James W. Wisler

A unique mechanism of β-blocker action: Carvedilol stimulates β-arrestin signaling

For many years, β-adrenergic receptor antagonists (β-blockers or βAR antagonists) have provided significant morbidity and mortality benefits in patients who have sustained acute myocardial infarction. More recently, β-adrenergic receptor antagonists have been found to provide survival benefits in patients suffering from heart failure, although the efficacy of different β-blockers varies widely in this condition. One drug, carvedilol, a nonsubtype-selective βAR antagonist, has proven particularly effective in the treatment of heart failure, although the mechanism(s) responsible for this are controversial. Here, we report that among 16 clinically relevant βAR antagonists, carvedilol displays a unique profile of in vitro signaling characteristics. We observed that in β2 adrenergic receptor (β2AR)-expressing HEK-293 cells, carvedilol has inverse efficacy for stimulating Gs-dependent adenylyl cyclase but, nonetheless, stimulates (i) phosphorylation of the receptors cytoplasmic tail on previously documented G protein-coupled receptor kinase sites; (ii) recruitment of β-arrestin to the β2AR; (iii) receptor internalization; and (iv) activation of extracellular regulated kinase 1/2 (ERK 1/2), which is maintained in the G protein-uncoupled mutant β2ART68F,Y132G,Y219A (β2ARTYY) and abolished by β-arrestin2 siRNA. Taken together, these data indicate that carvedilol is able to stabilize a receptor conformation which, although uncoupled from Gs, is nonetheless able to stimulate β-arrestin-mediated signaling. We hypothesize that such signaling may contribute to the special efficacy of carvedilol in the treatment of heart failure and may serve as a prototype for a new generation of therapeutic β2AR ligands.

β-Arrestin-biased Agonism at the β2-Adrenergic Receptor

Classically, the β2-adrenergic receptor (β2AR) and other members of the seven-transmembrane receptor (7TMR) superfamily activate G protein-dependent signaling pathways in response to ligand stimulus. It has recently been discovered, however, that a number of 7TMRs, including β2AR, can signal via β-arrestin-dependent pathways independent of G protein activation. It is currently unclear if among β2AR agonists there exist ligands that disproportionately signal via G proteins or β-arrestins and are hence “biased.” Using a variety of approaches that include highly sensitive fluorescence resonance energy transfer-based methodologies, including a novel assay for receptor internalization, we show that the majority of known β2AR agonists exhibit relative efficacies for β-arrestin-associated activities (β-arrestin membrane translocation and β2AR internalization) identical to the irrelative efficacies for G protein-dependent signaling (cyclic AMP generation). However, for three βAR ligands there is a marked bias toward β-arrestin signaling; these ligands stimulate β-arrestin-dependent receptor activities to a much greater extent than would be expected given their efficacy for G protein-dependent activity. Structural comparison of these biased ligands reveals that all three are catecholamines containing an ethyl substitution on the α-carbon, a motif absent on all of the other, unbiased ligands tested. Thus, these studies demonstrate the potential for developing a novel class of 7TMR ligands with a distinct bias for β-arrestin-mediated signaling.

MARCH2 promotes endocytosis and lysosomal sorting of carvedilol-bound β2-adrenergic receptors

The β2-adrenergic receptor antagonist carvedilol recruits MARCH2, a unique E3 ubiquitin ligase, to promote receptor endocytosis and lysosomal trafficking.

Conformationally selective RNA aptamers allosterically modulate the [beta]2-adrenoceptor

G-protein-coupled receptor (GPCR) ligands function by stabilizing multiple, functionally distinct receptor conformations. This property underlies how “biased agonists” activate specific subsets of a given receptor’s signaling profile. However, stabilization of distinct active GPCR conformations to enable structural characterization of mechanisms underlying GPCR activation remains difficult. These challenges have accentuated the need for receptor tools that allosterically stabilize and regulate receptor function via unique, previously unappreciated mechanisms. Here, utilizing a highly diverse RNA library combined with advanced selection strategies involving state-of-the-art next-generation sequencing and bioinformatics analyses, we identify RNA aptamers that bind a prototypical GPCR, β2-adrenoceptor (β2AR). Using biochemical, pharmacological, and biophysical approaches, we demonstrate that these aptamers bind with nanomolar affinity at defined surfaces of the receptor, allosterically stabilizing active, inactive, and ligand-specific receptor conformations. The discovery of RNA aptamers as allosteric GPCR modulators significantly expands the diversity of ligands available to study the structural and functional regulation of GPCRs.

Domain Structure and DNA Binding Regions of β Protein from Bacteriophage λ

β protein from bacteriophage λ promotes a single-strand annealing reaction that is central to Red-mediated recombination at double-strand DNA breaks and chromosomal ends. β protein binds most tightly to an intermediate of annealing formed by the sequential addition of two complementary oligonucleotides. Here we have characterized the domain structure of β protein in the presence and absence of DNA using limited proteolysis. Residues 1–130 form an N-terminal “core” domain that is resistant to proteases in the absence of DNA, residues 131–177 form a central region with enhanced resistance to proteases upon DNA complex formation, and the C-terminal residues 178–261 of β protein are sensitive to proteases in both the presence and absence of DNA. We probed the DNA binding regions of β protein further using biotinylation of lysine residues and mass spectrometry. Several lysine residues within the first 177 residues of β protein are protected from biotinylation in the DNA complex, whereas none of the lysine residues in the C-terminal portion are protected. The results lead to a model for the domain structure and DNA binding of β protein in which a stable N-terminal core and a more flexible central domain come together to bind DNA, whereas a C-terminal tail remains disordered. A fragment consisting of residues 1–177 of β protein maintains normal binding to sequentially added complementary oligonucleotides and has significantly enhanced binding to single-strand DNA.

Probing the DNA sequence specificity of Escherichia coli RECA protein

Escherichia coli RecA protein catalyzes the central DNA strand-exchange step of homologous recombination, which is essential for the repair of double-stranded DNA breaks. In this reaction, RecA first polymerizes on single-stranded DNA (ssDNA) to form a right-handed helical filament with one monomer per 3 nt of ssDNA. RecA generally binds to any sequence of ssDNA but has a preference for GT-rich sequences, as found in the recombination hot spot Chi (5′-GCTGGTGG-3′). When this sequence is located within an oligonucleotide, binding of RecA is phased relative to it, with a periodicity of three nucleotides. This implies that there are three separate nucleotide-binding sites within a RecA monomer that may exhibit preferences for the four different nucleotides. Here we have used a RecA coprotease assay to further probe the ssDNA sequence specificity of E.coli RecA protein. The extent of self-cleavage of a λ repressor fragment in the presence of RecA, ADP-AlF4 and 64 different trinucleotide-repeating 15mer oligonucleotides was determined. The coprotease activity of RecA is strongly dependent on the ssDNA sequence, with TGG-repeating sequences giving by far the highest coprotease activity, and GC and AT-rich sequences the lowest. For selected trinucleotide-repeating sequences, the DNA-dependent ATPase and DNA-binding activities of RecA were also determined. The DNA-binding and coprotease activities of RecA have the same sequence dependence, which is essentially opposite to that of the ATPase activity of RecA. The implications with regard to the biological mechanism of RecA are discussed.

The role of β-arrestin2-dependent signaling in thoracic aortic aneurysm formation in a murine model of Marfan syndrome

This manuscript demonstrates that β-arrestin2 mediates thoracic aortic aneurysm formation in a murine model of Marfan syndrome (MFS) by regulating proaneurysmal signaling. This work identifies a novel signaling cascade that contributes to aortic aneurysm formation as well as several potential, previously unappreciated therapeutic targets in MFS.

A guidance pathway for the selection of novel anticoagulants in the treatment of atrial fibrillation.

Oral anticoagulation with vitamin K antagonists has served as the primary treatment for the prevention of stroke and systemic embolization in patients with atrial fibrillation (AF) for decades. Over the past several years, multiple novel oral anticoagulants targeting key mediators of coagulation, including thrombin and factor Xa, have been developed. Specifically, agents targeting thrombin (dabigatran) and factor Xa (apixaban and rivaroxaban) have either reached late stages of clinical development (apixaban) or have received approval (dabigatran, rivaroxaban) by the US Food and Drug Administration for use in patients with nonvalvular AF. The promising results derived from large-scale clinical trials with these agents compared to warfarin expand the available therapeutic options for the prevention of stroke and systemic embolization in this rapidly increasing patient population. Here we present a general guidance pathway for the initiation and selection of oral anticoagulants in patients with AF.

Emerging paradigms in arterial thrombosis

A traditional perspective of arterial thrombosis begins with vessel wall injury and exposure of subendothelial proteins, including collagen and tissue factor, to circulating cellular and non-cellular components. Adhesion and activation of platelets, mediated by their interaction with von Willebrand protein and collagen, respectively, coupled with tissue factor-mediated activation of coagulation proteins, results in thrombin generation and fibrin formation. While this time-honored paradigm remains firm and soundly based, emerging evidence suggests that arterial thrombosis is much more complex and dynamic than originally believed. Several novel triggers, templates and facilitators, such as cell-free nucleic acids, histones, DNA-histone complexes, polyphosphates, and microvesicles have recently been identified and require inclusion in the expanding universe of thrombosis as a dominant phenotype of human disease. Because these mediators appear to have modest if any effect on physiologic hemostasis, they likely represent acquired and disease or condition-dependent processes that are highly attractive targets for pharmacologic intervention.

Oral factor Xa inhibitors for the long-term management of ACS

Despite considerable reductions in cardiovascular events in patients with an acute coronary syndrome (ACS) receiving dual antiplatelet therapy (DAPT), substantial residual risk persists. This unmet need has stimulated the development of anticoagulant drugs that target specific coagulation factors involved in the pathogenesis of thrombosis after atheromatous plaque disruption. Factor Xa is an attractive target for inhibition because of both its integral role in coagulation and its recognized participation in cellular proliferation and inflammation. Several oral, direct factor Xa inhibitors are undergoing investigation and large, phase III clinical trials of two agents, apixaban and rivaroxaban, in patients with an ACS have been completed. On the basis of the known pathobiology of ACS, one might anticipate that drugs in this class of anticoagulant would beneficially reduce ischemic and thrombotic events; however, a strategy of combined anticoagulant therapy and DAPT is likely to increase concomitant bleeding complications. The balance of benefit and risk will ultimately determine uptake in clinical practice. We review the available data on factor Xa inhibitors in the long-term management of patients with an ACS.