Nitric Oxide Modulates β2-Adrenergic Receptor Palmitoylation and Signaling

Abstract

To determine whether nitric oxide (NO) modulates the ␤-adrenergic signaling pathway, we treated cells expressing ␤ 2 -adrenergic receptors (␤ 2 AR) with the NO donors, 3-morpholinosydnonimine (SIN-1) and 1,2,3,4-oxatriazolium,5-amino-3-(3-chloro-2-methylphenyl)-chloride and determined the intracellular production of cAMP after exposure to ␤-adrenergic receptor agonists, cholera toxin and forskolin. NO significantly decreased the potency of the ␤-adrenergic agonist, isoproterenol, to stimulate cAMP production without affecting the stimulatory action of forskolin and cholera toxin, which directly activate adenylyl cyclase and G s , respectively. Treatment with the NO donor increased the guanyl nucleotide-sensitive high affinity constant for the agonist, isoproterenol, thus suggesting that it reduced functional coupling between the receptor and G s . Stimulation of endogenous NO production by lipopolysaccharide in RAW 264.7 macrophages also caused a significant increase in the EC 50 for isoproterenol-stimulated cAMP production. SIN-1 treatment also led to a reduction in both basal and isoproterenol-stimulated incorporation of [ 3 H]palmitate into the ␤ 2 AR. Signaling through the nonpalmitoylated, Gly 341 ␤ 2 AR mutant was unchanged by SIN-1 treatment. Given the link between ␤ 2 AR palmitoylation and its responsiveness to agonist, these results suggest that the primary action of NO was depalmitoylation of the ␤ 2 AR resulting in decreased signaling through the ␤ 2 AR. Nitric oxide (NO)1 is a biologic signal involved in vasodilatation, neurotransmission, and immune defense (1, 2). It is an unstable gas that diffuses across membranes, lacks specific receptors, and is rapidly inactivated by a chemical reaction. Its synthesis by the enzyme NO synthase (NOS) from arginine and oxygen is finely regulated and can be achieved through both calcium-dependent and calcium-independent pathways (3). Both the calcium-dependent endothelial NOS and the calciumindependent inducible NOS (iNOS or NOS2) have been shown to play an important role in the control of vascular tone in normal and inflammatory conditions (4 -8), respectively. NO can modulate the action of various vasoactive hormones and transmitters (7). In particular, NO-mediated decreases in ␤-adrenergic responsiveness have been described (6, 9). However, the biochemical processes underlying this effect remain largely unexplored.At the cellular level, the best characterized effect of NO is the activation of guanylate cyclase by the formation of a heme-NO complex that enhances the catalytic activity of the enzyme and increases cGMP production (10). Although several of the effects of NO have been attributed to cGMP formation, NO also leads to nitrosylation of thiol groups on cysteine residues (3, 11) that may contribute to the diverse physiological actions of this gaseous second messenger. Indeed, nitrosylation has been suggested to modulate protein function as a consequence of conformational changes (3, 11), facilitation of ADP-ribosylation (12), and inhibition of protein palm...

S-Nitrosocysteine Increases Palmitate Turnover on Ha-Ras in NIH 3T3 Cells

“…The nitric oxide donor compound SIN-1 has also been reported to alter protein palmitoylation (60). As with SNC, the effects of SIN-1 on palmitoylation were protein-selective; palmitate incorporation into the transmembrane ␤-adrenergic receptor was reduced by SIN-1 treatment, whereas palmitate labeling of the G s ␣-subunit coupled to the receptor was unaffected (60).…”

“…The nitric oxide donor compound SIN-1 has also been reported to alter protein palmitoylation (60). As with SNC, the effects of SIN-1 on palmitoylation were protein-selective; palmitate incorporation into the transmembrane ␤-adrenergic receptor was reduced by SIN-1 treatment, whereas palmitate labeling of the G s ␣-subunit coupled to the receptor was unaffected (60). It thus appears that at least certain palmitoylated proteins, including Ha-Ras, are susceptible to nitrosylating agents but that each will display an individualized response, presumably resulting from regulatory mechanisms distinct from each protein.…”

A novel thiol compound, N-acetylcysteine amide, attenuates allergic airway disease by regulating activation of NF-κB and hypoxia-inducible factor-1α

“…Although the lung has a well-developed antioxidant system (Rahman et al, 1996(Rahman et al, , 1997, overproduction of ROS or depression of the protective system results in epithelial cell damage, cell shedding, and bronchial hyperreactivity (Hulsmann et al, 1994;Cortijo et al, 1999). Studies with animal models have indicated that ROS contribute to airway hyperresponsiveness by increasing vagal tone due to damage of oxidant sensitive β-adrenergic receptors as well as decreasing mucociliary clearance (Owen et al, 1991;Adam et al, 1999). Consistent with these observations, our data have shown that generation of ROS in BAL cells was significantly increased after allergen challenge in OVA-inhaled mice and that administration of AD4 reduced the ROS generation and inhibited airway inflammation and hyperresponsiveness.…”

“…AD4 was prepared as previously described (Adam et al, 1999;Bahat-Stroomza et al, 2005;Bartov et al, 2006). AD4 (60 or 120 mg/kg body wt/day), dissolved in PBS, was administered i.p.…”

β-Arrestin-mediated activation of MAPK by inverse agonists reveals distinct active conformations for G protein-coupled receptors

“…Wild-type human ␤ 2 AR, V2R, and hemagglutinin (HA)-tagged (YPYDVPDYA) ␤ 2 AR sequences were subcloned into a modified pcDNA3 vector (Invitrogen) in which the cytomegalovirus promoter was replaced by a Rous sarcoma virus promoter as described (8). V2R-GFP and ␤2AR-GFP.…”