Recruitment of ß-arrestin 1 and 2 to the ß2-adrenoceptor: analysis of 65 ligands.

UNLABELLED: Beyond canonical signaling via Gαs and cAMP, the concept of functional selectivity at ß2-adrenoceptors (ß2ARs) describes the ability of adrenergic drugs to stabilize ligand-specific receptor conformations to initiate further signaling cascades comprising additional G-protein classes or ß-arrestins (ßarr). A set of 65 adrenergic ligands including 40 agonists and 25 antagonists in either racemic or enantiopure forms was used for ßarr recruitment experiments based on a split-luciferase assay in a cellular system expressing ß2AR. Many agonists showed only (weak) partial agonism regarding ßarr recruitment. Potencies and/or efficacies increased depending on the number of chirality centers in (R) configuration; no (S)-configured distomer was more effective at inducing ßarr recruitment other than the eutomer. ßarr2 was recruited more effectively than ßarr1. The analysis of antagonists revealed no significant effects on ßarr recruitment. Several agonists showed preference for activation of Gαs GTPase relative to ßarr recruitment, and no ßarr-biased ligand was identified. IN CONCLUSION: 1) agonists show strong bias for Gαs activation relative to ßarr recruitment; 2) agonists recruit ßarr1 and ßarr2 with subtle differences; and 3) there is no evidence for ßarr recruitment by antagonists.

Interaction of fenoterol stereoisomers with ß2-adrenoceptor-G sα fusion proteins: antagonist and agonist competition binding.

The specific interaction between G-protein-coupled receptors and ligand is the starting point for downstream signaling. Fenoterol stereoisomers were successfully used to probe ligand-specific activation (functional selectivity) of the ß2-adrenoceptor (ß2AR) (Reinartz et al. 2015). In the present study, we extended the pharmacological profile of fenoterol stereoisomers using ß2AR-Gsα fusion proteins in agonist and antagonist competition binding assays. Dissociations between binding affinities and effector potencies were found for (R,S')- and (S,S')-isomers of 4'-methoxy-1-naphthyl-fenoterol. Our data corroborate former studies on the importance of the aminoalkyl moiety of fenoterol derivatives for functional selectivity.

Structure-bias relationships for fenoterol stereoisomers in six molecular and cellular assays at the ß2-adrenoceptor.

Functional selectivity is well established as an underlying concept of ligand-specific signaling via G protein-coupled receptors (GPCRs). Functionally, selective drugs could show greater therapeutic efficacy and fewer adverse effects. Dual coupling of the ß2-adrenoceptor (ß2AR) triggers a signal transduction via Gsα and Giα proteins. Here, we examined 12 fenoterol stereoisomers in six molecular and cellular assays. Using ß2AR-Gsα and ß2AR-Giα fusion proteins, (R,S')- and (S,S')-isomers of 4'-methoxy-1-naphthyl-fenoterol were identified as biased ligands with preference for Gs. G protein-independent signaling via ß-arrestin-2 was disfavored by these ligands. Isolated human neutrophils constituted an ex vivo model of ß2AR signaling and demonstrated functional selectivity through the dissociation of cAMP accumulation and the inhibition of formyl peptide-stimulated production of reactive oxygen species. Ligand bias was calculated using an operational model of agonism and revealed that the fenoterol scaffold constitutes a promising lead structure for the development of Gs-biased ß2AR agonists.

AKT1 and AKT2 induce distinct phosphorylation patterns in HL-1 cardiac myocytes.

The protein kinase AKT is a central kinase in the heart and has a major impact on growth/hypertrophy, survival/apoptosis, and metabolism. To gain more insight into AKT isoform-specific signaling at the molecular level, we investigated the phosphoproteome of HL-1 cardiomyocytes carrying AKT1 or AKT2 isoform-specific knock down, respectively. We combined stable isotope labeling with high resolution mass spectrometry and identified 377 regulated phosphopeptides. Although AKT1 is expressed at 4-fold higher levels, insulin stimulation mainly activated AKT2, which might in part rely on a preferred interaction of AKT2 with the mammalian target of rapamycin complex 2. In line with this result, the highest number of regulated phosphopeptides was identified in the AKT2 knock down cells. Isoform-specific regulation of AKT targets not previously described could be observed, and specific regulation of indirect target sites allows a deeper insight into affected biological processes. In the myocardial context, we identified many phosphosites supporting a connection of AKT to excitation-contraction coupling. Phosphoproteins identified included L-type calcium channel, ryanodine receptor, junctophilin, histidine-rich calcium binding protein, phospholamban, heat shock protein beta-6, and Ca²âº/calmodulin-dependent kinase II. In conclusion, AKT isoform-specific knock down combined with quantitative phosphoproteomics provided a powerful strategy to unravel AKT isoform-specific signaling.

Systematic Analysis Reveals Elongation Factor 2 and α-Enolase as Novel Interaction Partners of AKT2.

AKT2 is one of the three isoforms of the protein kinase AKT being involved in the modulation of cellular metabolism. Since protein-protein interactions are one possibility to convey specificity in signal transduction, we performed AKT2-protein interaction analysis to elucidate their relevance for AKT2-dependent cellular functions. We identified heat shock protein 90 kDa (HSP90), Cdc37, heat shock protein 70 kDa (HSP70), 78 kDa glucose regulated protein (GRP78), tubulin, GAPDH, α-enolase and elongation factor 2 (EF2) as AKT2-interacting proteins by a combination of tandem affinity purification and mass spectrometry in HEK293T cells. Quantitative MS-analysis using stable isotope labeling by amino acids in cell culture (SILAC) revealed that only HSP90 and Cdc37 interact stably with AKT2, whereas the other proteins interact with low affinity with AKT2. The interactions of AKT2 with α-enolase and EF2 were further analyzed in order to uncover the functional relevance of these newly discovered binding partners. Despite the interaction of AKT2 and α-enolase, which was additionally validated by proximity ligation assay (PLA), no significant impact of AKT on α-enolase activity was detected in activity measurements. AKT stimulation via insulin and/or inhibition with the ATP-competitive inhibitor CCT128930 did not alter enzymatic activity of α-enolase. Interestingly, the direct interaction of AKT2 and EF2 was found to be dynamically regulated in embryonic rat cardiomyocytes. Treatment with the PI3-kinase inhibitor LY294002 before stimulation with several hormones stabilized the complex, whereas stimulation alone led to complex dissociation which was analyzed in situ with PLA. Taken together, these findings point to new aspects of AKT2-mediated signal transduction in protein synthesis and glucose metabolism.

Dissociations in the effects of ß2-adrenergic receptor agonists on cAMP formation and superoxide production in human neutrophils: support for the concept of functional selectivity.

In neutrophils, activation of the ß2-adrenergic receptor (ß2AR), a Gs-coupled receptor, inhibits inflammatory responses, which could be therapeutically exploited. The aim of this study was to evaluate the effects of various ß2AR ligands on adenosine-3',5'-cyclic monophosphate (cAMP) accumulation and N-formyl-L-methionyl-L-leucyl-L-phenylalanine (fMLP)-induced superoxide anion (O2(•-)) production in human neutrophils and to probe the concept of ligand-specific receptor conformations (also referred to as functional selectivity or biased signaling) in a native cell system. This is an important question because so far, evidence for functional selectivity has been predominantly obtained with recombinant systems, due to the inherent difficulties to genetically manipulate human native cells. cAMP concentration was determined by HPLC/tandem mass spectrometry, and O2(•-) formation was assessed by superoxide dismutase-inhibitable reduction of ferricytochrome c. ß2AR agonists were generally more potent in inhibiting fMLP-induced O2(•-) production than in stimulating cAMP accumulation. (-)-Ephedrine and dichloroisoproterenol were devoid of any agonistic activity in the cAMP assay, but partially inhibited fMLP-induced O2(•-) production. Moreover, (-)-adrenaline was equi-efficacious in both assays whereas the efficacy of salbutamol was more than two-fold higher in the O2(•-) assay. Functional selectivity was visualized by deviations of ligand potencies and efficacies from linear correlations for various parameters. We obtained no evidence for involvement of protein kinase A in the inhibition of fMLP-induced O2(•-) production after ß2AR-stimulation although cAMP-increasing substances inhibited O2(•-) production. Taken together, our data corroborate the concept of ligand-specific receptor conformations with unique signaling capabilities in native human cells and suggest that the ß2AR inhibits O2(•-) production in a cAMP-independent manner.

Endothelial NOS (NOS3) impairs myocardial function in developing sepsis.

Endothelial nitric oxide synthase (NOS)3-derived nitric oxide (NO) modulates inotropic response and diastolic interval for optimal cardiac performance under non-inflammatory conditions. In sepsis, excessive NO production plays a key role in severe hypotension and myocardial dysfunction. We aimed to determine the role of NOS3 on myocardial performance, NO production, and time course of sepsis development. NOS3(-/-) and C57BL/6 wildtype mice were rendered septic by cecum ligation and puncture (CLP). Cardiac function was analyzed by serial echocardiography, in vivo pressure and isolated heart measurements. Cardiac output (CO) increased to 160 % of baseline at 10 h after sepsis induction followed by a decline to 63 % of baseline after 18 h in wildtype mice. CO was unaltered in septic NOS3(-/-) mice. Despite the hyperdynamic state, cardiac function and mean arterial pressure were impaired in septic wildtype as early as 6 h post CLP. At 12 h, cardiac function in septic wildtype was refractory to catecholamines in vivo and respective isolated hearts showed impaired pressure development and limited coronary flow reserve. Hemodynamics remained stable in NOS3(-/-) mice leading to significant survival benefit. Unselective NOS inhibition in septic NOS3(-/-) mice diminished this survival benefit. Plasma NO( x )- and local myocardial NO( x )- and NO levels (via NO spin trapping) demonstrated enhanced NO( x )- and bioactive NO levels in septic wildtype as compared to NOS3(-/-) mice. Significant contribution by inducible NOS (NOS2) during this early phase of sepsis was excluded. Our data suggest that NOS3 relevantly contributes to bioactive NO pool in developing sepsis resulting in impaired cardiac contractility.

Computational and experimental analysis of the transmembrane domain 4/5 dimerization interface of the serotonin 5-HT(1A) receptor.

Experimental evidence suggests that most members of class A G-protein coupled receptors (GPCRs) can form homomers and heteromers in addition to functioning as single monomers. In particular, serotonin (5-HT) receptors were shown to homodimerize and heterodimerize with other GPCRs, although the details and the physiological role of the oligomerization has not yet been fully elucidated. Here we used computational modeling of the 5-HT(1A) receptor monomer and dimer to predict residues important for dimerization. Based on these results, we carried out rationally designed site-directed mutagenesis. The ability of the mutants to dimerize was evaluated using different FRET-based approaches. The reduced levels of acceptor photobleaching-Förster resonance energy transfer (FRET) and the lower number of monomers participating in oligomers, as assessed by lux-FRET, confirmed the decreased ability of the mutants to dimerize and the involvement of the predicted contacts (Trp175(4.64), Tyr198(5.41), Arg151(4.40), and Arg152(4.41)) at the interface. This information was reintroduced as constraints for computational protein-protein docking to obtain a high-quality dimer model. Analysis of the refined model as well as molecular dynamics simulations of wild-type (WT) and mutant dimers revealed compensating interactions in dimers composed of WT and W175A mutant. This provides an explanation for the requirement of mutations of Trp175(4.64) in both homomers for disrupting dimerization. Our iterative computational-experimental study demonstrates that transmembrane domains TM4/TM5 can form an interaction interface in 5-HT(1A) receptor dimers and indicates that specific amino acid interactions maintain this interface. The mutants and the optimized model of the dimer structure may be used in functional studies of serotonin dimers.

ß-Adrenergic signaling and response to pressure overload in transgenic mice with cardiac-specific overexpression of inducible NO synthase.

UNLABELLED: The role of iNOS induction in the context of cardiac hypertrophy and heart failure is still not fully understood. We have used transgenic mice with cardiac specific overexpression of iNOS (tg-iNOS) to investigate the consequences of high level NO formation on cardiac function in vivo and the response to chronic pressure overload. Conductance manometry was used to analyze cardiac function of wild type (WT) and tg-iNOS mice under basal conditions and ß-adrenergic stimulation. To investigate the influence of iNOS on cardiac function in hypertrophied hearts, transversal aortic constriction was performed. Despite a high level of cardiac NO formation tg-iNOS mice showed almost normal LV function under basal conditions. The cardiac response to ß-adrenergic stimulation, however, was completely abolished. Acute NOS inhibition led to an instantaneous recovery of the inotropic response to catecholamines in tg-iNOS mice. Chronic pressure overload induced a similar extent of cardiac hypertrophy in WT and tg-iNOS hearts. LV function, however, was more compromised in tg-iNOS hearts as revealed by a decreased contractility and cardiac output. IN CONCLUSION: a high level of cardiac NO formation does not induce heart failure per se but severely enhances the functional depression in response to pressure overload. This effect could be due to the tonic impairment of the cardiac ß-adrenergic response.

Active ester functional single core magnetic nanostructures as a versatile immobilization matrix for effective bioseparation and catalysis.

Multifunctional nanocarriers for amino functional targets with a high density of accessible binding sites are obtained in a single polymerization step by grafting from copolymerization of an active ester monomer from superparamagnetic cores. As a result of the brush-like structure of the highly dispersed shell, the nano-objects exhibit an available capture capacity for amines that is found to be up to 2 orders of magnitude higher than for commercial magnetic beads, and the functional brush shell can serve as a template for many types of pendant functional groups and molecules. As comonomer, oligo(ethylene glycol) methacrylate allows for excellent water solubility at room temperature, biocompatibility, and thermoflocculation. We demonstrate the biorelated applicability of the hybrid nanoparticles by two different approaches. In the first approach, the immobilization of trypsin to the core-shell nanoparticles results in highly active, nanoparticulate biocatalysts that can easily be separated magnetically. Second, we demonstrate that the obtained nanoparticles are suitable for the effective labeling of cell membranes, opening a novel pathway for the easy and effective isolation of membrane proteins.

Limitations of the colloidal silica method in mapping the endothelial plasma membrane proteome of the mouse heart.

The endothelial cell (EC) membrane is an important interface, which plays a crucial role in signal transduction. Our aim was to selectively purify luminal EC membrane proteins from the coronary vasculature of the isolated perfused mouse heart and analyze its composition with mass spectrometry (MS). To specifically label coronary ECs in the intact heart, the colloidal silica method was applied, which is based on the binding of positively charged colloidal silica to the surface of EC membranes. Transmission electron microscopy revealed the specific labeling of ECs of macro and microvessels. Two different methods of tissue homogenization (Teflon pestle and ultra blade) together with density centrifugation were used for membrane protein enrichment. Enrichment and purity was controlled by Western blot analysis using the EC-specific protein caveolin 1 and various intracellular marker proteins. The ultra blade method resulted in a tenfold enrichment of caveolin 1, while there was negligible contamination as judged by Western blot. However, protein yield was low and required pooling of ten hearts for MS. When enriched endothelial membrane proteins were digested with trypsin and analyzed by LC-MS, a total of 56 proteins could be identified, of which only 12 were membrane proteins. We conclude that coronary endothelial membranes can be conveniently labeled with colloidal silica. However, due to the ionic nature of interaction of colloidal silica with the EC membrane the shear rate required for cardiac homogenization resulted in a substantial loss of specificity.

Nitrosative stress leads to protein glutathiolation, increased s-nitrosation, and up-regulation of peroxiredoxins in the heart.

Nitric oxide (NO) is produced by different isoforms of nitric oxide synthases (NOSs) and operates as a mediator of important cell signaling pathways, such as the cGMP signaling cascade. Another mechanism by which NO exerts biological effects is mediated through S-nitrosation of target proteins. To explore thiol-based protein modifications in a situation of defined nitrosative stress, we used a transgenic mouse model with cardiac specific overexpression of inducible nitric oxide synthase (iNOS) and concomitant myoglobin deficiency (iNOS(+)/myo(-/-)). In comparison with the wild type hearts, protein glutathiolation detected by immunoblotting was significantly enhanced in iNOS(+)/myo(-/-) hearts, whereas protein S-nitrosation as measured by the biotin switch assay and two-dimensional PAGE revealed that nearly all of the detected proteins ( approximately 60) remained unchanged with the exception of three proteins. Tandem mass spectrometry revealed these proteins to be peroxiredoxins (Prxs), which are known to possess peroxidase activity, whereby hydrogen peroxide, peroxynitrite, and a wide range of organic hydroperoxides are reduced and detoxified. Immunoblotting with specific antibodies revealed up-regulation of Prx VI in the iNOS(+)/myo(-/-) hearts, whereas expression of Prx II and Prx III remained unchanged. Furthermore, the analysis of the cardiac S-nitrososubproteome identified several new proteins possibly being involved in NO-signaling pathways. Our data indicate that S-nitrosation and glutathiolation of cardiac proteins may contribute to the phenotype of NO-induced heart failure. The up-regulation of antioxidant proteins like Prx VI appears to be an additional mechanism to antagonize an excess of reactive oxygen/nitrogen species. Furthermore, S-nitrosation of Prxs may serve a new function in the signaling cascade of nitrosative stress.

Nitric oxide-mediated protein modification in cardiovascular physiology and pathology.

Nitric oxide (NO) is a key regulator of cardiovascular functions including the control of vascular tone, anti-inflammatory properties of the endothelium, cardiac contractility, and thrombocyte activation and aggregation. Numerous experimental data support the view that NO not only acts via cyclic guanosine monophosphate (cGMP)-dependent mechanisms but also modulates protein function by nitrosation, nitrosylation, glutathiolation, and nitration, respectively. To understand how NO regulates all of these diverse biological processes on the molecular level a comprehensive assessment of NO-mediated cGMP-dependent and independent targets is required. Novel proteomic approaches allow the simultaneous identification of large quantities of proteins modified in an NO-dependent manner and thereby will considerably deepen our understanding of the role NO plays in cardiovascular physiology and pathophysiology.