Frontiers in GPCR research.
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Chemistry, Pharmaceutical↗
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Ege A, a novel C2 domain containing protein, is essential for GPCR-mediated gene expression in dictyostelium.
During early stages of development, expression of aggregative genes in Dictyostelium is regulated by G protein-linked signaling pathways. We have isolated an aggregation-deficient mutant from a restriction enzyme-mediated insertional mutagenesis screen and have obtained its cDNA. Since the mutant expresses prestarvation genes but fails to express early genes, such as cAR1 and GP80, during development, we designated it early gene expression A (ege A). Ege A, encoding a cytosolic protein of 26 kDa, along with Ege B, belongs to a novel C2 domain-containing gene family. While Ege A mRNA is expressed during the first 2 h of development, Ege B is expressed at later stages. Ege A is not directly required for either G protein-mediated actin polymerization or activation of adenylyl cyclase. Ege A overexpressing and ege A(-) cells display similar phenotypes, suggesting that an optimal level of Ege A is required for proper function. Constitutive expression of a fully functional cAR1-YFP enables ege A(-) cells to form loose aggregates, but cAR1-YFP/ege A(-) cells are still unable to express GP80, suggesting that losses of gene expression were not solely due to a lack of cAR1. Overexpression of PKAcat, the constitutively active subunit of PKA, does not rescue the ege A(-) phenotype, suggesting that PKA is not located downstream from Ege A in the signaling pathway. We propose that Ege A is a novel cytosolic component required by early gene expression.
Adenylyl Cyclases↗
A hypothesis for GPCR activation.
Growing evidence that rhodopsin (RD) and related G protein-coupled receptors form functional dimers/oligomers, followed by direct proof (using atomic force microscopy) that in the retina disc membrane RD associates into a paracrystalline network of rows of dimers, need models of the RD-transducin (Gt) complex that would envision an optimal RD dimer/oligomer able to satisfy all well-documented interactions with Gt. Of the models proposed so far, only a few refer to RD dimers and only one of them proposes a complex of Gt with an RD oligomer (Filipek S, Krzyśko KA, Fotiadis D, Liang Y, Saperstein DA, Engel, A, Palczewski K Photochem Photobiol Sci 3: 628-638, 2004). This paper puts forward a hypothesis on another arrangement of RD monomers into the reported network of rows of dimers. Arguments for the compatibility of this set-up with interactions and activation of RD in the complex with Gt, in particular, with the well-documented movement of transmembrane helix 6 and cytosolic loop 3, which is vital for RD activation, are provided and discussed.
Dimerization↗
Farnesyl phosphates are endogenous ligands of lysophosphatidic acid receptors: inhibition of LPA GPCR and activation of PPARs.
Oligoprenyl phosphates are key metabolic intermediates for the biosynthesis of steroids, the side chain of ubiquinones, and dolichols and the posttranslational isoprenylation of proteins. Farnesyl phosphates are isoprenoid phosphates that resemble polyunsaturated fatty alcohol phosphates, which we have recently shown to be the minimal pharmacophores of lysophosphatidic acid (LPA) receptors. Here we examine whether farnesyl phosphates can interact with the cell surface and nuclear receptors for LPA. Both farnesyl phosphate and farnesyl diphosphate potently and specifically antagonized LPA-elicited intracellular Ca(2+)-mobilization mediated through the LPA(3) receptor, while causing only modest inhibition at the LPA(2) receptor and no measurable effect at the LPA(1) receptor. Farnesol also inhibited LPA(3) but was much less effective. The estimated dissociation constant of LPA(3) for farnesyl phosphate is 48+/-12 nM and 155+/-30 nM for farnesyl diphosphate. The transcription factor peroxisome proliferator-activated receptor gamma (PPARgamma) binds to and is activated by LPA and its analogs including fatty alcohol phosphates. We found that both farnesyl phosphate and diphosphate, but not farnesol, compete with the binding of the synthetic PPARgamma agonist [(3)H]rosiglitazone and activate the PPARgamma-mediated gene transcription. Farnesyl monophosphate at 1 microM, but not diphosphate, activated PPARalpha and PPARbeta/delta reporter gene expression. These results indicate new potential roles for the oligoprenyl phosphates as potential endogenous modulators of LPA targets and show that the polyisoprenoid chain is recognized by some LPA receptors.
Calcium Signaling↗
The challenge of drug discovery of a GPCR target: analysis of preclinical pharmacology of histamine H3 antagonists/inverse agonists.
Although the histamine H(3) receptor was identified pharmacologically in 1983, and despite widespread pharmaceutical interest in the target, no compound interacting specifically with this site has undergone successful clinical examination to develop the necessary proof-of-concept data. Therefore, clinical knowledge of the therapeutic potential of H(3) receptor antagonists in neuropsychiatric diseases, in metabolic diseases or in sleep disorders has yet to determine if the preclinical data that show broad efficacy in animal models of the aforementioned states are relevant to current unmet medical needs. H(3) receptors are complex, with species-related sequence differences that impact pharmacological responses. The receptors have a complex gene organization that provides opportunity for multiple slice isoforms, most of which remain poorly characterized even within a species. H(3) receptors are constitutively active, although the extent of this could vary either between species and/or receptor splice isoforms, both of which may provide opportunity for preferential coupling to different G-proteins. Thus, it is not surprising that the pharmacological effects of known H(3) ligands are complex and diverse, since these agents may act both as agonists and antagonists in different systems. Moreover, other compounds show inverse agonism in some models but neutral antagonist activity in others. Some of this diversity may be related to different ligand-dependent receptor activation states or to the effects of key amino acids important for ligand recognition. This commentary provides an overview of these complexities as applied to the H(3) receptor and the challenges these intricacies create for drug discovery.
Animals↗
Uncoupling of GPCR and RhoA-induced Ca2+-sensitization of chicken amnion smooth muscle lacking CPI-17.
Ca2+-sensitization of smooth muscle occurs through inhibition of myosin light chain phosphatase (MLCP) leading to an increase in the MLCK:MLCP activity ratio. MLCP is inhibited through phosphorylation of its regulatory subunit (MYPT-1) following activation of the RhoA/Rho kinase (ROK) pathway or through phosphorylation of the PP1c inhibitory protein, CPI-17, by PKC delta or ROK. Here, we explore the crosstalk between these two modes of MLCP inhibition in a smooth muscle of a natural CPI-17 knockout, chicken amnion. GTPgammaS elicited Ca2+-sensitized force which was relaxed by GDI or Y-27632, however, U46619, carbachol and phorbol ester failed to induce Ca2+-sensitized force, but were rescued by recombinant CPI-17, and were sensitive to Y-27632 inhibition. In the presence, but not absence, of CPI-17, U46619 also significantly increased GTP.RhoA. There was no affect on MYPT-1 phosphorylation at T695, however, T850 phosphorylation increased in response to GTPgammaS stimulation. Together, these data suggest a role for CPI-17 upstream of RhoA activation possibly through activation of another PP1 family member targeted by CPI-17.
15-Hydroxy-11 alpha,9 alpha-(epoxymethano)prosta-5↗
FRET analysis reveals a critical conformational change within the Na,K-ATPase alpha1 subunit N-terminus during GPCR-dependent endocytosis.
Dopamine is a major regulator of sodium reabsorption in proximal tubule epithelia. It induces the endocytosis of plasma membrane Na,K-ATPase molecules, and this results in a reduced capacity of the cells to transport sodium. Dopamine induces the phosphorylation of Ser-18 in the alpha1-subunit of Na,K-ATPase. Fluorescence resonance energy transfer analysis of cells expressing YFP-alpha1 and beta1-CFP reveals that treatment of the cells with dopamine increases energy transfer between CFP and YFP. This is consistent with a protein conformational change that results in the N-terminal end of alpha1 moving closer to the internal face of the plasma membrane.
Alkaloids↗
Drosophila GPCR Han is a receptor for the circadian clock neuropeptide PDF.
The pigment-dispersing factor (PDF) is a neuropeptide controlling circadian behavioral rhythms in Drosophila, but its receptor is not yet known. From a large-scale temperature preference behavior screen in Drosophila, we isolated a P insertion mutant that preferred different temperatures during the day and night. This mutation, which we named han, reduced the transcript level of CG13758. We found that Han was expressed specifically in 13 pairs of circadian clock neurons in the adult brain. han null flies showed arrhythmic circadian behavior in constant darkness. The behavioral characteristics of han null mutants were similar to those of pdf null mutants. We also found that PDF binds specifically to S2 cells expressing Han, which results in the elevation of cAMP synthesis. Therefore, we herein propose that Han is a PDF receptor regulating circadian behavioral rhythm through coordination of activities of clock neurons.
Amino Acid Sequence↗
Reverse pharmacology of orexin: from an orphan GPCR to integrative physiology.
Orexins, which were initially identified as endogenous peptide ligands for two orphan G-protein coupled receptors (GPCRs), have been shown to have an important role in the regulation of energy homeostasis. Furthermore, the discovery of orexin deficiency in narcolepsy patients indicated that orexins are highly important factors for the sleep/wakefulness regulation. The efferent and afferent systems of orexin-producing neurons suggest interactions between these cells and arousal centers in the brainstem as well as important feeding centers in the hypothalamus. Electrophysiological studies have shown that orexin neurons are regulated by humoral factors, including leptin, glucose, and ghrelin as well as monoamines and acetylcholin. Thus, orexin neurons have functional interactions with hypothalamic feeding pathways and monoaminergic/cholinergic centers to provide a link between peripheral energy balance and the CNS mechanisms that coordinate sleep/wakefulness states and motivated behavior such as food seeking.
Animals↗
GPCR-mediated transactivation of RTKs in the CNS: mechanisms and consequences.
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Animals↗
Techniques: plasmon-waveguide resonance (PWR) spectroscopy as a tool to study ligand-GPCR interactions.
Plasmon-waveguide resonance (PWR) spectroscopy can be applied to integral membrane proteins incorporated into a supported lipid bilayer without the need for labeling. With high sensitivity and wide dynamic range, this technique can be used to characterize the kinetics and thermodynamics of conformational events associated with the binding of ligands to G-protein-coupled receptors (GPCRs), and to directly examine the interactions of GPCRs with G proteins and other downstream effectors in signal transduction. This allows an easy distinction to be made between agonists, antagonists and inverse agonists, and provides a powerful new tool for studying membrane signaling and for drug development.
Ligands↗
The antibody-capture [(35)S]GTPgammaS scintillation proximity assay: a powerful emerging technique for analysis of GPCR pharmacology.
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Antibodies↗
Techniques: Recent developments in computer-aided engineering of GPCR ligands using the human adenosine A3 receptor as an example.
G-protein-coupled receptors (GPCRs) represent the largest known family of signal-transducing molecules, and convey signals for light and many extracellular regulatory molecules. GPCRs are dysfunctional or dysregulated in several human diseases and are estimated to be the targets of >40% of the drugs used in clinical medicine today. The crystal structure of rhodopsin provides the first information on the three-dimensional structure of GPCRs, which now supports homology modeling studies and structure-based drug-design approaches. In this article, we review recent work on adenosine receptors, a family of GPCRs, and, in particular, on adenosine A(3) receptor antagonists. We focus on an iterative, bi-directional approach in which models are used to generate hypotheses that are tested by experimentation; the experimental findings are, in turn, used to refine the model. The success of this approach is due to the synergistic interaction between theory and experimentation.
Animals↗
A renaissance in trace amines inspired by a novel GPCR family.
Trace amines (TAs) are endogenous compounds that are related to biogenic amine neurotransmitters and are present in the mammalian nervous system in trace amounts. Although their pronounced pharmacological effects and tight link to major human disorders such as depression and schizophrenia have been studied for decades, the understanding of their molecular mode of action remained incomplete because of the apparent absence of specialized receptors. However, the recent discovery of a novel family of G-protein-coupled receptors (GPCRs) that includes individual members that are highly specific for TAs indicates a potential role for TAs as vertebrate neurotransmitters or neuromodulators, although the majority of these GPCRs so far have not been demonstrated to be activated by TAs. The unique pharmacology and expression pattern of these receptors make them prime candidates for targets in drug development in the context of several neurological diseases. Current research focuses on dissecting their molecular pharmacology and on the identification of endogenous ligands for the apparently TA-insensitive members of this receptor family.
Animals↗
Principles: a model for the allosteric interactions between ligand binding sites within a dimeric GPCR.
Increasing evidence suggests that most, if not all, G-protein-coupled receptors have the propensity to dimerize. The dimerization process can lead to crosstalk between the protomers and can result in cooperative binding of ligands to these protomers. However, many results suggest that the crosstalk between protomers within a dimer is not permanent. Receptors oscillate between two states: one that enables binding sites to crosstalk and another that does not enable such crosstalk. In this article, the consequences of the equilibrium between these two states on the binding properties of a receptor are analyzed using the predictions of two mathematical models.
Algorithms↗
Techniques: how to boost GPCR mutagenesis studies using yeast.
G-protein-coupled receptors (GPCRs) are the major targets of today's medicines. To elucidate the mechanism of activation of GPCRs and the interaction of these receptors with their G proteins, mutagenesis studies have proven to be a powerful tool and have provided insight into the structure and function of GPCRs. Random mutagenesis is useful in this respect particularly when combined with a robust screening assay that is based on the functional properties of the mutants. In this article, the use of random mutagenesis combined with a functional screening assay in yeast is described and compared with alternative approaches such as site-directed mutagenesis per se, alanine/cysteine scanning and another screening assay, receptor selection and amplification technology (R-SAT). Screening in yeast of randomly mutated GPCRs has proven successful in the identification of ligands for orphan receptors and in high-throughput approaches. Moreover, it has provided substantial insight into G-protein coupling and receptor activation.
Animals↗
Recent developments in constitutive receptor activity and inverse agonism, and their potential for GPCR drug discovery.
The concept of constitutively active G-protein-coupled receptors is now firmly rooted in receptor pharmacology. Many independent research groups have contributed to its acceptance since its introduction by Costa and Herz in 1989. This concept necessitated a revised ligand classification, and a new category of inverse agonists was introduced alongside existing agonist and antagonist ligands. Initially, it was hoped that new therapeutic modalities would become available. However, the drug industry has not adopted inverse agonism as a design criterion and instead accepted that some compounds emerge as (neutral) antagonists in compound screening, whereas other compounds possess inverse agonistic activity. In this article, we summarize aspects of the impact of constitutive activity on the drug-discovery process: for example, its use in orphan receptor assays, its link with pharmacogenetics and genomics, and its relevance for currently marketed drugs.
Alternative Splicing↗
GPCR antagonists as an alternative to COX-2 inhibitors: a case for the PGE2 EP1 receptor.
A recent article supports the concept that prostaglandin (PG)E(2) EP(1)-receptor antagonists reduce stroke severity and cell damage; could these agents become a substitute for cyclooxygenase (COX)-2 inhibitors? The total activity of COXs--rate-limiting enzymes of PGE(2) synthesis--increases following acute neurological insult. Drugs that offer the beneficial anti-inflammatory and neuroprotective effects of PGs but that limit the negative effects of COX-2 inhibition could provide the next generation of treatment for acute neuronal damage.
Cyclooxygenase 2 Inhibitors↗