Exploring an Intracellular Allosteric Site of CC-Chemokine Receptor 4 from 3D Models, Probe Simulations, and Mutagenesis.

We applied our previously developed probe confined dynamic mapping protocol, which combines enhanced sampling molecular dynamics (MD) simulations and fragment-based approaches, to identify the binding site of GSK2239633A (-[[3-[[3-[(5-chlorothiophen-2-yl)sulfonylamino]-4-methoxyindazol-1-yl]methyl]phenyl]methyl]-2-hydroxy-2-methylpropanamide), a selective CC-chemokine receptor type 4 (CCR4) negative allosteric modulator, using CCR4 homology and AlphaFold models. By comparing the performance across five computational models, we identified conserved (K310 and Y304) and non-conserved (M243) residue hotspots for GSK2239633A binding, which were validated by mutagenesis and bioluminescence resonance energy transfer assay. Further analysis of 3D models and MD simulations highlighted the pair of residues 6.

Molecular mechanism of distinct chemokine engagement and functional divergence of the human Duffy antigen receptor.

The Duffy antigen receptor is a seven-transmembrane (7TM) protein expressed primarily at the surface of red blood cells and displays strikingly promiscuous binding to multiple inflammatory and homeostatic chemokines. It serves as the basis of the Duffy blood group system in humans and also acts as the primary attachment site for malarial parasite Plasmodium vivax and pore-forming toxins secreted by Staphylococcus aureus. Here, we comprehensively profile transducer coupling of this receptor, discover potential non-canonical signaling pathways, and determine the cryoelectron microscopy (cryo-EM) structure in complex with the chemokine CCL7.

β-Arrestin-dependent and -independent endosomal G protein activation by the vasopressin type 2 receptor.

The vasopressin type 2 receptor (VR) is an essential G protein-coupled receptor (GPCR) in renal regulation of water homeostasis. Upon stimulation, the VR activates Gα and Gα, which is followed by robust recruitment of β-arrestins and receptor internalization into endosomes. Unlike canonical GPCR signaling, the β-arrestin association with the VR does not terminate Gα activation, and thus, Gα-mediated signaling is sustained while the receptor is internalized.

Gα signalling from endosomes: A new conundrum.

G-protein-coupled receptors (GPCRs) constitute the largest family of membrane receptors, and are involved in the transmission of a variety of extracellular stimuli such as hormones, neurotransmitters, light and odorants into intracellular responses. They regulate every aspect of physiology and, for this reason, about one third of all marketed drugs target these receptors. Classically, upon binding to their agonist, GPCRs are thought to activate G-proteins from the plasma membrane and to stop signalling by subsequent desensitisation and endocytosis.

β-arrestin-dependent and -independent endosomal G protein activation by the vasopressin type 2 receptor.

The vasopressin type 2 receptor (VR) is an essential GPCR in renal regulation of water homeostasis. Upon stimulation, the VR activates Gα and Gα, which is followed by robust recruitment of β-arrestins and receptor internalization into endosomes. Unlike canonical GPCR signaling, the β-arrestin association with the VR does not terminate Gα activation, and thus, Gα-mediated signaling is sustained while the receptor is internalized.

Michaelis-Menten Quantification of Ligand Signaling Bias Applied to the Promiscuous Vasopressin V2 Receptor.

Activation of G protein-coupled receptors by agonists may result in the activation of one or more G proteins and recruitment of arrestins. The extent of the activation of each of these pathways depends on the intrinsic efficacy of the ligand. Quantification of intrinsic efficacy relative to a reference compound is essential for the development of novel compounds.

Identification of Key Regions Mediating Human Melatonin Type 1 Receptor Functional Selectivity Revealed by Natural Variants.

Melatonin is a hormone mainly produced by the pineal gland and MT is one of the two G protein-coupled receptors (GPCRs) mediating its action. Despite an increasing number of available GPCR crystal structures, the molecular mechanism of activation of a large number of receptors, including MT, remains poorly understood. The purpose of this study is to elucidate the structural elements involved in the process of MT's activation using naturally occurring variants affecting its function.

Human MC4R variants affect endocytosis, trafficking and dimerization revealing multiple cellular mechanisms involved in weight regulation.

The Melanocortin-4 Receptor (MC4R) plays a pivotal role in energy homeostasis. We used human MC4R mutations associated with an increased or decreased risk of obesity to dissect mechanisms that regulate MC4R function. Most obesity-associated mutations impair trafficking to the plasma membrane (PM), whereas obesity-protecting mutations either accelerate recycling to the PM or decrease internalization, resulting in enhanced signaling.

Circadian, Sleep and Caloric Intake Phenotyping in Type 2 Diabetes Patients With Rare Melatonin Receptor 2 Mutations and Controls: A Pilot Study.

Background: Melatonin modulates circadian rhythms in physiology and sleep initiation. Genetic variants of the locus, encoding the melatonin MT receptor, have been associated with increased type 2 diabetes (T2D) risk. Carriers of the common intronic rs10830963 T2D risk variant have modified sleep and circadian traits such as changes of the melatonin profile.

The constitutive activity of the viral-encoded G protein-coupled receptor US28 supports a complex signalling network contributing to cancer development.

US28 is a viral G protein-coupled receptor (GPCR) encoded by the human cytomegalovirus (HCMV). This receptor, expressed both during lytic replication and viral latency, is required for latency. US28 is binding to a wide variety of chemokines but also exhibits a particularly high constitutive activity robustly modulating a wide network of cellular pathways altering the host cell environment to benefit HCMV infection.

Emerging Role of Compartmentalized G Protein-Coupled Receptor Signaling in the Cardiovascular Field.

G protein-coupled receptors (GPCRs) are cell surface receptors that for many years have been considered to function exclusively at the plasma membrane, where they bind to extracellular ligands and activate G protein signaling cascades. According to the conventional model, these signaling events are rapidly terminated by β-arrestin (β-arr) recruitment to the activated GPCR resulting in signal desensitization and receptor internalization. However, during the past decade, emerging evidence suggest that many GPCRs can continue to activate G proteins from intracellular compartments after they have been internalized.

Translating biased signaling in the ghrelin receptor system into differential in vivo functions.

Biased signaling has been suggested as a means of selectively modulating a limited fraction of the signaling pathways for G-protein-coupled receptor family members. Hence, biased ligands may allow modulation of only the desired physiological functions and not elicit undesired effects associated with pharmacological treatments. The ghrelin receptor is a highly sought antiobesity target, since the gut hormone ghrelin in humans has been shown to increase both food intake and fat accumulation.

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

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.

Type 2 diabetes-associated variants of the MT melatonin receptor affect distinct modes of signaling.

Melatonin is produced during the night and regulates sleep and circadian rhythms. Loss-of-function variants in , which encodes the melatonin receptor MT, a G protein-coupled receptor (GPCR), are associated with an increased risk of type 2 diabetes (T2D). To identify specific T2D-associated signaling pathway(s), we profiled the signaling output of 40 MT variants by monitoring spontaneous (ligand-independent) and melatonin-induced activation of multiple signaling effectors.

Purinergic Receptor Transactivation by the -Adrenergic Receptor Increases Intracellular Ca in Nonexcitable Cells.

The adrenergic receptor (AR) increases intracellular Ca in a variety of cell types. By combining pharmacological and genetic manipulations, we reveal a novel mechanism through which the AR promotes Ca mobilization (pEC = 7.32 ± 0.

Discovery of G Protein-Biased Dopaminergics with a Pyrazolo[1,5-a]pyridine Substructure.

1,4-Disubstituted aromatic piperazines are privileged structural motifs recognized by aminergic G protein-coupled receptors. Connection of a lipophilic moiety to the arylpiperazine core by an appropriate linker represents a promising concept to increase binding affinity and to fine-tune functional properties. In particular, incorporation of a pyrazolo[1,5-a]pyridine heterocyclic appendage led to a series of high-affinity dopamine receptor partial agonists.

Distinct conformations of GPCR-β-arrestin complexes mediate desensitization, signaling, and endocytosis.

β-Arrestins (βarrs) interact with G protein-coupled receptors (GPCRs) to desensitize G protein signaling, to initiate signaling on their own, and to mediate receptor endocytosis. Prior structural studies have revealed two unique conformations of GPCR-βarr complexes: the "tail" conformation, with βarr primarily coupled to the phosphorylated GPCR C-terminal tail, and the "core" conformation, where, in addition to the phosphorylated C-terminal tail, βarr is further engaged with the receptor transmembrane core. However, the relationship of these distinct conformations to the various functions of βarrs is unknown.

GPCR-G Protein-β-Arrestin Super-Complex Mediates Sustained G Protein Signaling.

Classically, G protein-coupled receptor (GPCR) stimulation promotes G protein signaling at the plasma membrane, followed by rapid β-arrestin-mediated desensitization and receptor internalization into endosomes. However, it has been demonstrated that some GPCRs activate G proteins from within internalized cellular compartments, resulting in sustained signaling. We have used a variety of biochemical, biophysical, and cell-based methods to demonstrate the existence, functionality, and architecture of internalized receptor complexes composed of a single GPCR, β-arrestin, and G protein.

Mapping physiological G protein-coupled receptor signaling pathways reveals a role for receptor phosphorylation in airway contraction.

G protein-coupled receptors (GPCRs) are known to initiate a plethora of signaling pathways in vitro. However, it is unclear which of these pathways are engaged to mediate physiological responses. Here, we examine the distinct roles of Gq/11-dependent signaling and receptor phosphorylation-dependent signaling in bronchial airway contraction and lung function regulated through the M3-muscarinic acetylcholine receptor (M3-mAChR).

Constitutive activities and inverse agonism in dopamine receptors.

The concept of activation in the absence of agonists has been demonstrated for many GPCRs and is now solidified as one of the principal aspects of GPCR signaling. In this chapter, we review how dopamine receptors demonstrate this ability. Although difficult to prove in vivo due to the presence of endogenous dopamine and lack of subtype-selective inverse agonists and "pure" antagonists (neutral ligands), in vitro assays such as measuring intracellular cAMP, [(35)S]GTPγS binding, and [(3)H]thymidine incorporation have uncovered the constitutive activation of D1- and D2-class receptors.

Functional analysis of human D1 and D5 dopaminergic G protein-coupled receptors: lessons from mutagenesis of a conserved serine residue in the cytosolic end of transmembrane region 6.

In mammals, dopamine G protein-coupled receptors (GPCR) are segregated into two categories: D1-like (D1R and D5R) and D2-like (D2R(short), D2R(long), D3R, and D4R) subtypes. D1R and D5R are primarily coupled to stimulatory heterotrimeric GTP-binding proteins (Gs/olf) leading to activation of adenylyl cyclase and production of intracellular cAMP. D1R and D5R share high level of amino acid identity in transmembrane (TM) regions.

The third intracellular loop of D1 and D5 dopaminergic receptors dictates their subtype-specific PKC-induced sensitization and desensitization in a receptor conformation-dependent manner.

We previously showed that phorbol-12-myristate-13-acetate (PMA) mediates a robust PKC-dependent sensitization and desensitization of the highly homologous human Gs protein and adenylyl cyclase (AC)-linked D1 (hD1R) and D5 (hD5R) dopaminergic receptors, respectively. Here, we demonstrate using forskolin-mediated AC stimulation that PMA-mediated hD1R sensitization and hD5R desensitization is not associated with changes in AC activity. We next employed a series of chimeric hD1R and hD5R to delineate the underlying structural determinants dictating the subtype-specific regulation of human D1-like receptors by PMA.

Probing the constitutive activity among dopamine D1 and D5 receptors and their mutants.

Dopamine D1 and D5 receptors are prototypical cell-surface seven-transmembrane (TM) G protein-coupled receptors (GPCRs) mediating elevation of intracellular cAMP levels. The high level of constitutive activity of D5 receptor mediating intracellular cAMP production is one of the functional hallmarks distinguishing the closely related D1-like dopaminergic subtypes (D1 and D5). D1-like subtypes share over 80% identity within their TM regions.

Selective angiotensin II AT(2) receptor agonists with reduced CYP 450 inhibition.

Structural alterations to the benzylic position of the first drug-like selective angiotensin II AT(2) receptor agonist (1) have been performed, with the emphasis to reduce the CYP 450 inhibitory property of the initial structure. The imidazole moiety, responsible for the CYP 450 inhibitory effect in 1, was replaced with various heterocycles. In addition, the modes of attachment of the heterocycles, that is, carbon versus nitrogen attachment, and introduction of carbonyl functionalities to the benzylic position have been evaluated.