixed by 10 min incubation in 4%paraformaldehyde and permeabilized with 0.2 M NH4Cl/phosphate buffered saline(PBS) plus 0.2% Triton X-100. After blocking with 3% BSA/PBS, cells werestained with various combinations of primary antibodies and corresponding FITC-or TRITC-conjugated secondary antibodies. Fluorescent images were obtained byZeiss Laser Scanning Confocal Microscope (LSM7 DUO) with a 406 oilimmersion objective (Zeiss Plan-Neofluar), and analyzed with Zen 2009 LEsoftware.

Ubiquitylation assaysAt 48 h after transfection, HEK293T cells were pretreated with MG132 (10 mM)or DMSO for 3 h at 37 C and lysed for 40 min in ubiquitylation lysis buffer A(8 M urea, 100 mM NaH2PO4, 1% Triton X-100, 10 mM Tris, pH 8.0). Thesupernatant was incubated with Ni2+-nitrilotriacetic acid-agarose (Qiagen) for 2 h.After washing twice with buffer A and twice with buffer B (same as buffer Aexcept for 0.5% Triton X-100 and pH 6.3), the proteins were eluted twice withbuffer C (same as buffer A except for 0.1% Triton X-100 and pH 4.5). All theprocedures were performed at room temperature.

Luciferase reporter assaysHEK293T cells were co-transfected with 180 ng 36 NFkB luciferase reportervector, 20 ng b-gal vector as an indicator of transfection efficiency, and 600 ng ofvarious expression vectors. After 36–48 h, the supernatants of whole cell extractswere harvested for luciferase assays with a kit (Tropix, Bedford, MA) andluciferase activities were quantified by FLUOstar OPTIMA microplate reader(BMG Lab technologies, Germany) and normalized to parallel b-gal intensity tocorrect for differences in transfection efficiency. Protein concentration wasmeasured using Bio-Rad DC Protein Assay (Bio-Rad Laboratories).

NFkB and AP1 activation assaysNuclear proteins from HEK293T cells were prepared using the TransAMTM

transcription assay kit (Active Motif). Nuclear extracts (5 mg; prepared asinstructed by the manufacturer) were used to measure p65 and p50 nucleartranslocation with TransAMTM NFkB Family kit, following the manufacturer’sprotocol. Nuclear c-Fos was analyzed with the TransAMTM AP1 c-Fos kit (ActiveMotif).

Mouse tissue isolation and cytokine assaysMesenteric arteries were removed from A2BAR KO and control 8-week-old malemice (strain-matched) and flash frozen. The tissue was homogenized and lysed onice in radioimmunoprecipitation assay (RIPA) buffer A (50 mM Tris-HCl pH 8.0,150 mM NaCl, 1% NP-40, 0.5% sodium deoxycholate, 0.1% SDS) with proteinaseinhibitor cocktail. Lysates were vortexed for 10 min at 4 C and then placed on icefor 30 min. The lysates were then frozen in liquid nitrogen and then thawed at37 C. This freeze-thaw cycle was repeated. The samples were centrifuged at 800 gfor 10 min at 4 C and the infranatant removed. This was repeated to clear thesample of all cell debris and remaining fat tissue. Following protein determinationwith the BioRad assay, samples were subjected to western blotting (75 mg/lane).To upregulate the expression of the A2BAR in other tissues, 6–8-week-old A2BARKO and wild-type mice were challenged with Lipopolysaccharides (LPS)administered intraperitoneally (10 mg/kg, Sigma 026:B6). For immunoblotting,mice were sacrificed 5 h after LPS treatment, different tissues were homogenizedin RIPA buffer B (50 mM Tris-HCl, 1.0 mM EDTA, 150 mM NaCl, 0.1% SDS,1% NP-40, 0.25% sodium deoxycholate, 1 mM PMSF, pH 7.4). After

centrifugation at 16,000 g for 10 min, the supernatant was collected forimmunoblot analysis. For cytokine assays, mouse serum was collected atdifferent time points from 1–10 h after LPS challenge, a time frame in whichcytokine release is maximal (Cao et al., 2006). The levels of TNFa, IL-10, and IL-12 were measured using ELISA kits from eBioscience (San Diego, CA).

Peritoneal macrophage isolation

Peritoneal macrophages were collected from 12-week-old mice as non activatedand seeded into plates at 16106 cells/ml in Macrophage serum free medium(Invitrogen, 12065), supplemented with 0.1% penicillin-streptomycin for 2 hoursat 37 C. Cells were then collected in RIPA buffer A (see above) for western blotanalysis. This yields a low number of macrophages, all used for western blotting(50 mg protein/lane).

Quantification of western blots and statistics

Quantification of protein bands in western blots was carried out with ImageJsoftware. All data are expressed as means 6 SE. Unless indicated otherwise,Student’s two-tailed t-test was used for statistical analysis. P,0.05 was consideredas statistically significant.

AcknowledgementsWe thank Dr Robert J. Lefkowitz (Duke University) and Dr Gang Pei(Shanghai Institutes for Biological Sciences Chinese Academy ofSciences) for kindly providing b-arrestin WT and KO MEF celllines, Dr Shanthi V. Sitaraman (Emory University) for A2BARcDNA, Dr Gerd Walz (University of Freiburg) for flag-taggedubiquitin vector, and Dr Xiandi Gong (Nanyang TechnologicalUniversity) for pIRES2-EGFP-CFTR vector. We are also grateful toMr Kalun So and Ms Quan You Li for technical assistance and DrWei Zhang for help in figure preparation. Y.S. and P.H. conceivedthe study and wrote the manuscript; P.H. directed the study; Y.S.performed and analyzed most of the work; Y.D. performed initialyeast two-hybrid screening and contributed to experimental designand data analysis in other yeast two-hybrid assays, A.S.E. performedanalyses in mouse mesenteric artery and macrophages; W.H.performed macrophage assays; A.Q. performed initial yeast two-hybrid screening; W.K.L. performed pair-wise pull down assays;Y.W. partially contributed to ubiquitylation and A2BAR knockdown experiments; W.Z. provided reagents and contributed toexperimental design; K.R. provided the A2BAR knockout mice,participated in the analyses of arteries and macrophages, providedconceptual insights, and participated in manuscript writing.

FundingThis work was supported by Hong Kong Research Grants Council[grant number GRF661008 to P.H.]; and by the National Heart,Lung, and Blood Institute (NHLBI) [grant number HL093149 toK.R.]. Deposited in PMC for release after 12 months.

Supplementary material available online at

http://jcs.biologists.org/lookup/suppl/doi:10.1242/jcs.105023/-/DC1

ReferencesBeinke, S. and Ley, S. C. (2004). Functions of NF-kappaB1 and NF-kappaB2 in

immune cell biology. Biochem. J. 382, 393-409.

Blackburn, M. R., Vance, C. O., Morschl, E. and Wilson, C. N. (2009). Adenosinereceptors and inflammation. Handb. Exp. Pharmacol. 193, 215-269.

Blackwell, T. S. and Christman, J. W. (1997). The role of nuclear factor-kappa B incytokine gene regulation. Am. J. Respir. Cell Mol. Biol. 17, 3-9.

Cao, S., Zhang, X., Edwards, J. P. and Mosser, D. M. (2006). NF-kappaB1 (p50)homodimers differentially regulate pro- and anti-inflammatory cytokines inmacrophages. J. Biol. Chem. 281, 26041-26050.

Ciechanover, A., Gonen, H., Bercovich, B., Cohen, S., Fajerman, I., Israel, A.,

Mercurio, F., Kahana, C., Schwartz, A. L., Iwai, K. et al. (2001). Mechanisms ofubiquitin-mediated, limited processing of the NF-kappaB1 precursor protein p105.Biochimie 83, 341-349.

Cohen, S., Orian, A. and Ciechanover, A. (2001). Processing of p105 is inhibited bydocking of p50 active subunits to the ankyrin repeat domain, and inhibition isalleviated by signaling via the carboxyl-terminal phosphorylation/ubiquitin-ligasebinding domain. J. Biol. Chem. 276, 26769-26776.

Csoka, B., Nemeth, Z. H., Rosenberger, P., Eltzschig, H. K., Spolarics, Z., Pacher, P.,

Selmeczy, Z., Koscso, B., Himer, L., Vizi, E. S. et al. (2010). A2B adenosine receptors

Journal of Cell Science 125 (19)4516

Journ

alof

Cell

Scie

nce

protect against sepsis-induced mortality by dampening excessive inflammation. J.

Immunol. 185, 542-550.Diaz-Meco, M. T., Dominguez, I., Sanz, L., Dent, P., Lozano, J., Municio, M. M.,

Berra, E., Hay, R. T., Sturgill, T. W. and Moscat, J. (1994). zeta PKC inducesphosphorylation and inactivation of I kappa B-alpha in vitro. EMBO J. 13, 2842-2848.

Donoso, M. V., Lopez, R., Miranda, R., Briones, R. and Huidobro-Toro, J. P.(2005). A2B adenosine receptor mediates human chorionic vasoconstriction andsignals through arachidonic acid cascade. Am. J. Physiol. Heart Circ. Physiol. 288,H2439-H2449.

Eckle, T., Faigle, M., Grenz, A., Laucher, S., Thompson, L. F. and Eltzschig, H. K.

(2008a). A2B adenosine receptor dampens hypoxia-induced vascular leak. Blood 111,2024-2035.

Eckle, T., Grenz, A., Laucher, S. and Eltzschig, H. K. (2008b). A2B adenosinereceptor signaling attenuates acute lung injury by enhancing alveolar fluid clearancein mice. J. Clin. Invest. 118, 3301-3315.

Feoktistov, I. and Biaggioni, I. (1995). Adenosine A2b receptors evoke interleukin-8secretion in human mast cells. An enprofylline-sensitive mechanism with implicationsfor asthma. J. Clin. Invest. 96, 1979-1986.

Fiebich, B. L., Biber, K., Gyufko, K., Berger, M., Bauer, J. and van Calker, D.

(1996). Adenosine A2b receptors mediate an increase in interleukin (IL)-6 mRNA andIL-6 protein synthesis in human astroglioma cells. J. Neurochem. 66, 1426-1431.

Fredholm, B. B. (2007). Adenosine, an endogenous distress signal, modulates tissuedamage and repair. Cell Death Differ. 14, 1315-1323.

Fredholm, B. B., IJzerman, A. P., Jacobson, K. A., Klotz, K. N. and Linden, J.

(2001). International Union of Pharmacology. XXV. Nomenclature and classificationof adenosine receptors. Pharmacol. Rev. 53, 527-552.

Frick, J. S., MacManus, C. F., Scully, M., Glover, L. E., Eltzschig, H. K. andColgan, S. P. (2009). Contribution of adenosine A2B receptors to inflammatoryparameters of experimental colitis. J. Immunol. 182, 4957-4964.

Gao, H., Sun, Y., Wu, Y., Luan, B., Wang, Y., Qu, B. and Pei, G. (2004).Identification of beta-arrestin2 as a G protein-coupled receptor-stimulated regulator ofNF-kappaB pathways. Mol. Cell 14, 303-317.

Grenz, A., Osswald, H., Eckle, T., Yang, D., Zhang, H., Tran, Z. V., Klingel, K.,

Ravid, K. and Eltzschig, H. K. (2008). The reno-vascular A2B adenosine receptorprotects the kidney from ischemia. PLoS Med. 5, e137.

Hart, M. L., Jacobi, B., Schittenhelm, J., Henn, M. and Eltzschig, H. K. (2009).Cutting Edge: A2B Adenosine receptor signaling provides potent protection duringintestinal ischemia/reperfusion injury. J. Immunol. 182, 3965-3968.

Hayden, M. S. and Ghosh, S. (2004). Signaling to NF-kappaB. Genes Dev. 18, 2195-2224.

Heissmeyer, V., Krappmann, D., Hatada, E. N. and Scheidereit, C. (2001). Sharedpathways of IkappaB kinase-induced SCF(betaTrCP)-mediated ubiquitination anddegradation for the NF-kappaB precursor p105 and IkappaBalpha. Mol. Cell. Biol. 21,1024-1035.

Hua, X., Kovarova, M., Chason, K. D., Nguyen, M., Koller, B. H. and Tilley, S. L.

(2007). Enhanced mast cell activation in mice deficient in the A2b adenosinereceptor. J. Exp. Med. 204, 117-128.

Ishikawa, H., Claudio, E., Dambach, D., Raventos-Suarez, C., Ryan, C. and Bravo,

R. (1998). Chronic inflammation and susceptibility to bacterial infections in micelacking the polypeptide (p)105 precursor (NF-kappaB1) but expressing p50. J. Exp.

Med. 187, 985-996.Kolachala, V., Asamoah, V., Wang, L., Obertone, T. S., Ziegler, T. R., Merlin, D.

and Sitaraman, S. V. (2005). TNF-alpha upregulates adenosine 2b (A2b) receptorexpression and signaling in intestinal epithelial cells: a basis for A2bR overexpressionin colitis. Cell. Mol. Life Sci. 62, 2647-2657.

Kong, T., Westerman, K. A., Faigle, M., Eltzschig, H. K. and Colgan, S. P. (2006).HIF-dependent induction of adenosine A2B receptor in hypoxia. FASEB J. 20, 2242-2250.

Koscso, B., Csoka, B., Selmeczy, Z., Himer, L., Pacher, P., Virag, L. and Hasko, G.(2012). Adenosine augments IL-10 production by microglial cells through an A2Badenosine receptor-mediated process. J. Immunol. 188, 445-453.

Liu, F., Wan, Q., Pristupa, Z. B., Yu, X. M., Wang, Y. T. and Niznik, H. B. (2000).Direct protein-protein coupling enables cross-talk between dopamine D5 and gamma-aminobutyric acid A receptors. Nature 403, 274-280.

Matharu, A. L., Mundell, S. J., Benovic, J. L. and Kelly, E. (2001). Rapid agonist-induced desensitization and internalization of the A(2B) adenosine receptor ismediated by a serine residue close to the COOH terminus. J. Biol. Chem. 276, 30199-30207.

Milano, C. A., Allen, L. F., Rockman, H. A., Dolber, P. C., McMinn, T. R., Chien,

K. R., Johnson, T. D., Bond, R. A. and Lefkowitz, R. J. (1994). Enhancedmyocardial function in transgenic mice overexpressing the beta 2-adrenergic receptor.Science 264, 582-586.

Nehring, R. B., Horikawa, H. P., El Far, O., Kneussel, M., Brandstatter, J. H.,Stamm, S., Wischmeyer, E., Betz, H. and Karschin, A. (2000). The metabotropicGABAB receptor directly interacts with the activating transcription factor 4. J. Biol.

Chem. 275, 35185-35191.

Orian, A., Schwartz, A. L., Israel, A., Whiteside, S., Kahana, C. and Ciechanover,A. (1999). Structural motifs involved in ubiquitin-mediated processing of the NF-kappaB precursor p105: roles of the glycine-rich region and a downstreamubiquitination domain. Mol. Cell. Biol. 19, 3664-3673.

Parameswaran, N., Pao, C. S., Leonhard, K. S., Kang, D. S., Kratz, M., Ley, S. C.

and Benovic, J. L. (2006). Arrestin-2 and G protein-coupled receptor kinase 5interact with NFkappaB1 p105 and negatively regulate lipopolysaccharide-stimulatedERK1/2 activation in macrophages. J. Biol. Chem. 281, 34159-34170.

Pereira, S. G. and Oakley, F. (2008). Nuclear factor-kappaB1: regulation and function.Int. J. Biochem. Cell Biol. 40, 1425-1430.

Rees, D. A., Lewis, B. M., Lewis, M. D., Francis, K., Scanlon, M. F. and Ham, J.

(2003). Adenosine-induced IL-6 expression in pituitary folliculostellate cells ismediated via A2b adenosine receptors coupled to PKC and p38 MAPK. Br. J.

Pharmacol. 140, 764-772.

Ryzhov, S., Goldstein, A. E., Matafonov, A., Zeng, D., Biaggioni, I. and Feoktistov,

I. (2004). Adenosine-activated mast cells induce IgE synthesis by B lymphocytes: anA2B-mediated process involving Th2 cytokines IL-4 and IL-13 with implications forasthma. J. Immunol. 172, 7726-7733.

Ryzhov, S., Zaynagetdinov, R., Goldstein, A. E., Novitskiy, S. V., Blackburn, M. R.,Biaggioni, I. and Feoktistov, I. (2008a). Effect of A2B adenosine receptor geneablation on adenosine-dependent regulation of proinflammatory cytokines. J.

Pharmacol. Exp. Ther. 324, 694-700.

Ryzhov, S., Zaynagetdinov, R., Goldstein, A. E., Novitskiy, S. V., Dikov, M. M.,

Blackburn, M. R., Biaggioni, I. and Feoktistov, I. (2008b). Effect of A2B adenosinereceptor gene ablation on proinflammatory adenosine signaling in mast cells. J.

Immunol. 180, 7212-7220.

Sauter, K., Grampp, T., Fritschy, J. M., Kaupmann, K., Bettler, B., Mohler, H. andBenke, D. (2005). Subtype-selective interaction with the transcription factor CCAAT/enhancer-binding protein (C/EBP) homologous protein (CHOP) regulates cell surfaceexpression of GABA(B) receptors. J. Biol. Chem. 280, 33566-33572.

Schulte, G. and Fredholm, B. B. (2000). Human adenosine A(1), A(2A), A(2B), andA(3) receptors expressed in Chinese hamster ovary cells all mediate thephosphorylation of extracellular-regulated kinase 1/2. Mol. Pharmacol. 58, 477-482.

Schulte, G. and Fredholm, B. B. (2003). Signalling from adenosine receptors tomitogen-activated protein kinases. Cell. Signal. 15, 813-827.

St Hilaire, C., Koupenova, M., Carroll, S. H., Smith, B. D. and Ravid, K. (2008).TNF-alpha upregulates the A2B adenosine receptor gene: The role of NAD(P)Hoxidase 4. Biochem. Biophys. Res. Commun. 375, 292-296.

Sun, Y., Wu, F., Sun, F. and Huang, P. (2008). Adenosine promotes IL-6 release inairway epithelia. J. Immunol. 180, 4173-4181.

Wang, L., Radu, C. G., Yang, L. V., Bentolila, L. A., Riedinger, M. and Witte, O. N.

(2005). Lysophosphatidylcholine-induced surface redistribution regulates signaling ofthe murine G protein-coupled receptor G2A. Mol. Biol. Cell 16, 2234-2247.

Wang, Y., Fu, X., Gaiser, S., Kottgen, M., Kramer-Zucker, A., Walz, G. and

Wegierski, T. (2007). OS-9 regulates the transit and polyubiquitination of TRPV4 inthe endoplasmic reticulum. J. Biol. Chem. 282, 36561-36570.

White, J. H., McIllhinney, R. A., Wise, A., Ciruela, F., Chan, W. Y., Emson, P. C.,

Billinton, A. and Marshall, F. H. (2000). The GABAB receptor interacts directlywith the related transcription factors CREB2 and ATFx. Proc. Natl. Acad. Sci. USA

97, 13967-13972.

Witherow, D. S., Garrison, T. R., Miller, W. E. and Lefkowitz, R. J. (2004). beta-Arrestin inhibits NF-kappaB activity by means of its interaction with the NF-kappaBinhibitor IkappaBalpha. Proc. Natl. Acad. Sci. USA 101, 8603-8607.

Xaus, J., Mirabet, M., Lloberas, J., Soler, C., Lluis, C., Franco, R. and Celada, A.(1999). IFN-gamma up-regulates the A2B adenosine receptor expression inmacrophages: a mechanism of macrophage deactivation. J. Immunol. 162, 3607-3614.

Yaar, R., Jones, M. R., Chen, J. F. and Ravid, K. (2005). Animal models for the studyof adenosine receptor function. J. Cell. Physiol. 202, 9-20.

Yang, D., Zhang, Y., Nguyen, H. G., Koupenova, M., Chauhan, A. K., Makitalo, M.,

Jones, M. R., St Hilaire, C., Seldin, D. C., Toselli, P. et al. (2006). The A2Badenosine receptor protects against inflammation and excessive vascular adhesion. J.

Clin. Invest. 116, 1913-1923.

Yang, D., Koupenova, M., McCrann, D. J., Kopeikina, K. J., Kagan, H. M.,Schreiber, B. M. and Ravid, K. (2008). The A2b adenosine receptor protects againstvascular injury. Proc. Natl. Acad. Sci. USA 105, 792-796.

Zhong, H., Voll, R. E. and Ghosh, S. (1998). Phosphorylation of NF-kappa B p65 byPKA stimulates transcriptional activity by promoting a novel bivalent interaction withthe coactivator CBP/p300. Mol. Cell 1, 661-671.

Zhong, H., Belardinelli, L., Maa, T., Feoktistov, I., Biaggioni, I. and Zeng, D. (2004).A(2B) adenosine receptors increase cytokine release by bronchial smooth musclecells. Am. J. Respir. Cell Mol. Biol. 30, 118-125.

Zhou, Y., Mohsenin, A., Morschl, E., Young, H. W., Molina, J. G., Ma, W., Sun,C. X., Martinez-Valdez, H. and Blackburn, M. R. (2009). Enhanced airwayinflammation and remodeling in adenosine deaminase-deficient mice lacking the A2Badenosine receptor. J. Immunol. 182, 8037-8046.

A2BAR binds to and stabilizes p105 4517