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At least 685 records · Page 38Linked to original sources
PubMed2006

Molecular dynamics of a biophysical model for beta2-adrenergic and G protein-coupled receptor activation.

This study analyzes 16 molecular dynamic simulations of a biophysical model for beta(2)-adrenergic (B2AR) and G protein-coupled receptor (GPCR) activation. In this model, a highly conserved cysteine residue, C106 (C3.25 or CysIII:01), provides a free sulfhydryl or thiol group in an acid-base equilibrium between uncharged (RSH) and charged (RS(-)) states that functions as an electrostatic molecular switch for receptor activation. The transition of C106 in the B2AR between acid and base states significantly changes the helical/transmembrane (TM) domain interactions and the electrostatic interaction energy differences (DeltaDeltaE(EL)). The DeltaDeltaE(EL) changes correlate well with the experimentally observed ligand efficacies. The TM interaction energies display patterns compatible with those previously recognized as responsible for GPCR activation. Key differences between the agonist, epinephrine, and the antagonist, pindolol, are seen for the TM3 x 6, TM3 x 4, TM6 x 7 and TM1 x 7 interaction energies. Pindolol also produces a weaker DeltaDeltaE(EL) interaction and less TM interaction energy changes, which are important differences between the agonist and antagonist ligands. The D115E mutant with pindolol displays a greater DeltaDeltaE(EL) and TM interactions than for the wild-type B2AR with pindolol. This explains the higher activity of pindolol in the D115E mutant. The constitutively active D130A mutant displays TM interaction patterns similar to those for the activating ligands implying a common pattern for receptor activation. These findings support the broad concept of protean agonism and demonstrate the potential for allosteric modulation. They also demonstrate that this two-state model agrees with many previous experimental and theoretical observations of GPCRs.

Amino Acid Sequence↗
PubMed2003

The [35S]GTPgammaS binding assay: approaches and applications in pharmacology.

Receptors of the of seven transmembrane spanning, heterotrimeric G protein coupled family (GPCR) play crucial roles in regulating physiological functions and consequently are targets for the action of many classes of drugs. Activation of receptor by agonist leads to the dissociation of GDP from Galpha of the Galphabetagamma heterotrimer, followed by the binding of GTP to Galpha and subsequent modulation of downstream effectors. The G protein heterotrimer is reformed by GTPase activity of the Galpha subunit, forming Galpha-GDP and so allowing Galpha and Gbetagamma to recombine. The [35S]GTPgammaS assay measures the level of G protein activation following agonist occupation of a GPCR, by determining the binding of the non-hydrolyzable analog [35S]GTPgammaS to Galpha subunits. Thus, the assay measures a functional consequence of receptor occupancy at one of the earliest receptor-mediated events. The assay allows for traditional pharmacological parameters of potency, efficacy and antagonist affinity, with the advantage that agonist measures are not subjected to amplification or other modulation that may occur when analyzing parameters further downstream of the receptor. In general the assay is experimentally more feasible for receptors coupled to the abundant G(i/o) proteins. Nevertheless, [35S]GTPgammaS binding assays are used with GPCRs that couple to the G(s) and G(q) families of G proteins, especially in artificial expression systems, or using receptor-Galpha constructs or immunoprecipitation of [35S]GTPgammaS-labeled Galpha. The relative simplicity of the assay has made it very popular and its use is providing insights into contemporary pharmacological topics including the roles of accessory proteins in signaling, constitutive activity of receptors and agonist specific signaling.

Animals↗
PubMed2003

Homo- and hetero-oligomerization of G protein-coupled receptors.

G protein-coupled receptors (GPCRs) form homo-oligomeric and hetero-oligomeric complexes. This understanding has prompted a re-evaluation of many aspects of GPCR biology, however the concept of receptor complexes has not been fully integrated into the current thinking about GPCR structure and function. Nevertheless, receptor oligomerization is a pivotal aspect of the structure and function of GPCRs that has been shown to have implications for receptor trafficking, signaling, and pharmacology and more intricate models for understanding the physiological roles of these receptors are emerging. Here, we summarize some of the advances made in understanding the structural basis and the functional roles of homo- and hetero- oligomerization in this important group of receptors. Although this discussion focuses primarily on the dopamine receptors, particularly the D2 dopamine receptor, and the opioid and serotonin receptors, we discuss the principles governing the oligomerization of all rhodopsin-like GPCRs and potentially of the entire superfamily of these receptors.

Animals↗
PubMed2004

Novel roles for arrestins in the post-endocytic trafficking of G protein-coupled receptors.

G protein-coupled receptors (GPCRs) represent the largest family of transmembrane signaling molecules in the human genome. As such, they interact with numerous intracellular molecules, which can act either to propagate or curtail signaling from the receptor. Their primary mode of cellular activation occurs through heterotrimeric G proteins, which in turn can activate a wide spectrum of effector molecules, including phosphodiesterases, phospholipases, adenylyl cyclases and ion channels. Active GPCRs are also the target of G protein-coupled receptor kinases, which phosphorylate the receptors culminating in the binding of the protein arrestin. This results in rapid desensitization through inhibition of G protein binding, as well as novel mechanisms of cellular activation that involve the scaffolding of cellular kinases to GPCR-arrestin complexes. Arrestins can also serve to mediate the internalization of certain GPCRs, a process which plays an important role in regulating cellular activity both by mediating long-term desensitization through down regulation (degradation) of receptors and by recycling desensitized receptors back to the cell surface to initiate additional rounds of signaling. The mechanisms that regulate the subsequent intracellular trafficking of GPCRs following internalization are largely unknown. Recently however, it has become clear that the pattern of receptor phosphorylation and subsequent binding of arrestin play a critical role in the intracellular trafficking of internalized receptors, thereby dictating the ultimate fate of the receptor. In addition, arrestins have now been shown to be required for the recycling of GPCRs that are capable of internalizing through arrestin-independent mechanisms. This review will summarize recent advances in our understanding of the roles of arrestins in post-endocytic GPCR trafficking.

Animals↗
PubMed2005

Cannabinoid receptor homo- and heterodimerization.

CB1 cannabinoid receptors mediate the psychoactive effects of Delta(9)THC and actions of the endogenous cannabinoids [Howlett, A.C., Barth, F., Bonner, T.I., Cabral, G., Casellas, P., Devane, W.A., Felder, C.C., Herkenham, M., Mackie, K., Martin, B.R., Mechoulam, R., Pertwee, R.G., 2002. International Union of Pharmacology: XXVII. Classification of cannabinoid receptors. Pharmacological Reviews 54 (2) 161-202.]. CB1 receptors belong to the G protein-coupled receptor (GPCR) superfamily. In recent years, it has become apparent that many GPCRs exist as multimers--either of like or unlike receptors [Kroeger, K.M., Pfleger, K.D., Eidne, K.A., 2003. G-protein coupled receptor oligomerization in neuroendocrine pathways. Frontiers of Neuroendocrinology 24 (4) 254-278; Milligan, G., 2004. G protein-coupled receptor dimerization: function and ligand pharmacology. Molecular Pharmacology 66 (1) 1-7.]. Importantly, GPCR multimerization plays a key role in enriching the signaling repertoire of these receptors. In this review, the evidence for CB1 multimerization will be presented, the implications for cannabinoid signaling discussed, and possible future directions for this research considered.

Dimerization↗
PubMed2004

Human follitropin receptor (FSHR) interacts with the adapter protein 14-3-3tau.

The human follitropin (follicle stimulating hormone, FSH) receptor (FSHR) is a G protein-coupled receptor (GPCR). To identify cytoplasmic proteins that may regulate FSHR function, a yeast-based interaction trap was performed. A linked construct of the first and second intracellular loops (iL1-iL2 bait) of FSHR was used as bait and a human ovarian cDNA library was used as prey. Among the proteins identified that interacted with the bait was 14-3-3tau, a member of a family of homodimeric cytoplasmic adapter proteins. Human granulosa cells, the site of FSHR expression in the ovary, were found to contain 14-3-3tau. Importantly, 14-3-3tau co-immunoprecipitated with FSHR stably expressed in HEK 293 cells. Its association with FSHR was follitropin-dependent. Over-expression of 14-3-3tau resulted in a modest decrease of follitropin-induced cAMP accumulation. Collectively, these data support a role for 14-3-3tau in follitropin action. The finding that 14-3-3tau interacts with FSHR is novel and should lead to new insights into the regulation of GPCR in general and FSHR specifically.

14-3-3 Proteins↗
PubMed2006

A role for Fyn in Trk receptor transactivation by G-protein-coupled receptor signaling.

Signaling through Trk receptor tyrosine kinases can occur in the absence of neurotrophins through certain G-protein-coupled receptors (GPCRs). It has previously been suggested that GPCR-mediated Trk activation occurs on intracellular membranes and involves several second messengers, including Src family kinases and intracellular calcium. Here, we describe a novel role for the Src family kinase, Fyn, in regulating signaling events between GPCRs and Trk. We find that Fyn expression is sufficient to allow transactivation of Trk by adenosine and that Fyn and Trk are colocalized in a juxtanuclear membrane compartment. Adenosine activation of Fyn results in direct phosphorylation of Trk in vitro and follows a delayed time course that coincides with Trk activation. These results indicate that Fyn is activated by GPCR stimulation and is responsible for transactivation of Trk receptors on intracellular membranes.

Adenosine↗
PubMed2005

PDF receptor signaling in Drosophila contributes to both circadian and geotactic behaviors.

The neuropeptide Pigment-Dispersing Factor (PDF) is a principle transmitter regulating circadian locomotor rhythms in Drosophila. We have identified a Class II (secretin-related) G protein-coupled receptor (GPCR) that is specifically responsive to PDF and also to calcitonin-like peptides and to PACAP. In response to PDF, the PDF receptor (PDFR) elevates cAMP levels when expressed in HEK293 cells. As predicted by in vivo studies, cotransfection of Neurofibromatosis Factor 1 significantly improves coupling of PDFR to adenylate cyclase. pdfr mutant flies display increased circadian arrhythmicity, and also display altered geotaxis that is epistatic to that of pdf mutants. PDFR immunosignals are expressed by diverse neurons, but only by a small subset of circadian pacemakers. These data establish the first synapse within the Drosophila circadian neural circuit and underscore the importance of Class II peptide GPCR signaling in circadian neural systems.

Adrenomedullin↗
PubMed2006

RGS expression rate-limits recovery of rod photoresponses.

Signaling through G protein-coupled receptors (GPCRs) underlies many cellular processes, yet it is not known which molecules determine the duration of signaling in intact cells. Two candidates are G protein-coupled receptor kinases (GRKs) and Regulators of G protein signaling (RGSs), deactivation enzymes for GPCRs and G proteins, respectively. Here we investigate whether GRK or RGS governs the overall rate of recovery of the light response in mammalian rod photoreceptors, a model system for studying GPCR signaling. We show that overexpression of rhodopsin kinase (GRK1) increases phosphorylation of the GPCR rhodopsin but has no effect on photoresponse recovery. In contrast, overexpression of the photoreceptor RGS complex (RGS9-1.Gbeta5L.R9AP) dramatically accelerates response recovery. Our results show that G protein deactivation is normally at least 2.5 times slower than rhodopsin deactivation, resolving a long-standing controversy concerning the mechanism underlying the recovery of rod visual transduction.

Animals↗
PubMed2005

Exceptional total and functional yields of the human adenosine (A2a) receptor expressed in the yeast Saccharomyces cerevisiae.

The yeast Saccharomyces cerevisiae was used to express a medically relevant G-protein coupled receptor (GPCR), the human adenosine (A2a) receptor, with a C-terminal green fluorescent protein (GFP) fusion tag. In prior studies, we established an expression system for A2a-GFP. Here, we quantified the total A2a-GFP expression levels by correlating GFP levels as detected by fluorescence and densitometry to A2a-GFP molecules overexpressed in the system. We also quantified A2a-GFP functional levels by classical radioligand binding assays. Approximately, 120,000 functional A2a-GFP molecules per cell were present on the plasma membrane as determined by radioligand binding. Using whole cell GFP fluorescence, 340,000 A2a-GFP molecules per cell were detected; approximately 70% of those molecules were plasma membrane localized, as determined by using confocal microscopy analysis. These results show that a significant portion of the total expressed protein is functional. In addition, the quick and inexpensive whole cell fluorescence appears to provide a good approximation of functional receptor numbers for this case. Importantly, the amount of functionally expressed A2a-GFP per culture ( approximately 4 mg/L) is among the highest reported for any GPCR in any expression system.

Green Fluorescent Proteins↗
PubMed2005

Large-scale purification and characterization of human parathyroid hormone-1 receptor stably expressed in HEK293S GnTI- cells.

Human parathyroid hormone-1 receptor (hPTHR1) belongs to class II of the G protein-coupled receptor (GPCR) family, whose members all contain a seven-transmembrane helix domain. The receptor regulates bone metabolism through interactions with its ligand, human parathyroid hormone (hPTH). For structural studies of the hPTHR1/hPTH complex, we constructed a mammalian cell line to stably express recombinant hPTHR1 in large-scale. The receptor was solubilized with dodecyl maltoside and purified with affinity chromatography. The purified receptor displayed restricted N-glycosylation as expected. Functionality was demonstrated: the hPTHR1 retained affinity for bPTH-(1-34) and specifically cross-linked to a radioiodinated bPTH-(1-34) analog. This work describes an approach for preparing milligram-scale quantities of receptor for elucidation of the structural biology of this seven-transmembrane GPCR.

Bioreactors↗
PubMed2004

The alpha-factor mating pheromone of Saccharomyces cerevisiae: a model for studying the interaction of peptide hormones and G protein-coupled receptors.

Mating in Saccharomyces cerevisiae is initiated by the secretion of diffusible peptide pheromones that are recognized by G protein-coupled receptors (GPCR). This review summarizes the use of the alpha-factor (WHWLQLKPGQPMY)--GPCR (Ste2p) interaction as a paradigm to understand the recognition between medium-sized peptide hormones and their cognate receptors. Studies over the past 15 years have indicated that the alpha-factor is bent around the center of the pheromone and that residues near the amine terminus play a central role in triggering signal transduction. The bend in the center appears not to be rigid and this flexibility is likely necessary for conformational changes that occur as the receptor switches from the inactive to active state. The results of synthetic, biological, biochemical, molecular biological, and biophysical analyses have led to a preliminary model for the structure of the peptide bound to its receptor. Antagonists for Ste2p have changes near the N-terminus of alpha-factor, and mutated forms of Ste2p were discovered that appear to favor binding of these antagonists relative to agonists. Many features of this yeast recognition system are relevant to and have counterparts in mammalian cells.

Alanine↗
PubMed2003

Fluorescent ligands, antibodies, and proteins for the study of receptors.

Fluorescent molecules bound to receptors can show their location and, if binding is reversible, can provide pharmacological information such as affinity and proximity between interacting molecules. The spatial precision offered by visualisation transcends the diverse localisation and low molecular concentration of receptor molecules. Consequently, the relationships between receptor location and function and life cycles of receptors have become better understood as a result of fluorescent labeling. Each of these aspects contributes new insights to drug action and potential new targets. The relationships between spatial distribution of receptor and function are largely unknown. This is particularly apparent for native receptors expressed in their normal host tissues where communication between heterogeneous cell types influences receptor distribution and function. In cultured cell systems, particularly for G-protein-coupled receptors (GPCR), fluorescence-based methods have enabled the visualisation of the cycle of agonist-stimulated receptor clustering, endocytic internalisation to the perinuclear region, degradation of the receptor-ligand complex, and recycling back to the surface membrane. Using variant forms of green fluorescent protein (GFP), antibodies, or fluorescent ligands, it is possible to detect or visualise the formation of oligomeric receptor complexes. Careful selection of fluorescent molecules based on their spectral properties enables resonance energy transfer and multilabel visualisation with colocalisation studies. Fluorescent agonist and antagonist ligands are now being used in parallel with GFP to study receptor cycling in live cells. This review covers how labeling and visualisation technologies have been applied to the study of major pharmacologically important receptors and illustrates this by giving examples of recent techniques that have relied on GFP, antibodies, or fluorescent ligands alone or in combination for the purpose of studying GPCR.

Animals↗
PubMed2004

A glance at G-protein-coupled receptors for lipid mediators: a growing receptor family with remarkably diverse ligands.

A plethora of lipid-like molecules known to act as intracellular second messengers are now recognized to signal cells through plasma membrane 7 transmembrane G-protein-coupled receptors (GPCRs). This has been the result of a decade-long genetic hunt for novel sequences encoding 7 transmembrane receptor proteins and the efforts to pair novel sequences with biologically active substances of (partly) unknown molecular mechanism of action. Identification of novel GPCR ligand pairs represents the first step to shed more light into the mode of action of novel cellular signaling molecules in human health and disease and might represent a fruitful source for the development of new drugs, judged on the successful history of GPCR as drug targets. Since 2000, more than 16 reports became available on lipid mediators--as diverse as lysophospholipids, arachidonic acid metabolites, short-, medium-, and long-chain fatty acids as well as steroid-like molecules--exerting their effects as extracellular mediators via rhodopsin-like family GPCRs. These reports have opened new avenues for research in human lipid receptor physiology and pharmacology. Here, the current knowledge on the recently deorphanized lipid receptors, including their isolation, expression pattern, function, and possible physiological or pathological roles will be reviewed.

Animals↗
PubMed2006

Regulation of metabotropic glutamate receptor signaling, desensitization and endocytosis.

Metabotropic glutamate receptors (mGluRs) comprise a unique family of G protein-coupled receptors (GPCR) that can be classified into 3 groups based on G protein coupling specificity and sequence similarity. Group I mGluRs (mGluR1 and mGluR5) are coupled to the heterotrimeric G protein Galpha(q/11) and trigger the release of calcium from intracellular stores. In the present review, we discuss the molecular mechanisms involved in the desensitization and endocytosis of group I mGluRs. Group I mGluRs desensitize in response to both second-messenger-dependent protein kinases and G protein-coupled receptor kinases (GRK). However, GRK2-mediated mGluR1 desensitization appears to be both phosphorylation- and beta-arrestin-independent. In addition to GRK-mediated uncoupling of mGluRs from heterotrimeric G proteins, the huntingtin-interacting protein, optineurin, also contributes to mGluR1 and mGluR5 desensitization. The G protein-uncoupling activity of optineurin appears to be facilitated by the presence of polyglutamine-expanded mutant huntingtin but not wild-type huntingtin. Group I mGluRs also undergo both agonist-dependent and -independent endocytosis in both heterologous cell expression systems and primary neuronal cultures. The present review overviews the current understanding of the contribution of second messenger-dependent protein kinases, beta-arrestins and a novel Ral/phospholipase D2 (PLD2)-mediated endocytic pathway to the regulation of Group I mGluR endocytosis. Overall, the regulation of Group I mGluR desensitization and endocytosis appears to be mediated by the same molecular intermediates as have been described for more typical GPCR such as the beta(2)-adrenergic receptor. However, there appears to be subtle, but important, differences in the mechanisms by which these intermediates are employed to regulate Group I mGluR desensitization and endocytosis.

Animals↗
PubMed2006

Proteome analysis to study signal transduction of G protein-coupled receptors.

G protein-coupled receptors (GPCR) play an important role in drug development. Although many classical signal transduction pathways have been elucidated, more and more cross-talk to other cascades, e.g. MAP-kinase have been reported. In order to identify the overall function of receptor stimulation in a specific cell type or under certain conditions proteome analysis has been shown to be a very successful and powerful approach. Here, we will summarize the current state of the art of proteome analysis applied to GPCR.

Animals↗
PubMed2005

Gene ancestry of the cannabinoid receptor family.

Genome sequencing projects, and their available resources, have revealed two distinct genes encoding cannabinoid receptors, CB(1) and CB(2). Biochemical evidence in support of a third cannabinoid receptor includes signal transduction events and vasodilation in the vasculature of cannabinoid receptor knockout mice after exposure to the endogenous cannabinoid, anandamide. In addition, a nonpsychoactive ingredient in marijuana, abnormal cannabidiol, which does not activate the two characterized cannabinoid receptor homologues, has been shown to induce vasodilation in the endothelium. Our work distinguishes the biochemical differences by way of a phylogenetic analysis of cannabinoid receptors. Recently a putative orthologue to CB(1) and CB(2) has been identified in the urochordate, Ciona intestinalis, indicating the presence of cannabinoid receptors previous to the evolution of vertebrates. Moreover, the Ciona sequence shares equal identity to both cannabinoid paralogous sequences and no other GPCR sequence identified in an exhaustive database search is as similar. We propose that, although an alternate cannabinergic-activating pathway may be present, it does not include a GPCR (or other receptor type) phylogenetically related to the CB(1)/CB(2)Ciona lineage.

Animals↗
PubMed2005

Chemical approaches to the lysophospholipid receptors.

Both ligand-based and GPCR privileged scaffold chemical tools have recently emerged to provide new insights into the function and physiology of the GPCR lysophospholipid receptors both in vitro and in vivo. Both rational, design-based approaches as well as hybrid approaches where high throughput screening has been coupled to an understanding of critical molecular interactions have been productive in advancing understanding of physiology and potential therapeutics in this field. It is now feasible to identify reasonably potent and selective small molecules that provide chemical proof-of-concept in vivo directly from high throughput screening. These developments, coupled with the availability of receptor knock-out mice, presage rapid progress in the field.

Animals↗