Showing papers by "Robert J. Lefkowitz published in 2018"

Biased signalling: from simple switches to allosteric microprocessors

Abstract: A given G protein-coupled receptor can signal through a range of downstream transducers depending on the stimulating ligand, enabling biased signalling towards different biological outcomes. Lefkowitz and colleagues describe the latest advances in the field, including efforts to harness biased signalling for improved therapeutic outcomes. G protein-coupled receptors (GPCRs) are the largest class of receptors in the human genome and some of the most common drug targets. It is now well established that GPCRs can signal through multiple transducers, including heterotrimeric G proteins, GPCR kinases and β-arrestins. While these signalling pathways can be activated or blocked by 'balanced' agonists or antagonists, they can also be selectively activated in a 'biased' response. Biased responses can be induced by biased ligands, biased receptors or system bias, any of which can result in preferential signalling through G proteins or β-arrestins. At many GPCRs, signalling events mediated by G proteins and β-arrestins have been shown to have distinct biochemical and physiological actions from one another, and an accurate evaluation of biased signalling from pharmacology through physiology is crucial for preclinical drug development. Recent structural studies have provided snapshots of GPCR–transducer complexes, which should aid in the structure-based design of novel biased therapies. Our understanding of GPCRs has evolved from that of two-state, on-and-off switches to that of multistate allosteric microprocessors, in which biased ligands transmit distinct structural information that is processed into distinct biological outputs. The development of biased ligands as therapeutics heralds an era of increased drug efficacy with reduced drug side effects.

Manifold roles of β-arrestins in GPCR signaling elucidated with siRNA and CRISPR/Cas9

Abstract: G protein-coupled receptors (GPCRs) use diverse mechanisms to regulate the mitogen-activated protein kinases ERK1/2. β-Arrestins (βArr1/2) are ubiquitous inhibitors of G protein signaling, promoting GPCR desensitization and internalization and serving as scaffolds for ERK1/2 activation. Studies using CRISPR/Cas9 to delete βArr1/2 and G proteins have cast doubt on the role of β-arrestins in activating specific pools of ERK1/2. We compared the effects of siRNA-mediated knockdown of βArr1/2 and reconstitution with βArr1/2 in three different parental and CRISPR-derived βArr1/2 knockout HEK293 cell pairs to assess the effect of βArr1/2 deletion on ERK1/2 activation by four Gs-coupled GPCRs. In all parental lines with all receptors, ERK1/2 stimulation was reduced by siRNAs specific for βArr2 or βArr1/2. In contrast, variable effects were observed with CRISPR-derived cell lines both between different lines and with activation of different receptors. For β2 adrenergic receptors (β2ARs) and β1ARs, βArr1/2 deletion increased, decreased, or had no effect on isoproterenol-stimulated ERK1/2 activation in different CRISPR clones. ERK1/2 activation by the vasopressin V2 and follicle-stimulating hormone receptors was reduced in these cells but was enhanced by reconstitution with βArr1/2. Loss of desensitization and receptor internalization in CRISPR βArr1/2 knockout cells caused β2AR-mediated stimulation of ERK1/2 to become more dependent on G proteins, which was reversed by reintroducing βArr1/2. These data suggest that βArr1/2 function as a regulatory hub, determining the balance between mechanistically different pathways that result in activation of ERK1/2, and caution against extrapolating results obtained from βArr1/2- or G protein-deleted cells to GPCR behavior in native systems.

Biased G Protein-Coupled Receptor Signaling: Changing the Paradigm of Drug Discovery.

Abstract: G protein–coupled receptors (GPCRs) are a family of 7 transmembrane-spanning proteins that collectively serve as the largest group of therapeutic targets. Within cardiology, GPCRs such as α-and β-adrenergic receptors, the angiotensin type I receptor, and the P2Y12 receptor as well, are the targets of a variety of widely used medications. Ligands for GPCRs have been characterized canonically as agonists, which promote or stabilize conformational changes in the receptor that result in the activation of heterotrimeric G proteins and the generation of second-messenger systems, or antagonists that block such activation. Work over the past 2 decades, however, has found that ligands can induce distinct active receptor conformations that activate only specific subsets of a given receptor’s functional repertoire.1 In particular, ligands have been identified that exhibit bias or functional selectivity toward specific G proteins or even other signal transducers such as β-arrestins. Further exploration of this biased signaling biology has led to important changes in the way pharmacological agents are developed and screened. Although these terms have been used interchangeably to characterize a variety of GPCR signaling and biological functions, for the purpose of this review, we will use the terms bias or functional selectivity to refer specifically to the ability of a GPCR ligand to stimulate signaling through 1 signal transducer over another (eg, β-arrestin versus G protein). Beyond the recognition and development of these biased agonists, researchers have discovered novel mechanisms by which these ligands interact with receptors and engender unique functional profiles (Figure). The majority of pharmacological GPCR ligands target orthosteric binding sites, …

Small-Molecule Positive Allosteric Modulators of the β2-Adrenoceptor Isolated from DNA-Encoded Libraries.

Abstract: Conventional drug discovery efforts at the β2-adrenoceptor (β2AR) have led to the development of ligands that bind almost exclusively to the receptor’s hormone-binding orthosteric site. However, targeting the largely unexplored and evolutionarily unique allosteric sites has potential for developing more specific drugs with fewer side effects than orthosteric ligands. Using our recently developed approach for screening G protein–coupled receptors (GPCRs) with DNA-encoded small-molecule libraries, we have discovered and characterized the first β2AR small-molecule positive allosteric modulators (PAMs)—compound (Cmpd)-6 [(R)-N-(4-amino-1-(4-(tert-butyl)phenyl)-4-oxobutan-2-yl)-5-(N-isopropyl-N-methylsulfamoyl)-2-((4-methoxyphenyl)thio)benzamide] and its analogs. We used purified human β2ARs, occupied by a high-affinity agonist, for the affinity-based screening of over 500 million distinct library compounds, which yielded Cmpd-6. It exhibits a low micro-molar affinity for the agonist-occupied β2AR and displays positive cooperativity with orthosteric agonists, thereby enhancing their binding to the receptor and ability to stabilize its active state. Cmpd-6 is cooperative with G protein and β-arrestin1 (a.k.a. arrestin2) to stabilize high-affinity, agonist-bound active states of the β2AR and potentiates downstream cAMP production and receptor recruitment of β-arrestin2 (a.k.a. arrestin3). Cmpd-6 is specific for the β2AR compared with the closely related β1AR. Structure–activity studies of select Cmpd-6 analogs defined the chemical groups that are critical for its biologic activity. We thus introduce the first small-molecule PAMs for the β2AR, which may serve as a lead molecule for the development of novel therapeutics. The approach described in this work establishes a broadly applicable proof-of-concept strategy for affinity-based discovery of small-molecule allosteric compounds targeting unique conformational states of GPCRs.

Sortase ligation enables homogeneous GPCR phosphorylation to reveal diversity in β-arrestin coupling.

Abstract: The ability of G protein-coupled receptors (GPCRs) to initiate complex cascades of cellular signaling is governed by the sequential coupling of three main transducer proteins, G protein, GPCR kinase (GRK), and β-arrestin. Mounting evidence indicates these transducers all have distinct conformational preferences and binding modes. However, interrogating each transducer’s mechanism of interaction with GPCRs has been complicated by the interplay of transducer-mediated signaling events. For example, GRK-mediated receptor phosphorylation recruits and induces conformational changes in β-arrestin, which facilitates coupling to the GPCR transmembrane core. Here we compare the allosteric interactions of G proteins and β-arrestins with GPCRs’ transmembrane cores by using the enzyme sortase to ligate a synthetic phosphorylated peptide onto the carboxyl terminus of three different receptors. Phosphopeptide ligation onto the β 2 -adrenergic receptor (β 2 AR) allows stabilization of a high-affinity receptor active state by β-arrestin1, permitting us to define elements in the β 2 AR and β-arrestin1 that contribute to the receptor transmembrane core interaction. Interestingly, ligation of the identical phosphopeptide onto the β 2 AR, the muscarinic acetylcholine receptor 2 and the μ-opioid receptor reveals that the ability of β-arrestin1 to enhance agonist binding relative to G protein differs substantially among receptors. Furthermore, strong allosteric coupling of β-arrestin1 correlates with its ability to attenuate, or “desensitize,” G protein activation in vitro. Sortase ligation thus provides a versatile method to introduce complex, defined phosphorylation patterns into GPCRs, and analogous strategies could be applied to other classes of posttranslationally modified proteins. These homogeneously phosphorylated GPCRs provide an innovative means to systematically study receptor–transducer interactions.

G protein–coupled receptor kinases (GRKs) orchestrate biased agonism at the β2-adrenergic receptor

Abstract: Biased agonists of G protein-coupled receptors (GPCRs), which selectively activate either G protein- or β-arrestin-mediated signaling pathways, are of major therapeutic interest because they have the potential to show improved efficacy and specificity as drugs. Efforts to understand the mechanistic basis of this phenomenon have focused on the hypothesis that G proteins and β-arrestins preferentially couple to distinct GPCR conformations. However, because GPCR kinase (GRK)-dependent receptor phosphorylation is a critical prerequisite for the recruitment of β-arrestins to most GPCRs, GRKs themselves may play an important role in establishing biased signaling. We showed that an alanine mutant of the highly conserved residue tyrosine 219 (Y219A) in transmembrane domain five of the β2-adrenergic receptor (β2AR) was incapable of β-arrestin recruitment, receptor internalization, and β-arrestin-mediated activation of extracellular signal-regulated kinase (ERK), whereas it retained the ability to signal through G protein. We found that the impaired β-arrestin recruitment in cells was due to reduced GRK-mediated phosphorylation of the β2AR Y219A C terminus, which was recapitulated in vitro with purified components. Furthermore, in vitro ligation of a synthetically phosphorylated peptide onto the C terminus of β2AR Y219A rescued both the initial recruitment of β-arrestin and its engagement with the intracellular core of the receptor. These data suggest that the Y219A mutation generates a G protein-biased state primarily by conformational selection against GRK coupling, rather than against β-arrestin. Together, these findings highlight the importance of GRKs in modulating the biased agonism of GPCRs.

β-arrestin 1 regulates β2-adrenergic receptor-mediated skeletal muscle hypertrophy and contractility.

Abstract: β2-adrenergic receptors (β2ARs) are the target of catecholamines and play fundamental roles in cardiovascular, pulmonary, and skeletal muscle physiology. An important action of β2AR stimulation on skeletal muscle is anabolic growth, which has led to the use of agonists such as clenbuterol by athletes to enhance muscle performance. While previous work has demonstrated that β2ARs can engage distinct signaling and functional cascades mediated by either G proteins or the multifunctional adaptor protein, β-arrestin, the precise role of β-arrestin in skeletal muscle physiology is not known. Here, we tested the hypothesis that agonist activation of the β2AR by clenbuterol would engage β-arrestin as a key transducer of anabolic skeletal muscle growth. The contractile force of isolated extensor digitorum longus muscle (EDL) and calcium signaling in isolated flexor digitorum brevis (FDB) fibers were examined from the wild-type (WT) and β-arrestin 1 knockout mice (βarr1KO) followed by chronic administration of clenbuterol (1 mg/kg/d). Hypertrophic responses including fiber composition and fiber size were examined by immunohistochemical imaging. We performed a targeted phosphoproteomic analysis on clenbuterol stimulated primary cultured myoblasts from WT and βarr1KO mice. Statistical significance was determined by using a two-way analysis with Sidak’s or Tukey’s multiple comparison test and the Student’s t test. Chronic administration of clenbuterol to WT mice enhanced the contractile force of EDL muscle and calcium signaling in isolated FDB fibers. In contrast, when administered to βarr1KO mice, the effect of clenbuterol on contractile force and calcium influx was blunted. While clenbuterol-induced hypertrophic responses were observed in WT mice, this response was abrogated in mice lacking β-arrestin 1. In primary cultured myoblasts, clenbuterol-stimulated phosphorylation of multiple pro-hypertrophy proteins required the presence of β-arrestin 1. We have identified a previously unappreciated role for β-arrestin 1 in mediating β2AR-stimulated skeletal muscle growth and strength. We propose these findings could have important implications in the design of future pharmacologic agents aimed at reversing pathological conditions associated with skeletal muscle wasting.

GPCR signaling: conformational activation of arrestins.

Abstract: The arrestins, a small family of versatile adapter proteins, play central roles in G-protein coupled receptor (GPCR) desensitization, endocytosis, and signal transduction. Two papers by Latorraca et al. and Eichel et al. published recently in Nature, dissect the independent and concerted mechanisms by which the phosphorylated GPCR tail and transmembrane core conformationally enable arrestins to perform their diverse functions. G-protein coupled receptors (GPCRs), also known as seven transmembrane receptors (7 TMRs) are the largest family of cell surface proteins, with ~800 members identified in the human genome. They represent the commonest targets of therapeutic drugs used for the treatment of a wide range of health problems. Their remarkably diverse actions are mediated and modulated by the extracellular stimulus-driven interaction of the receptors with three families of proteins: the heterotrimeric guanine nucleotidebinding (G) proteins, the G protein-coupled receptor kinases (GRKs), and the arrestins. G proteins were discovered as the paradigmatic relay switches connecting the receptors to membrane effectors, such as second messenger-generating enzymes. GRKs and arrestins were discovered as a two-component system, which turns off the receptors. The activated receptors are phosphorylated by the kinases, typically on multiple sites on the cytoplasmic C-terminal tail. This leads to binding of the arrestins, initially to the phosphorylated receptor C tail, and then to the agonist-modified conformation of the 7 TM core. This occludes the G-protein-binding site on the receptor thus diminishing receptor G-protein signaling, a process known as receptor desensitization. There are four members of the arrestin family including two visual arrestins (arrestins 1 and 4), which are confined to the retina, and two universally expressed non-visual forms, arrestins 2 and 3, generally referred to as β-arrestin-1 and β-arrestin-2. Recently two papers have shed new light on the distinct roles of the phosphorylated receptor C-terminus and the 7 TM core in mediating arrestin interaction with and activation by GPCRs. Protein members of the versatile arrestin family contain two major domains (N and C), which consist largely of β-sheets and connecting loops. While initially discovered in the context of receptor desensitization, it was subsequently discovered that β-arrestins also serve as multifunctional adapters mediating receptor interaction with several components of the clathrincoated pit endocytic machinery, thus mediating receptor internalization and trafficking. They also bind to a long list of signaling proteins thus enabling β-arrestins to serve as signaling intermediates in their own right, acting either independently of or in concert with G-proteins to modulate cellular activities. The remarkable diversity of functions carried out by arrestins after their interaction with the receptors has engendered much interest in the mechanisms of GPCR-β-arrestin interactions and how these are translated into specific cellular consequences. Confirming decades-long ideas, recent structural studies of both visual arrestin interaction with rhodopsin and β-arrestin interaction with other GPCRs, have revealed two types of interactions within receptor-arrestin complexes: one involving the phosphorylated cytoplasmic tail of the GPCR (RP-tail), referred to as the ‘tail’ interaction, and one involving the transmembrane core of the GPCR, referred to as the ‘core’ interaction. It has been suggested that these are in turn mediated by two distinct sites on arrestins — a ‘phosphorylation sensor’ and an ‘activation sensor’. In the past, studies have largely focused on activation of arrestins by the RP-tail and its functional outcomes, 9 while the role of the receptor transmembrane core, if any, has remained obscure. However, the two new papers illuminate the distinct roles of both the RP-tail and the 7 TM core in mediating arrestin interaction with and activation by GPCRs. Specifically, these studies focus a much brighter light on the mechanisms of arrestin activation by the GPCR core. Using atomic-level molecular dynamic simulations of arrestin and β-arrestin, Latorraca and colleagues investigated the structural mechanisms by which the RP-tail, and the transmembrane core, in particular, regulate arrestin activation. In the simulations, the authors used a key conformational change in arrestin that occurs upon activation (a twist of the C domain of about 20° relative to the N domain) as a primary indicator of arrestin activation. According to current models, the initial step of arrestin activation requires the displacement of the long C tail of arrestin (which is folded back on the N domain in the inactive conformation) by the RP-tail of an activated GPCR. The authors show that this displacement of the arrestin C tail leads to prolongation in active state conformations of arrestin, even when not bound to the receptor. This may explain the previously noted curious finding that β-arrestin remains active and continues signaling following its dissociation from the activating GPCR. Using the rhodopsin-arrestin structure as a template for their simulations, the authors demonstrate that the receptor core and RP-tail each individually stabilize active conformations of arrestin (Fig. 1). The simultaneous binding of both regions as occurs with a full-length receptor adds further stabilization. According to their simulations, the key contacts responsible for the core interaction are between the receptor’s intracellular loops (ICLs), e.g., ICL-2 and ICL-3, and the C-loop and back loop in the C domain of arrestin, respectively. These computational findings are supported by fluorescence spectroscopic studies on arrestin mutants, which monitor conformational changes both at the RP-tail interface (the arrestin gate loop) and

A Serendipitous Scientist

Abstract: Growing up in a middle-class Jewish home in the Bronx, I had only one professional goal: to become a physician. However, as with most of my Vietnam-era MD colleagues, I found my residency training interrupted by the Doctor Draft in 1968. Some of us who were academically inclined fulfilled this obligation by serving in the US Public Health Service as commissioned officers stationed at the National Institutes of Health. This experience would eventually change the entire trajectory of my career. Here I describe how, over a period of years, I transitioned from the life of a physician to that of a physician-scientist; my 50 years of work on cellular receptors; and some miscellaneous thoughts on subjects as varied as Nobel prizes, scientific lineages, mentoring, publishing, and funding.