Robert J. Lefkowitz

Conformational Basis of Functionally Selective Allosteric Modulation of the Angiotensin II type 1 Receptor by Small Molecules

Blockade of signaling through the angiotensin II type 1 receptor (AT1R), a prototypical G protein-coupled receptor (GPCR), by angiotensin receptor blockers (ARBs) is a major therapeutic approach to treating a wide variety of cardiovascular and renal diseases 1 . Like most GPCRs, the AT1R signals through two transducers, G proteins and β-arrestins 2,3 . Previous reports have described β-arrestin-biased peptide orthosteric agonists for the AT1R with potential therapeutic advantages over currently available unbiased ARBs 4–6 . Here we report the DNA- encoded library screening-guided isolation and pharmacological characterization of the first small molecule AT1R allosteric ligands. We use cryo-electron microscopy, double electron- electron resonance spectroscopy, molecular dynamics simulations, and targeted mutagenesis to determine their binding sites, binding modes and conformational mechanisms driving their unique and divergent modulatory effects on G protein and β-arrestin pathways. Our findings uncover new mechanisms for precisely controlling the dynamic behavior of the AT1R with implications for drug development targeting this pathophysiologically important receptor family.

L-162,313 engages peptide-like AT1R activation switches, enabling design of more efficacious small-molecule agonists.

May inform design of oral AT1R agonists; leaves open human translation and therapeutic utility.

MAY 2018WislerCirculation

Biased GPCR ligands promise more targeted therapy with fewer side effects than unbiased ligands.

This review highlights the therapeutic potential of biased GPCR ligands in cardiovascular medicine, which can selectively target beneficial signaling pathways while avoiding those responsible for adverse effects.

APR 2000LefkowitzCirculation

Catecholamines, Cardiac β-Adrenergic Receptors, and Heart Failure

Small-molecule modulation of β-arrestins

Identifies small-molecule inhibitors targeting β-arrestins, revealing therapeutic potential.

Phenylephrine drives severe hypertrophy and reduces survival in mice overexpressing alpha1B adrenergic receptors.

In a transgenic mouse model, betaARK1 appears to be a key molecule in the transition from myocardial hypertrophy to heart failure during chronic sympathetic activation.

Structure and dynamics determine G protein coupling specificity at a class A GPCR

G protein–coupled receptors (GPCRs) exhibit varying degrees of selectivity for different G protein isoforms. Despite the abundant structures of GPCR–G protein complexes, little is known about the mechanism of G protein coupling specificity. The β 2 -adrenergic receptor is an example of GPCR with high selectivity for Gαs, the stimulatory G protein for adenylyl cyclase, and much weaker for the Gαi family of G proteins inhibiting adenylyl cyclase. By developing a Gαi-biased agonist (LM189), we provide structural and biophysical evidence supporting that distinct conformations at ICL2 and TM6 are required for coupling of the different G protein subtypes Gαs and Gαi. These results deepen our understanding of G protein specificity and bias and can accelerate the design of ligands that select for preferred signaling pathways.

Small-molecule modulation of β-arrestins

β-arrestins are multifunctional regulators of G protein-coupled receptor (GPCR) signaling, orchestrating diverse downstream signaling events and physiological responses across the vast GPCR superfamily. While GPCR pharmacology has advanced to target orthosteric and allosteric sites, as well as G proteins and GRKs, comparable chemical tools to study β-arrestins remain lacking. Here, we report the discovery of small-molecule inhibitors that selectively target β-arrestins and delineate their mechanism of action through integrated pharmacological, biochemical, biophysical, and structural analyses. These inhibitors disrupt β-arrestin-engagement with agonist-activated GPCRs, impairing desensitization, internalization, and β-arrestin-dependent functions while sparing G protein-receptor coupling. Cryo-EM, MD simulations, and structure-guided mutagenesis reveal that one modulator, Cmpd-5, engages a cryptic pocket formed by the middle, C-, and lariat loops of β-arrestin1-a critical receptor-binding interface-stabilizing a distinct conformation incompatible with GPCR engagement. Together, these findings provide a mechanistic framework for β-arrestin modulation, introducing transducer-targeted strategies to fine-tune GPCR signaling and guide the development of pathway-specific therapeutics.

Beta-arrestin 1 mediated Src activation via Src SH3 domain revealed by cryo-electron microscopy

Cryo-electron microscopy analysis reveals how beta-arrestin 1 binds the Src SH3 domain at two distinct sites, indicating that displacement of this domain relieves kinase autoinhibition.

G Protein-Coupled Receptors: A Century of Research and Discovery

GPCRs (G protein-coupled receptors), also known as 7 transmembrane domain receptors, are the largest receptor family in the human genome, with ≈800 members. GPCRs regulate nearly every aspect of human physiology and disease, thus serving as important drug targets in cardiovascular disease. Sharing a conserved structure comprised of 7 transmembrane α-helices, GPCRs couple to heterotrimeric G-proteins, GPCR kinases, and β-arrestins, promoting downstream signaling through second messengers and other intracellular signaling pathways. GPCR drug development has led to important cardiovascular therapies, such as antagonists of β-adrenergic and angiotensin II receptors for heart failure and hypertension, and agonists of the glucagon-like peptide-1 receptor for reducing adverse cardiovascular events and other emerging indications. There continues to be a major interest in GPCR drug development in cardiovascular and cardiometabolic disease, driven by advances in GPCR mechanistic studies and structure-based drug design. This review recounts the rich history of GPCR research, including the current state of clinically used GPCR drugs, and highlights newly discovered aspects of GPCR biology and promising directions for future investigation. As additional mechanisms for regulating GPCR signaling are uncovered, new strategies for targeting these ubiquitous receptors hold tremendous promise for the field of cardiovascular medicine.

Structure and dynamics determine G protein coupling specificity at a class A GPCR

G protein coupled receptors (GPCRs) exhibit varying degrees of selectivity for different G protein isoforms. Despite the abundant structures of GPCR-G protein complexes, little is known about the mechanism of G protein coupling specificity. The β2-adrenergic receptor is an example of GPCR with high selectivity for Gαs, the stimulatory G protein for adenylyl cyclase, and much weaker for the Gαi family of G proteins inhibiting adenylyl cyclase. By developing a new Gαi-biased agonist (LM189), we provide structural and biophysical evidence supporting that distinct conformations at ICL2 and TM6 are required for coupling of the different G protein subtypes Gαs and Gαi. These results deepen our understanding of G protein specificity and bias and can accelerate the design of ligands that select for preferred signaling pathways.

G protein–coupled receptors: from radioligand binding to cellular signaling

Radioligand binding techniques facilitated the identification and study of G-protein coupled receptors that now represent the largest class of targets for therapeutic drugs.

NOV 2023LefkowitzNature Communications

The authors argue that the conclusions by Benkel et al. regarding carvedilol signaling through β2AR-mediated G protein activation are incorrect and should not guide clinical decisions.

This editorial challenges a recent study's claim that carvedilol activates β2-adrenoceptors, cautioning against applying those findings to clinical practice.

Signal transduction at GPCRs: Allosteric activation of the ERK MAPK by β-arrestin

β-arrestins are multivalent adaptor proteins that bind active phosphorylated G protein-coupled receptors (GPCRs) to inhibit G protein signaling, mediate receptor internalization, and initiate alternative signaling events. β-arrestins link agonist-stimulated GPCRs to downstream signaling partners, such as the c-Raf-MEK1-ERK1/2 cascade leading to ERK1/2 activation. β-arrestins have been thought to transduce signals solely via passive scaffolding by facilitating the assembly of multiprotein signaling complexes. Recently, however, β-arrestin 1 and 2 were shown to activate two downstream signaling effectors, c-Src and c-Raf, allosterically. Over the last two decades, ERK1/2 have been the most intensely studied signaling proteins scaffolded by β-arrestins. Here, we demonstrate that β-arrestins play an active role in allosterically modulating ERK kinase activity in vitro and within intact cells. Specifically, we show that β-arrestins and their GPCR-mediated active states allosterically enhance ERK2 autophosphorylation and phosphorylation of a downstream ERK2 substrate, and we elucidate the mechanism by which β-arrestins do so. Furthermore, we find that allosteric stimulation of dually phosphorylated ERK2 by active-state β-arrestin 2 is more robust than by active-state β-arrestin 1, highlighting differential capacities of β-arrestin isoforms to regulate effector signaling pathways downstream of GPCRs. In summary, our study provides strong evidence for a new paradigm in which β-arrestins function as active "catalytic" scaffolds to allosterically unlock the enzymatic activity of signaling components downstream of GPCR activation.

Allosteric modulator potentiates β2AR agonist–promoted bronchoprotection in asthma models

Asthma is a chronic inflammatory disease associated with episodic airway narrowing. Inhaled β2-adrenergic receptor (β2AR) agonists (β2-agonists) promote - with limited efficacy - bronchodilation in asthma. All β2-agonists are canonical orthosteric ligands that bind the same site as endogenous epinephrine. We recently isolated a β2AR-selective positive allosteric modulator (PAM), compound-6 (Cmpd-6), which binds outside of the orthosteric site and modulates orthosteric ligand functions. With the emerging therapeutic potential of G-protein coupled receptor allosteric ligands, we investigated the impact of Cmpd-6 on β2AR-mediated bronchoprotection. Consistent with our findings using human β2ARs, Cmpd-6 allosterically potentiated β2-agonist binding to guinea pig β2ARs and downstream signaling of β2ARs. In contrast, Cmpd-6 had no such effect on murine β2ARs, which lack a crucial amino acid in the Cmpd-6 allosteric binding site. Importantly, Cmpd-6 enhanced β2 agonist-mediated bronchoprotection against methacholine-induced bronchoconstriction in guinea pig lung slices, but - in line with the binding studies - not in mice. Moreover, Cmpd-6 robustly potentiated β2 agonist-mediated bronchoprotection against allergen-induced airway constriction in lung slices obtained from a guinea pig model of allergic asthma. Cmpd-6 similarly enhanced β2 agonist-mediated bronchoprotection against methacholine-induced bronchoconstriction in human lung slices. Our results highlight the potential of β2AR-selective PAMs in the treatment of airway narrowing in asthma and other obstructive respiratory diseases.

Loss of biased signaling at a G protein-coupled receptor in overexpressed systems

G protein-coupled receptors (GPCRs) regulate cellular signaling pathways by coupling to two classes of transducers: heterotrimeric G proteins and β-arrestins. [Sarcosine1Ile4Ile8]-angiotensin II (SII), an analog of the endogenous ligand angiotensin II (AngII) for the angiotensin II type 1 receptor (AT1R), fails to activate G protein in physiologically relevant models. Despite this, SII and several derivatives induce cellular signaling outcomes through β-arrestin-2-dependent mechanisms. However, studies reliant on exogenous AT1R overexpression indicate that SII is a partial agonist for G protein signaling and lacks β-arrestin-exclusive functional specificity. We investigated this apparent discrepancy by profiling changes in functional specificity at increasing expression levels of AT1R using a stably integrated tetracycline-titratable expression system stimulated with AngII, SII, and four other AngII analogs displaying different signaling biases. Unbiased and G protein-biased ligands activated dose-dependent calcium responses at all tested receptor concentrations. In contrast, β-arrestin-biased ligands induced dose-dependent calcium signaling only at higher AT1R overexpression levels. Using inhibitors of G proteins, we demonstrated that both Gi and Gq/11 mediated overexpression-dependent calcium signaling by β-arrestin-biased ligands. Regarding β-arrestin-mediated cellular events, the β-arrestin-biased ligand TRV026 induced receptor internalization at low physiological receptor levels insufficient for it to initiate calcium signaling. In contrast, unbiased AngII exhibited no relative preference between these outcomes under such low receptor conditions. However, with high receptor overexpression, TRV026 lost its functional selectivity. These results suggest receptor overexpression misleadingly distorts the bias of AT1R ligands and highlight the risks of using overexpressed systems to infer the signaling bias of GPCR ligands in physiologically relevant contexts.

GPCR-mediated β-arrestin activation deconvoluted with single-molecule precision

β-arrestins bind G protein-coupled receptors to terminate G protein signaling and to facilitate other downstream signaling pathways. Using single-molecule fluorescence resonance energy transfer imaging, we show that β-arrestin is strongly autoinhibited in its basal state. Its engagement with a phosphopeptide mimicking phosphorylated receptor tail efficiently releases the β-arrestin tail from its N domain to assume distinct conformations. Unexpectedly, we find that β-arrestin binding to phosphorylated receptor, with a phosphorylation barcode identical to the isolated phosphopeptide, is highly inefficient and that agonist-promoted receptor activation is required for β-arrestin activation, consistent with the release of a sequestered receptor C tail. These findings, together with focused cellular investigations, reveal that agonism and receptor C-tail release are specific determinants of the rate and efficiency of β-arrestin activation by phosphorylated receptor. We infer that receptor phosphorylation patterns, in combination with receptor agonism, synergistically establish the strength and specificity with which diverse, downstream β-arrestin-mediated events are directed.

Translating science to medicine: The case for physician-scientists

As the number of physician-scientists continues to decline, action must be taken to support them as they embark on their careers.

The GPCR–β-arrestin complex allosterically activates C-Raf by binding its amino terminus

G protein–coupled receptors (GPCRs) convert external stimuli into cellular signals through heterotrimeric guanine nucleotide-binding proteins (G-proteins) and β-arrestins (βarrs). In a βarr-dependent signaling pathway, βarrs link GPCRs to various downstream signaling partners, such as the Raf–mitogen-activated protein kinase extracellular signal–regulated kinase–extracellular signal-regulated kinase cascade. Agonist-stimulated GPCR–βarr complexes have been shown to interact with C-Raf and are thought to initiate the mitogen-activated protein kinase pathway through simple tethering of these signaling partners. However, recent evidence shows that in addition to canonical scaffolding functions, βarrs can allosterically activate downstream targets, such as the nonreceptor tyrosine kinase Src. Here, we demonstrate the direct allosteric activation of C-Raf by GPCR–βarr1 complexes in vitro. Furthermore, we show that βarr1 in complex with a synthetic phosphopeptide mimicking the human V2 vasopressin receptor tail that binds and functionally activates βarrs also allosterically activates C-Raf. We reveal that the interaction between the phosphorylated GPCR C terminus and βarr1 is necessary and sufficient for C-Raf activation. Interestingly, the interaction between βarr1 and C-Raf was considerably reduced in the presence of excess activated H-Ras, a small GTPase known to activate C-Raf, suggesting that H-Ras and βarr1 bind to the same region on C-Raf. Furthermore, we found that βarr1 interacts with the Ras-binding domain of C-Raf. Taken together, these data suggest that in addition to canonical scaffolding functions, GPCR–βarr complexes directly allosterically activate C-Raf by binding to its amino terminus. This work provides novel insights into how βarrs regulate effector molecules to activate downstream signaling pathways. G protein–coupled receptors (GPCRs) convert external stimuli into cellular signals through heterotrimeric guanine nucleotide-binding proteins (G-proteins) and β-arrestins (βarrs). In a βarr-dependent signaling pathway, βarrs link GPCRs to various downstream signaling partners, such as the Raf–mitogen-activated protein kinase extracellular signal–regulated kinase–extracellular signal-regulated kinase cascade. Agonist-stimulated GPCR–βarr complexes have been shown to interact with C-Raf and are thought to initiate the mitogen-activated protein kinase pathway through simple tethering of these signaling partners. However, recent evidence shows that in addition to canonical scaffolding functions, βarrs can allosterically activate downstream targets, such as the nonreceptor tyrosine kinase Src. Here, we demonstrate the direct allosteric activation of C-Raf by GPCR–βarr1 complexes in vitro. Furthermore, we show that βarr1 in complex with a synthetic phosphopeptide mimicking the human V2 vasopressin receptor tail that binds and functionally activates βarrs also allosterically activates C-Raf. We reveal that the interaction between the phosphorylated GPCR C terminus and βarr1 is necessary and sufficient for C-Raf activation. Interestingly, the interaction between βarr1 and C-Raf was considerably reduced in the presence of excess activated H-Ras, a small GTPase known to activate C-Raf, suggesting that H-Ras and βarr1 bind to the same region on C-Raf. Furthermore, we found that βarr1 interacts with the Ras-binding domain of C-Raf. Taken together, these data suggest that in addition to canonical scaffolding functions, GPCR–βarr complexes directly allosterically activate C-Raf by binding to its amino terminus. This work provides novel insights into how βarrs regulate effector molecules to activate downstream signaling pathways. G protein–coupled receptors (GPCRs), also called 7-transmembrane receptors, are the largest group of membrane proteins that regulate a multitude of physiological processes (1Pierce K.L. Premont R.T. Lefkowitz R.J. Seven-transmembrane receptors.Nat. Rev. Mol. Cell Biol. 2002; 3: 639-650Crossref PubMed Scopus (2164) Google Scholar, 2Rockman H.A. Koch W.J. Lefkowitz R.J. Seven-transmembrane-spanning receptors and heart function.Nature. 2002; 415: 206-212Crossref PubMed Scopus (797) Google Scholar). Approximately 35% of all Food and Drug Administration–approved drugs target GPCRs (3Sriram K. Insel P.A. G protein-coupled receptors as targets for approved drugs: How many targets and how many drugs?.Mol. Pharmacol. 2018; 93: 251-258Crossref PubMed Scopus (662) Google Scholar). In response to various external stimuli, such as light, hormones, and neurotransmitters, GPCRs initiate diverse cellular signaling processes through G protein–dependent and β-arrestin (βarr)-dependent pathways (4Hilger D. Masureel M. Kobilka B.K. Structure and dynamics of GPCR signaling complexes.Nat. Struct. Mol. Biol. 2018; 25: 4-12Crossref PubMed Scopus (519) Google Scholar, 5DeWire S.M. Ahn S. Lefkowitz R.J. Shenoy S.K. Beta-arrestins and cell signaling.Annu. Rev. Physiol. 2007; 69: 483-510Crossref PubMed Scopus (1194) Google Scholar). In the classical G protein–dependent pathway, agonist-bound GPCRs activate heterotrimeric guanine nucleotide-binding proteins (G proteins), leading to cellular response through a range of second messengers and other effectors (6Oldham W.M. Hamm H.E. Heterotrimeric G protein activation by G-protein-coupled receptors.Nat. Rev. Mol. Cell Biol. 2008; 9: 60-71Crossref PubMed Scopus (856) Google Scholar, 7Gilman A.G. G proteins: Transducers of receptor-generated signals.Annu. Rev. Biochem. 1987; 56: 615-649Crossref PubMed Scopus (5333) Google Scholar). The C-tail of GPCRs is phosphorylated by GPCR kinases (8Pitcher J.A. Freedman N.J. Lefkowitz R.J. G protein-coupled receptor kinases.Annu. Rev. Biochem. 1998; 67: 653-692Crossref PubMed Scopus (1082) Google Scholar). βarrs then bind to the phosphorylated receptor tail and engage with the receptor core, sterically block G protein coupling, and mediate the internalization of GPCRs (9Benovic J.L. Kuhn H. Weyand I. Codina J. Caron M.G. Lefkowitz R.J. Functional desensitization of the isolated beta-adrenergic receptor by the beta-adrenergic receptor kinase: Potential role of an analog of the retinal protein arrestin (48-kDa protein).Proc. Natl. Acad. Sci. U. S. A. 1987; 84: 8879-8882Crossref PubMed Scopus (409) Google Scholar, 10Lohse M.J. Benovic J.L. Codina J. Caron M.G. Lefkowitz R.J. beta-Arrestin: a protein that regulates beta-adrenergic receptor function.Science. 1990; 248: 1547-1550Crossref PubMed Scopus (931) Google Scholar). Furthermore, βarrs coupling to the activated GPCR initiates alternative signaling pathways independent of or in concert with G proteins (5DeWire S.M. Ahn S. Lefkowitz R.J. Shenoy S.K. Beta-arrestins and cell signaling.Annu. Rev. Physiol. 2007; 69: 483-510Crossref PubMed Scopus (1194) Google Scholar, 11Thomsen A.R.B. Plouffe B. Cahill 3rd, T.J. Shukla A.K. Tarrasch J.T. Dosey A.M. Kahsai A.W. Strachan R.T. Pani B. Mahoney J.P. Huang L. Breton B. Heydenreich F.M. Sunahara R.K. Skiniotis G. et al.GPCR-G protein-beta-arrestin super-complex mediates sustained G protein signaling.Cell. 2016; 166: 907-919Abstract Full Text Full Text PDF PubMed Scopus (374) Google Scholar). βarrs thus serve as adaptors and scaffold proteins that link GPCR to numerous signaling molecules, such as components of mitogen-activated protein kinase (MAPK) cascades and nonreceptor tyrosine kinase Src, and regulate various cellular functions, such as chemotaxis and apoptosis (12Luttrell L.M. Ferguson S.S. Daaka Y. Miller W.E. Maudsley S. Della Rocca G.J. Lin F. Kawakatsu H. Owada K. Luttrell D.K. Caron M.G. Lefkowitz R.J. Beta-arrestin-dependent formation of beta2 adrenergic receptor-Src protein kinase complexes.Science. 1999; 283: 655-661Crossref PubMed Scopus (1287) Google Scholar, 13Luttrell L.M. Roudabush F.L. Choy E.W. Miller W.E. Field M.E. Pierce K.L. Lefkowitz R.J. Activation and targeting of extracellular signal-regulated kinases by beta-arrestin scaffolds.Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 2449-2454Crossref PubMed Scopus (712) Google Scholar, 14DeFea K.A. Zalevsky J. Thoma M.S. Dery O. Mullins R.D. Bunnett N.W. beta-arrestin-dependent endocytosis of proteinase-activated receptor 2 is required for intracellular targeting of activated ERK1/2.J. Cell Biol. 2000; 148: 1267-1281Crossref PubMed Scopus (697) Google Scholar, 15Peterson Y.K. Luttrell L.M. The diverse roles of arrestin scaffolds in G protein-coupled receptor signaling.Pharmacol. Rev. 2017; 69: 256-297Crossref PubMed Scopus (287) Google Scholar). MAPK cascades play a critical role in transmitting environmental signals into cellular responses (16Davis R.J. The mitogen-activated protein kinase signal transduction pathway.J. Biol. Chem. 1993; 268: 14553-14556Abstract Full Text PDF PubMed Google Scholar). A typical MAPK cascade consists of three types of kinases: an MAP kinase kinase kinase, an MAP kinase kinase, and an MAPK (17Plotnikov A. Zehorai E. Procaccia S. Seger R. The MAPK cascades: Signaling components, nuclear roles and mechanisms of nuclear translocation.Biochim. Biophys. Acta. 2011; 1813: 1619-1633Crossref PubMed Scopus (668) Google Scholar). In the typical Raf–MAPK extracellular signal–regulated kinase (MEK)–extracellular signal-regulated kinase (ERK) module, activated C-Raf (MAP kinase kinase kinase) phosphorylates downstream MEK1 (also known as MAP kinase kinase), which then phosphorylates the ERK (MAPK). ERK then phosphorylates numerous effector proteins and regulates diverse physiological processes, such as cell growth, proliferation, and differentiation (18Seger R. Krebs E.G. The MAPK signaling cascade.FASEB J. 1995; 9: 726-735Crossref PubMed Scopus (3254) Google Scholar). C-Raf is the most upstream kinase in the cascade, and its activation is crucial for MAPK signaling. C-Raf consists of three conserved regions (CRs) (Fig. 1A). CR1, on the amino terminus, contains a Ras-binding domain (RBD) and a cysteine-rich domain (CRD); CR2 corresponds to a region rich in serine/threonine residues, whereas CR3 includes the catalytic domain on the carboxyl terminus (19Lavoie H. Therrien M. Regulation of RAF protein kinases in ERK signalling.Nat. Rev. Mol. Cell Biol. 2015; 16: 281-298Crossref PubMed Scopus (426) Google Scholar). The activation of C-Raf is a complex process that involves Ras binding, regulatory phosphorylation, and protein–lipid interactions (19Lavoie H. Therrien M. Regulation of RAF protein kinases in ERK signalling.Nat. Rev. Mol. Cell Biol. 2015; 16: 281-298Crossref PubMed Scopus (426) Google Scholar, 20Terrell E.M. Morrison D.K. Ras-mediated activation of the Raf family kinases.Cold Spring Harb. Perspect. Med. 2019; 9a033746Crossref PubMed Scopus (97) Google Scholar). GPCR–βarr complexes interact with C-Raf to initiate MAPK cascade signaling (13Luttrell L.M. Roudabush F.L. Choy E.W. Miller W.E. Field M.E. Pierce K.L. Lefkowitz R.J. Activation and targeting of extracellular signal-regulated kinases by beta-arrestin scaffolds.Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 2449-2454Crossref PubMed Scopus (712) Google Scholar, 14DeFea K.A. Zalevsky J. Thoma M.S. Dery O. Mullins R.D. Bunnett N.W. beta-arrestin-dependent endocytosis of proteinase-activated receptor 2 is required for intracellular targeting of activated ERK1/2.J. Cell Biol. 2000; 148: 1267-1281Crossref PubMed Scopus (697) Google Scholar). Both the N and C domains of βarrs have been known to interact with C-Raf (21Song X. Coffa S. Fu H. Gurevich V.V. How does arrestin assemble MAPKs into a signaling complex?.J. Biol. Chem. 2009; 284: 685-695Abstract Full Text Full Text PDF PubMed Scopus (139) Google Scholar). However, the βarr-binding site in C-Raf has not been determined. βarrs have been demonstrated to act like typical scaffold proteins that bring together the components of MAPK cascade to facilitate signal transduction (13Luttrell L.M. Roudabush F.L. Choy E.W. Miller W.E. Field M.E. Pierce K.L. Lefkowitz R.J. Activation and targeting of extracellular signal-regulated kinases by beta-arrestin scaffolds.Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 2449-2454Crossref PubMed Scopus (712) Google Scholar, 14DeFea K.A. Zalevsky J. Thoma M.S. Dery O. Mullins R.D. Bunnett N.W. beta-arrestin-dependent endocytosis of proteinase-activated receptor 2 is required for intracellular targeting of activated ERK1/2.J. Cell Biol. 2000; 148: 1267-1281Crossref PubMed Scopus (697) Google Scholar). Surprisingly, recent studies showed that in addition to scaffolding functions, GPCR–βarr complexes may serve as allosteric modulators for downstream signaling partners such as kinase Src (22Pakharukova N. Masoudi A. Pani B. Staus D.P. Lefkowitz R.J. Allosteric activation of proto-oncogene kinase Src by GPCR-beta-arrestin complexes.J. Biol. Chem. 2020; 295: 16773-16784Abstract Full Text Full Text PDF PubMed Scopus (16) Google Scholar, 23Yang F. Xiao P. Qu C.X. Liu Q. Wang L.Y. Liu Z.X. He Q.T. Liu C. Xu J.Y. Li R.R. Li M.J. Li Q. Guo X.Z. Yang Z.Y. He D.F. et al.Allosteric mechanisms underlie GPCR signaling to SH3-domain proteins through arrestin.Nat. Chem. Biol. 2018; 14: 876-886Crossref PubMed Scopus (44) Google Scholar). However, it is unclear whether βarrs can also allosterically activate C-Raf. Here, we show that βarr1, activated via either phosphorylated GPCR or the GPCR surrogate V2Rpp (a vasopressin receptor 2 phosphorylated C-terminal peptide with eight phosphates), binds C-Raf and allosterically activates it. Therefore, GPCR–βarr complexes not only function just as typical scaffold proteins for the C-Raf/MEK/ERK module but also play an active role in regulating MAPK signaling. To identify the βarr1-binding elements within C-Raf, we performed glutathione-S-transferase (GST) pull-down assays using GST-C-Raf fusion proteins and βarr1 (Fig. 1A). βarr1 with the GST-C-Raf amino terminus, and βarr1 was for GST-C-Raf carboxyl terminus (Fig. show that does not interact with βarr1 βarr1 and C-Raf amino terminus interact Both and active βarr1 bind C-Raf amino the binding was in the presence of V2Rpp and the The amino terminus of C-Raf the and To the roles of domains in the and contains the and as proteins to the with βarr1, binds βarr1 (Fig. that the domain of C-Raf directly interacts with In the presence of the domain for and of the thus to the binding of Interestingly, H-Ras, a small GTPase known to activate C-Raf, also interacts with the same domains of C-Raf S. S. Raf domains mediate interaction with Biol. Chem. 1995; Full Text Full Text PDF PubMed Scopus Google Scholar). To whether binding for H-Ras and βarr1 have we an H-Ras with (a analog of can with βarr1 to bind an excess of activated H-Ras, the interaction between and βarr1 was reduced (Fig. with binding of βarr1 and H-Ras to the same region of C-Raf. Therefore, these data suggest that βarr1 binds to the amino terminus of C-Raf as GPCR–βarr complexes have been demonstrated to allosterically activate signaling partners such as the tyrosine kinase Src (22Pakharukova N. Masoudi A. Pani B. Staus D.P. Lefkowitz R.J. Allosteric activation of proto-oncogene kinase Src by GPCR-beta-arrestin complexes.J. Biol. Chem. 2020; 295: 16773-16784Abstract Full Text Full Text PDF PubMed Scopus (16) Google Scholar, 23Yang F. Xiao P. Qu C.X. Liu Q. Wang L.Y. Liu Z.X. He Q.T. Liu C. Xu J.Y. Li R.R. Li M.J. Li Q. Guo X.Z. Yang Z.Y. He D.F. et al.Allosteric mechanisms underlie GPCR signaling to SH3-domain proteins through arrestin.Nat. Chem. Biol. 2018; 14: 876-886Crossref PubMed Scopus (44) Google Scholar). To whether GPCR–βarr complexes allosterically regulate C-Raf we an to the C-Raf in (Fig. To the scaffolding function of βarr1, we the of protein of the physiological by C-Raf. We a receptor with a phosphorylated C-tail of the vasopressin 2 receptor that binding to was in membrane scaffold protein as a and it a complex on the D.P. H. M.J. L.M. Lefkowitz R.J. Skiniotis G. Structure of the complex in a 2020; PubMed Scopus Google Scholar). GPCR–βarr complexes such as the are by a synthetic The C-Raf catalytic in the presence of the as shown in (Fig. and as (Fig. of C-Raf The and or to the C-Raf To whether the complex activates C-Raf, we a in kinase using to the of by C-Raf in the or the presence of We found that the complex the C-Raf in with (Fig. Therefore, reveal that the complex allosterically activates kinase C-Raf. we an excess of the C-Raf amino terminus to its to the allosteric activation of C-Raf by The allosteric activation of C-Raf is by excess C-Raf amino terminus (Fig. reveal that the complex allosterically activates C-Raf by with its amino terminus. data also demonstrate that βarr1 binds to the amino terminus of C-Raf. studies that GPCR–βarr complexes in the βarrs bind to the phosphorylated C terminus and the of in the βarrs bind only to the phosphorylated GPCR C terminus A.W. Pani B. Lefkowitz R.J. GPCR activation of 2018; PubMed Scopus Google Scholar). studies show that the of βarr1 interacts with the and as a membrane (Fig. D.P. H. M.J. L.M. Lefkowitz R.J. Skiniotis G. Structure of the complex in a 2020; PubMed Scopus Google Scholar, B. J. M.E. of arrestin function as a membrane 2017; PubMed Scopus Google Scholar). the βarr1 which is required for interactions with the receptor core, we a complex in the 3rd, T.J. Tarrasch J.T. Plouffe B. Yang F. Huang L.Y. Kahsai A.W. S. Shukla A.K. B. et of GPCR-beta-arrestin complexes mediate and Natl. Acad. Sci. U. S. A. 2017; PubMed Scopus Google Scholar). in the of βarr1, we a complex that interactions with the (Fig. Both and complexes showed to C-Raf with the complex (Fig. suggest that the interactions between the GPCR and βarr1 between the of βarr1 and the are necessary for the allosteric activation of C-Raf by suggest that the binding of the phosphorylated GPCR C terminus to βarr1 a critical role in the allosteric activation of C-Raf. To V2Rpp was to activate βarr1 in vitro. We found that the active βarr1 allosterically activate C-Raf, whereas βarr1 to also show that V2Rpp was sufficient to activate C-Raf, whereas not have a to (Fig. demonstrate that the interaction between the phosphorylated GPCR C terminus and βarr1 is necessary and sufficient for the allosteric activation of C-Raf. H-Ras a critical role in the activation process of C-Raf (19Lavoie H. Therrien M. Regulation of RAF protein kinases in ERK signalling.Nat. Rev. Mol. Cell Biol. 2015; 16: 281-298Crossref PubMed Scopus (426) Google Scholar, 20Terrell E.M. Morrison D.K. Ras-mediated activation of the Raf family kinases.Cold Spring Harb. Perspect. Med. 2019; 9a033746Crossref PubMed Scopus (97) Google Scholar). 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R. J. L. data Y. and R. J. L. N. Y. A. and R. J. L. and Y. R. J. L. R. J. L. was by the of R. J. R. J. L. is a The is the of the and does not the of the of

Unique Positive Cooperativity Between the β-Arrestin–Biased β-Blocker Carvedilol and a Small Molecule Positive Allosteric Modulator of the β2-Adrenergic Receptor

Among b-blockers that are clinically prescribed for heart failure, carvedilol is a first-choice agent with unique pharmacological properties. Carvedilol is distinct from other b-blockers in its ability to elicit b-arrestin-biased agonism, which has been suggested to underlie its cardioprotective effects. Augmenting the pharmacologic properties of carvedilol thus holds the promise of developing more efficacious and/or biased b-blockers. We recently identified compound-6 (cmpd-6), the first small molecule positive allosteric modulator of the b2-adrenergic receptor (b2AR). Cmpd-6 is positively cooperative with orthosteric agonists at the b2AR and enhances agonistmediated transducer (G-protein and b-arrestin) signaling in an unbiased manner. Here, we report that cmpd-6, quite unexpectedly, displays strong positive cooperativity only with carvedilol among a panel of structurally diverse b-blockers. Cmpd-6 enhances the binding affinity of carvedilol for the b2AR and augments its ability to competitively antagonize agonist-induced cAMP generation. Cmpd-6 potentiates b-arrestin1but not Gs-protein-mediated high-affinity binding of carvedilol at the b2AR and b-arrestin-mediated cellular functions in response to carvedilol including extracellular signal-regulated kinase phos-phorylation, receptor endocytosis, and trafficking into lysosomes.