In I, n=3; *p 0

In I, n=3; *p 0.05 versus arr2 WT. (J) There are no significant differences between any conditions in ISO-induced (10 M, 2 min) cAMP production, a signature of G protein-mediated 2AR signaling; n=4-6. (K and L) Suppression of ISO-induced (10 M, 5 min) ERK phosphorylation by n/iNOS is dependent upon C253 within arr2 in HEK293 cells stably expressing 2AR-TYY, which can couple to arrs but cannot activate G proteins (in L, n=3; *p 0.05 versus WT without n/iNOS and C253S). Confocal immunofluorescence microscopy of W9 cells transiently transfected with either WT or C253S arr2, and either nNOS or iNOS, or without transfection of NOS showed that both WT and C253S arr2 were Px-104 predominantly cytoplasmic in the absence of ISO (Figure S3C) and that stimulation of the 2AR with ISO resulted in the re-distribution of both WT and C253S arr2 from cytoplasm to the plasma membrane and co-localization with the 2AR (Figure S3C). inhibits the canonical function of -arrestins to promote preferential G protein Px-104 signaling (bias) via G protein-coupled receptors. This general mechanism for biased signaling profoundly impacts the severity of heart failure, and provides a novel function for nitric oxide broadly. Ligand-induced stimulation of G protein-coupled receptors (GPCRs) activates heterotrimeric G proteins to initiate a broad range of intracellular signaling cascades. G protein-mediated signaling through most GPCRs is usually truncated by recruitment of -arrestins (arr1 and arr2; also designated arrestin-2 and arrestin-3), resulting in both receptor desensitization and down-regulation by internalization (Ahn et al., 2003). The functional purview of arrs was greatly expanded by the discovery that arrs serve to scaffold elements mediating signals that may be largely impartial of G proteins, best characterized in the case of MAPK-based transduction (Lefkowitz and Shenoy, 2005; Shenoy et al., 2006). Furthermore, arrs interact with cytoplasmic partners impartial of G proteins, and thereby exhibit diverse functionality (Lefkowitz and Shenoy, 2005; Ma and Pei, 2007). Analysis of GPCR-dependent transduction induced by synthetic ligands including multiple therapeutically relevant drugs has shown that man-made ligands can elicit biased signaling, i.e. differential activation of G protein-versus arr-mediated pathways, and biased agonism remains the subject of intensive pharmacological analysis with Px-104 broad potential clinical applicability (Wisler et al., 2014). The ability of synthetic ligands to elicit biased signaling by GPCRs points to the possibility that endogenous mechanisms might operate to bias signaling. Examples exist of endogenous allosteric modulators that interact directly with GPCRs to bias signaling through particular GPCRs (van der Westhuizen et al., 2015). However, the physiological relevance of receptor allostery that may impact biased signaling is not well established, and general mechanisms that might bias signaling through GPCRs have not been described. We considered that signaling bias might be endogenously regulated by a mechanism common to multiple GPCRs, and in particular that post-translational regulation of the arrs Px-104 by the ubiquitous signaling molecule nitric oxide (NO) might provide such a mechanism. NO generation is usually coupled to stimulation of multiple GPCRs and regulates receptor-affiliated proteins through S-nitrosylation (Haldar and Stamler, 2013; Hess et al., 2005; Ozawa et al., 2008; Wang et al., 2006; Whalen et al., 2007). Prior analysis has shown that eNOS-mediated S-nitrosylation of arr2 at the C-terminus Cys410 facilitates conformational changes in arr2 that enhance receptor internalization (Ozawa et al., 2008). Here we report that S-nitrosylation by neuronal (nNOS) or inducible NOS (iNOS) of a newly Rabbit Polyclonal to IRF-3 (phospho-Ser386) discovered site that is conserved in arr1/2, Cys251/253, provides a general mechanism to bias GPCR signaling by suppressing canonical arr-based function but not G protein-mediated signaling. Moreover, S-nitrosylation of Cys251/253 alters cytoplasmic interactions of arrs, enabling arrs to function independently and providing an additional signature of bias. These results provide a novel role for NO in cellular signaling and new understanding of arr-based function, with potential implications for physiology and disease, in particular heart failure, where -adrenergic receptor (AR) compromise may be newly understood in terms of arr versus G protein bias. Results arr1 and arr2 Bind and Are S-nitrosylated by both nNOS and iNOS at a Single Common Cys In human embryonic kidney (HEK293) cells stably transfected with rat nNOS (HEK-nNOS) or human iNOS (HEK-iNOS) and transiently transfected with rat arr1 or arr2, both arr1 and arr2 were S-nitrosylated by both nNOS (Physique 1A) and iNOS (Physique 1B), as assessed by the SNO-RAC method (Forrester et al., 2009). In addition, endogenous arr2 was S-nitrosylated by endogenous iNOS in RAW264.7 cells following iNOS induction by bacterial lipopolysaccharide and interferon- (LPS/IF) (Determine 1C) (see Figures S1D and S1E for validation of antibodies). Open in a separate window Physique 1 S-nitrosylation by nNOS and by iNOS of Cys 251/253 within arr1/2 and Association of n/iNOS with arr1/2(A and B) Both arr1 and arr2 are S-nitrosylated by (A) nNOS and (B) iNOS in HEK cells stably transfected with rat/human n/iNOS and transiently transfected with rat arr1/2. Assay by SNO-RAC followed by Western blotting; omission of ascorbate serves as a control for specificity. (C).