Agonist- and H O - mediated oxidation of the 2 adrenergic...

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Agonist- and H2O2- mediated oxidation of the 2 adrenergic receptor: evidence of receptor

S-sulfenation as detected by a modified biotin switch assay.

Rebecca N. Burns and Nader H. Moniri1

Department of Pharmaceutical Sciences

College of Pharmacy and Health Sciences

Mercer University

3001 Mercer University Drive

Atlanta, GA 30341

JPET Fast Forward. Published on September 13, 2011 as DOI:10.1124/jpet.111.185975

Copyright 2011 by the American Society for Pharmacology and Experimental Therapeutics.

This article has not been copyedited and formatted. The final version may differ from this version.JPET Fast Forward. Published on September 13, 2011 as DOI: 10.1124/jpet.111.185975

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Running title: Agonist- and H2O2- mediated S-sulfenation of the 2AR.

Corresponding Author1:

Nader H. Moniri, Ph.D.



Mercer University


Atlanta, GA 30341

(678)-547-6246 (tel)

(678)-547-6423 (fax)

[email protected]

# of text pages: 27

# of figures: 5

# of references: 35

# of words in abstract: 249

# of words in introduction: 647

# of words in discussion: 1499

Recommended Section Assignment: Cellular and Molecular

Abbreviations: ROS, reactive oxygen species; 2AR, 2-adrenergic receptor; GPCR, G protein-

coupled receptor; HEK293, human embryonic kidney cells; H2O2, hydrogen peroxide; MMTS,

methyl methanethiosulfonate; ISO, isoproterenol; SOH, sulfenic acid; NAC, N-acetyl-L-cysteine.


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Abstract

Reactive oxygen species (ROS), including hydrogen peroxide (H2O2), have recently been shown

to be generated upon agonism of several members of the G protein-coupled receptor (GPCR)

superfamily, including 2-adrenergic receptors (2AR). Previously, we have demonstrated that

inhibition of intracellular ROS generation mitigates 2AR signaling, suggesting that 2AR-

mediated ROS generation is capable of feeding back to regulate receptor function. Given that

ROS, specifically H2O2, are able to post-translationally oxidize protein cysteine sulfhydryls

(Cys-SH) to cysteine-sulfenic acids (Cys-SOH), the goal of the current study was to assess

whether ROS are capable of S-sulfenating 2AR. Using a modified biotin-switch assay which is

selective for cysteine-sulfenic acids, our results demonstrate for the first time that H2O2 treatment

facilitates S-sulfenation of transiently overexpressed 2AR in HEK293 cells. Importantly,

stimulation of cells with the -agonist isoproterenol produces both dose- and time-dependent S-

sulfenation of 2AR, an effect which is receptor dependent, and which demonstrates that

receptor-generated ROS are also capable of oxidizing the 2AR. Receptor-dependent S-

sulfenation was inhibited by the chemoselective sulfenic acid alkylator dimedone, and the

cysteine anti-oxidant N-acetyl-L-cysteine. Moreover, our results reveal that receptor oxidation

occurs in cells which endogenously express physiologically relevant levels of 2AR, as treatment

of human alveolar epithelial A549 cells with either H2O2 or the 2-selective agonist formoterol

promoted receptor S-sulfenation. These findings provide the first evidence, to our knowledge,

that a mammalian GPCR can be oxidized by S-sulfenation, and signify an important first step

towards shedding light on the overlooked role of ROS in the regulation of 2AR function.


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Introduction

The beta-2-adrenergic receptor (2AR) is the prototypical and best characterized member

of the G protein-coupled receptor (GPCR) superfamily, mediating physiological responses upon

binding of the catecholamines epinephrine and norepinephrine. Agonism of 2AR facilitates

generation of intracellular second messengers, most notably, adenosine 3’,5’-cyclic

monophosphate (cAMP) which activates protein kinase A (PKA) leading to cellular responses.

Termination of agonist-occupied receptor signaling occurs upon phosphorylation of 2AR by

members of the G protein-coupled receptor kinase family, facilitating receptor desensitization

and initiating a cascade of G-protein independent signaling through the recruitment of -arrestin

partner proteins. While cAMP and its effectors, as well as-arrestins have been viewed as chief

mediators of cellular responses upon 2AR activation, our laboratory has been examining a novel

aspect of 2AR signaling, specifically, the role of reactive oxygen species (ROS) in regulation of

2AR function.

ROS, which include hydrogen peroxide (H2O2), superoxide (O2-), and hydroxyl radical

(OH), have traditionally been viewed as cytotoxic byproducts of cellular respiration and

metabolism. However, a growing body of evidence has also demonstrated that ROS play central

roles in transducing intracellular signal events due to their high reactivity within the cellular

milieu. In this regard, it is well described that ROS can post-translationally modify proteins

through modification of cysteine sulfhydryl (-SH) groups, yielding oxidized cysteine-sulfenic

acids (-SOH). This reversible post-translational modification can lead to formation of higher

order redox states such as sulfinic (-SO2H) or sulfonic (-SO3H) acids, disulfides (S-S), or upon

reaction with nitrogen species, S-nitrosothiols (-SNO), any of which can lead to altered protein

function (Charles et al., 2007; Hess et al., 2005; Reddie and Carroll, 2008).


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We have recently demonstrated that agonism of the 2AR facilitates generation of

intracellular ROS, in particular superoxide and hydrogen peroxide (H2O2) (Moniri and Daaka,

2007). These results have been corroborated by other groups using cardiomyocyte models of

cardiomyopathy and heart failure, suggesting that the ROS-2AR link is physiologically

significant and if unregulated, may be involved in pathophysiological states (Li et al., 2010; Xu

et al., 2011). Interestingly, agonism of 2AR in human neutrophils facilitates a decrease in

formyl-Met-Leu-Phe-mediated ROS generation (Opdahl et al., 1993; Anderson, et al., 1996;

Barnett et al., 1997), and others have shown that 2AR agonism specifically decreases only

extracellular ROS from these cells (Kopprasch et al., 1997). Together, these results suggest that

2AR agonism may have cell type-specific differential effects on modulating ROS levels. Our

previous results demonstrate that 2AR-mediated ROS generation was abrogated by the ROS

scavenger and cysteine residue antioxidant N-acetyl-L-cysteine (NAC), the flavin-containing

(NADPH) oxidase inhibitor diphenyliodonium chloride (DPI), and the Rac1 inhibitor

NSC23766, suggesting that 2 receptors can signal through one of the Rac1 dependent NADPH

oxidase isoforms to generate intracellular ROS (Moniri and Daaka, 2007). Recent results

confirm this view, whereby 2AR agonism was shown to stimulate ROS generation via -

arrestin/Rac1-dependent activation of NADPH oxidases (Gong et al., 2008). Most importantly,

our previous work demonstrates that inhibition of ROS markedly abrogates 2AR-mediated

cAMP formation and PKA activity, as well as receptor phosphorylation and internalization, but

does not affect ligand binding (Moniri and Daaka, 2007). These results suggested that oxidants

are indispensible for receptor signaling, and that ROS are likely able to regulate 2AR function.

Based on these aggregate data, we hypothesized that ROS which are generated upon β2AR

agonism can feedback to oxidize the receptor, and that this modification allows β2AR to maintain


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functionally competent conformations which allow for proper signaling. Since ROS such as

H2O2 are capable of oxidizing cysteine residues, initially forming sulfenic acids, we

hypothesized that one or more β2AR cysteine residues could be oxidized to cysteine sulfenic

acids. In this study, we have developed a modified biotin switch assay to determine if exogenous

oxidant (i.e., H2O2) or receptor agonism could mediate direct oxidation of the 2AR. Our results

demonstrate, for the first time, that ROS are capable of S-sulfenating the 2AR and that receptor

agonism also facilitates this oxidative modification.


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Materials and Methods

Chemicals and reagents: Isoproterenol hydrochloride and (S)-propranolol were obtained from

MP Biomedicals (Solon, OH) and Sigma-Aldrich (St. Louis, MO), respectively. (±)-Formoterol

hemifumarate was obtained from Tocris Bioscience (Ellisville, MO). Dimedone (5,5-Dimethyl-

1,3-cyclohexanedione), N-acetyl-L-cysteine, and 9.7M H2O2 were obtained from Sigma-Aldrich.

All other chemicals used were obtained at the highest available purity from Thermo Fisher

Scientific (Waltham, MA) or Sigma-Aldrich. The FLAG-epitope tagged pcDNA3-FLAG-β2AR

construct (Tang et al., 1999) was a kind gift from Dr. Robert J. Lefkowitz (Duke University

Medical Center).

Cell culture, transfection, and treatment: Cell growth media and supplements were obtained

from Hyclone (Thermo Fisher Scientific, Waltham, MA). Human embryonic kidney-293 cells

(HEK293) from the American Type Culture Collection (Manassas, VA) were grown at 37°C in a

humidified atmosphere of 5% CO2 in Dulbecco’s Modified Eagle Medium (DMEM)

supplemented with 10% FBS and 1% penicillin/streptomycin. At approximately 90%

confluency, cells grown in 100 mm dishes were transfected with 5μg of pcDNA3-FLAG-β2AR

plasmid using LipoD293 according to the manufacturer’s instructions (SignaGen, Rockville,

MD). Cells were subcultured accordingly on collagenized (rat tail type-I) 6-well plates at a

density of approximately 2 x 106

cells/well for all experiments, and were serum-starved

overnight prior to assay. The human alveolar epithelial cell line, A549, which endogenously

express 2AR, was a kind gift from Dr. Philip Moos (University of Utah). A549 cells were

cultured exactly as described above except they were grown to 70% confluency in 100 mm

dishes and serum-starved for four hours for all experiments (Szentendrei et al., 1992).


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Sulfenic acid selective biotin-switch assay: Cells were treated with appropriate agents for the

indicated times. For propranolol experiments, cells were pre-incubated with 10 M (S)-

propranolol for 5 minutes prior to agonist stimulation. Micromolar concentrations were used to

ensure that biased -arrestin agonism, which has been demonstrated to occur by propranolol at

the related 1AR, did not contribute to S-sulfenation. For experiments with NAC, cells were

preincubated with 10 mM NAC in starvation media for four hours, as we have described

previously (Moniri and Daaka, 2007). Following stimulation, cells were immediately placed on

ice and washed twice with ice-cold phosphate buffered saline (PBS). Whole cells were

resuspended in ice-cold PBS and collected by centrifugation at 3,000 rpm for 8 minutes at 4°C.

Cells were then lysed in HENS buffer (25 mM HEPES pH 7.6, 1 mM EDTA, 1% SDS, 0.01 mM

neocuproine), homogenized and cleared of cellular debris by centrifugation at 14,000 x g for 15

minutes. Protein concentrations were standardized by DC Protein Assay (Bio-Rad, Hercules,

CA) and the cysteine-reactive methylator methyl methanethiosulfonate (MMTS) (Pierce,

Rockford, IL) was added (20 mM) to methylate free cysteine sulfhydryl groups, thereby

protecting them from further chemical modification, as performed for detection of S-

nitrosylation (Figure 1, step 1) (Jaffrey et al., 2001). Sodium dodecyl sulfate (SDS) was also

added to a final concentration of 2.5% (v/v) to ensure protein denaturation, and lysates were

incubated at 50°C for 30 minutes with frequent agitation followed by acetone precipitation of the

protein. Acetone-precipitated proteins were resuspended in HENS buffer and 20 mM sodium

arsenite (RICCA, Arlington, TX) was added to selectively reduce cysteine sulfenic acids (-SOH)

groups back to the cysteine sulfhydryl (-SH), as demonstrated by Saurin and colleagues (2004)

(Figure 1, step 2). The ability of NaAsO2 to selectively reduce sulfenic acids, and not higher

order sulfinic acids (-SO2H), sulfonic acids (-SO3H), disulfides (S-S), or S-nitrosothiols (-SNO)


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provides sulfenic-acid selectivity to this assay (Saurin et al., 2004). Reduction of sulfenic acids

to free sulfhydryls, coupled with MMTS-mediated protection of initial sulfhydryls, allows for

selective biotinylation of the formerly oxidized sulfenic acids with the sulfhydryl-reactive

biotinylating agent biotin-HPDP (400 M) (Pierce, Rockford, IL) (Figure 1, step 3), as described

for biotin-switch assays which detect protein S-nitrosothiols (Jaffrey et al., 2001). For

experiments testing the specificity of S-sulfenation, 5 mM dimedone was included per reaction

for 30 minutes prior to biotin-switch, as described by others (Saurin et al., 2004). Following

biotin-switch of sulfenic acids, lysates were acetone precipitated and resuspended in HENS/10

(1:10 HENS/H2O) and an aliquot was reserved to determine reaction protein input. The

remaining sample was neutralized with neutralization buffer (20 mM HEPES pH 7.6, 1 mM

EDTA, 100 mM NaCl, 0.5% Triton-X-100) and 30 l of streptavidin-agarose (Pierce, Rockford,

IL) was added to pull-down biotinylated proteins (Figure 1, step 4). After overnight tumbling at

4°C, beads were washed three times in wash buffer (20 mM HEPES pH 7.6, 1 mM EDTA, 600

mM NaCl, 0.5% Triton-X-100) and proteins eluted in Laemmli buffer with 2.5% 2-

mercaptoethanol. Samples, as well as the corresponding reserved inputs were subjected to SDS-

PAGE, transferred to PVDF membranes and immunoblotted with anti-FLAG antibody (M2,

Sigma-Aldrich, St. Louis, MO) for transfected HEK293 cells or with anti-β2AR antibody

(GeneTex Inc., Irvine, CA) for endogenous β2AR in A549 cells (Figure 1, step 5).

Immunoblotted reaction bands correspond to ‘biotin-switched’ S-sulfenated FLAG-2AR or

2AR, and were detected in the appropriate range (~50 kDa) similar to input lysates using ECL.

Data analysis: Resulting data were quantified by densitometric analysis using NIH Image J

(Bethesda, MD) and Graphpad Prism 3.0 (San Diego, CA). Data are expressed as mean ± S.E.M


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for representative experiments repeated at least three independent times. Where not visible, error

bars fall within the symbol size. Statistical analysis was performed, as appropriate, either by

one-way analysis of variance and post-hoc Tukey’s test or by Student’s t-test using Graphpad

Instat (San Diego, CA). Statistical significance is represented as * p < 0.05, ** p < 0.01, *** p <

0.001.


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Results

H2O2 and Isoproterenol stimulate sulfenic acid oxidation of 2AR

Our previous results suggested that agonism of 2AR with the non-selective -receptor

agonist isoproterenol (ISO) lead to marked generation of intracellular ROS. Since inhibition of

ROS abrogated 2AR function, we hypothesized that ROS were capable of directly regulating

the 2AR. Given that ROS are well known to modify protein cysteine residues by oxidizing

them, at least initially, to sulfenic acids, we hypothesized that ROS could directly oxidize the

2AR yielding sulfenic acids. To test this hypothesis, we utilized the biotin switch assay shown

in Figure 1, which has been modified for selective detection of sulfenic acids. As shown in

Figure 2A, treatment of HEK293 cells transiently expressing FLAG-epitope tagged 2AR with

exogenous ROS in the form of H2O2 (100 M) yielded rapid and marked oxidation of 2AR.

Full length immunoblots, shown as supplemental figures, demonstrate that the biotin-switch

signal detected with the anti-FLAG M2 antibody is highly selective for a FLAG immunoreactive

band at approximately 50 kDa, which represents the expected size of FLAG-2AR. Non-specific

bands in the 95-180 kDa range appeared in an exposure length-dependent manner and are likely

to represent aggregated receptors, an effect which is highly typical upon heating and elution of

GPCRs in SDS-sample buffer (supplemental figures). The H2O2-induced S-sulfenation signal

peaked at 1 minute following addition of the oxidant and yielded a 350 ± 20% increase in

oxidation compared to untreated control cells (p < 0.001 compared to control) (Figure 2A-B,

Supplemental Figure 1). Moreover, H2O2-mediated oxidation of 2AR was sustained through the

time course of treatment (5-30 minutes) but began to decline to basal levels after 30 minutes

(Figure 2A-B, Supplemental Figure 1). 2AR S-sulfenation was also detected at lower H2O2

concentrations, and it is noteworthy that while treatment with H2O2 for 1-30 minutes did not


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affect cell viability, treatment for 60 minutes often facilitated detachment of approximately 30%

of HEK293 cells (data not shown), which is likely promoted by the relatively weak adherence of

this cell line. Furthermore, control experiments in which biotin-labeling was performed in the

absence of NaAsO2 demonstrated no appreciable biotin switch signal (data not shown).

Since our and others previous results have shown that agonism of 2AR with ISO

promotes robust generation of intracellular ROS, we examined whether agonism of 2AR with

ISO could facilitate sulfenic acid oxidation of 2AR, similar to that seen upon treatment with

exogenous H2O2. Results in Figure 2C-D, as well as Supplemental Figure 2, demonstrate that

agonism of 2AR with ISO (10 M) facilitates rapid and significant sulfenic acid oxidation of

2AR. Following one minute of agonism, receptor sulfenic acid oxidation was 175 ± 22 % of

control, and this increased to 252 ± 24 % of control after 5 minutes of agonism (p < 0.01 for both

time points versus unstimulated control). Unlike that seen with H2O2, the ISO-mediated sulfenic

acid oxidation of 2AR peaked 15 minutes following agonism, yielding a 306 ± 25 % increase in

oxidation compared to untreated control cells (p < 0.001 versus unstimulated control), and

decreased toward basal levels thereafter (114 ± 5 and 128 ± 11 % of control at 30 and 60

minutes, respectively) (Figure 2C-D, Supplemental Figure 2). Significantly, this timeframe

correlates precisely with our previous results which showed that the height of ISO-mediated

generation of intracellular ROS occurs within 20 minutes of receptor agonism and declines

thereafter (Moniri and Daaka, 2007), and suggests that agonist-mediated ROS generation within

this timeframe modulates 2AR S-sulfenation. As seen in full-length immunoblots in

Supplemental Figures 1-2, the anti-FLAG immunoreactivity was similar in both H2O2 and ISO

treated conditions, with only the major 2AR-specific band (ca. 50 kDa) and the non-specific

aggregation towards the top of the blot (95-180 kDa) being recognized. In order to establish if


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the ISO-mediated receptor oxidation is dose-dependent, we performed the biotin-switch assay

with increasing concentrations of ISO. Initial dose-responses were performed at the peak time

point seen in Figure 2C-D (i.e., 15 min), while a lower oxidative signal was revealed at

concentrations of 10-100 nM, a strong signal was revealed at micromolar concentrations,

suggesting that the oxidative signal could be saturated in the presence of agonist during this

duration (data not shown). Meanwhile, a potent and saturable dose-dependent sulfenic acid

oxidation of 2AR was observed following 5 minutes of agonism with ISO (Figure 2E-F,

Supplemental Figure 3), demonstrating a dose-response relationship at earlier time points. Non-

linear regression analysis of the dose-response graph resulted in a pEC50 of -7.4 ± 0.3 (R2 =

0.90), reaching plateau at 1 M (365 ± 10 % of control, p < 0.001 versus unstimulated control).

It is noteworthy that the ISO-dose response produces a shallow curve with a calculated Hill slope

of approximately 0.7, implying that agonist cooperativity may play a role in the S-sulfenation

effect. While our previous data demonstrated that inhibition of ROS does not affect agonist

binding to the 2AR in membrane preparations (Moniri and Daaka, 2007), results presented here

cannot discount the possibility that agonist-mediated receptor S-sulfenation could indeed affect

binding of subsequent agonist molecules in situ. Moreover, S-sulfenation can be influenced by

intracellular ROS pools, which are increased by 2AR agonism in HEK293 cells, as well as by

favorable reducing conditions which constitutively exist inside cells. These factors could

contribute a ROS-specific component to the extended ternary model of agonist binding and

thereby possibly influence dose-response relationships in regards to any particular agonist’s

potential to induce receptor S-sulfenation.

Isoproterenol-mediated 2AR S-sulfenation is inhibited by 2AR antagonism


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Our previous results demonstrated that 2AR-mediated ROS generation was directly

dependent on receptor agonism, as it was abolished in the presence of the -receptor antagonist

propranolol (Moniri and Daaka, 2007). To determine if 2AR S-sulfenation was receptor-

dependent, we preformed the biotin-switch assay in cells pretreated for 5 minutes with (S)-

propranolol in the absence or presence of ISO. (S)-propranolol alone had no significant effect on

stimulating 2AR S-sulfenation throughout a 60 minute time course, as demonstrated in Figure

3A and Supplemental Figure 4. In the presence of ISO, (S)-propranolol significantly abrogated

the ISO-mediated oxidation of 2AR at 1, 5, and 15 minutes to 110 ± 20%, 89 ± 7, and 86% ±

4% of control, respectively (Figure 3B-C, Supplemental Figure 5) (p < 0.05, p < 0.001, and p <

0.001 versus the ISO-stimulated conditions, respectively). These results demonstrate that

receptor agonism facilitates the oxidation of the 2AR, and suggest that 2AR-mediated ROS

generation, which peaks within 20 minutes of agonism, is capable of facilitating S-sulfenation of

the receptor.

2AR S-sulfenation is inhibited by dimedone and N-acetyl-L-cysteine

In order to confirm the nature of the 2AR oxidative product, we utilized the cyclic

diketone nucleophile 5,5-dimethyl-1,3-cyclohexanedione (dimedone), which selectively and

irreversibly reacts with S-sulfenic acids forming a covalent adduct that prevents reduction of the

sulfenic acid group by sodium arsenite (Benitez and Allison, 1974; Ellis and Poole, 1997; Saurin

et al., 2004). In the presence of dimedone, the biotin switch signal would be expected to be

attenuated due to nucleophillic attack and ‘trapping’ of the S-sulfenic acid by dimedone. As

shown in figure 4, ISO-mediated S-sulfenation (262 ± 10% at 10 M for 15 minutes) was

significantly abrogated by dimedone treatment (105 ± 13.5%) (p <0.01 versus ISO) (Figure 4A-

B, Supplemental Figure 6). Likewise, H2O2-mediated S-sulfenation (376 ± 9%) was also


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attenuated in the presence of dimedone (142 ± 35%) (p <0.05 versus (Figure 4A-B,

Supplemental Figure 6), however complete abolishment of this signal was not seen, likely due to

overoxidation which becomes insensitive to dimedone upon treatment with H2O2, as described

by others (Gutscher et al., 2009). These results demonstrate that the 2AR oxidant species

induced by receptor agonism and exogenous oxidant treatment is indeed a sulfenic acid, at least

for an initial stable period. We also utilized the membrane permeable thiol antioxidant NAC,

which is extensively used in redox studies to assess the effect of thiol-excess in oxidation. The

effects of NAC on H2O2-mediated oxidation are well described, such that NAC impedes

oxidative processes by acting as a ROS scavenger. Specifically, the free thiol of NAC behaves

as a martyr, taking on the brunt of oxidation by ROS to become oxidized itself, thereby reducing

ROS’ ability to modify other thiols. As such, NAC has been described as a cysteine-residue

oxidation protectant, and as shown in figure 4C-D and Supplemental Figure 7, ISO-mediated S-

sulfenation of 2AR (270 ± 10% at 10 M for 15 minutes) was markedly attenuated in cells

pretreated for four hours with NAC (138 ± 14%) (p < 0.01 versus ISO). These results are

consistent with our previously published results demonstrating that NAC decreases 2AR-

mediated ROS generation and inhibits 2AR function (Moniri and Daaka, 2007), and

demonstrate that the ISO-mediated S-sulfenation is specific to ROS.

Agonist- and H2O2- mediated 2AR S-sulfenation occurs in human alveolar cells

Since our initial characterization of 2AR S-sulfenation here is performed in a clonal cell

model of forced 2AR overexpression, and is dependent on an epitope-tagged receptor, we

wished to examine if 2AR S-sulfenation occurred in a physiologically relevant system. Given

that 2-receptors are physiologically critical for pulmonary function, and that 2-receptor

agonists are foundational therapeutic agents used to dilate the bronchial and alveolar epithelium


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in pulmonary disorders such as asthma and chronic obstructive pulmonary disease, we examined

whether H2O2 or 2-agonism would stimulate 2AR S-sulfenation in human lung alveolar

epithelial cells. Here, we utilized the well characterized immortalized A549 human lung alveolar

epithelial cell line, which endogenously expresses both 2 and 1 receptors (Szentendrei T, et al.,

1992). Using the biotin-switch assay described in figure 1 in conjunction with a 2AR-specific

antibody raised against the C-terminal tail of the receptor, our results demonstrate that

stimulation of A549 cells with H2O2 facilitated significant S-sulfenation of 2AR compared to

unstimulated control (Figure 5A, Supplemental Figure 8). Since A549 cells also express 1AR

and ISO is a non-selective -receptor agonist, we utilized the highly 2-selective agonist

formoterol, which exhibits 300-fold greater potency for 2AR versus 1AR (Anderson 1993), to

avoid concurrent stimulation of 1AR in these cells. Results of these experiments demonstrate

that despite its relatively low intrinsic efficacy (Hanania et al., 2010) and the low expression

levels of 2AR on A549 cells (Szentendrei et al., 1992), formoterol (1 M, 15 minutes)

stimulates S-sulfenation of 2AR compared to unstimulated control (Figure 5B, Supplemental

Figure 8). Taken together, these results suggest that 2AR S-sulfenation by ROS or receptor

agonism occurs in physiologically relevant tissues such as the alveolar epithelium, where 2AR

agonism is the foundational therapy to achieve bronchodilation in airway disorders such as

asthma.


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Discussion

Over the last half-decade, it has been demonstrated and is now well accepted that

agonism of the 2AR leads to generation of ROS. These results have been obtained from a

variety of tissue and cell types including cardiomyocytes (Li et al., 2010; Xu et al., 2011),

microglia (Qian et al., 2009), as well as our own and other’s studies in embryonic kidney cells

(Moniri and Daaka, 2007; Gong et al., 2008). In each case, ROS were not only shown to be

generated upon 2AR agonism, but were also shown to serve purposeful roles in regulating 2AR

signaling. Our previous results demonstrated that 2AR-mediated ROS generation was

accompanied by a definitive requirement of ROS for proper 2AR signaling, suggesting that

some degree of ROS are indispensible for receptor function. Xu and colleagues recently

showed that mice transgenically overexpressing 2AR exhibited a greater degree of cardiac ROS

production, which in of itself was not surprising, but this effect markedly contributed to cardiac

inflammation and failure, suggesting that overexertion of the 2AR-ROS link may have

pathophysiological consequences (Xu et al., 2011). These aggregate studies imply that low

degrees of ROS are required for 2AR signaling, while higher levels of ROS lead to detrimental

effects, similar to the current paradigm which suggests micromolar H2O2 levels may regulate

signaling while higher levels facilitate unfavorable oxidative stress responses (Valko et al.,

2007). One of the primary ROS species reported to be generated following 2AR agonism is

superoxide, which occurs via the action of NADPH-oxidase enzymes and is subsequently

dismutated yielding H2O2 (Gong et al., 2008; Qian et al., 2009; Li at al., 2010; Xu et al., 2011).

This two-electron oxidant species’ chief biological activity comes from its ability to readily

oxidize thiol groups of protein cysteine residues, and the initial product of this reaction is an S-

sulfenic acid (S-OH). The overall purpose of this study was to determine if ROS are capable of


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oxidizing the 2AR to form S-sulfenic acids. Using a biotin-switch assay which had been

modified to assess S-sulfenation by way of the well characterized sulfenic acid reducing agent

sodium arsenite, the present study demonstrates for the first time that ROS are capable of S-

sulfenating the 2AR. We also reveal that 2AR S-sulfenation occurs upon receptor agonism

with the classical -receptor catecholamine agonist isoproterenol as well as the 2-selective

agonist formoterol, and importantly, the observation that this effect is receptor-dependent (i.e.,

blocked by propranolol) suggests that 2AR-generated ROS contribute to S-sulfenation.

The sulfenic acid, unlike higher order sulfinic and sulfonic acids, is the simplest of the

organosulfur oxyacids, and as a consequence these species are predicted to be unstable and

reactive. Despite these characteristics, previous work has demonstrated that limited solvent

access and a lipophilic environment, along with a lack of other reactive sulfhydryl groups in the

vicinity of the site of S-sulfenation allows for marked stabilization of protein S-sulfenic acids

(Allison, 1976; Liu, 1977). While our results demonstrate that an S-sulfenated 2AR species can

be stabilized upon treatment of cells with 2-agonist as well as physiologically relevant

concentrations of H2O2, we cannot rule out the possibility that this modification could be further

oxidized to higher order species, either concurrent with or following S-sulfenic formation.

Nevertheless, to date, over two-hundred proteins accounting for vast biochemical functions that

include metabolism, transcription, DNA repair, nuclear transport, intracellular trafficking,

immune regulation, and cell signaling, amongst others have been identified as at least being

initially S-sulfenated (Leonard et al., 2009), and to our knowledge, this study represents the first

time that a mammalian G protein-coupled receptor has been recognized as being oxidized to an

S-sulfenic acid. While our results suggest a role for S-sulfenation in 2AR regulation, an

absolute physiological role of protein S-sulfenation remains elusive, making it difficult to gauge


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what role, if any, S-sulfenation has on 2AR physiology. However, our observation that 2AR

S-sulfenation occurs in human lung alveolar epithelial cells suggests that this modification

occurs endogenously upon agonism of physiologically relevant levels of receptor.

A notable feature of the ISO-mediated oxidation is its apparent transient nature, which

saturates at 15 minutes following agonism. This observation is of pharmacological interest due

to the temporal dependence of ISO-stimulated ROS generation, which also peaks approximately

15-20 minutes after agonist addition in these cells (Moniri and Daaka, 2007). While the decrease

in receptor S-sulfenation seen upon treatment with ISO may be attributed to reducing-favorable

intracellular conditions, we cannot rule out the possibility that further modification of the

receptor by phosphorylation, ubiquitination, or signal-dependent events such as sequestration and

internalization may play a role in desensitizing the S-sulfenation signal. Meanwhile, the kinetics

of ISO-mediated S-sulfenation is in accordance with previously reported G protein-dependent

2AR signaling events such as cAMP formation, and correlate precisely to our previous data on

the ROS-dependence of 2AR signaling (Moniri and Daaka, 2007). Interestingly, since -

arrestin recruitment and 2AR-mediated G-protein-independent signaling are known to be

temporally regulated by GRK-mediated phosphorylation, specifically occurring after 10-15

minutes of agonism (Shenoy et al., 2006), our kinetic data with ISO may also suggest a role for

-arrestin in the desensitization of the S-sulfenation signal.

Whereas ISO-mediated ROS generation has been well described, ROS generation

induced by other 2AR agonists has not been characterized to a great extent. Importantly, our

laboratory has also detected S-sulfenation induced by other -agonists including epinephrine,

albuterol, and formoterol. However, interpretation of these S-sulfenating effects must be made

with caution as the ROS-generating effects of these agents remain virtually unstudied. Our


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results here should also be interpreted under the context of several other important factors which

may influence -agonist-mediated S-sulfenation. These include the well described rapid auto-

oxidation of the catecholamine-like -agonists, a consequence which leads to loss of agonist

function (Misra and Fridovitch, 1972). This effect may be significant given that many cell-based

investigations employ addition of antioxidants (e.g., ascorbic acid) to catecholamine-agonist

drug stocks in order to prevent catechol oxidation. However, by sequestering ROS pools, this

approach could conceivably also hinder receptor S-sulfenation and possibly influence receptor

function. Additionally, many 2-agonists, including albuterol, formoterol, and salmeterol have

atypical binding kinetics which induce slow receptor dissociation (Teschemacher and Lemoine,

1999), and it is possible that this unique pharmacological property could affect ROS generation

and receptor S-sulfenation. Furthermore, the well described variable intrinsic efficacies of -

receptor agonists could be expected to contribute to differing abilities of these agents to promote

ROS generation and receptor S-sulfenation (Hanania et al., 2002; Hanania et al, 2010). In this

regard, ISO and epinephrine are reported to have full agonist efficacies while formoterol and

albuterol exhibit one-fifth and one-twentieth of the respective responses. Further studies

underway in our laboratory will seek to address these issues and will aid in assessing the

contribution of S-sulfenation on 2AR conformations, signaling, and physiological function.

Our future studies will also address the localization of the exact site(s) of 2AR S-

sulfenation. Whereas many studies which aim to decipher sites of thiol modification utilize

expression of mutant protein constructs in which cysteines are mutated to non-reactive residues,

these studies often lead to biological responses which are divergent from wild-type. In this

regard, mutation of several cytosolic-facing or helix-associated 2AR cysteines has been shown

to lead to loss of expression or function, which would confound interpretation of such results.


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Additionally, though the biotin-switch assay shown here effectively demonstrates 2AR S-

sulfenation, the method is limited due to its dependence on SDS denaturing conditions, which

may destabilize modified thiols, decreasing the sensitivity when used to localize sites of

modification based on site-directed mutagenesis (Charles et al., 2007). Based on these issues, we

are employing a proteomic approach to identifying the sites of oxidation using mass

spectrometry methodology coupled with fluorescent dimedone congeners which allow for

quantification of S-sulfenation and localization of the site(s) of oxidation (Poole et al., 2005).

The human 2AR contains thirteen cysteine residues and several investigations have examined

the importance of various cysteine residues in 2AR structure and function. Our efforts to

identify the site(s) of oxidation will focus on the intracellular-exposed cysteines with particular

emphasis on Cys341, which creates a fourth intracellular pseudo-loop and is required for creation

of a high affinity agonist-coupling state (O’Dowd et al., 1989; Moffett el al., 1993), as well as

residues within transmembrane helices VI and VII, and the C-terminal tail, which have been

shown to be involved in rotation and conformational changes upon agonism (Ghanouni et al.,

2001; Shi et al., 2002; Granier et al., 2007).

In summary, the current study demonstrates for the first time that the human 2AR can be

oxidized by ROS to form receptor-S-sulfenic acids, and importantly S-sulfenation occurs upon

receptor agonism, which has previously been shown to induce intracellular ROS generation.

Since 2AR is the prototype of the Class-A GPCRs and it is often used as a model for the study

of other rhodopsin-like receptors, some of which have been linked to ROS generation, our results

constitute an important first step towards characterization of the ROS/GPCR relationship, and

may have broader implications in the overall study of the role of ROS in regulation of GPCRs.


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Acknowledgements

The authors wish to thank Mrs. Vivienne Oder for providing excellent secretarial assistance, and

Dr. Robert J. Lefkowitz (Duke University Medical Center) and Dr. Philip Moos (University of

Utah) for the noted reagents.


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Authorship contributions

Participated in research design: Burns and Moniri

Conducted experiments: Burns and Moniri

Contributed new reagents or analytic tools: Burns and Moniri

Performed data analysis: Burns and Moniri

Wrote or contributed to the writing of the manuscript: Burns and Moniri


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References

Allison WS. Formation and reactions of sulfenic acids in proteins. (1976) Accts Chem Res. 9,

293-299

Anderson GP. Formoterol: pharmacology, molecular basis of agonism, and mechanism of long

duration of a highly potent and selective beta 2-adrenoceptor agonist bronchodilator. (1993) Life

Sci. 52(26):2145-60.

Anderson R, Feldman C, Theron AJ, Ramafi G, Cole PJ, Wilson R. Anti-inflammatory,

membrane-stabilizing interactions of salmeterol with human neutrophils in vitro. (1996) Br J

Pharmacol. 117(7):1387-94.

Barnett CC, Moore EE, Partrick DA, Silliman CC. Beta-adrenergic stimulation down-regulates

neutrophil priming for superoxide generation, but not elastase release. (1997) J Surg Res.

70(2):166-70.

Benitez LV, Allison WS. The inactivation of the acyl phosphatase activity catalyzed by the

sulfenic acid form of glyceraldehyde 3-phosphate dehydrogenase by dimedone and olefins.

(1974) J Biol Chem. 249(19):6234-6243.

Charles RL, Schroder E, May G, Free P, Gaffney PR, Wait R, Begum S, Heads RJ, Eaton R.

Protein sulfenation as a redox sensor. (2007) Mol Cell Proteomics. 6(9):1473-1484.


at ASPE



ownloaded from


JPET #185975

25

Ellis HR, Poole LB. Novel application of 7-chloro-4-nitrobenzo-2-oxa-1,3-diazole to identify

cysteine sulfenic acid in the AhpC component of alkyl hydroperoxide reductase. (1997)

Biochemistry. 36(48):15013-15018.

Ghanouni P, Steenhuis JJ, Farrens DL, Kobilka BK. Agonist-induced conformational changes in

the G-protein-coupling domain of the beta 2 adrenergic receptor. (2001) Proc Natl Acad Sci

USA. 98(11):5997-6002.

Gong K, Li Z, Xu M, Du J, Lv Z, Zhang Y. A novel protein kinase A-independent, beta-

arrestin-1-dependent signaling pathway for p38 mitogen-activated protein kinase activation by

beta2-adrenergic receptors. (2008) J Biol Chem. 283:29028-36.

Granier S, Kim S, Shafer AM, Ratnala VR, Fung JJ, Zare RN, Kobilka B. Structure and

conformational changes in the C-terminal domain of the beta2-adrenoceptor: insights from

fluorescence resonance energy transfer studies. (2007) J Biol Chem. 282(18):13895-13905.

Gutscher M, Sobotta MC, Wabnitz GH, Ballikaya S, Meyer AJ, Samstag Y, Dick TP.

Proximity-based protein thiol oxidation by H2O2-scavenging peroxidases. (2009) J Biol Chem.

284(46):31532-31540.

Hanania NA, Sharafkhaneh A, Barber R, Dickey BF. Beta-agonist intrinsic efficacy:

measurement and clinical significance. (2002) Am J Respir Crit Care Med. 165(10):1353-1358.


at ASPE



ownloaded from


JPET #185975

26

Hanania NA, Dickey BF, Bond RA. Clinical implications of the intrinsic efficacy of beta-

adrenoceptor drugs in asthma: full, partial and inverse agonism. (2010) Curr Opin Pulm Med.

16(1):1-5.

Hess DT, Matsumoto A, Kim SO, Marshall HE, Stamler JS. Protein S-nitrosylation: purview

and parameters. (2005) Nat Rev Mol Cell Biol. 6:150-66.

Jaffrey SR, Erdjument-Bromage H, Ferris CD, Tempst P, Snyder SH. Protein S-nitrosylation: a

physiological signal for neuronal nitric oxide. (2001) Nat Cell Biol. 3:193-7.

Kopprasch S, Gatzweiler A, Graessler J, Schröder HE. Beta-adrenergic modulation of FMLP-

and zymosan-induced intracellular and extracellular oxidant production by polymorphonuclear

leukocytes. (1997) Mol Cell Biochem .168(1-2):133-9.

Leonard SE, Reddie KG, Carroll KS. Mining the thiol proteome for sulfenic acid modifications

reveals new targets for oxidation in cells. (2009) ACS Chem Biol. 18;4(9):783-799.

Li J, Yan B, Huo Z, Liu Y, Xu J, Sun Y, Liu Y, Liang D, Peng L, Zhang Y, Zhou ZN, Shi J, Cui

J, Chen YH. Beta2- but not beta1-adrenoceptor activation modulates intracellular oxygen

availability. (2010) J Physiol. 588:2987-98

Liu TY. The role of sulfur in proteins. (1977) In: The proteins (Neurath H. and Hill RL., eds).

Vol 3, Third ed., pp 239-402, Academic Press, New York.


at ASPE



ownloaded from

http://www.ncbi.nlm.nih.gov/pubmed/9062902




JPET #185975

27

Misra HP and Fridovitch L. The role of superoxide anion in the autooxidation of epinephrine

and a simple assay for superoxide dismutase. (1972) J Biol Chem. 247:3170-3175.

Moffett S, Mouillac B, Bonin H, Bouvier M. Altered phosphorylation and desensitization

patterns of a human beta 2-adrenergic receptor lacking the palmitoylated Cys341. (1993)

EMBO J. 12(1):349-356.

Moniri NH and Daaka Y. Agonist-stimulated reactive oxygen species formation regulates beta2-

adrenergic receptor signal transduction. (2007) Biochem Pharmacol. 74:64-73.

O'Dowd BF, Hnatowich M, Caron MG, Lefkowitz RJ, Bouvier M. Palmitoylation of the human

beta 2-adrenergic receptor. Mutation of Cys341 in the carboxyl tail leads to an uncoupled

nonpalmitoylated form of the receptor. (1989) J Biol Chem. 5;264(13):7564-7569.

Opdahl H, Benestad HB, Nicolaysen G. Effect of beta-adrenergic agents on human neutrophil

granulocyte activation with N-formyl-methionyl-leucyl-phenylalanine and phorbol myristate

acetate. (1993) Pharmacol Toxicol. 72(4-5):221-8.

Poole LB, Zeng BB, Knaggs SA, Yakubu M, King SB. Synthesis of chemical probes to map

sulfenic acid modifications on proteins. (2005) Bioconjug Chem.16(6):1624-1628.


at ASPE



ownloaded from


JPET #185975

28

Qian L, Hu X, Zhang D, Snyder A, Wu HM, Li Y, Wilson B, Lu RB, Hong JS, Flood PM. beta2

Adrenergic receptor activation induces microglial NADPH oxidase activation and dopaminergic

neurotoxicity through an ERK-dependent/protein kinase A-independent pathway. (2009) Glia.

57(15):1600-1609.

Reddie KG, Carroll KS. Expanding the functional diversity of proteins through cysteine

oxidation. (2008) Curr Opin Chem Biol. 12:746-54.

Saurin AT, Neubert H, Brennan JP, Eaton P. Widespread sulfenic acid formation in tissues in

response to hydrogen peroxide. (2004) Proc Natl Acad Sci U S A. 101:17982-7.

Shenoy SK, Drake MT, Nelson CD, Houtz DA, Xiao K, Madabushi S, Reiter E, Premont RT,

Lichtarge O, Lefkowitz RJ. Beta-arrestin-dependent, G protein-independent ERK1/2 activation

by the beta2 adrenergic receptor. (2006) J. Biol. Chem. 281:1261–1273.

Shi L, Liapakis G, Xu R, Guarnieri F, Ballesteros JA, Javitch JA. Beta2 adrenergic receptor

activation. Modulation of the proline kink in transmembrane 6 by a rotamer toggle switch.

(2002) J Biol Chem. 277(43):40989-40996.

Szentendrei T, Lazar-Wesley E, Nakane T, Virmani M, Kunos G. Selective regulation of beta 2-

adrenergic receptor gene expression by interleukin-1 in cultured human lung tumor cells. (1992)

J Cell Physiol. 152:478-85.


at ASPE



ownloaded from


JPET #185975

29

Tang Y, Hu LA, Miller WE, Ringstad N, Hall RA, Pitcher JA, DeCamilli P, Lefkowitz RJ.

Identification of the endophilins (SH3p4/p8/p13) as novel binding partners for the beta1-

adrenergic receptor. (1999) Proc Natl Acad Sci U S A. 96:12559-64.

Teschemacher A, Lemoine H. Kinetic analysis of drug-receptor interactions of long-acting beta2

sympathomimetics in isolated receptor membranes: evidence against prolonged effects of

salmeterol and formoterol on receptor-coupled adenylyl cyclase. (1999) J Pharmacol Exp Ther.

288(3):1084-1092.

Valko M, Leibfritz D, Moncol J, Cronin MT, Mazur M, Telser J. Free radicals and antioxidants

in normal physiological functions and human disease. (2007) Int J Biochem Cell Biol. 39(1):44-

84.

Xu Q, Dalic A, Fang L, Kiriazis H, Ritchie RH, Sim K, Gao XM, Drummond G, Sarwar M,

Zhang YY, Dart AM, Du XJ. Myocardial oxidative stress contributes to transgenic β₂-

adrenoceptor activation-induced cardiomyopathy and heart failure. (2011) Br J Pharmacol.

162:1012-28.


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Footnotes

This study was funded, in part, by a New Investigator Program grant (to N.H.M.) from the

American Association of Colleges of Pharmacy. Mrs. Burns is supported by a predoctoral

fellowship from the American Foundation for Pharmaceutical Education. Portions of this study

were presented at the 2011 Experimental Biology meeting in Washington, D.C.

Submit reprint requests to:

Nader H. Moniri, Ph.D.



Mercer University


Atlanta, GA 30341

(678)-547-6246 (tel)

(678)-547-6423 (fax)

[email protected]


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Figure Legends

Figure 1: Modified biotin-switch to detect S-sulfenation of 2AR. Cell lysates were denatured

in SDS and proteins, depicted as containing either cysteine sulfhydryls (S-H), cysteine sulfenic

acids (S-OH), or cysteine disulfides (S-S), are treated with the sulfhydryl methylating agent

methyl methanethiosulfonate (MMTS) to protect initial sulfhydryl groups (Step 1). Samples are

then treated with NaAsO2 to selectively reduce the sulfenic acids to sulfhydryls (Step 2). The

newly created sulfhydryl groups (formerly sulfenic acids) are the labeled with the sulfhydryl-

reactive biotinylating agent biotin-HPDP (Step 3). All biotinylated proteins in the samples are

pulled-down with streptavadin-agarose beads (Step 4), resolved by SDS-PAGE, transferred to

PVDF membranes, and immunoblotted with either anti-FLAG M2 antibody (for HEK293 cells

overexpressing FLAG-2AR) or anti-2AR antibody (for A549 cells endogenously expressing

2AR) (Step 5).

Figure 2: H2O2 and isoproterenol induced S-sulfenation of 2AR. HEK293 cells expressing

FLAG-2AR were stimulated with H2O2 or ISO for the indicated times and concentrations, cells

were lysed and lysates subjected to the biotin-switch method described. H2O2 (100 M)

stimulates the rapid and sustained S-sulfenation of 2AR (A-B). Receptor agonism with ISO (10

M) stimulates S-sulfenation of 2AR in a time-dependent manner, with the signal peaking at 15

minutes (C-D). Agonism with ISO was also dose-dependent and non-linear regression analysis

(not shown) resulted in a pEC50 of 7.4 ± 0.3 for the dose-response (E-F). Input lysates


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demonstrate equivalent protein input in each reaction. Statistical significance is represented as *

p < 0.05, ** p < 0.01, *** p < 0.001 compared to respective unstimulated control condition.

Figure 3: Isoproterenol induced S-sulfenation of 2AR is receptor mediated. HEK293 cells

expressing FLAG-2AR were stimulated with (S)-propranolol alone or preincubated with (S)-

propranolol for 5 minutes prior to addition of ISO (10 M), cells were then lysed and lysates

subjected to the biotin-switch method described. By itself, (S)-propranolol had no effect on

2AR S-sulfenation (A,C). Preincubation of (S)-propranolol prior to ISO stimulation markedly

inhibited ISO-mediated S-sulfenation of 2AR at all time points tested (B-C). Input lysates

demonstrate equivalent protein input in each reaction. Statistical significance is represented as *

p < 0.05 and *** p < 0.001 compared to respective unstimulated control condition.

Figure 4: The selective and irreversible S-sulfenic acid alkylator dimedone and the cysteine

protecting antioxidant N-acetyl-L-cysteine inhibit 2AR S-sulfenation. HEK293 cells expressing

FLAG-2AR were stimulated with H2O2 (100 M) or ISO (10 M), lysed and lysates subjected

to the biotin-switch method with 5 mM dimedone preceding the biotinylating step. ISO and

H2O2-mediated S-sulfenation of 2AR was significantly abrogated in the presence of dimedone,

demonstrating the selective formation of S-sulfenic acids on the 2AR (A-B). HEK293 cells

expressing FLAG-2AR were treated with N-acetyl-L-cysteine (NAC, 10 mM) for four hours and

stimulated with ISO (10 M), cells were then lysed and lysates subjected to the biotin-switch

method as described. ISO-mediated S-sulfenation of 2AR was significantly reduced in the

presence of NAC (C-D). Input lysates demonstrate equivalent protein input in each reaction.


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Statistical significance is represented as * p < 0.05, ** p < 0.01, *** p < 0.001 compared to

shown condition.

Figure 5: H2O2 and agonist-stimulated 2AR S-sulfenation occurs in human alveolar epithelial

cells. The human alveolar epithelial cell line A549, which endogenously expresses 2AR was

stimulated for 15 minutes with H2O2 (100 M) (A) or the 2-selective agonist formoterol (1 M)

(B), cells were then lysed and lysates subjected to the biotin-switch method using a 2AR-

specific antibody raised against the C-terminal tail of the receptor. Both H2O2 and formoterol

facilitate S-sulfenation of 2AR compared to unstimulated controls in these cells. Input lysates

demonstrate equivalent protein input in each reaction. Indicated bands represent the major

2AR-immunoreactive bands at approximately 50 kDa.


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