Phosphorylation patterns in the AT1R C-terminal tail specify distinct downstream signaling pathways.

Different ligands stabilize specific conformations of the angiotensin II type 1 receptor (AT1R) that direct distinct signaling cascades mediated by heterotrimeric G proteins or β-arrestin. These different active conformations are thought to engage distinct intracellular transducers because of differential phosphorylation patterns in the receptor C-terminal tail (the "barcode" hypothesis). Here, we identified the AT1R barcodes for the endogenous agonist AngII, which stimulates both G protein activation and β-arrestin recruitment, and for a synthetic biased agonist that only stimulates β-arrestin recruitment.

Nanobody-Mediated Dualsteric Engagement of the Angiotensin Receptor Broadens Biased Ligand Pharmacology.

Dualsteric G protein-coupled receptor (GPCR) ligands are a class of bitopic ligands that consist of an orthosteric pharmacophore, which binds to the pocket occupied by the receptor's endogenous agonist, and an allosteric pharmacophore, which binds to a distinct site. These ligands have the potential to display characteristics of both orthosteric and allosteric ligands. To explore the signaling profiles that dualsteric ligands of the angiotensin II type 1 receptor (AT1R) can access, we ligated a 6e epitope tag-specific nanobody (single-domain antibody fragment) to angiotensin II (AngII) and analogs that show preferential allosteric coupling to Gq (TRV055, TRV056) or -arrestin (TRV027).

Antibodies Expand the Scope of Angiotensin Receptor Pharmacology.

G protein-coupled receptors (GPCRs) are key regulators of human physiology and are the targets of many small molecule research compounds and therapeutic drugs. While most of these ligands bind to their target GPCR with high affinity, selectivity is often limited at the receptor, tissue, and cellular level. Antibodies have the potential to address these limitations but their properties as GPCR ligands remain poorly characterized.

Nanobodies as Probes and Modulators of Cardiovascular G Protein-Coupled Receptors.

Understanding the activation of G protein-coupled receptors (GPCRs) is of paramount importance to the field of cardiovascular medicine due to the critical physiological roles of these receptors and their prominence as drug targets. Although many cardiovascular GPCRs have been extensively studied as model receptors for decades, new complexities in their regulation continue to emerge. As a result, there is an ongoing need to develop novel approaches to monitor and to modulate GPCR activation.

Rapid generation of potent antibodies by autonomous hypermutation in yeast.

The predominant approach for antibody generation remains animal immunization, which can yield exceptionally selective and potent antibody clones owing to the powerful evolutionary process of somatic hypermutation. However, animal immunization is inherently slow, has poor compatibility with certain antigens ( . .

Synthetic nanobodies as angiotensin receptor blockers.

There is considerable interest in developing antibodies as functional modulators of G protein-coupled receptor (GPCR) signaling for both therapeutic and research applications. However, there are few antibody ligands targeting GPCRs outside of the chemokine receptor group. GPCRs are challenging targets for conventional antibody discovery methods, as many are highly conserved across species, are biochemically unstable upon purification, and possess deeply buried ligand-binding sites.

Conformational Basis of G Protein-Coupled Receptor Signaling Versatility.

G protein-coupled receptors (GPCRs) are privileged structural scaffolds in biology that have the versatility to regulate diverse physiological processes. Interestingly, many GPCR ligands exhibit significant 'bias' - the ability to preferentially activate subsets of the many cellular pathways downstream of these receptors. Recently, complementary information from structural and spectroscopic approaches has made significant inroads into understanding the mechanisms of these biased ligands.

Angiotensin and biased analogs induce structurally distinct active conformations within a GPCR.

Biased agonists of G protein-coupled receptors (GPCRs) preferentially activate a subset of downstream signaling pathways. In this work, we present crystal structures of angiotensin II type 1 receptor (AT1R) (2.7 to 2.

Molecular mechanism of biased signaling in a prototypical G protein-coupled receptor.

Biased signaling, in which different ligands that bind to the same G protein-coupled receptor preferentially trigger distinct signaling pathways, holds great promise for the design of safer and more effective drugs. Its structural mechanism remains unclear, however, hampering efforts to design drugs with desired signaling profiles. Here, we use extensive atomic-level molecular dynamics simulations to determine how arrestin bias and G protein bias arise at the angiotensin II type 1 receptor.

Structure of the M2 muscarinic receptor-β-arrestin complex in a lipid nanodisc.

After activation by an agonist, G-protein-coupled receptors (GPCRs) recruit β-arrestin, which desensitizes heterotrimeric G-protein signalling and promotes receptor endocytosis. Additionally, β-arrestin directly regulates many cell signalling pathways that can induce cellular responses distinct from that of G proteins. In contrast to G proteins, for which there are many high-resolution structures in complex with GPCRs, the molecular mechanisms underlying the interaction of β-arrestin with GPCRs are much less understood.

Detergent- and phospholipid-based reconstitution systems have differential effects on constitutive activity of G-protein-coupled receptors.

A hallmark of G-protein-coupled receptors (GPCRs) is the conversion of external stimuli into specific cellular responses. In this tightly-regulated process, extracellular ligand binding by GPCRs promotes specific conformational changes within the seven transmembrane helices, leading to the coupling and activation of intracellular "transducer" proteins, such as heterotrimeric G proteins. Much of our understanding of the molecular mechanisms that govern GPCR activation is derived from experiments with purified receptors reconstituted in detergent micelles.

Distinctive Activation Mechanism for Angiotensin Receptor Revealed by a Synthetic Nanobody.

The angiotensin II (AngII) type 1 receptor (AT1R) is a critical regulator of cardiovascular and renal function and is an important model for studies of G-protein-coupled receptor (GPCR) signaling. By stabilizing the receptor with a single-domain antibody fragment ("nanobody") discovered using a synthetic yeast-displayed library, we determined the crystal structure of active-state human AT1R bound to an AngII analog with partial agonist activity. The nanobody binds to the receptor's intracellular transducer pocket, stabilizing the large conformational changes characteristic of activated GPCRs.

Angiotensin Analogs with Divergent Bias Stabilize Distinct Receptor Conformations.

"Biased" G protein-coupled receptor (GPCR) agonists preferentially activate pathways mediated by G proteins or β-arrestins. Here, we use double electron-electron resonance spectroscopy to probe the changes that ligands induce in the conformational distribution of the angiotensin II type I receptor. Monitoring distances between 10 pairs of nitroxide labels distributed across the intracellular regions enabled mapping of four underlying sets of conformations.

G protein-coupled receptor kinases (GRKs) orchestrate biased agonism at the β-adrenergic receptor.

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.

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

Conventional drug discovery efforts at the -adrenoceptor (AR) 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 AR small-molecule positive allosteric modulators (PAMs)-compound (Cmpd)-6 [()--(4-amino-1-(4-(-butyl)phenyl)-4-oxobutan-2-yl)-5-(-isopropyl--methylsulfamoyl)-2-((4-methoxyphenyl)thio)benzamide] and its analogs.

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

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.

Multidimensional Tracking of GPCR Signaling via Peroxidase-Catalyzed Proximity Labeling.

G-protein-coupled receptors (GPCRs) play critical roles in regulating physiological processes ranging from neurotransmission to cardiovascular function. Current methods for tracking GPCR signaling suffer from low throughput, modification or overexpression of effector proteins, and low temporal resolution. Here, we show that peroxidase-catalyzed proximity labeling can be combined with isobaric tagging and mass spectrometry to enable quantitative, time-resolved measurement of GPCR agonist response in living cells.

Allosteric "beta-blocker" isolated from a DNA-encoded small molecule library.

The β-adrenergic receptor (βAR) has been a model system for understanding regulatory mechanisms of G-protein-coupled receptor (GPCR) actions and plays a significant role in cardiovascular and pulmonary diseases. Because all known β-adrenergic receptor drugs target the orthosteric binding site of the receptor, we set out to isolate allosteric ligands for this receptor by panning DNA-encoded small-molecule libraries comprising 190 million distinct compounds against purified human βAR. Here, we report the discovery of a small-molecule negative allosteric modulator (antagonist), compound 15 [([4-((2)-3-((()-3-(3-bromophenyl)-1-(methylamino)-1-oxopropan-2-yl)amino)-2-(2-cyclohexyl-2-phenylacetamido)-3-oxopropyl)benzamide], exhibiting a unique chemotype and low micromolar affinity for the βAR.

Allosteric nanobodies reveal the dynamic range and diverse mechanisms of G-protein-coupled receptor activation.

G-protein-coupled receptors (GPCRs) modulate many physiological processes by transducing a variety of extracellular cues into intracellular responses. Ligand binding to an extracellular orthosteric pocket propagates conformational change to the receptor cytosolic region to promote binding and activation of downstream signalling effectors such as G proteins and β-arrestins. It is well known that different agonists can share the same binding pocket but evoke unique receptor conformations leading to a wide range of downstream responses (‘efficacy’).

Conformationally selective RNA aptamers allosterically modulate the β2-adrenoceptor.

G-protein-coupled receptor (GPCR) ligands function by stabilizing multiple, functionally distinct receptor conformations. This property underlies the ability of 'biased agonists' to activate specific subsets of a given receptor's signaling profile. However, stabilizing distinct active GPCR conformations to enable structural characterization of mechanisms underlying GPCR activation remains difficult.