Structural Basis of the Activation of Heterotrimeric Gs-Protein by Isoproterenol-Bound ß1-Adrenergic Receptor.

Cardiac disease remains the leading cause of morbidity and mortality worldwide. The ß1-adrenergic receptor (ß1-AR) is a major regulator of cardiac functions and is downregulated in the majority of heart failure cases. A key physiological process is the activation of heterotrimeric G-protein Gs by ß1-ARs, leading to increased heart rate and contractility. Here, we use cryo-electron microscopy and functional studies to investigate the molecular mechanism by which ß1-AR activates Gs. We find that the tilting of α5-helix breaks a hydrogen bond between the sidechain of His373 in the C-terminal α5-helix and the backbone carbonyl of Arg38 in the N-terminal αN-helix of Gαs. Together with the disruption of another interacting network involving Gln59 in the α1-helix, Ala352 in the ß6-α5 loop, and Thr355 in the α5-helix, these conformational changes might lead to the deformation of the GDP-binding pocket. Our data provide molecular insights into the activation of G-proteins by G-protein-coupled receptors.

Molecular basis of ß-arrestin coupling to formoterol-bound ß1-adrenoceptor.

The ß1-adrenoceptor (ß1AR) is a G-protein-coupled receptor (GPCR) that couples1 to the heterotrimeric G protein Gs. G-protein-mediated signalling is terminated by phosphorylation of the C terminus of the receptor by GPCR kinases (GRKs) and by coupling of ß-arrestin 1 (ßarr1, also known as arrestin 2), which displaces Gs and induces signalling through the MAP kinase pathway2. The ability of synthetic agonists to induce signalling preferentially through either G proteins or arrestins-known as biased agonism3-is important in drug development, because the therapeutic effect may arise from only one signalling cascade, whereas the other pathway may mediate undesirable side effects4. To understand the molecular basis for arrestin coupling, here we determined the cryo-electron microscopy structure of the ß1AR-ßarr1 complex in lipid nanodiscs bound to the biased agonist formoterol5, and the crystal structure of formoterol-bound ß1AR coupled to the G-protein-mimetic nanobody6 Nb80. ßarr1 couples to ß1AR in a manner distinct to that7 of Gs coupling to ß2AR-the finger loop of ßarr1 occupies a narrower cleft on the intracellular surface, and is closer to transmembrane helix H7 of the receptor when compared with the C-terminal α5 helix of Gs. The conformation of the finger loop in ßarr1 is different from that adopted by the finger loop of visual arrestin when it couples to rhodopsin8. ß1AR coupled to ßarr1 shows considerable differences in structure compared with ß1AR coupled to Nb80, including an inward movement of extracellular loop 3 and the cytoplasmic ends of H5 and H6. We observe weakened interactions between formoterol and two serine residues in H5 at the orthosteric binding site of ß1AR, and find that formoterol has a lower affinity for the ß1AR-ßarr1 complex than for the ß1AR-Gs complex. The structural differences between these complexes of ß1AR provide a foundation for the design of small molecules that could bias signalling in the ß-adrenoceptors.

PtdIns(4,5)P2 stabilizes active states of GPCRs and enhances selectivity of G-protein coupling.

G-protein-coupled receptors (GPCRs) are involved in many physiological processes and are therefore key drug targets1. Although detailed structural information is available for GPCRs, the effects of lipids on the receptors, and on downstream coupling of GPCRs to G proteins are largely unknown. Here we use native mass spectrometry to identify endogenous lipids bound to three class A GPCRs. We observed preferential binding of phosphatidylinositol-4,5-bisphosphate (PtdIns(4,5)P2) over related lipids and confirm that the intracellular surface of the receptors contain hotspots for PtdIns(4,5)P2 binding. Endogenous lipids were also observed bound directly to the trimeric Gαsßγ protein complex of the adenosine A2A receptor (A2AR) in the gas phase. Using engineered Gα subunits (mini-Gαs, mini-Gαi and mini-Gα12)2, we demonstrate that the complex of mini-Gαs with the ß1 adrenergic receptor (ß1AR) is stabilized by the binding of two PtdIns(4,5)P2 molecules. By contrast, PtdIns(4,5)P2 does not stabilize coupling between ß1AR and other Gα subunits (mini-Gαi or mini-Gα12) or a high-affinity nanobody. Other endogenous lipids that bind to these receptors have no effect on coupling, highlighting the specificity of PtdIns(4,5)P2. Calculations of potential of mean force and increased GTP turnover by the activated neurotensin receptor when coupled to trimeric Gαißγ complex in the presence of PtdIns(4,5)P2 provide further evidence for a specific effect of PtdIns(4,5)P2 on coupling. We identify key residues on cognate Gα subunits through which PtdIns(4,5)P2 forms bridging interactions with basic residues on class A GPCRs. These modulating effects of lipids on receptors suggest consequences for understanding function, G-protein selectivity and drug targeting of class A GPCRs.

The Isoleucine at Position 118 in Transmembrane 2 Is Responsible for the Selectivity of Xamoterol, Nebivolol, and ICI89406 for the Human ß1-Adrenoceptor.

Known off-target interactions frequently cause predictable drug side-effects (e.g., ß1-antagonists used for heart disease, risk ß2-mediated bronchospasm). Computer-aided drug design would improve if the structural basis of existing drug selectivity was understood. A mutagenesis approach determined the ligand-amino acid interactions required for ß1-selective affinity of xamoterol and nebivolol, followed by computer-based modeling to provide possible structural explanations. 3H-CGP12177 whole cell binding was conducted in Chinese hamster ovary cells stably expressing human ß1, ß2, and chimeric ß1/ß2-adrenoceptors (ARs). Single point mutations were investigated in transiently transfected cells. Modeling studies involved docking ligands into three-dimensional receptor structures and performing molecular dynamics simulations, comparing interaction frequencies between apo and holo structures of ß1 and ß2-ARs. From these observations, an ICI89406 derivative was investigated that gave further insights into selectivity. Stable cell line studies determined that transmembrane 2 was crucial for the ß1-selective affinity of xamoterol and nebivolol. Single point mutations determined that the ß1-AR isoleucine (I118) rather than the ß2 histidine (H93) explained selectivity. Studies of other ß1-ligands found I118 was important for ICI89406 selective affinity but not that for betaxolol, bisoprolol, or esmolol. Modeling studies suggested that the interaction energies and solvation of ß1-I118 and ß2-H93 are factors determining selectivity of xamoterol and ICI89406. ICI89406 without its phenyl group loses its high ß1-AR affinity, resulting in the same affinity as for the ß2-AR. The human ß1-AR residue I118 is crucial for the ß1-selective affinity of xamoterol, nebivolol, and ICI89406 but not all ß1-selective compounds. SIGNIFICANCE STATEMENT: Some ligands have selective binding affinity for the human ß1 versus the ß2-adrenoceptor; however, the molecular/structural reason for this is not known. The transmembrane 2 residue isoleucine I118 is responsible for the selective ß1-binding of xamoterol, nebivolol, and ICI89406 but does not explain the selective ß1-binding of betaxolol, bisoprolol, or esmolol. Understanding the structural basis of selectivity is important to improve computer-aided ligand design, and targeting I118 in ß1-adrenoceptors is likely to increase ß1-selectivity of drugs.

Backbone NMR reveals allosteric signal transduction networks in the ß1-adrenergic receptor.

G protein-coupled receptors (GPCRs) are physiologically important transmembrane signalling proteins that trigger intracellular responses upon binding of extracellular ligands. Despite recent breakthroughs in GPCR crystallography, the details of ligand-induced signal transduction are not well understood owing to missing dynamical information. In principle, such information can be provided by NMR, but so far only limited data of functional relevance on few side-chain sites of eukaryotic GPCRs have been obtained. Here we show that receptor motions can be followed at virtually any backbone site in a thermostabilized mutant of the turkey ß1-adrenergic receptor (ß1AR). Labelling with [(15)N]valine in a eukaryotic expression system provides over twenty resolved resonances that report on structure and dynamics in six ligand complexes and the apo form. The response to the various ligands is heterogeneous in the vicinity of the binding pocket, but gets transformed into a homogeneous readout at the intracellular side of helix 5 (TM5), which correlates linearly with ligand efficacy for the G protein pathway. The effect of several pertinent, thermostabilizing point mutations was assessed by reverting them to the native sequence. Whereas the response to ligands remains largely unchanged, binding of the G protein mimetic nanobody NB80 and G protein activation are only observed when two conserved tyrosines (Y227 and Y343) are restored. Binding of NB80 leads to very strong spectral changes throughout the receptor, including the extracellular ligand entrance pocket. This indicates that even the fully thermostabilized receptor undergoes activating motions in TM5, but that the fully active state is only reached in presence of Y227 and Y343 by stabilization with a G protein-like partner. The combined analysis of chemical shift changes from the point mutations and ligand responses identifies crucial connections in the allosteric activation pathway, and presents a general experimental method to delineate signal transmission networks at high resolution in GPCRs.

ß1-adrenergic receptor N-terminal cleavage by ADAM17; the mechanism for redox-dependent downregulation of cardiomyocyte ß1-adrenergic receptors.

ß1-adrenergic receptors (ß1ARs) are the principle mediators of catecholamine action in cardiomyocytes. We previously showed that the ß1AR extracellular N-terminus is a target for post-translational modifications that impact on signaling responses. Specifically, we showed that the ß1AR N-terminus carries O-glycan modifications at Ser37/Ser41, that O-glycosylation prevents ß1AR N-terminal cleavage, and that N-terminal truncation influences ß1AR signaling to downstream effectors. However, the site(s) and mechanism for ß1AR N-terminal cleavage in cells was not identified. This study shows that ß1ARs are expressed in cardiomyocytes and other cells types as both full-length and N-terminally truncated species and that the truncated ß1AR species is formed as a result of an O-glycan regulated N-terminal cleavage by ADAM17 at R31↓L32. We identify Ser41 as the major O-glycosylation site on the ß1AR N-terminus and show that an O-glycan modification at Ser41 prevents ADAM17-dependent cleavage of the ß1-AR N-terminus at S41↓L42, a second N-terminal cleavage site adjacent to this O-glycan modification (and it attenuates ß1-AR N-terminal cleavage at R31↓L32). We previously reported that oxidative stress leads to a decrease in ß1AR expression and catecholamine responsiveness in cardiomyocytes. This study shows that redox-inactivation of cardiomyocyte ß1ARs is via a mechanism involving N-terminal truncation at R31↓L32 by ADAM17. In keeping with the previous observation that N-terminally truncated ß1ARs constitutively activate an AKT pathway that affords protection against doxorubicin-dependent apoptosis, overexpression of a cleavage resistant ß1AR mutant exacerbates doxorubicin-dependent apoptosis. These studies identify the ß1AR N-terminus as a structural determinant of ß1AR responses that can be targeted for therapeutic advantage.

The role of ß-adrenergic system remodeling in human heart failure: A mechanistic investigation.

ß-adrenergic receptor antagonists (ß-blockers) are extensively used to improve cardiac performance in heart failure (HF), but the electrical improvements with these clinical treatments are not fully understood. The aim of this study was to analyze the electrophysiological effects of ß-adrenergic system remodeling in heart failure with reduced ejection fraction and the underlying mechanisms. We used a combined mathematical model that integrated ß-adrenergic signaling with electrophysiology and calcium cycling in human ventricular myocytes. HF remodeling, both in the electrophysiological and signaling systems, was introduced to quantitatively analyze changes in electrophysiological properties due to the stimulation of ß-adrenergic receptors in failing myocytes. We found that the inotropic effect of ß-adrenergic stimulation was reduced in HF due to the altered Ca2+ dynamics resulting from the combination of structural, electrophysiological and signaling remodeling. Isolated cells showed proarrhythmic risk after sympathetic stimulation because early afterdepolarizations appeared, and the vulnerability was greater in failing myocytes. When analyzing coupled cells, ß-adrenergic stimulation reduced transmural repolarization gradients between endocardium and epicardium in normal tissue, but was less effective at reducing these gradients after HF remodeling. The comparison of the selective activation of ß-adrenergic isoforms revealed that the response to ß2-adrenergic receptors stimulation was blunted in HF while ß1-adrenergic receptors downstream effectors regulated most of the changes observed after sympathetic stimulation. In conclusion, this study was able to reproduce an altered ß-adrenergic activity on failing myocytes and to explain the mechanisms involved. The derived predictions could help in the treatment of HF and guide in the design of future experiments.

ß2-Adrenergic Stimulation Compartmentalizes ß1 Signaling Into Nanoscale Local Domains by Targeting the C-Terminus of ß1-Adrenoceptors.

RATIONALE: ßARs (ß-adrenergic receptors) are prototypical GPCRs (G protein-coupled receptors) that play a pivotal role in sympathetic regulation. In heart cells, ß1AR signaling mediates a global response, including both l-type Ca2+ channels in the sarcolemma/T tubules and RyRs (ryanodine receptors) in the SR (sarcoplasmic reticulum). In contrast, ß2AR mediates local signaling with little effect on the function of SR proteins. OBJECTIVE: To investigate the signaling relationship between ß1ARs and ß2ARs. METHOD AND RESULTS: Using whole-cell patch-clamp analyses combined with confocal Ca2+ imaging, we found that the activation of compartmentalized ß2AR signaling was able to convert the ß1AR signaling from global to local mode, preventing ß1ARs from phosphorylating RyRs that were only nanometers away from sarcolemma/T tubules. This offside compartmentalization was eliminated by selective inhibition of ß2AR, GRK2 (GPCR kinase-2), ßarr1 (ß-arrestin-1), and phosphodiesterase-4. A knockin rat model harboring mutations of the last 3 serine residues of the ß1AR C terminus, a component of the putative ßarr1 binding site and GRK2 phosphorylation site, eliminated the offside compartmentalization conferred by ß2AR activation. CONCLUSIONS: ß2AR stimulation compartmentalizes ß1AR signaling into nanoscale local domains in a phosphodiesterase-4-dependent manner by targeting the C terminus of ß1ARs. This finding reveals a fundamental negative feed-forward mechanism that serves to avoid the cytotoxicity of circulating catecholamine and to sharpen the transient ß1AR response of sympathetic excitation.

Screening of ß1- and ß2-Adrenergic Receptor Modulators through Advanced Pharmacoinformatics and Machine Learning Approaches.

Cardiovascular diseases (CDs) are a major concern in the human race and one of the leading causes of death worldwide. ß-Adrenergic receptors (ß1-AR and ß2-AR) play a crucial role in the overall regulation of cardiac function. In the present study, structure-based virtual screening, machine learning (ML), and a ligand-based similarity search were conducted for the PubChem database against both ß1- and ß2-AR. Initially, all docked molecules were screened using the threshold binding energy value. Molecules with a better binding affinity were further used for segregation as active and inactive through ML. The pharmacokinetic assessment was carried out on molecules retained in the above step. Further, similarity searching of the ChEMBL and DrugBank databases was performed. From detailed analysis of the above data, four compounds for each of ß1- and ß2-AR were found to be promising in nature. A number of critical ligand-binding amino acids formed potential hydrogen bonds and hydrophobic interactions. Finally, a molecular dynamics (MD) simulation study of each molecule bound with the respective target was performed. A number of parameters obtained from the MD simulation trajectories were calculated and substantiated the stability between the protein-ligand complex. Hence, it can be postulated that the final molecules might be crucial for CDs subjected to experimental validation.

Autoantibody profiling on a plasmonic nano-gold chip for the early detection of hypertensive heart disease.

The role of autoimmunity in cardiovascular (CV) diseases has been increasingly recognized. Autoimmunity is most commonly examined by the levels of circulating autoantibodies in clinical practices. Measurement of autoantibodies remains, however, challenging because of the deficiency of reproducible, sensitive, and standardized assays. The lack of multiplexed assays also limits the potential to identify a CV-specific autoantibody profile. To overcome these challenges, we developed a nanotechnology-based plasmonic gold chip for autoantibody profiling. This approach allowed simultaneous detection of 10 CV autoantibodies targeting the structural myocardial proteins, the neurohormonal regulatory proteins, the vascular proteins, and the proteins associated with apoptosis and coagulation. Autoantibodies were measured in four groups of participants across the continuum of hypertensive heart diseases. We observed higher levels of all 10 CV autoantibodies in hypertensive subjects (n = 77) compared with healthy participants (n = 30), and the autoantibodies investigated were related to each other, forming a highly linked network. In addition, we established that autoantibodies to troponin I, annexin-A5, and beta 1-adrenegic receptor best discriminated hypertensive subjects with adverse left ventricular (LV) remodeling or dysfunction (n = 49) from hypertensive subjects with normal LV structure and function (n = 28). By further linking these three significant CV autoantibodies to the innate and growth factors, we revealed a positive but weak association between autoantibodies to troponin I and proinflammatory cytokine IL-18. Overall, we demonstrated that this platform can be used to evaluate autoantibody profiles in hypertensive subjects at risk for heart failure.

High Pressure Shifts the ß1-Adrenergic Receptor to the Active Conformation in the Absence of G Protein.

G protein-coupled receptors (GPCRs) are versatile chemical sensors, which transmit the signal of an extracellular binding event across the plasma membrane to the intracellular side. This function is achieved via the modulation of highly dynamical equilibria of various conformational receptor states. Here we have probed the effect of pressure on the conformational equilibria of a functional thermostabilized ß1-adrenergic GPCR (ß1AR) by solution NMR. High pressure induces a large shift in the conformational equilibrium (midpoint ∼600 bar) from the preactive conformation of agonist-bound ß1AR to the fully active conformation, which under normal pressure is only populated when a G protein or a G protein-mimicking nanobody (Nb) binds to the intracellular side of the ß1AR·agonist complex. No such large effects are observed for an antagonist-bound ß1AR or the ternary ß1AR·agonist·Nb80 complex. The detected structural changes of agonist-bound ß1AR around the orthosteric ligand binding pocket indicate that the fully active receptor occupies an ∼100 Å3 smaller volume than that of its preactive form. Most likely, this volume reduction is caused by the compression of empty (nonhydrated) cavities in the ligand binding pocket and the center of the receptor, which increases the ligand receptor interactions and explains the ∼100-fold affinity increase of agonists in the presence of G protein. The finding that isotropic pressure induces a directed motion from the preactive to the fully active GPCR conformation provides evidence of the high mechanical robustness of this important functional switch.

Novel Paradigms Governing ß1-Adrenergic Receptor Trafficking in Primary Adult Rat Cardiac Myocytes.

The ß1-adrenergic receptor (ß1-AR) is a major cardiac G protein-coupled receptor, which mediates cardiac actions of catecholamines and is involved in genesis and treatment of numerous cardiovascular disorders. In mammalian cells, catecholamines induce the internalization of the ß1-AR into endosomes and their removal promotes the recycling of the endosomal ß1-AR back to the plasma membrane; however, whether these redistributive processes occur in terminally differentiated cells is unknown. Compartmentalization of the ß1-AR in response to ß-agonists and antagonists was determined by confocal microscopy in primary adult rat ventricular myocytes (ARVMs), which are terminally differentiated myocytes with unique structures such as transverse tubules (T-tubules) and contractile sarcomeres. In unstimulated ARVMs, the fluorescently labeled ß1-AR was expressed on the external membrane (the sarcolemma) of cardiomyocytes. Exposing ARVMs to isoproterenol redistributed surface ß1-ARs into small (∼225-250 nm) regularly spaced internal punctate structures that overlapped with puncta stained by Di-8 ANEPPS, a membrane-impermeant T-tubule-specific dye. Replacing the ß-agonist with the ß-blocker alprenolol, induced the translocation of the wild-type ß1-AR from these punctate structures back to the plasma membrane. This step was dependent on two barcodes, namely, the type-1 PDZ binding motif and serine at position 312 of the ß1-AR, which is phosphorylated by a pool of cAMP-dependent protein kinases anchored at the type-1 PDZ of the ß1-AR. These data show that redistribution of the ß1-AR in ARVMs from internal structures back to the plasma membrane was mediated by a novel sorting mechanism, which might explain unique aspects of cardiac ß1-AR signaling under normal or pathologic conditions.

Site-specific O-Glycosylation by Polypeptide N-Acetylgalactosaminyltransferase 2 (GalNAc-transferase T2) Co-regulates ß1-Adrenergic Receptor N-terminal Cleavage.

The ß1-adrenergic receptor (ß1AR) is a G protein-coupled receptor (GPCR) and the predominant adrenergic receptor subtype in the heart, where it mediates cardiac contractility and the force of contraction. Although it is the most important target for ß-adrenergic antagonists, such as ß-blockers, relatively little is yet known about its regulation. We have shown previously that ß1AR undergoes constitutive and regulated N-terminal cleavage participating in receptor down-regulation and, moreover, that the receptor is modified by O-glycosylation. Here we demonstrate that the polypeptide GalNAc-transferase 2 (GalNAc-T2) specifically O-glycosylates ß1AR at five residues in the extracellular N terminus, including the Ser-49 residue at the location of the common S49G single-nucleotide polymorphism. Using in vitro O-glycosylation and proteolytic cleavage assays, a cell line deficient in O-glycosylation, GalNAc-T-edited cell line model systems, and a GalNAc-T2 knock-out rat model, we show that GalNAc-T2 co-regulates the metalloproteinase-mediated limited proteolysis of ß1AR. Furthermore, we demonstrate that impaired O-glycosylation and enhanced proteolysis lead to attenuated receptor signaling, because the maximal response elicited by the ßAR agonist isoproterenol and its potency in a cAMP accumulation assay were decreased in HEK293 cells lacking GalNAc-T2. Our findings reveal, for the first time, a GPCR as a target for co-regulatory functions of site-specific O-glycosylation mediated by a unique GalNAc-T isoform. The results provide a new level of ß1AR regulation that may open up possibilities for new therapeutic strategies for cardiovascular diseases.

Co-translational formation and pharmacological characterization of beta1-adrenergic receptor/nanodisc complexes with different lipid environments.

G protein-coupled receptors are of key significance for biomedical research. Streamlined approaches for their efficient recombinant production are of pivotal interest in order to explore their intrinsic conformational dynamics and complex ligand binding behavior. We have systematically optimized the co-translational association and folding of G protein-coupled receptors with defined membranes of nanodiscs by cell-free expression approaches. Each optimization step was quantified and the ligand binding active fraction of the receptor samples could drastically be improved. The strategy was exemplified with a stabilized and a non-stabilized derivative of the turkey beta1-adrenergic receptor. Systematic lipid screens with preformed nanodiscs revealed that generation of ligand binding active conformations of the analyzed beta1-adrenergic receptors strongly depends on lipid charge, flexibility and chain length. The lipid composition of the nanodisc membranes modulates the affinities to a variety of ligands of both receptor derivatives. In addition, the thermostabilization procedure had a significant impact on specific ligand affinities of the receptor and abolished or reduced the binding of certain antagonists. Both receptors were highly stable after purification with optimized nanodisc membranes. The procedure avoids any detergent contact of the receptors and sample production takes less than two days. Moreover, even non-stabilized receptors can be analyzed and their prior purification is not necessary for the formation of nanodisc complexes. The established process appears therefore to be suitable as a new platform for the functional or even structural characterization of recombinant G protein-coupled receptors associated with defined lipid environments.

Beta2-adrenergic receptor homodimers: Role of transmembrane domain 1 and helix 8 in dimerization and cell surface expression.

Even though there are hundreds of reports in the published literature supporting the hypothesis that G protein-coupled receptors (GPCR) form and function as dimers this remains a highly controversial area of research and mechanisms governing homodimer formation are poorly understood. Crystal structures revealing homodimers have been reported for many different GPCR. For adrenergic receptors, a potential dimer interface involving transmembrane domain 1 (TMD1) and helix 8 (H8) was identified in crystal structures of the beta1-adrenergic (ß1-AR) and ß2-AR. The purpose of this study was to investigate a potential role for TMD1 and H8 in dimerization and plasma membrane expression of functional ß2-AR. Charged residues at the base of TMD1 and in the distal portion of H8 were replaced, singly and in combination, with non-polar residues or residues of opposite charge. Wild type and mutant ß2-AR, tagged with YFP and expressed in HEK293 cells, were evaluated for plasma membrane expression and function. Homodimer formation was evaluated using bioluminescence resonance energy transfer, bimolecular fluorescence complementation, and fluorescence correlation spectroscopy. Amino acid substitutions at the base of TMD1 and in the distal portion of H8 disrupted homodimer formation and caused receptors to be retained in the endoplasmic reticulum. Mutations in the proximal region of H8 did not disrupt dimerization but did interfere with plasma membrane expression. This study provides biophysical evidence linking a potential TMD1/H8 interface with ER export and the expression of functional ß2-AR on the plasma membrane. This article is part of a Special Issue entitled: Interactions between membrane receptors in cellular membranes edited by Kalina Hristova.

Identification of phosphorylation sites on ß1-adrenergic receptor in the mouse heart.

ß1-adrenergic receptor (Adrb1) belongs to the superfamily of G-protein-coupled receptors (GPCRs) and plays a critical role in the regulation of heart rate and myocardial contraction force. GPCRs are phosphorylated at multiple sites to regulate distinct signal transduction pathways in different tissues. However, little is known about the location and function of distinct phosphorylation sites of Adrb1 in vivo. To clarify the mechanisms underlying functional regulation associated with Adrb1 phosphorylation in vivo, we aimed to identify Adrb1 phosphorylation sites in the mouse heart using phosphoproteomics techniques with nano-flow liquid chromatography/tandem mass spectrometry (LC-MS/MS). We revealed the phosphorylation residues of Adrb1 to be Ser274 and Ser280 in the third intracellular loop and Ser412, Ser417, Ser450, Ser451, and Ser462 at the C-terminus. We also found that phosphorylation at Ser274, Ser280, and Ser462 was enhanced in response to stimulation with an Adrb1 agonist. This is the first study to identify Adrb1 phosphorylation sites in vivo. These findings will provide novel insights into the regulatory mechanisms mediated by Adrb1 phosphorylation.

The structural basis for agonist and partial agonist action on a ß(1)-adrenergic receptor.

ß-adrenergic receptors (ßARs) are G-protein-coupled receptors (GPCRs) that activate intracellular G proteins upon binding catecholamine agonist ligands such as adrenaline and noradrenaline. Synthetic ligands have been developed that either activate or inhibit ßARs for the treatment of asthma, hypertension or cardiac dysfunction. These ligands are classified as either full agonists, partial agonists or antagonists, depending on whether the cellular response is similar to that of the native ligand, reduced or inhibited, respectively. However, the structural basis for these different ligand efficacies is unknown. Here we present four crystal structures of the thermostabilized turkey (Meleagris gallopavo) ß(1)-adrenergic receptor (ß(1)AR-m23) bound to the full agonists carmoterol and isoprenaline and the partial agonists salbutamol and dobutamine. In each case, agonist binding induces a 1 Å contraction of the catecholamine-binding pocket relative to the antagonist bound receptor. Full agonists can form hydrogen bonds with two conserved serine residues in transmembrane helix 5 (Ser(5.42) and Ser(5.46)), but partial agonists only interact with Ser(5.42) (superscripts refer to Ballesteros-Weinstein numbering). The structures provide an understanding of the pharmacological differences between different ligand classes, illuminating how GPCRs function and providing a solid foundation for the structure-based design of novel ligands with predictable efficacies.

Negative cooperativity across ß1-adrenoceptor homodimers provides insights into the nature of the secondary low-affinity CGP 12177 ß1-adrenoceptor binding conformation.

At the ß1-adrenoceptor, CGP 12177 potently antagonizes agonist responses at the primary high-affinity catecholamine conformation while also exerting agonist effects of its own through a secondary low-affinity conformation. A recent mutagenesis study identified transmembrane region (TM)4 of the ß1-adrenoceptor as key for this low-affinity conformation. Others suggested that TM4 has a role in ß1-adrenoceptor oligomerization. Here, assessment of the dissociation rate of a fluorescent analog of CGP 12177 [bordifluoropyrromethane-tetramethylrhodamine-(±)CGP 12177 (BODIPY-TMR-CGP)] at the human ß1-adrenoceptor expressed in Chinese hamster ovary cells revealed negative cooperative interactions between 2 distinct ß1-adrenoceptor conformations. The dissociation rate of 3 nM BODIPY-TMR-CGP was 0.09 ± 0.01 min(-1) in the absence of competitor ligands, and this was enhanced 2.2- and 2.1-fold in the presence of 1 µM CGP 12177 and 1 µM propranolol, respectively. These effects on the BODIPY-TMR-CGP dissociation rate were markedly enhanced in ß1-adrenoceptor homodimers constrained by bimolecular fluorescence complementation (9.8- and 9.9-fold for 1 µM CGP 12177 and 1 µM propranolol, respectively) and abolished in ß1-adrenoceptors containing TM4 mutations vital for the second conformation pharmacology. This study suggests that negative cooperativity across a ß1-adrenoceptor homodimer may be responsible for generating the low-affinity pharmacology of the secondary ß1-adrenoceptor conformation.

Single-molecule analysis of fluorescently labeled G-protein-coupled receptors reveals complexes with distinct dynamics and organization.

G-protein-coupled receptors (GPCRs) constitute the largest family of receptors and major pharmacological targets. Whereas many GPCRs have been shown to form di-/oligomers, the size and stability of such complexes under physiological conditions are largely unknown. Here, we used direct receptor labeling with SNAP-tags and total internal reflection fluorescence microscopy to dynamically monitor single receptors on intact cells and thus compare the spatial arrangement, mobility, and supramolecular organization of three prototypical GPCRs: the ß(1)-adrenergic receptor (ß(1)AR), the ß(2)-adrenergic receptor (ß(2)AR), and the γ-aminobutyric acid (GABA(B)) receptor. These GPCRs showed very different degrees of di-/oligomerization, lowest for ß(1)ARs (monomers/dimers) and highest for GABA(B) receptors (prevalently dimers/tetramers of heterodimers). The size of receptor complexes increased with receptor density as a result of transient receptor-receptor interactions. Whereas ß(1)-/ß(2)ARs were apparently freely diffusing on the cell surface, GABA(B) receptors were prevalently organized into ordered arrays, via interaction with the actin cytoskeleton. Agonist stimulation did not alter receptor di-/oligomerization, but increased the mobility of GABA(B) receptor complexes. These data provide a spatiotemporal characterization of ß(1)-/ß(2)ARs and GABA(B) receptors at single-molecule resolution. The results suggest that GPCRs are present on the cell surface in a dynamic equilibrium, with constant formation and dissociation of new receptor complexes that can be targeted, in a ligand-regulated manner, to different cell-surface microdomains.

Synthesis and Pharmacological Activity of a New Series of 1-(1H-Indol-4-yloxy)-3-(2-(2-methoxyphenoxy)ethylamino)propan-2-ol Analogs.

ß-Adrenergic receptor antagonists are important therapeutics for the treatment of cardiovascular disorders. In the group of ß-blockers, much attention is being paid to the third-generation drugs that possess important ancillary properties besides inhibiting ß-adrenoceptors. Vasodilating activity of these drugs is produced through different mechanisms, such as nitric oxide (NO) release, ß2 -agonistic action, α1 -blockade, antioxidant action, and Ca(2+) entry blockade. Here, a study on evaluation of the cardiovascular activity of five new compounds is presented. Compound 3a is a methyl and four of the tested compounds (3b-e) are dimethoxy derivatives of 1-(1H-indol-4-yloxy)-3-(2-(2-methoxyphenoxy)ethylamino)propan-2-ol. The obtained results confirmed that the methyl and dimethoxy derivatives of 1-(1H-indol-4-yloxy)-3-(2-(2-methoxyphenoxy)ethylamino)propan-2-ol and their enantiomers possess α1 - and ß1 -adrenolytic activities and that the antiarrhythmic and hypotensive effects of the tested compounds are related to their adrenolytic properties.