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Summary Ligands targeting G protein-coupled receptors (GPCRs) are currently classified as either orthosteric, allosteric, or dualsteric/bitopic. Here, we introduce a new pharmacological concept for GPCR functional modulation: sequential receptor activation . A hallmark feature of this is a stepwise ligand binding mode with transient activation of a first receptor site followed by sustained activation of a second topographically distinct site. We identify 4-CMTB (2-(4-chlorophenyl)-3-methyl-N-(thiazol-2-yl)butanamide), previously classified as a pure allosteric agonist of the free fatty acid receptor 2, as the first sequential activator and corroborate its two-step activation in living cells by tracking integrated responses with innovative label-free biosensors that visualize multiple signaling inputs in real time. We validate this unique pharmacology with traditional cellular readouts, including mutational and pharmacological perturbations along with computational methods, and propose a kinetic model applicable to the analysis of sequential receptor activation. We envision this form of dynamic agonism as a common principle of nature to spatiotemporally encode cellular information. [ABSTRACT FROM AUTHOR]

The biased agonism of the G protein-coupled receptors (GPCRs), where in addition to a traditional G protein-signaling pathway a GPCR promotes intracellular signals though β-arrestin, is a novel paradigm in pharmacology. Biochemical and biophysical studies have suggested that a GPCR forms a distinct ensemble of conformations signaling through the G protein and β-arrestin. Here we report on the dynamics of the β2adrenergic receptor bound to the β-arrestin and G protein-biased agonists and the empty receptor to further characterize the receptor conformational changes caused by biased agonists. We use conventional andaccelerated molecular dynamics (aMD) simulations to explore the conformational transitions of the GPCR from the active state to die inactive state. We found that aMD simulations enable monitoring of the transition within the nanosecond time scale while capturing the known microscopic characteristics of the inactive states, such as the ionic lock, the inward position of F6.44, and water clusters. Distinct conformational states are shown to be stabilized by each biased agonist In particular, in simulations of the receptor with the β-arrestin-biased agonist N-cyclopentylbutanepherine, we observe a different pattern of motions in helix 7 when compared to simulations with the G protein-biased agonist salbutamol that involves perturbations of the network of interactions within the NPxxY motif. Understanding the network of interactions induced by biased ligands and die subsequent receptor conformational shifts will lead to development of more efficient drugs. [ABSTRACT FROM AUTHOR]

When it is difficult to develop selective ligands within a family of related G-protein-coupled receptors (GPCRs), chemically engineered receptors activated solely by synthetic ligands (RASSLs) are useful alternatives for probing receptor function. In the present work, we explored whether a RASSL of the free fatty acid receptor 2 (FFA2) could be developed on the basis of pharmacological variation between species orthologs. For this, bovine FFA2 was characterized, revealing distinct ligand selectivity compared with human FFA2. Homology modeling mid mutational analysis demonstrated a single mutation in human FFA2 of C4.57G resulted in a human FFA2 receptor with ligand selectivity similar to the bovine receptor. This was exploited to generate human FFA2- RASSL by the addition of a second mutation at a known orthosteric ligand interaction site, H6.55Q. The resulting FFA2-RASSL displayed a >100-fold loss of activity to endogenous ligands, while responding to the distinct ligand sorbic acid with pECk0 values for inhibition of cAMP, 5.83 ± 0.11; Ca2+ mobilization, 4.63 ± 0.05; ERK phosphorylation, 5.61 ± 0.06; and dynamic mass redistribution, 5.35 - 0.06. This FFA2-RASSL will be useful in future studies on this receptor and demonstrates that exploitation of pharmacological variation between species orthologs is a powerful method to generate novel chemically engineered GPCRs. [ABSTRACT FROM AUTHOR]

Free fatty acid receptors 2 and 3 (FFA2 and FFA3) are G protein- coupled receptors for short chain free fatty acids (SCFAs). They respond to the same set of endogenous ligands but with distinct rank-order of potency such that acetate (C2) has been described as FFA2-selective, whereas propionate (C3) is nonselective. Although C2 was confirmed to be selective for human FFA2 over FFA3, this ligand was not selective between the mouse orthologs. Moreover, although C3 was indeed not selective between the human orthologs, it displayed clear selectivity for mouse FFA3 over mouse FFA2. This altered selectivity to C2 and C3 resulted from broad differences in SCFAs potency at the mouse orthologs. In studies to define the molecular basis for these observations, marked variation in ligand-independent constitutive activity was identified using a [35S]GTPγS assay. The orthologs with higher potency for the SCFAs, human FFA2 and mouse FFA3, displayed high constitutive activity in this assay, whereas the orthologs with lower potency for the agonist ligands, mouse FFA2 and human FFA3, did not. Sequence alignments of the second extracellular loop identified single negatively charged residues in FFA2 and FFA3 not conserved between species and predicted to form ionic lock interactions with arginine residues within the FFA2 or FFA3 agonist binding pocket to regulate constitutive activity and SCFA potency. Reciprocal mutation of these residues between species orthologs resulted in the induction (or repression) of constitutive activity and in most cases also yielded corresponding changes in SCFA potency. [ABSTRACT FROM AUTHOR]

Background: Previous findings indicate that susceptibility to Leishmania (Viannia) panamensis infection of monocyte-derived macrophages from patients and asymptomatically infected individuals were associated with the adaptive immune response and clinical outcome. Methodology/Principal Findings: To understand the basis for this difference we examined differential gene expression of human monocyte-derived macrophages following exposure to L. (V.) panamensis. Gene activation profiles were determined using macrophages from healthy volunteers cultured with or without stationary phase promastigotes of L. (V.) panamensis. Significant changes in expression (>1.5-fold change; p<0.05; up- or down-regulated) were identified at 0.5, 4 and 24 hours. mRNA abundance profiles varied over time, with the highest level of activation occurring at earlier time points (0.5 and 4 hrs). In contrast to observations for other Leishmania species, most significantly changed mRNAs were up- rather than down-regulated, especially at early time points. Up-regulated transcripts over the first 24 hours belonged to pathways involving eicosanoid metabolism, oxidative stress, activation of PKC through G protein coupled receptors, or mechanism of gene regulation by peroxisome proliferators via PPARα. Additionally, a marked activation of Toll-receptor mediated pathways was observed. Comparison with published microarray data from macrophages infected with L. (Leishmania) chagasi indicate differences in the regulation of genes involved in signaling, motility and the immune response. Conclusions: Results show that the early (0.5 to 24 hours) human monocyte-derived macrophage response to L. (Viannia) panamensis is not quiescent, in contrast to published reports examining later response times (48–96 hours). Early macrophage responses are important for the developing cellular response at the site of infection. The kinetics and the mRNA abundance profiles induced by L. (Viannia) panamensis illustrate the dynamics of these interactions and the distinct biologic responses to different Leishmania species from the outset of infection within their primary host cell. Author Summary: Leishmania parasites cause a spectrum of diseases (cutaneous, visceral and the deforming forms—chronic cutaneous and mucocutaneous) known as leishmaniasis. The macrophage, a key cell in the immune system, is the cellular target of Leishmania parasites in the mammalian host. Previous studies showed the responses of monocytederived macrophages from naturally infected humans to infection with Leishmania (Viannia) panamensis were key to adaptive immune responses and clinical outcome. Consequently, an mRNA microarray approach was employed to assess the changes in macrophage gene expression over time (0.5 to 24 hours) induced by L. panamensis. The highest level of gene expression induction occurred early (0.5–4 hours); the early pathways (groups of genes) activated included those involved in the innate immune response (signaling, phagocytosis, TLR activation, and inflammatory). Early gene activation is presumed to be important for the developing cellular milieu at the site of infection. By 24 hours post-infection the dominant pathways involved metabolic functions. However, a comparison of the macrophage response to L. (V.) panamensis to that of L. (L.) chagasi (causative agent of visceral leishmaniasis) at 24 hours revealed a differential up-regulation of genes (cell adhesion, signaling, and inflammation) in response to these species. These observations underscore the distinct biology of different Leishmania species from the outset of infection. [ABSTRACT FROM AUTHOR]

From the molecular mechanism of antagonist unbinding in the β1 and β2 adrenoceptors investigated by steered molecular dynamics, we attempt to provide further possibilities of ligand subtype and subspecies selectivity. We have simulated unbinding of β1-selective Esmolol and β2-selective ICI-118551 from both receptors to the extracellular environment and found distinct molecular features of unbinding. By calculating work profiles, we show different preference in antagonist unbinding pathways between the receptors, in particular, perpendicular to the membrane pathway is favourable in the β1 adrenoceptor, whereas the lateral pathway involving helices 5, 6 and 7 is preferable in the β2 adrenoceptor. The estimated free energy change of unbinding based on the preferable pathway correlates with the experimental ligand selectivity. We then show that the non-conserved K347 (6.58) appears to facilitate in guiding Esmolol to the extracellular surface via hydrogen bonds in the β1 adrenoceptor. In contrast, hydrophobic and aromatic interactions dominate in driving ICI-118551 through the easiest pathway in the β2 adrenoceptor. We show how our study can stimulate design of selective antagonists and discuss other possible molecular reasons of ligand selectivity, involving sequential binding of agonists and glycosylation of the receptor extracellular surface. [ABSTRACT FROM AUTHOR]

The vasopressin type 2 receptor (V2R) is an essential G protein-coupled receptor (GPCR) in renal regulation of water homeostasis. Upon stimulation, the V2R activates Gas and Gaq/11, which is followed by robust recruitment of ß-arrestins and receptor internalization into endosomes. Unlike canonical GPCR signaling, the ß-arrestin association with the V2R does not terminate Gas activation, and thus, Gas-mediated signaling is sustained while the receptor is internalized. Here, we demonstrate that this V2R ability to co-interact with G protein/ß-arrestin and promote endosomal G protein signaling is not restricted to Gas, but also involves Gaq/11. Furthermore, our data imply that ß-arrestins potentiate Gas/Gaq/11 activation at endosomes rather than terminating their signaling. Surprisingly, we found that the V2R internalizes and promote endosomal G protein activation independent of ß-arrestins to a minor degree. These new observations challenge the current model of endosomal GPCR signaling and suggest that this event can occur in both ß-arrestin-dependent and -independent manners. [ABSTRACT FROM AUTHOR]

Crystallization and determination of the high resolution three-dimensional structure of the β2-adrenergic receptor in 2007 was followed by structure elucidation of a number of other receptors, including those for neurotensin and glucagon. These major advances foster the understanding of structure–activity relationship of these receptors and structure-based rational design of new ligands having more predictable activity. At present, structure determination of gut hormone receptors in complex with their ligands (natural, synthetic) and interacting signalling proteins, for example, G-proteins, arrestins, represents a challenge which promises to revolutionize gut hormone endocrinonology. [ABSTRACT FROM AUTHOR]

GPR84 is an immune cell-expressed, proinflammatory receptor currently being assessed as a therapeutic target in conditions including fibrosis and inflammatory bowel disease. Although it was previously shown that the orthosteric GPR84 activators 2-HTP and 6-OAU promoted its interactions with arrestin-3, a G protein-biased agonist DL-175 did not. Here, we show that replacement of all 21 serine and threonine residues within i-loop 3 of GPR84, but not the two serines in the C-terminal tail, eliminated the incorporation of [32P] and greatly reduced receptor-arrestin-3 interactions promoted by 2-HTP. GPR84 was phosphorylated constitutively on residues Ser221 and Ser224, while various other amino acids are phosphorylated in response to 2-HTP. Consistent with this, an antiserum able to identify pSer221/pSer224 recognized GPR84 from cells treated with and without activators, whereas an antiserum able to identify pThr263/pThr264 only recognized GPR84 after exposure to 2-HTP and not DL-175. Two distinct GPR84 antagonists as well as inhibition of G protein-coupled receptor kinase 2/3 prevented phosphorylation of pThr263/pThr264, but neither strategy affected constitutive phosphorylation of Ser221/Ser224. Furthermore, mutation of residues Thr263 and Thr264 to alanine generated a variant of GPR84 also limited in 2-HTP-induced interactions with arrestin-2 and -3. By contrast, this mutant was unaffected in its capacity to reduce cAMP levels. Taken together, these results define a key pair of threonine residues, regulated only by subsets of GPR84 small molecule activators and by GRK2/3 that define effective interactions with arrestins and provide novel tools to monitor the phosphorylation and functional status of GPR84. [ABSTRACT FROM AUTHOR]

The free fatty acid receptor 1 (FFA1), a G protein-coupled receptor (GPCR) naturally activated by long-chain fatty acids is a novel target for the treatment of metabolic diseases. The basic amine spirocyclic periphery of Eli Lilly’s drug candidate LY2881835 for treatment of type 2 diabetes mellitus (which reached phase I clinical trials) inspired a series of novel FFA1 agonists. These were designed to incorporate the 3-[4-(benzyloxy)phenyl]propanoic acid pharmacophore core decorated with a range of spirocyclic motifs. The latter were prepared via the Prins cyclization and subsequent modification of the 4-hydroxytetrahydropyran moiety in the Prins product. Here, we synthesize 19 compounds and test for FFA1 activity. Within this pilot set, a nanomolar potency (EC 50 = 55 nM) was reached. Four lead compounds (EC 50 range 55–410 nM) were characterized for aqueous solubility, metabolic stability, plasma protein binding and Caco-2 permeability. While some instability in the presence of mouse liver microsomes was noted, mouse pharmacokinetic profile of the compound having the best overall ADME properties was evaluated to reveal acceptable bioavailability ( F = 10.3%) and plasma levels achieved on oral administration. [ABSTRACT FROM AUTHOR]

FFA2 is a G protein-coupled receptor that responds to short chain fatty acids and has generated interest as a therapeutic target for metabolic and inflammatory conditions. However, definition of its functions has been slowed by a dearth of selective ligands that can distinguish it from the closely related FFA3. At present, the only selective ligands described for FFA2 suffer from poor potency, altered signaling due to allosteric modes of action, or a lack of function at non-human orthologs of the receptor. To address the need for novel selective ligands, we synthesized two compounds potentially having FFA2 activity and examined the molecular basis of their function. These compounds were confirmed to be potent and selective orthosteric FFA2 agonists. A combination of ligand structure-activity relationship, pharmacological analysis, homology modeling, species ortholog comparisons, and mutagenesis studies were then employed to define the molecular basis of selectivity and function of these ligands. From this, we identified key residues within both extracellular loop 2 and the transmembrane domain regions of FFA2 critical for ligand function. One of these ligands was active with reasonable potency at rodent orthologs of FFA2 and demonstrated the role of FFA2 in inhibition of lipolysis and glucagon-like peptide-1 secretion in murine-derived 3T3-L1 and STC-1 cell lines, respectively. Together, these findings describe the first potent and selective FFA2 orthosteric agonists and demonstrate key aspects of ligand interaction within the binding site of FFA2 that will be invaluable in future ligand development at this receptor. [ABSTRACT FROM AUTHOR]

Seven-transmembrane receptors (7TMRs), also termed G protein-coupled receptors (GPCRs), form the largest class of cell surface membrane receptors, involving several hundred members in the human genome. Nearly 30% of marketed pharmacological agents target 7TMRs. 7TMRs adopt multiple conformations upon agonist binding. Biased agonists, in contrast to non-biased agonists, are believed to stabilize conformations preferentially activating either G-protein- or β-arrestin-dependent signaling pathways. However, proof that cognate conformations of receptors display structural differences within their binding site where biased agonism initiates, are still lacking. Here, we show that a non-biased agonist, cholecystokinin (CCK) induces conformational states of the CCK2R activating Gq-protein-dependent pathway (CCK2RG) or recruiting β-arrestin2 (CCK2Rβ) that are pharmacologically and structurally distinct. Two structurally unrelated antagonists competitively inhibited both pathways. A third ligand (GV150013X) acted as a high affinity competitive antagonist on CCK2RG but was nearly inefficient as inhibitor of CCK2Rβ. Several structural elements on both GV150013X and in CCK2R binding cavity, which hinder binding of GV150013X only to the CCK2Rβ were identified. At last, proximity between two conserved amino acids from transmembrane helices 3 and 7 interacting through sulfur-aromatic interaction was shown to be crucial for selective stabilization of the CCK2Rβ state. These data establish structural evidence for distinct conformations of a 7TMR associated with β-arrestin-2 recruitment or G-protein coupling and validate relevance of the design of biased ligands able to selectively target each functional conformation of 7TMRs. [ABSTRACT FROM AUTHOR]

• GIP receptor is expressed in neuroendocrine tumours with high incidence and density • GIP receptor rapidly and abundantly internalizes upon GIP stimulation • Internalized GIP receptor continues to stimulate production of cyclic AMP from early endosomes • Amino acids involved in either ligand binding, receptor activation, G protein binding, or both are identified. GIP is well known as a peptide regulating metabolic functions. In this review paper, we summarize a series of data on GIP receptor (GIPR). First, expression study of GIPR in human neuroendocrine tumours showed a very high incidence (nearly 100%) and a high density in both functional and non functional pancreatic tumours, ileal tumours, bronchial tumours and medullary thyroid carcinomas. Then, data on internalization of GIPR following stimulation by GIP are reported. Rapid and abundant internalization of GIPR also found in tumor pancreatic endocrine cells opens the possibility of tumor imaging and eradication using radiolabeled GIP. Interestingly, internalized GIPR continues to signal in early endosomes stimulating production of cAMP and activation of PKA, thus, supporting the view that GIPR signals from both plasma membrane and vesicles of internalization. At last, we summarize data from studies using in synergy molecular modeling and site-directed mutagenesis, which identified crucial amino acids of transmembrane domains of GIPR involved in GIPR binding site of GIP and/or in its activation and coupling to Gs protein. All together, these last molecular data may help to better understand structure-activity relationship data on GIP and GIPR. [ABSTRACT FROM AUTHOR]