G-protein Coupled Cell Surface Receptors Research Articles

Monitoring the Reversibility of GPCR Signaling by Combining Photochromic Ligands with Label-free Impedance Analysis.

G protein-coupled cell surface receptors (GPCR) trigger complex intracellular signaling cascades upon agonist binding. Classic pharmacological assays provide information about binding affinities, activation or blockade at different stages of the signaling cascade, but real time dynamics and reversibility of these processes remain often disguised. We show that combining photochromic NPY receptor ligands, which can be toggled in their receptor activation ability by irradiation with light of different wavelengths, with whole cell label-free impedance assays allows observing the cell response to receptor activation and its reversibility over time. The concept demonstrated on NPY receptors may be well applicable to many other GPCRs providing a deeper insight into the time course of intracellular signaling processes.

Sphingosine-1-phosphate related signalling pathways manipulating virus replication.

Viruses can create a unique cellular environment that facilitates replication and transmission. Sphingosine kinases (SphKs) produce sphingosine-1-phosphate (S1P), a bioactive sphingolipid molecule that performs both physiological and pathological effects primarily by activating a subgroup of the endothelial differentiation gene family of G-protein coupled cell surface receptors known as S1P receptors (S1PR1-5). A growing body of evidence indicates that the SphK/S1P axis is crucial for regulating cellular activities in virus infections like respiratory viruses, enteroviruses, hepatitis viruses, herpes viruses, and arboviruses replicate. Depending on the type of virus, pro- or anti-viral activities of the SphK/S1P axis sometimes rely on the host immune system and sometimes directly through intracellular signalling pathways or cell proliferation. Recent research has shown novel roles of S1P and SphK in viral replication. Sphingosine kinase isoforms (SphK1 and SphK2) levels can be manipulated by several viruses to promote the effects that are expected. Regulation of cellular signalling pathways plays a significant role in the mechanism. The purpose of this review is to provide insight of the characters played by the SphK/S1P axis throughout diverse viral infection processes. We then assess potential therapeutic methods that are based on S1P signalling and metabolism during viral infections.

Using the novel HiBiT tag to label cell surface relaxin receptors for BRET proximity analysis.

Relaxin family peptide 1 (RXFP1) is the receptor for relaxin a peptide hormone with important therapeutic potential. Like many G protein‐coupled receptors (GPCRs), RXFP1 has been reported to form homodimers. Given the complex activation mechanism of RXFP1 by relaxin, we wondered whether homodimerization may be explicitly required for receptor activation, and therefore sought to determine if there is any relaxin‐dependent change in RXFP1 proximity at the cell surface. Bioluminescence resonance energy transfer (BRET) between recombinantly tagged receptors is often used in GPCR proximity studies. RXFP1 targets poorly to the cell surface when overexpressed in cell lines, with the majority of the receptor proteins sequestered within the cell. Thus, any relaxin‐induced changes in RXFP1 proximity at the cell surface may be obscured by BRET signal originating from intracellular compartments. We therefore, utilized the newly developed split luciferase system called HiBiT to specifically label the extracellular terminus of cell surface RXFP1 receptors in combination with mCitrine‐tagged receptors, using the GABAB heterodimer as a positive control. This demonstrated that the BRET signal detected from RXFP1‐RXFP1 proximity at the cell surface does not appear to be due to stable physical interactions. The fact that there is also no relaxin‐mediated change in RXFP1‐RXFP1 proximity at the cell surface further supports these conclusions. This work provides a basis by which cell surface GPCR proximity and expression levels can be specifically studied using a facile and homogeneous labeling technique such as HiBiT.

Chemokines from a Structural Perspective.

Chemokines are a family of small, highly conserved cytokines that mediate various biological processes, including chemotaxis, hematopoiesis, and angiogenesis, and that function by interacting with cell surface G-Protein Coupled Receptors (GPCRs). Because of their significant involvement in various biological functions and pathologies, chemokines and their receptors have been the focus of therapeutic discovery for clinical intervention. There are several sub-families of chemokines (e.g., CXC, CC, C, and CX3C) defined by the positions of sequentially conserved cysteine residues. Even though all chemokines also have a highly conserved, three-stranded β-sheet/α-helix tertiary structural fold, their quarternary structures vary significantly with their sub-family. Moreover, their conserved tertiary structures allow for subunit swapping within and between sub-family members, thus promoting the concept of a “chemokine interactome”. This review is focused on structural aspects of CXC and CC chemokines, their functional synergy and ability to form heterodimers within the chemokine interactome, and some recent developments in structure-based chemokine-targeted drug discovery.

Regulation of α2B-Adrenergic Receptor Cell Surface Transport by GGA1 and GGA2.

The molecular mechanisms that control the targeting of newly synthesized G protein-coupled receptors (GPCRs) to the functional destinations remain poorly elucidated. Here, we have determined the role of Golgi-localized, γ-adaptin ear domain homology, ADP ribosylation factor-binding proteins 1 and 2 (GGA1 and GGA2) in the cell surface transport of α2B-adrenergic receptor (α2B-AR), a prototypic GPCR, and studied the underlying mechanisms. We demonstrated that knockdown of GGA1 and GGA2 by shRNA and siRNA significantly reduced the cell surface expression of inducibly expressed α2B-AR and arrested the receptor in the perinuclear region. Knockdown of each GGA markedly inhibited the dendritic expression of α2B-AR in primary cortical neurons. Consistently, depleting GGA1 and GGA2 attenuated receptor-mediated signal transduction measured as ERK1/2 activation and cAMP inhibition. Although full length α2B-AR associated with GGA2 but not GGA1, its third intracellular loop was found to directly interact with both GGA1 and GGA2. More interestingly, further mapping of interaction domains showed that the GGA1 hinge region and the GGA2 GAE domain bound to multiple subdomains of the loop. These studies have identified an important function and revealed novel mechanisms of the GGA family proteins in the forward trafficking of a cell surface GPCR.

The Human Microbiome in Hematologic Malignancies

The significant role of gut micro biome in human physiology, immune modification and nutrients supply has been recognized and is advancing. Scientific reports suggest association of the human micro biome with the risk of various diseases including hematologic malignancies. Metabolites (such as short chain fatty acids (SCFAs)) released into the gut as a result of bacterial fermentation can enter the circulation and change the systemic metabolome. This can influence hematopoiesis and the nature of cells recruited to sites of inflammation. Bacterial metabolites especially SCFAs have an important role in host metabolism and serve to modulate immune responses. They have potential to activate several G protein-coupled cell surface receptors (GPCRs) expressed on various immune cells. SCFAs have been shown to suppress nuclear factor Kappa B (NF-kB) and inflammatory cytokines like IL-6 and tumor necrosis factor α (TNF-α) and they can also induce IL-10 production consequently affecting the generation of Th1 and Th17 cells and drive proliferation of T cells and B cells. The intimate, bidirectional relationship between microbes and host immune responses suggests the association of the micro biome with lymphoma and blood cancer. The role of pathogenic organisms such as Borrelia burgdorferi, Chlamydophila psittaci, Epstein-Barr virus(EBV), hepatitis C virus (HCV), Helicobacter pyloriand human immunodeficiency virus(HIV) has been suggested to be associated with hematologic malignancies. Changes in the micro biome can result in excessive infection, tissue damage and pathology as well as in the development of hematologic malignancies and subsequently can have direct impact on overall patient outcome.

G Protein-Coupled Receptor 87: a Promising Opportunity for Cancer Drug Discovery.

G protein-coupled receptors (GPRs) constitute one of the largest families of membrane proteins encoded by the human genome. Upon binding to various ligands, these seven-transmembrane receptors play an essential role in many physiological processes, including neurotransmission, immunity, inflammation, regulation of mood and behavior. In view of their important functions, aberrant expression and activity of GPRs have been implicated in a wide spectrum of diseases, including tumorigenesis. GPR87, a cell surface GPR related to the LPA receptor family, is overexpressed in diverse carcinomas and plays an essential role in tumor cell survival. In our recent work, we uncovered that GPR87 expression is regulated by the tumor suppressor p53 and by DNA damage in a p53-dependent manner. Moreover, we found that a lack of GPR87 triggers an increase in p53, concomitant with a decrease in Akt, which results in the sensitization of tumor cells to DNA damage-induced apoptosis and growth suppression. Altogether, we uncovered an essential function for GPR87 in p53-dependent cell survival in response to stress signals. Due to their unique structure, localization and ligand binding ability, GPRs have been extensively used for drug development and are the most common targets of commercial drugs. Although studies are required to determine GPR87 natural ligand(s) and signaling pathways, GPR87 is undoubtedly a very promising novel target for cancer prevention and treatment.

ENDOSOMAL GENERATION OF cAMP in GPCR SIGNALING

It has been widely assumed that the production of the ubiquitous second messenger cAMP mediated by cell surface G protein-coupled receptors (GPCR) and its termination take place exclusively at the plasma membrane. Recent studies are revealing that diverse GPCRs do not always follow this conventional paradigm. In the new model GPCRs mediate G protein signaling not only from the plasma membrane, but also from endosomal membranes. This model proposes that following ligand binding and activation, cell surface GPCRs internalize and redistribute into early endosomes where trimeric G proteins signaling can be maintained for an extended period of time. This presentation will discuss the molecular and cellular mechanistic subtleties as well as the physiological consequences and potential medical applications of this unexpected process, which is drastically changing how we think about GPCR signaling and regulation, and how we study drugs that target this receptor family.

Endosomal generation of cAMP in GPCR signaling.

It has been widely assumed that the production of the ubiquitous second messenger cyclic AMP, which is mediated by cell surface G protein–coupled receptors (GPCRs), and its termination take place exclusively at the plasma membrane. Recent studies reveal that diverse GPCRs do not always follow this conventional paradigm. In the new model, GPCRs mediate G-protein signaling not only from the plasma membrane but also from endosomal membranes. This model proposes that following ligand binding and activation, cell surface GPCRs internalize and redistribute into early endosomes, where trimeric G protein signaling can be maintained for an extended period of time. This Perspective discusses the molecular and cellular mechanistic subtleties as well as the physiological consequences of this unexpected process, which is considerably changing how we think about GPCR signaling and regulation and how we study drugs that target this receptor family.

RETRACTED: Agonist-Induced GPCR Shedding from the Ciliary Surface Is Dependent on ESCRT-III and VPS4

Membrane trafficking of G protein-coupled receptors (GPCRs) is crucial for temporal and spatial control of cell-surface GPCR signaling. Receptor internalization is a well-documented method cells use for regulating a wide variety of GPCRs following their exposure to agonists. We report that, upon agonist stimulation, a GPCR called vasoactive intestinal peptide receptor 2 (VPAC2) is shed, rather than being internalized, in vitro and in vivo, from the membrane of primary cilia--solitary hair-like organelles that project from the cell surface. VPAC2 is released into the extracellular milieu in the form of ciliary ectosomes that are devoid of exosome markers. The agonist-induced VPAC2 shedding is selective, as shown by the fact that other ciliary membrane proteins including two ciliary GPCRs are not shed with VPAC2. VPAC2 ectosome shedding is dependent on several components of endosomal sorting complexes required for transport (ESCRT), including a subset of ESCRT-III, VPS4, and LIP5. Agonist-stimulated VPAC2 is important for ciliary-ectosome generation because it allows VPS4 and LIP5 to transiently accumulate in primary cilia. Shedding of VPAC2 from the ciliary surface results in termination of intracellular VPAC2 signaling. Agonist-induced GPCR shedding from the ciliary surface may represent an additional mode of GPCR trafficking and signal regulation.

Arresting times for PTEN

Arresting times for PTEN

The FPR2-induced rise in cytosolic calcium in human neutrophils relies on an emptying of intracellular calcium stores and is inhibited by a gelsolin-derived PIP2-binding peptide

BackgroundThe molecular basis for neutrophil recognition of chemotactic peptides is their binding to specific G-protein-coupled cell surface receptors (GPCRs). Human neutrophils express two pattern recognition GPCRs, FPR1 and FPR2, which belong to the family of formyl peptide receptors. The high degree of homology between these two receptors suggests that they share many functional and signal transduction properties, although they exhibit some differences with respect to signaling. The aims of this study were to determine whether FPR2 triggers a unique signal that allows direct influx of extracellular calcium without the emptying of intracellular calcium stores, and whether the gelsolin-derived PIP2-binding peptide, PBP10, selectively inhibits FPR2-mediated transient rise in intracellular Ca2+.ResultsThe transient rise in intracellular Ca2+ induced by agonists for FPR1 or FPR2 in human neutrophils occurred also in the presence of a chelator of Ca2+ (EGTA). PBP10 inhibited not only FPR2-induced oxidase activity, but also the transient rise in intracellular Ca2+.ConclusionsCa2+ signaling mediated via FPR2 follows the same route as FPR1, which involves initial emptying of the intracellular stores. PBP10 inhibits selectively the signals generated by FPR2, both with respect to NADPH-oxidase activity and the transient rise in intracellular Ca2+ induced by agonist exposure.

Activation of heterotrimeric G-proteins independent of a G-protein coupled receptor and the implications for signal processing

Heterotrimeric G-proteins are key transducers for signal transfer from outside the cell, mediating signals emanating from cell-surface G-protein coupled receptors (GPCR). Many, if not all, subtypes of heterotrimeric G-proteins are also regulated by accessory proteins that influence guanine nucleotide binding, guanosine triphosphate (GTP) hydrolysis, or subunit interactions. One subgroup of such accessory proteins (activators of G-protein signaling; AGS proteins) refer to a functionally defined group of proteins that activate selected G-protein signaling systems in the absence of classical G-protein coupled receptors. AGS and related proteins provide unexpected insights into the regulation of the G-protein activation–deactivation cycle. Different AGS proteins function as guanine nucleotide exchange factors or guanine nucleotide dissociation inhibitors and may also influence subunit interactions by interaction with Gβγ. These proteins play important roles in the generation or positioning of signaling complexes and of the regulation of GPCR signaling, and as alternative binding partners for G-protein subunits. Perhaps of even broader impact is the discovery that AGS proteins provide a foundation for the concept that heterotrimeric G-protein subunits are processing signals within the cell involving intrinsic cues that do not involve the classical signal input from a cell surface GPCR.

Rapid, Nongenomic Responses to Ecdysteroids and Catecholamines Mediated by a NovelDrosophilaG-Protein-Coupled Receptor

Nongenomic response pathways mediate many of the rapid actions of steroid hormones, but the mechanisms underlying such responses remain controversial. In some cases, cell-surface expression of classical nuclear steroid receptors has been suggested to mediate these effects, but, in a few instances, specific G-protein-coupled receptors (GPCRs) have been reported to be responsible. Here, we describe the activation of a novel, neuronally expressed Drosophila GPCR by the insect ecdysteroids ecdysone (E) and 20-hydroxyecdysone (20E). This is the first report of an identified insect GPCR interacting with steroids. The Drosophila melanogaster dopamine/ecdysteroid receptor (DmDopEcR) shows sequence homology with vertebrate beta-adrenergic receptors and is activated by dopamine (DA) to increase cAMP levels and to activate the phosphoinositide 3-kinase pathway. Conversely, E and 20E show high affinity for the receptor in binding studies and can inhibit the effects of DA, as well as coupling the receptor to a rapid activation of the mitogen-activated protein kinase pathway. The receptor may thus represent the Drosophila homolog of the vertebrate "gamma-adrenergic receptors," which are responsible for the modulation of various activities in brain, blood vessels, and pancreas. Thus, DmDopEcR can function as a cell-surface GPCR that may be responsible for some of the rapid, nongenomic actions of ecdysteroids, during both development and signaling in the mature adult nervous system.

Signaling and cellular mechanisms in cardiac protection by ischemic and pharmacological preconditioning.

Ischemic preconditioning (IPC) is a defensive adaptive cellular phenomenon. Brief ischemic stimuli render the heart resistant to subsequent similar stress. Signaling for IPC and pharmacologically induced preconditioning involves several G-protein coupled cell surface receptors, second messengers, specific Ser-Thr-protein kinase-C isoforms, Tyr-kinases, and finally, results in activation of ATP-dependent potassium channels (inward rectifiers) at the sarcolemma and in the mitochondria. In cardiomyocytes these channels regulate cytosolic and mitochondrial Ca2+ levels. K+ influx into mitochondria proves to be a key factor for keeping the mitochondrial permeability transition pore closed. This ensures continuous energy production and prevents cell death by apoptosis or necrosis. Molecular structure, function, and pharmacological properties of the ATP-dependent potassium channels and of the mitochondrial permeability transition pore are discussed. Channel activating agents mimic IPC and also affect reactive oxygen species producing enzymes involved in mitochondrial respiration. Volatile anesthetics, among other drugs, mimic the cardioprotective effects of IPC. Their intracellular signaling and clinical application are briefly discussed.

Molecular cloning and characterization of the canine prostaglandin E receptor EP2 subtype

Prostaglandin E 2 (PGE 2) binds to four G-protein coupled cell surface receptors (EP1–EP4) and has been implicated as a local mediator of bone anabolism via a cyclic AMP mediated pathway following activation of the EP2 and/or EP4 receptor subtype. A canine kidney cDNA library was screened using a human EP2 probe, and a clone with an open reading frame of 1083 bp, potentially encoding a protein of 361 amino acids, was characterized. This open reading frame has 89% identity to the human EP2 cDNA at the nucleotide level and 87% identity at the predicted protein level. Scatchard analysis of a CHO cell line stably transfected with canine EP2 yielded a dissociation constant of 22 nM for PGE 2. Competition binding studies, using 3H-PGE 2 as ligand, demonstrated specific displacement by PGE 2, Prostaglandin E 1, Prostaglandin A 3, and butaprost (an EP2 selective ligand), but not by ligands with selectivity for the related DP, FP, IP, or TP receptors. Specific ligand binding also resulted in increased levels of cAMP in EP2 transfected cells with no evidence of short-term, ligand-induced desensitization. Northern blot analysis revealed two transcripts of 3300 and 2400 bp in canine lung, and reverse-transcription polymerase chain reaction showed expression in all tissues examined. Southern blot analysis suggests the presence of a single-copy gene for EP2 in the dog.

Interaction of PAK with Rac: determination of a minimum binding domain on PAK.

Rac and Cdc42 are both members of the Rho family of GTP binding proteins involved in signal transduction pathways linking activation of growth-factor or G-protein coupled cell surface receptors to cytoskeletal re-organisation and activation of the stress responsive JNKk3APK pathway [l]. p21 activated kinase (PAK) is a serin&nine kinase with a molecular mass of 65kDa which is activated by autophosphorylation following interaction with the GTP forms of Rac and Cdc42 [2]. Sequence homology studies suggested that PAK residues 75-94 contain a motif required for Rac binding, however using a filter binding assay the smallest region of PAK reported to bind strongly to Rac is 67-150 [3,4]. We have studied the Rac/PAK interaction using a scintillation proximity assay designed to measure protein-protein interactions [5] with the aim of determining the smallest PAK domain required for binding to Rac.