Balancing G protein selectivity and efficacy in the adenosine A receptor.

The adenosine A receptor (AR) engages several G proteins, notably G and its cognate G protein. This coupling promiscuity is facilitated by a dynamic ensemble, revealed by F nuclear magnetic resonance imaging of AR and G protein. Two transmembrane helix 6 (TM6) activation states, formerly associated with partial and full agonism, accommodate the differing volumes of G and G.

Mapping the conformational landscape of the stimulatory heterotrimeric G protein.

Heterotrimeric G proteins serve as membrane-associated signaling hubs, in concert with their cognate G-protein-coupled receptors. Fluorine nuclear magnetic resonance spectroscopy was employed to monitor the conformational equilibria of the human stimulatory G-protein α subunit (Gα) alone, in the intact Gαβγ heterotrimer or in complex with membrane-embedded human adenosine A receptor (AR). The results reveal a concerted equilibrium that is strongly affected by nucleotide and interactions with the βγ subunit, the lipid bilayer and AR.

Allosteric modulation of the adenosine A receptor by cholesterol.

Cholesterol is a major component of the cell membrane and commonly regulates membrane protein function. Here, we investigate how cholesterol modulates the conformational equilibria and signaling of the adenosine A receptor (AR) in reconstituted phospholipid nanodiscs. This model system conveniently excludes possible effects arising from cholesterol-induced phase separation or receptor oligomerization and focuses on the question of allostery.

Structures and Dynamics of Native-State Transmembrane Protein Targets and Bound Lipids.

Membrane proteins work within asymmetric bilayers of lipid molecules that are critical for their biological structures, dynamics and interactions. These properties are lost when detergents dislodge lipids, ligands and subunits, but are maintained in native nanodiscs formed using styrene maleic acid (SMA) and diisobutylene maleic acid (DIBMA) copolymers. These amphipathic polymers allow extraction of multicomponent complexes of post-translationally modified membrane-bound proteins directly from organ homogenates or membranes from diverse types of cells and organelles.

Advances in the study of GPCRs by F NMR.

Crystallography and cryo-electron microscopy have advanced atomic resolution perspectives of inactive and active states of G protein-coupled receptors (GPCRs), alone and in complex with G proteins or arrestin. F NMR can play a role in ascertaining activation mechanisms and understanding the complete energy landscape associated with signal transduction. Fluorinated reporters are introduced biosynthetically via fluorinated amino acid analogs or chemically, via thiol-specific fluorinated reporters.

Structural Insight into G Protein-Coupled Receptor Signaling Efficacy and Bias between Gs and β-Arrestin.

G protein-coupled receptors (GPCRs) form the largest family of membrane proteins involved in signal transduction. Because of their ability to regulate a wide range of cellular responses and their dysregulation being associated with many diseases, GPCRs remain a key therapeutic target for several clinical indications. In recent years, it has been demonstrated that ligands for a given receptor can engage distinct pathways with different relative efficacies, a concept known as biased signaling or functional selectivity.

Hybridization of β-Adrenergic Agonists and Antagonists Confers G Protein Bias.

Starting from the β-adrenoceptor agonist isoprenaline and beta-blocker carvedilol, we designed and synthesized three different chemotypes of agonist/antagonist hybrids. Investigations of ligand-mediated receptor activation using bioluminescence resonance energy transfer biosensors revealed a predominant effect of the aromatic head group on the intrinsic activity of our ligands, as ligands with a carvedilol head group were devoid of agonistic activity. Ligands composed of a catechol head group and an antagonist-like oxypropylene spacer possess significant intrinsic activity for the activation of Gα, while they only show weak or even no β-arrestin-2 recruitment at both β- and β-AR.

Bioluminescence resonance energy transfer-based imaging of protein-protein interactions in living cells.

Bioluminescence resonance energy transfer (BRET) is a transfer of energy between a luminescence donor and a fluorescence acceptor. Because BRET occurs when the distance between the donor and acceptor is <10 nm, and its efficiency is inversely proportional to the sixth power of distance, it has gained popularity as a proximity-based assay to monitor protein-protein interactions and conformational rearrangements in live cells. In such assays, one protein of interest is fused to a bioluminescent energy donor (luciferases from Renilla reniformis or Oplophorus gracilirostris), and the other protein is fused to a fluorescent energy acceptor (such as GFP or YFP).

Publisher Correction: Structural insights into binding specificity, efficacy and bias of a βAR partial agonist.

In the version of this paper originally published, the structure for epinephrine shown in Figure 1a was redrawn with an extra carbon. The structure has been replaced in the HTML and PDF versions of the article. The original and corrected versions of the structure are shown below.

Structural insights into binding specificity, efficacy and bias of a βAR partial agonist.

Salmeterol is a partial agonist for the β adrenergic receptor (βAR) and the first long-acting βAR agonist to be widely used clinically for the treatment of asthma and chronic obstructive pulmonary disease. Salmeterol's safety and mechanism of action have both been controversial. To understand its unusual pharmacological action and partial agonism, we obtained the crystal structure of salmeterol-bound βAR in complex with an active-state-stabilizing nanobody.

Bioluminescence resonance energy transfer-based biosensors allow monitoring of ligand- and transducer-mediated GPCR conformational changes.

G protein-coupled receptors (GPCRs) are seven-transmembrane proteins that mediate a variety of cellular response which make them a target of choice for drug development in many indications. It is now well established that GPCRs can adopt several distinct conformations that can be differentially stabilized by various ligands resulting in different biological outcomes, a concept known as functional selectivity. However, due to the highly hydrophobic nature of GPCRs, tools to monitor these conformational ensembles are limited and addressing their conformation dynamics remains a challenge with current structural biology approaches.

Evolutionary action and structural basis of the allosteric switch controlling βAR functional selectivity.

Functional selectivity of G-protein-coupled receptors is believed to originate from ligand-specific conformations that activate only subsets of signaling effectors. In this study, to identify molecular motifs playing important roles in transducing ligand binding into distinct signaling responses, we combined in silico evolutionary lineage analysis and structure-guided site-directed mutagenesis with large-scale functional signaling characterization and non-negative matrix factorization clustering of signaling profiles. Clustering based on the signaling profiles of 28 variants of the β-adrenergic receptor reveals three clearly distinct phenotypical clusters, showing selective impairments of either the Gi or βarrestin/endocytosis pathways with no effect on Gs activation.

Multimodal and Polymorphic Interactions between Anillin and Actin: Their Implications for Cytokinesis.

Cytokinesis of animal cells requires the assembly of a contractile ring, which promotes daughter cell splitting. Anillin is a conserved scaffold protein involved in organizing the structural components of the contractile ring including filamentous actin (F-actin), myosin, and septins and in forming the subsequent midbody ring. Like other metazoan homologs, Drosophila anillin contains a conserved domain that can bind and bundle F-actin, but the importance and molecular details of its interaction with F-actin remain unclear.