Agonist-stimulated reactive oxygen species formation regulates β2-adrenergic receptor signal transduction

Abstract

Generation of reactive oxygen species (ROS) can occur upon agonist stimulation of surface receptors to modulate downstream signaling processes. Here, we show that activation of the β2 adrenergic receptor (β2AR) by stimulation with the agonist isoproterenol leads to generation of ROS that is required for β2AR signal transduction. Specifically, we show that inhibition of NADPH oxidase with diphenyliodonium chloride, inhibition of the small GTPase Rac1 with NSC23766, and inhibition of formed ROS with the antioxidant N-acetyl-l-cysteine decreases β2AR-mediated cAMP formation, protein kinase A activation, and receptor phosphorylation and internalization, but does not impact ligand binding. The results also show that inhibition of ROS attenuates active β2AR-mediated binding of GTP to α subunits of heterotrimeric G proteins. Based on these results, we propose that agonist-dependent ROS formation is needed for β2AR signal transduction, perhaps through stabilization of active receptor conformers by redox-mediated modification of receptor and/or Gα proteins cysteine residues.

Introduction

G protein-coupled receptors (GPCRs) represent the largest and most diverse family of surface receptors, transducing a myriad of intracellular signaling cascades that control, among other things, cell proliferation, survival and migration [1]. Upon agonist ligand binding, GPCRs activate distinct effector enzymes through specific signaling cascades that are dependent upon heterotrimeric GTP-binding proteins (G proteins). β2-Adrenergic receptor (β2AR), the best characterized member of the GPCR family, mediates physiological responses upon binding to epinephrine and norepinephrine by coupling to heterotrimeric G proteins, leading to formation of the second messenger adenosine 3′,5′-cyclic monophosphate (cAMP) through activation of adenylyl cyclases [2]. Protein kinase A (PKA) is activated by cAMP, and mediates the cellular response to β2AR agonist ligand binding. Signal termination (i.e. desensitization) of β2AR occurs upon phosphorylation of receptor by members of the G protein-coupled receptor kinase (GRK) family (e.g. GRK2), leading to generation of high affinity binding of phosphorylated receptor and cytosolic β-arrestin proteins [3]. The binding of β-arrestins to receptor not only inhibits the further activation of G proteins, but also initiates events that lead to receptor internalization [4] and ‘second wave’ signaling [5].

Reactive oxygen species (ROS), which include free radicals, hydroxyl radicals, superoxide anions, as well as non-radicals such as hydrogen peroxide (H₂O₂), can play central roles in transducing intracellular signaling events [6], [7]. Oxidant-generating molecules such as H₂O₂ are capable of modifying amino acid residues (e.g. methionine and cysteine), leading to modulation of protein function and protein-protein interactions [8]. The intracellular synthesis of ROS occurs primarily through NADPH oxidase system, activation of which requires Rac and Rap [7], members of the Ras superfamily of small G proteins. ROS generation can transpire in response to a variety of stimuli, including peptide growth factors and cytokines [9]. For example, the epidermal growth factor (EGF) receptor has been shown to be involved in acute and rapid production of ROS upon autophosphorylation following ligand binding. Here, ROS production attenuates EGF-mediated activation of ERK MAP kinases, leading to the suggestion that ROS production is an intrinsic signal transduction desensitizer [10]. Distinctly, engagement of B cell receptors with immunoglobulin in lymphoma cells promotes ROS-dependent amplification of the cell signal, leading to the conclusion that ROS is, rather, a signal transducer [11]. In the case of GPCRs, the stimulation of respective receptors with serotonin, acetylcholine, glutamate and angiotensin, for example, has also been linked to formation of ROS, although the signaling consequences that result remain unknown [9]. A study by Marques and Bicho [12] showed that β2AR-mediated signaling was inhibited by sulfhydryl reagents, and the authors concluded that ROS exerts its effects by acting on cysteine residues in the G protein and within the receptor-G protein complex.

Herein, we have examined the role of ROS on the ligand binding and function of β2AR in HEK293 cells. Initial results, consistent with those in the literature [13], [14], [15], showed that agonist stimulation of β2AR leads to increased ROS generation. To extend these observations, we tested the hypothesis that ROS effects signaling by the β2AR. Accordingly, we used inhibitors of specific steps involved in the ROS-signaling cascade to assess the effects of ROS inhibition on β2AR signal transduction. Selective pharmacological inhibition of ROS generation using the NADPH oxidase inhibitor diphenyleneiodonium chloride (DPI), the Rac1 inhibitor NSC23766 (NSC), or the antioxidant N-aceytl-l-cysteine (NAC) all led to impairment of agonist-promoted cellular response, including attenuation of β2AR phosphorylation and internalization, as well as reduction in second messenger formation and GTP-γ-³⁵S binding to Gα proteins. These results implicate ROS as key modulators of intracellular β2AR-associated signal transduction processes.