Abstract

At least 55 types of GPCRs are known to directly mediate neuronal and endocrine regulation of cardiac and vascular responses, and many more influence cardiovascular functions indirectly. In spite of this apparent abundance of potential GPCR drug targets, currently only p-adrenoceptor antagonists (p-blockers) and antagonists of angiotensin II type 1 receptors (AT1 antagonists) are in widespread use in clinical cardiovascular therapeutics. In addition, some other types of receptors for mono-amine...

Neuronal Cell Environment

Neuronal cell environments are highly differentiated and exert strong influences on GPCR function. The study of ligand-receptor interactions in a system that closely mimics the native environment of the receptor in vivo represents an ongoing challenge. Overexpression of GPCRs in recombinant systems can exaggerate agonist efficacy of ligands such that compounds with partial agonist activity in vivo appear to be full agonists. Therefore, the true differentiation of full and partial agonism must...

Platelet Activating Factor Receptor

PAF is an important phospholipid second messenger known to activate platelets, neutrophils, monocytes, and lymphocytes.22 PAF, through activation of its receptor, induces, among other functions, endothelial cell migration and promotes in vivo angiogenesis, thus acting as a mediator of vascularization for tumor growth and metastasis in breast cancer.23,24 Activation of the Gaq-linked PAF receptor in endothelial cells results in stimulation of Src and focal adhesion kinase (Fak), both critical...

Contents

10.1 Introduction Not All 7TMRs are 10.2 Receptor Classification by Ligand 10.2.1 ABC(DEF) Classification of 10.2.2 Four Classes of Ligands Charged, Simple, Flexible, and 10.3 Ligand Diversity Classification Conforms to Receptor Phylogeny 196 10.3.1 GRAFS Classification of 10.3.2 Rhodopsin Subfamilies Bind Different Classes of Ligand 197 10.4 Structural Flexibilities of Membrane 10.4.1 Lack of Hydrophobic Drive in 10.4.2 Cytochrome C Oxidase, Subunit 10.5 Molecular Bioinformatics in Drug 10.5.1...

Concluding Remarks

The cardiovascular therapeutic applications of most GPCR ligands to date are based on two fundamental premises. First, the drugs target the extracellular domains and specifically the ligand binding sites of the receptors, either mimicking the actions of the natural agonist ligands or blocking their access to the binding sites. Second, the receptor proteins have been considered molecular entities invariable in the human population. Both assumptions have recently been challenged. The...

Low Density Lipoprotein Receptor Related Proteins as CoReceptors for FzRs

The low density lipoprotein receptor-related proteins 5 and 6 (LPR5 and LPR6) serve as coreceptors with FzRs in the p-catenin pathway. LPR5 and LPR6 may also bind to secreted Dickkopf (Dkk) proteins that act as antagonists on the FzR-LRP complex by the formation of LPR5 and LPR6 complexes with another recently identified transmembrane receptor class (Kremen) which are internalized, making LPR5 and LPR6 unavailable for complexing with FzRs.105 As a consequence of the binding of Wnt to FzR-LRP...

Calcium Sensing Receptors

The pituitary tumor transforming gene (PTTG) is an oncogene that is normally expressed in few tissues although it is abundantly expressed in the testis. PTTG is widely expressed in cancer,91 and its expression has been directly correlated with both proliferation and angiogenesis. Recently, extracellular calcium (Ca2+) acting on the calcium-sensing receptor (CaR) has been shown to induce a rapid and prolonged upregulation of PTTG mRNA in a nonmetastasizing model for humoral hypercalcemia of...

Homogeneous Non Radioactive Binding Assays

Fluorescent assays offer the advantage of eliminating the use of radioactivity. Peptide and protein ligands are most suitable for fluorescent labeling, whereas maintaining a functional fluorescently labeled small molecule can prove quite a challenge. Nonetheless, certain fluorescent small molecules have been synthesized (e.g., muscarinic receptor ligands are available from Invitrogen). A wide range of fluorophores is available for labeling, for example, BODIPY, Alexa and Cy dyes. BODIPY and Cy...

Orexin System

Orexins modulate food intake, energy balance, and arousal and sleep patterns as a consequence of activation of two GPCRs (OX1R and OX2R) by two ligands (OXA and OXB).7374 Components of the orexin system are similarly distributed anatomically but remain functionally distinct. The ultimate physiological outcomes are determined by the specific receptor-ligand pairings OX1R appears to bind OXA exclusively, whereas both orexin ligands have affinity for OX2R. Extensive studies have been conducted...

Introduction

G protein-coupled receptors (GPCRs) comprise the largest and most diverse family of integral membrane proteins responsible for signaling across cellular membranes. The initial signaling events are mediated by a wide variety of stimuli (including photons, Ca2+ ions, odorants, tasting molecules, amino acids, nucleotides, peptides, and proteins). The signal is transmitted to the cytoplasm, where downstream signaling is induced by receptor-mediated activation of G proteins. Based on pharmacological...

Mammalian Expression

The optimal hosts for mammalian GPCR expression are obviously mammalian cells due to the capabilities of their more native post-translational modification mechanisms and the presence of mammalian G proteins for functional activity assessments. Transient expression has been achieved for many GPCRs, such as adrenergic, dopamine, muscarinic, serotonin, and somatostatin receptors with Bmax values in the range of 10 to 20 pmol mg. Although the expression levels have been acceptable, the transfection...

CAMP Assays

Production of cAMP is regulated through AC an effector enzyme whose activity can be modulated by different GTP binding proteins. Ten different isoforms of AC have been identified, based on primary sequence, tissue distribution, and regulation.38 Forskolin, a diterpene, can be used to directly activate AC to increase cAMP production. AC enzymes can be regulated by a variety of input signals (Ca2+, calmodulin, GaS, Gai, PKC) and should be considered as signal integrators. Subsequent to Gs-coupled...

Editors

Lundstrom, Ph.D. earned his Ph.D. from the University of Helsinki, Finland on overexpression of viral membrane proteins in Bacillus subtilis. He conducted postdoctoral research at Cetus Corporation in California on antisense expression and PCR technologies. Dr. Lundstrom then returned to his native Finland where he was appointed a senior scientist at Orion Pharmaceuticals where he worked on cloning, expression, and structural studies of catechol-o-methyltransferases. From 1992...

Ligands Devoid of Sulfur

Over 25 years ago, investigation of polyamines in vivo established that the tetramine disulfide, benextramine, was an a-adrenergic receptor (a-AR) antagonist. Further work established that inhibition by benextramine was irreversible, time-dependent, and essentially specific for a-ARs (muscarinic and nicotinic acetylcholine receptors were also affected, although these were considered ionic interactions). A hypothesis was proposed that a buried thiol group in the receptor was unmasked by...

TM7 and the NPxxY Motif

The high conservation of the NP*xx(x)Y motif in the rhodopsin family receptors is reflected in the reference position for rhodopsin of Pro303750, which is identified in the motif by an asterisk. Like all of the TM helices, except TM3, helix TM7 is not contiguous and is kinked. The location of the kink is maintained by Cys264647, which acts like a peg upon which the TM7 helix is hung and closes a circle of interaction with retinal (Figure 10.6A). The g-sulfur of Cys264647 lies adjacent to...

Redox Control Of Receptor Activation

Over the years, the sensitivity of ligand-receptor interactions to redox reagents has been demonstrated for most 7TMR systems. The roles of 7TMR and G protein cysteines have been extensively investigated using redox reagents and SDM, both alone and in combination. Ligand interactions with and activations of 7TMRs are sensitive to mild oxidation (generally beneficial) and reducing agents (generally destructive), as well as divalent metal ions and other typical cysteine-reactive entities....

Conclusions

Critical analysis of GPCRs implicated in metabolic disease reveals that, despite apparent selectivity at the level of ligand binding, few receptors prove exquisitely specific when subjected to exhaustive pharmacological analysis in cellular and animal models. This review has highlighted several targets with demonstrated potential for therapeutic intervention, by virtue of medical need and tractability. Ultimate success, as measured by amelioration of human maladies, will depend upon the ability...

Four Classes of Ligands Charged Simple Flexible And Complex

Representative ligands of the human family A receptors fall into four major classes when listed according to their molecular weights (Figure 10.1). The small hydro-philic monoamines are charged and superseded by larger and more amphipathic molecules constituting an amorphous group, the more rigid members of which are hydrophobic and behave in a simple fashion according to Law of Mass Action kinetics. The extremely amphiphilic peptide hormones are flexible in everything they do and lead...

Other Prokaryotic Systems

Halobacterium salinarum, a member of Archaebacteria, has a purple-colored plasma membrane that contains a complex of the bacterio-opsin protein (Bop) and its chromophore retinal in a 1 1 ratio.23 This complex, known as bacteriorhodopsin, forms highly ordered two-dimensional structures for which a high resolution structure was determined.24 Heterologous expression systems based on bacteriorhodopsin have been established for H. salinarum.25 Introduction of the human muscarinic M1 receptor and the...

In Vitro Pharmacological Screening for Potency Selectivity

Optimized Lead as Potential Human Therapeutic Optimized Lead as Potential Human Therapeutic FIGURE 3.2 Flowchart of GPCR drug discovery research. of GPCRs. Family C shares structural similarity to the metabotropic glutamate receptor and is characterized by an extracellular ligand-binding domain. It is comprised of 11 liganded and about 5 orphan GPCRs and is represented by the metabotropic glutamate and GABAb receptors. The most recently described and less characterized Family F S is composed of...

Initiation of Wnt Frizzled Signaling

Wnt Frizzled signaling has been extensively studied in embryogenesis and development of vertebrates and invertebrates.94 Wnt and its Frizzled receptor govern the FIGURE 5.2 Simplified scheme of the Hedgehog and Frizzled signal transduction pathways. Patched (Ptc) is an inhibitor of the Smoothened (Smo) protein. In the absence of Hedgehog (Hh) protein-binding to Ptc, the transcription factor Ci is tethered to microtubules by Cos2 and Fused (Fu) proteins, allowing Ci to be cleaved into a...

Angiotensin Receptors

Angiotensin receptors couple to signal transduction pathways linked to cell proliferation. Increased expression of AT(1) has been reported in human prostate cancer tissue. The angiotensin II (A-II) receptor blocker (ARB), an antihypertensive agent, was shown to inhibit proliferation of prostate cancer cells through suppression of MAPK and STAT3 phosphorylation.53 Oral administration of ARB inhibited the growth of prostate cancer xerographs in both androgen-dependent and androgen-independent...

Ghrelin Receptors

Ghrelin is a gastrointestinal peptide that stimulates food intake and growth hormone secretion.57 In certain tumors, specifically pituitary adenomas, gastrointestinal car-cinoids, and endocrine tumors of the pancreas, production of ghrelin has been detected.5859 The actions of ghrelin are mediated by specific receptors (GHS-R types 1a and b) that regulate GH release-related functions and have also been shown to regulate growth control activities in tumor cells.61 Recent reports indicate that...

Melanophore Based Receptor Functional Activity

Melanophore cells derived from the neural crest of Xenopus laevis offer a highly sensitive system for the screening of ligands for GPCRs. The amphibian mela-nophores contain pigmented organelles called melanosomes, which are filled with the dark pigment melanin. Many Gs- and Grcoupled receptors have been shown to influence pigment dispersion and aggregation after stimulation with the appropriate ligands. The state of pigment distribution within a cell reflects activation of AC or phospholipase...

Comparison Of Expression Systems

As indicated in the introduction, no single expression system is ideal for all GPCRs. Table 8.1 summarizes the different expression systems and lists the advantages, disadvantages, and examples of GPCRs expressed. Generally, the cell-free expression system has provided only poor expression of GPCRs the only examples to date are unpublished studies of human p2 AR. However, the simplicity and speed of cellfree translation and the flexibility that allows evaluation of expression from PCR products...

Sequence And Structural Searches For An Activation Mechanism

The conventional view of ligand-induced allosteric regulation of G protein activation assumes some form of TM helix movement, involving some combination of trans-lational or rotational movement, in whichever plane, and by bending, extension or uncoiling of a helix. Being able to capture this activation event would provide a desirable read-out of agonist activity and might also distinguish between other flavors of partial or inverse agonism, allosteric modulation, and antagonism. The conserved...

Frizzled Receptors

Wnt constitutes a family of genes encoding ligands essential for signaling in early development.93 These secreted glycoproteins, which resemble other secreted glycoprotein ligands such as gonadotropins, act via members of the Frizzled gene family. The Wnt signaling transduction pathway is essentially a network of a number of separate but interacting pathways. Aberrant activation and upregulation of the Wnt pathway is a key feature of many cancers. Despite extensive investigation of the role of...

Receptor Binding Assays General Considerations

Receptor binding assays have become increasingly popular since the introduction of multiple filtration platforms for automation. Recent advances have also enabled researchers to perform receptor binding assays in a homogeneous format so that no additional separation steps are required. The selection of an ideal radioligand for a binding assay depends on ligand stability, specific activity and selectivity higher values for all of these properties are preferred. The affinity of the ligand for the...

Rhodopsin Structure Revelations

The most conserved residue in each of the seven helices is the basis of a comparative numbering scheme, so that a particular receptor TM sequence can be compared with all others, e.g., rhodopsin Pro3037-50 is the conserved residue in the NPxx(x)Y motif of TM7.16 The derivation of GPCR models from MSAs of the known sequences has a long tradition. A particularly comprehensive attempt,17 performed prior to the elucidation of the rhodopsin crystal structure, used distance geometry derived from the...

Schizophrenia

Schizophrenia and schizoaffective disorders are characterized by an imbalance of the dopaminergic system resulting from or consequently affecting glutaminergic, GABA, and serotonergic pathway transmission, with genetic, environmental, and developmental components.72 GPCRs play a major role in the symptomatic treatment of schizophrenia and form the basis for the only available drug treatment, but these drugs unfortunately have no demonstrated abilities to affect disease progression. Most of our...

Resensitization

The mechanism of regaining responsiveness to ligands varies significantly from one GPCR to another. Certain internalized receptors are rapidly recycled to the plasma membrane in a fully resensitized state, whereas other GPCRs are slowly recycled or not recycled at all.41 In the case of the p2-adrenergic receptor, the dephosphory-lation of GRK-phosphorylated receptors in early endosomes seems critical for resen-sitization.42 The balance between protein kinase and protein phosphatase activities...

Purinergic Receptors

The adenosine A3 receptor was found to be expressed in different tumor cell lines, including Jurkat T, pineal gland, astrocytoma, melanoma, and colon and prostate carcinoma cells,86 suggesting it may serve as a target for tumor growth inhibition. Activation of the A3 receptor, however, was shown to attenuate growth of melano-cytes both in vitro and in vivo as well as colon carcinoma growth in mice through modulation of the Wnt signaling pathway linked to the TCF LEF p-catenin path-way.87...

Cannabinoid System

The cannabinoids affect both feeding responses and energy balance through mechanisms that are distinctive from those of the melanocortins. The cannabinoid system19 includes two GPCRs, cannabinoid receptors-1 (CB1) and -2 (CB2) transporters and hydrolyzing enzymes responsible for metabolizing lipophilic natural ligands and the endocannabinoids, anandamide and 2-arachidonoyl glycerol.2021 These endogenous ligands exert potent orexigenic effects and impact reward centers in the brain, thereby...

Cytochrome C Oxidase Subunit III

Cytochrome C oxidase is an integral membrane complex of up to 13 components in bacteria and mitochondria of which subunit III (designated chain C in 2occ, Figure 10.3A) is a 7TM receptor of unknown function. The helices of subunit III can be over 30 residues long, have no features in common with GPCRs, and form a topography similar to a Greek-key spiral when viewed from the amino terminal face. The void between TM2 and TM3 is partially filled by phosphatidylcholine aligned along TM3 in the...

Chemokine Receptors and Neurodegeneration

The blood-brain barrier isolates the healthy CNS from circulating cells of the immune system. Within the CNS, microglia act as immune cells and when activated they are capable of releasing proinflammatory and cytotoxic substances including chemokines. More than 50 human chemokines have been identified and grouped into four subfamilies (a, p, g, and 5) based on the pattern of two cysteine residues in the N-terminal domain, each with a corresponding family of GPCRs (Table 7.2). Receptors within...

Cholecytostokinin2 Receptor

The cholecytostokinin-2 receptor (CCK2R) shows growth-promoting effects on normal and neoplastic gastrointestinal cells when stimulated by gastrin. It is highly expressed in many cancers of different origins including medullary thyroid carcinomas, small cell lung cancers, astrocytomas, and ovarian cancers but is not present in the corresponding tissues.40 A constitutively active CCK2R mutant in which the conserved glutamate (E) within the (E)-aspartate (D)-arginine (R)-tyrosine (Y) motif (E DRY...

Yeast Expression

Various yeasts representing lower eukaryotic organisms are commonly used for recombinant protein expression. For instance, Baker's yeast, Saccharomyces cere-visiae, has been subjected to GPCR expression. The yeast a-factor Ste2p was expressed with C-terminal FLAG and His6 tags at yields up to 1 mg.29 Purified Ste2p receptor could be reconstituted into artificial phospholipid vesicles. Nevertheless, restoration of ligand-binding activity required the presence of solubilized yeast membranes. In...

Metabotropic Glutamate Receptors

Studies in a new melanoma mouse model recently implicated the ectopic expression of the Gq 11-coupled metabotropic glutamate receptor 1 (mGluRl, Grml), a member of the C family of GPCRs, in melanomagenesis and metastasis.8889 Alterations in the expression of Grml are sufficient to promote mouse melanocyte transformation in vivo.88 In addition to the coupling to Gq 11 proteins, Grml may act to activate Gs proteins in transfected cells.90

Receptor Classification By Ligand Size

The crystal structure of bovine rhodopsin2 is considered to be applicable to all of the GPCR superfamily, with the a-helices traversing the lipid bilayer seven times. Bacteriorhodopsin and the many other prokaryotic opsins are not GPCRs, although their sequences and structures imply relatedness. A recent genomics survey of 16 species of Archaea and 96 species of bacteria identified two widespread families of novel 7TMRs that exhibit a bewildering array of amino and carboxyl terminal...

Microphysiometer Assays

The microphysiometer assay system is the most generic assay available for measuring the metabolic activation of cells expressing GPCRs following activation by an agonist. The cellular acidification rate response induced by agonists can be monitored by a cytosensor microphysiometer. The cytosensor system uses a silicon-based light addressable potential sensor (LAPS) to monitor changes in pH. Differences in pH under basal, stimulated conditions due to an agonist and inhibited stage caused by an...

The Essence of Agonism

The different categories of smells and aromatic substances can be readily distinguished. Pleasant and fruity odors are generally esters, ketones, and alcohols distinguished by the presence of an oxygen, often double-bonded. Distinctive and less pleasant smells are associated with nitrogen, such as the fishy smells of quaternary amines. The word aromatic conjures up a mental image of ring structures or p-orbitals and a wholesome smell or whiff of a volatile organic compound. The worst smell of...

References

HTS where next Drug Disc World. 2001, 2, 25-30. 2. Kell, D. Screensavers trends in high-throughput analysis. Trends Biotechnol. 1999, 17, 89-91. 3. Lavery, P. Brown, M.J. Pope, A.J. Simple absorbance-based assays for ultra-high throughput screening. J Biomol Screen. 2001, 6, 3-9. 4. Burns, D.J Kofron, J.L. Warrior, U. Beutel, B.A. Well-less, gel permeation formats for ultra-HTS. Drug Disc Today. 2001, 6, S40-S47. 5. Hodgson, J. Receptor screening and the search for new...

Opioid Receptors

GPCRs interacting with morphine and other analgesic opiate ligands were first defined pharmacologically in various tissues. The cloning of the m, 5, and k opioid receptors (designated MOR, DOR and KOR by International Union of Pharmacology nomenclature) in the 1990s gave real promise that selective agents with fewer side effects would be developed. Morphine is a MOR-selective ligand with very low potencies at both KOR and DOR. Overwhelming evidence, including lack of morphine-induced analgesia...

Sulfur Charges Rings

The presence of a positive charge appears to be a minimal requirement in monoamine agonist ligands and an aromatic structure is common. The long wavelength absor-bance and intrinsic fluorescence of aromatic amino acids are much appreciated in the study of proteins, and the potent quenching ability of adjacent sulfhydryl and disulfide moieties is a frequent and annoying feature, although it is also evidence of a close affinity between aromatic and cysteine residues. The association of sulfur and...

Source Material And Assay Diversity

Historically, drug discovery focused on native tissues which involved assays such as neurotransmitter or hormone release measurements and radioactive binding assays. Researchers now have a plethora of reagents and assays available to screen for selective lead molecules. As sources of receptors, researchers can choose native tissue, recombinant or nonrecombinant cell lines (see Chapter 8). Issues such as specificities of native tissues due to expression of multiple receptor subtypes,...

Structural Flexibilities Of Membrane Receptors

The seven individual helices for the five GRAFS families can be aligned (except for TM2 and TM7 from the G and F families) and each helix is self-consistent across the five families, providing no evidence of reordering and the production of a different folding topology.8 Based on this remarkable degree of consistency, it is difficult to appreciate why 7TMRs are so resistant to structural elucidation attempts, assuming oligomeric complexing, coreceptors and or other integral components are not...

Synaptic Transmission and Neurotransmitter Pathways

The function of a GPCR in the CNS must be seen in the context of its neuronal cell environment, which is specialized to receive, integrate, and transmit electrical and chemical signals, and its neuroanatomical localization. A wide range of neurotransmitter (NT) molecules that signal through GPCRs have been identified and include amino acids, biogenic amines, peptides, and lipids. NT pathways in the CNS have been mapped according to distribution and connectivity of cells signaling through these...

Summary

In conclusion, GPCRs have long been highly desired drug discovery targets by the pharmaceutical industry, resulting in the development of many of the best-selling drugs on the market today that account for nearly 25 of total drug sales and prescriptions. Drugs targeting these receptors have a diverse variety of therapeutic indications including allergies, cancer, pain management, and diseases affecting cardiovascular, central nervous system, gastrointestinal, and metabolic functions. Developing...

Gpcr Desensitization

Because GPCR signaling potentiates many important cellular functions, it is essential that the mechanism of activation is initiated and terminated rapidly, specifically, and precisely even when agonist stimulation is continued. This phenomenon of desen-sitization can function both on the GPCR and G protein levels. Rapid termination of GPCR function can occur through receptor phosphorylation mediated by PKA or PKC (protein kinase C). Receptor phosphorylation is a typical example of negative...

General Principles of cAMP Assays Identifying Active Compounds

For Gas-coupled receptors, an agonist will stimulate production of cAMP, while an antagonist will inhibit agonist-induced cAMP production. For Gai-coupled receptors, the effect of an agonist whose binding results in the inhibition of AC cannot easily be measured because the basal cAMP is too low. Cells are first stimulated to produce cAMP with forskolin. An agonist will inhibit forskolin-induced cAMP production and this is measurable. Gai-coupled receptor antagonist cells are stimulated to...

Inverse Agonists And Constitutive Activities

Inverse agonists represent an established drug class possessing the property of reducing receptor-mediated constitutive activity of GPCRs. It is the ability of GPCRs to undergo an agonist-independent conversion from the inactive state of the receptor to the activated form that makes them capable of activating G proteins.7879 Arena pharmaceuticals (San Diego, California) developed a technology to identify inverse agonists. Its constitutively activated receptor technology (CART) is based on the...

Ligands Containing Cysteine

It has been known for many years that the glycosylated trophic hormones LH and FSH exert intrinsic thioredoxin-like activities.53 Thioredoxin uses a localized disul-fide bond to form mixed disulfides with cysteine-containing proteins, promoting either the cleavage of preformed disulfide bridges or the formation of cystine bridges from reduced thiols. Thioredoxin activity is usually measured by its ability to refold denatured RNAse, a property expressed by LH and FSH that is stronger than that...

Melanocortin System

The hypothalamic melanocortin system is perhaps the most extensively documented family of GPCRs (melanocortin receptors or MCRs) and ligands implicated in the central regulation of feeding behavior and energy homeostasis.1,2 Mounting pharmacologic and genetic data indicate that MC3R and MC4R are the key modulators of this system.34 The melanocortin system is distinctive in that both natural agonists and antagonists have been identified. Bioactive peptides (melanocyte-stimulating hormones or...

Cannabinoid Receptors

Activation of cannabinoid CB1 receptors has been shown to inhibit growth of xenograph tumors of thyroid origin via attenuation of p21ras activity.26 Stimulation of this receptor with the metabolically stable endocannabinoid analog (Met-F-AEA) resulted in inhibition of tumor angiogenesis and metastasis via suppression of VEGF and its receptor (Flt-1) and enhancement of p27kip1 levels in rat thyroid metastatic cells and in abrogation of formation of metastatic nodules in vivo.26 Effects could be...

Aquaporin

The 3D structures of the aquaporin and glycerol facilitator family members do not seem related to 7TMRs at first glance,14 but the particular arrangement of a-helices means that they should not be ignored. These water and substrate channel proteins consist of 6TM helices and two half-TM helices that are apposed amino-terminally in the 3D structure to produce a seventh helical TM span (like an extended arrangement of the antibiotic half-helix dimer, gramicidin A, 1jno). The final topography of...

Expression Systems for GPCRs

E. coli wheat germ Rapid, PCR applicable Rapid, inexpensive, high yields, easy scale-up Relatively easy to use, high biomass, scaleable Relatively Toxic to cells, no post-translational modifications Low yields Requires refolding Cloning complicated, no post-translational modifications, fusion proteins No post-translation modifications Hyperglycosylation, clone selection, thick cell wall Thick cell wall More expensive, virus stock prep slow Time-consuming Human LPTHR1 Human LGLP-1 Human...

Psychiatric Diseases

Psychiatric disorders include schizophrenia, depression, anxiety, and bipolar disease and represent a major focus of drug discovery efforts in the CNS area. The most effective treatments have highly complex pharmacologies, but emerging therapies now target specific GPCRs in the hope of increasing efficacy while reducing unwanted side effects. It is widely accepted that the major mental illnesses are polygenetic and result from the interactions of both genetic and environmental components. The...

Molecular Bioinformatics In Drug Discovery

Being the only representative structure, rhodopsin is the basis of homology modeling for the 7TMRs in many studies, of which some have spawned attempts at virtual screening. For many years, the molecular modeling community based their analyses on the low resolution electron microscopy (EM) structures of bacteriorhodopsin. Others attempted to derive models from the less well resolved bovine rhodopsin EM sources, using distance constraints from experimental data, MSAs, ligand specificities and...

Preface

The impacts of G protein-coupled receptors (GPCRs) from social, therapeutic, and economic points of view are enormous. The broad range of GPCRs covers all areas of modern medicine. The effects of point mutations, overexpression or perturbed regulation of GPCR function, and their overstimulation or inhibition affect such areas as cardiovascular, metabolic, neurodegenerative, psychiatric, and viral diseases and cancers. Currently, more than 50 of drug targets are based on GPCRs and annual...

Antibody Dependent cAMP Detection Assays

These assays are based on the competition between the cellular cAMP and the labeled cAMP with the anti-cAMP antibody. To achieve the best results possible from cAMP assays, it is essential to determine the optimal number of cells per well. The optimal cell concentration should produce a relatively low basal level of cAMP, which will yield a good response to a relatively low concentration of stimulator. When evaluating the inhibitory compounds, the level of stimulator will be as low as possible...

Heterogeneous Receptor Binding Assays

Heterogeneous binding assays can be performed with radio-labeled or lanthanidelabeled ligands. Virtually every heterogeneous binding assay utilizes a filtration step to remove unbound labeled ligand. This technology has the advantage of removing compounds that could interfere with the detection of the bound tracer. The main disadvantage of this nonhomogeneous assay is the difficulty in automating the separation step, which results in lower throughput of the screen. Fluorescent labeling of...

Families Of Gpcrs

The overall amino acid sequence homology among GPCRs is rather low. Certain homologous regions arise from sequence alignment of the 7TMs within the GPCR families. The largest group of GPCRs is designated family A or class 1 and consists of light receptors (rhodopsin), adrenaline (adrenergic) receptors, and olfactory receptors. Every member shows a highly conserved arginine in the Asp-Arg-Tyr (DRY) motif6 located at the cytoplasmic side of the third transmembrane domain (TM3). Approximately 25...

TM3 and the Conserved Cystine Bridge

The extracellular dithiol bridge between TM3 and the second outer loop is the only motif common to the 7TMR superfamily, apart from the ubiquitous presence of the 7TM helices. The second extracellular loop in rhodopsin produces a small region of antiparallel p-sheet, strands p3 and p4, which is extended by strands p1 and p2 from the amino terminal chain to produce a plug over retinal in the binding site (Figure 10.6). Retinal is located against TM3 and surrounded by a p-bonded cage of 12...

Secreted Frizzled Related Proteins

The secreted Frizzled-related proteins (sFRPs) comprise a family of five secreted glycoproteins that may antagonize Wnt signaling through the binding and sequestering of Wnt ligands, and thus may have important implications in tumorigenesis. Both the downregulation and upregulation of sFRPs have been correlated with cancers.99 100 Moreover, in addition to genetic alterations, changes in the status of DNA methylation of the sFRP2 gene, known as epigenetic alterations, can be used as markers for...

Chemokine Receptors and Their Ligands

Cysteine Number of chemokine ligands 13 17 11 Receptors Number of C cysteine residue. X any amino acid residue. Chemokine receptors are expressed on neurons, microglia, and glia and may modulate various aspects of neuroinflammation and neurodegeneration.191 For example, neuronal presynaptic chemokine receptors have been shown to inhibit glutamate release and may thereby modulate excitotoxicity.192 Additional effects of chemokines directly on neurons may also play a role in disease progression....

Lack of Hydrophobic Drive in 7TMRs

Water-soluble globular proteins adopt reasonably restrained formal structures during the folding process because of hydrophobic drive.9 The generally hydrophobic nature of zwitterionic amino acids is worsened by the elimination of water during peptide bond formation in the production of extended chains of residues. Hydrophobic collapse ensures that the more hydrophilic residues are surface-exposed and many secondary modifications are exploited to increase aqueous solubility, including...

Frizzled Smoothened Family of GPCRs

Signaling through the G protein-coupled frizzled smoothened receptors plays a crucial role in a diverse array of developmental processes (Figure 5.2). The ten frizzled receptors (FzRs) known in humans may be activated by a plethora of Wingless (Wnt) ligands identified in mammals. These ligands belong to a family of protooncogenes. The Wnt denotes the relationship of this family to the Droso-phila segment polarity wingless gene and to its vertebrate ortholog, murine mammary tumor virus...

Other GPCRs

The NK1 receptor and its endogenous peptide agonist, substance P (SP), have been studied as potential targets for the treatment of pain for many years. Although strong evidence from animal models demonstrated analgesic properties of the available selective NK1 antagonists, they have not proven effective in humans. SP is found in small unmyelinated sensory fibers associated with nociception.165 NK1 receptors are expressed in the dorsal horn of the spinal cord where SP is released following...

Other Signaling Pathways

Receptor Mediated Caveolin Endocytosis

In addition to coupling to G proteins, GPCRs have been shown to mediate signal transduction events through interactions with other cellular proteins (Figure 2.2). For example, c-Src tyrosine kinase interacts with the proline-rich SH3 (Src homol-ogy-3) domain in the third intracellular loop of the p3-adrenergic receptor, which leads to the activation of the extracellular signal-regulated kinase (ERK)-mitogen-activated protein kinase (MAPK) cascade.15 It has also been demonstrated that the...

Endothelin Receptors

Endothelins and their receptors have been implicated in the development and progression of prostatic, ovarian, and cervical cancers and melanomas45 through autocrine and paracrine signaling pathways. The three isopeptides that constitute the endothelin family serve as potent mitogens of these tumors. In ovarian carcinoma xenographs, in which the endothelin A receptor (EtAR) is highly expressed, the selective bioavailable antagonist ABT-627 (atrasentan) was shown to significantly inhibit tumor...

High Throughput Screening

The development of robust and sensitive assays for finding inhibitors or allosteric modulators of ligand binding and or receptor activity is critical to screening that will allow the discovery of new leads. Traditional screening methods used membrane preparations and specific, radio-labeled ligands. Compounds that competed with a labeled ligand were identified after the separation of the unbound radioactivity by filtration. Even though receptor-radioligand binding screens are robust, they do...

Functional Gpcr Assays

Whether inactive GPCRs are pre-coupled to G proteins and how GPCRs can activate heterotrimeric G proteins are issues subject to a lot of speculation. In general, activated GPCRs stimulate heterotrimeric G proteins in membranes in a manner that involves GDP release (a rate-limiting step) and then GTP binding. GTP binding to the a subunit of the G protein leads to a conformational change in its switch regions and promotes dissociation of the G protein complex into a- and pg-subunits. Both...

Protease Activated Receptors

A link between coagulation factors and tumor progression and metastasis is recognized, but the molecular basis remains poorly understood.62 Thrombin, the main effector protease of the coagulation cascade, elicits its response through activation of G protein-coupled protease activated receptors (PARs) designated PAR-1 through PAR-4.63 Activation of PAR-1 induces proliferate signaling and migratory responses in a variety of cells. Increased PAR-1 mRNA and protein expression has been associated...

Advent of Recombinant Expression and Purification Technology

In contrast to other GPCRs, rhodopsin is readily available from native sources (bovine retina) in very high quantities and this source proved useful in limited early structural studies.31 Significant structural information was obtained also from unpu-rified systems expressing GPCRs, and such studies have played important roles in studies of ligand binding pockets of GPCRs other than rhodopsin. However, high resolution structural studies require significant amounts of recombinant pure protein. A...

GTP Binding Assays

A GTPgS binding assay detects the level of G protein activation following agonist stimulation. In the resting stage, G proteins exist as Ga(GDP)pg heterotrimers with GDP bound to Ga there is a low rate of GDP-GTP exchange. Subsequent to agonist stimulation of the receptor, GDP dissociates from Ga, which allows GTP to bind to Ga. Agonist-mediated GPCR activation increases the rate of guanine nucleotide exchange and allows the dissociated G protein subunits to interact with effector proteins to...

Introduction Not All 7tmrs Are Gpcrs

A common system of receptor activation in G protein-coupled receptors (GPCRs) has been contemplated often, primarily because of the conserved heptahelical structural motif and the shared signaling through G proteins. However, the great diversity in ligands has been used as a counter-argument against such considerations. The prevailing modern view is that no single lock exists for all agonist keys in GPCRs and that each ligand-receptor system has its own mechanistic binding and activation...

Neuroendocrine Function

In the CNS, hormone secretion from the pituitary is regulated by neurosecretory cells in the hypothalamus. GPCRs are abundant in the hypothalamus, many of which interact with peptide neurotransmitters to regulate hunger, thirst, water balance, body temperature, and blood pressure. Table 7.1 shows a large number of receptors expressed in hypothalamus, and additional examples include somatostatin, orexin, and angiotensin receptors. The hypothalamus has been an area of major interest in the study...

TM3 And The Dry Motif

In the rhodopsin family receptors, the highly conserved Arg350 in the DR*Y sequence at the cytosolic end of TM3 has been proposed to operate as an arginine switch (Figure 10.4B). The highly positive, large side chain was hypothesized to swing away from the receptor upon ligand binding, possibly being released from the conserved TM2 aspartate, Asp250, and to switch the G protein into an active conformation.40 The motif equivalent to the DRY sequence (ERY in rhodopsin) is EGLY in the secretin...

Somatostatin Receptors

The five somatostatin receptors (SSTR1 through SSTR5) belonging to the family of neuroendocrine receptors are widely expressed in pituitary gland, spleen, and the gastrointestinal tract.27 Somatostatin exists as two biologically active forms (SS-14 and SS-28) and inhibits secretion of growth hormone (GH) from the pituitary gland. The pituitary gland is crucial for the maintenance of several homeostatic functions including growth, metabolism, and reproduction. Since pituitary tumors are...

Receptor Subtypes As Novel Targets

Adrenoceptors constitute an established family of therapeutic drug targets and demonstrate how accumulating knowledge of receptor subtypes has gradually led to more refined therapeutic approaches in cardiovascular diseases. Mammals have nine subtypes of adrenoceptors. Norepinephrine, the sympathetic neurotransmitter, and epinephrine, an adrenomedullary hormone, regulate cardiovascular functions through most, or perhaps even all of these subtypes. The first division of adrenoceptors into two...

Receptor Gene Polymorphisms

Most human GPCR genes appear to be polymorphic, and the significance of receptor mutations for cardiovascular disease risk and as determinants of therapeutic responses constitutes another rapidly expanding area of research. Genetic polymorphism is defined as the occurrence within a population of two or more allelic variants of a given gene sequence, in such proportions that the rarest cannot be maintained merely by recurrent mutations. Genetic variation may influence one or more aspects of the...

Foreword

The most widely used and safest medicines today are ligands to GPCRs. The first generation of antihypertensives p-blocked the p-adrenergic receptor, the later generations include blockers of the angiotensin receptor, both GPCRs. Antihistamines to fight hay fever and allergy, antipsychotics, and some antidepressant drugs are also ligands to GPCRs. Furthermore, the analgesic actions of morphine and its analogs are exerted at GPCRs and lately antiviral agents blocking HIV entry into cells are also...

Cellfree Translation

E. coli lysates containing coupled transcription-translation reaction reagents with continuous supplies of nucleotides, amino acids, and other essential components have been developed for cell-free translation.8 The system applied for rapid trans lation of simple soluble proteins can generate yields of tens of milligrams.9 Technology development has allowed the production of isotopically labeled proteins for nuclear magnetic resonance (NMR) and x-ray crystallography in cell-free translation...

TABLE 81 Continued Expression Systems for GPCRs

High expression Safety concerns Broad host range Relatively expensive MB, membrane bound IB, inclusion bodies SFV, semliki forest virus. a Unpublished results, M. Shirouzu, RIKEN Institute, Yokohama, Japan. b Unpublished results, R. Wagner, University of Strasbourg, France. satisfactory levels in bacteria due to the toxic effects they cause to host cells and the difficulties in refolding when expressed in inclusion bodies. Despite these problems, some success has...

Depression

The clinical description of depression is complex, covering a broad range of symptoms that lack a unifying biological hypothesis. Depression has both genetic and environmental components, with linkage studies suggesting it is a polygenic disor-der.131 Here again, effective therapeutics are contributing to an understanding of its underlying molecular mechanisms.132 Modern treatment for depression, which focuses exclusively on agents that modulate monoamine neurotransmission, began with a...

Nutrient Sensing Receptors

A small family of GPCRs with low homologies to other receptors has recently been identified.167 GPRs 40, 41, and 43 share 30 to 40 sequence identities to one another. Ligands for these receptors have been identified as long-chain fatty acids (GPR40) and short-chain carboxylic acids (GPR41 and GPR43).168-171 Long-chain fatty acids serve as energy sources and also have the potential to be converted to signaling molecules within cells. Excess long-chain fatty acids are known to induce insulin...

Distribution of Prominent GPCR Families with Known Endogenous Ligands in the Central Nervous System

Amygdala Cortex Hippocampus Hypothalamus Cord Ganglia a Based on published rat and human mRNA distribution. b Includes autoradiography data. FIGURE 7.1 History of psychiatric drug development. Timeline shows emergence of selected drug mechanisms (solid arrows) and therapeutic agents (plain text) with relevance to schizophrenia (above the line) and depression anxiety (below the line). S2 D2 serotonin 5HT2A + dopamine D2 receptor antagonists. TCA tricyclic antidepressants. MAOI monoamine oxidase...

Peripheral Energy Metabolism 6321 p3Adrenergic Receptor

Many of the peptide hormones described exert significant effects on energy metabolism via modulation of nutrient utilization or partitioning of fuel stores, and through effects on the hypothalamic feeding and energy circuits. Peripheral regulation of energy metabolism also occurs the most significant example is the effect of sympathetic activation on p-adrenergic receptors. A number of well-characterized adrenergic receptor subtypes exist, and the signal transduction pathways activated...

Melanin Concentrating Hormone Receptors

Melanin-concentrating hormone (MCH) is a 19-amino acid cyclic, orexigenic neuropeptide, expressed predominantly in the lateral hypothalamus where it binds a cognate GPCR, MCHR1, and signals through complex neural networks to promote feeding and reduce energy expenditure.42-46 Infusion of MCH into rodents stimulates feeding and, on a chronic level, causes moderate weight gain.47-52 Increased expression of MCH in genetic models of obesity and the finding that MCH neurons express the leptin...

Engineering of Fusion Proteins

Particularly for bacterial expression, fusion to prokaryotic proteins such as the maltose binding protein (MBP),5 glutathione S-transferase (GST),6 and bacteriophage PRD1 membrane protein7 has produced favorable results because the fusion partner has been demonstrated to improve yields and provide more stable proteins. E. coli Wheat germ 1 day PCR product FIGURE 8.1 Procedure and time requirements for recombinant GPCR production in various expression systems. Key steps in different processes...

Family A Gpcrs 521 Monoaminergic Receptors

The serotonin 5HT1C and 5HT2B, a1b-adrenergic and muscarinic ml, m3, and m5 acetylcholine receptors appear to harbor oncogenic potential. These receptors are able to activate signaling pathways associated with proliferation. They can trans form NIH 3T3 cells in an agonist-dependent manner.8-11 Injection of cells derived from transformed foci expressing these receptors into nude mice results in the generation of tumors, indicating that these receptors may function as protoonco-genes when...

FzRs as GPCRs

Although the FzRs exhibit several hallmarks of GPCRs, including presumed heptahelical structures, and exhibit Pertussis toxin- and specific G protein Ga or Gpg subunit-dependent signaling, their G protein activation capabilities have been investigated only minimally, and the FzRs are often still regarded as new members of the GPCR superfamily.112 Some FzRs have been shown to activate hetero-trimeric guanine nucleotide-binding (G) proteins.112122 The receptors appear to couple to members of the...

Scope Of Book

This book describes the principal mechanisms for signal transduction through activation of GPCRs and their interactions with G proteins. Fairly recently, it was discovered that GPCRs can also signal through alternative pathways without interactions with G proteins. This finding has cast doubts on the correctness of naming these receptors GPCRs. Rather it has been suggested that the scientific community refer to them as seven transmembrane (7TM) receptors based on their common structural...

Virally Encoded GPCRs

GPCRs encoded by viruses of the herpes virus family have drawn considerable attention recently because some may provide causative links with respect to the onset or progression of cancer. Various herpes viruses and pox viruses contain DNA sequences encoding proteins with homologies to cellular chemokine receptors.156 Since GPCRs and chemokine receptors play key roles in cellular communications and appear to play roles in cancer, the virally encoded GPCRs are believed to be of great importance...

Pharmacology And Therapeutics

GPCRs are involved in cardiovascular regulation in numerous ways. At least 55 types of GPCRs are known to directly mediate neuronal and endocrine regulation of cardiac and vascular responses, and many more influence cardiovascular functions indirectly, via primary effects on the neuronal, endocrine, and metabolic regulatory mechanisms that control various aspects of the functioning of cardiac and vascular cells and tissues. These receptors are widely expressed in different cell types of the...

Chemokine Receptors

The family of chemokines is involved in the control and regulation of the immune system by directing the migration and activation of leukocytes during homeostasis through interactions with their respective GPCRs.3,70 However, chemokines appear also to be involved in the leukocyte infiltration of tumors, a characteristic of cancer, and to influence metastatic potential and site-specific spread of tumor cells.3,71 In addition to their chemotactic properties, chemokines and their receptors play a...

Conformational Changes Of Rhodopsin

11.2.1 Early Magnetic Resonance Studies of Chromophore AND PROTEIN-LlPID INTERACTIONS The earliest documented experiments applying nuclear magnetic resonance (NMR) to the analysis of rhodopsin were limited to studying the isolated solubilized visual pigment chromophores. Conformations of the polyene chains of all-trans, 9-cis, 11-cis and 13-cis retinal, as well as model forms of Schiff Base all-trans analogs, were solved by 1H and natural abundance 13C solution NMR providing details about the...

Series Editor Andrew Carmen

Johnson & Johnson PRD, LLC San Diego, California, U.S.A. 1. Virtual Screening in Drug Discovery, edited by Juan Alvarez and Brian Shoichet 2. Industrialization of Drug Discovery From Target Selection Through Lead Optimization, edited by Jeffrey S. Handen, Ph.D. 3. Phage Display in Biotechnology and Drug Discovery, edited by Sachdev S. Sidhu 4. G Protein-Coupled Receptors in Drug Discovery, edited by Kenneth H. Lundstrom and Mark L. Chiu C ProteinCoupled Receptors in Drug Discovery Boca Raton...

Thyroid Stimulating Hormone Receptor

The growth and function of the thyroid are controlled by thyrotropin through activation of the thyroid-stimulating hormone (TSH) receptor.36 Mutations in TSH receptors are found in approximately 30 of all human thyroid adenomas.37 In recent years, various constitutively active mutations within the TSH receptor were identified and shown to be associated with continuous stimulation of the cAMP pathway, causing hyperthyroidism and thyroid hyperplasia. Similarly, in Graves' disease, autoantibodies...

LPA Receptors

Lysophosphatidic acid (LPA) is an extracellular lipid mediator that evokes growth factor-like responses, inducing cell proliferation, migration and survival, indicating a role in the initiation or progression of malignant disease.18 Increased levels of LPA have been detected in malignant effusions. Its receptors, particularly LPA2 and LPA3, are aberrantly expressed in several human cancers, particularly in ovarian and prostate cancer cells.19 In addition, autotaxin, an ectoenzyme known to be...