A study on the correlation of G-protein-coupled receptor types with amino acid composition

G-protein-coupled receptors have become a target in utilizing bioinformatics and genomics technology to facilitate drug discovery for psychiatric diseases. In this study the covariant-discriminant algorithm [Chou and Elrod (1999) Protein Eng., 12, 107-118] has been used to analyze the correlation between the types of G-protein-coupled receptors and the amino acid composition. It has been found that different types of G-protein-coupled receptors are quite closely correlated with the amino acid composition, implying that the types of G-protein-coupled receptors are predictable to a considerably accurate extent if a good training data set can be established for that purpose. The method derived here can be also used to do preliminary classification of orphan G-protein-coupled receptors. This will significantly expedite the process of identifying proper G-protein-coupled receptors for drug discovery.     

Acetylcholine receptors.

Alpha-Bungarotoxin is one of a class of proteins, isolated from snake venoms, which antagonize the action of acetylcholine at vertebrate neuromuscular junctions and 'electroplaques' of electric fish. Alpha-Bungarotoxin blocks acetylcholine action irreversibly and may be labelled with either 125I or 3H. This irreversible binding is used as the basis of an in vitro assay for acetylcholine receptors, whether in intact tissue, membrane fragments or solubilized preparations. Acetylcholine receptors from Torpedo and denervated skeletal muscle have been solutilized and substantially purified using affinity chromatography. The distribution of acetylcholine receptors in several tissues has been determined, and an auto-immune response, induced by injection of purified Torpedo receptors, has been studied.     

Methods for Studying Fc Receptor Expression

In the past two decades, rapid progress has been made in the field of Fc receptors. One of the reasons for this development has been the advances and improvements in the methodologies used to detect and analyze Fc receptors. This review describes the methods used to detect the murine and human Fc receptors of the various immunoglobulin isotypes at the RNA, protein, and functional level. We have given special attention to the binding assays used to detect Fc receptors through immunoglobulins or anti-Fc receptor monoclonal antibodies as probes. The various Fc receptor types exhibit a marked heterogeneity and we present a list of reagents that can differentiate between the different Fc receptor forms. These methods can detect Fc receptors on the cell membrane, as well as the cytoplasmic and secreted forms. Recent advances in the use of PCR and RNA/DNA analyses to study Fc receptor detection, expression, and function are also reviewed. The advantages and drawbacks of the various experimental approaches are presented. In addition, an appendix provides detailed protocols for the detection of Fc receptors by confocal immunofluorescence microscopy, rosetting, cytofluorometry, immunoprecipitation, and PCR     

Endocytosis in signalling and development

After a long period of neglect, endocytosis in plants is finally coming of age. The constitutive recycling of plasma membrane proteins has been well established in the past few years, and recent studies report the ligand-induced endocytosis of receptors and other plasma membrane proteins. Signalling by ligand-bound receptors from endosomes has not, however, been demonstrated in plants. Although novel markers have been used to map endocytic pathways, the functional compartmentalisation of endosomes is still controversial. It is thus not clear where and how cargo proteins such as receptors are sorted towards either recycling to the plasma membrane or targeting to the vacuole for degradation.     

Identification and functional roles of metabotropic glutamate receptor-interacting proteins

In the mammalian brain, a majority of excitatory synapses use glutamate as a neurotransmitter. Glutamate activates ligand-gated channels (ionotropic receptors) and G protein-coupled (metabotropic) receptors. During the past decade, a number of intracellular proteins have been described to interact with these receptors. These proteins not only scaffold the glutamate receptors at the pre- and post-synaptic membranes, but also regulate their subcellular targeting and intracellular signaling. Thus, identification of these proteins has been essential for further understanding the functions of glutamate receptors. Here we will focus on those proteins that interact with the subgroup of metabotropic glutamate (mGlu) receptors, and review the methods used for their identification, as well as their functional roles in neurons.     

Eleven new putative aminergic G-protein coupled receptors from Amphioxus (<Emphasis Type="Italic">Branchiostoma floridae</Emphasis>): identification,sequence analysis and phylogenetic relationship

We have identified eleven novel aminergic-like G-protein coupled receptor (GPCRs) sequences (named AmphiAmR1-11) by searching the genomic trace sequence database for the amphioxus species, Branchiostoma floridae. They share many of the structural motifs that have been used to characterize vertebrate and invertebrate aminergic GPCRs. A preliminary classification of these receptors has been carried out using both BLAST and Hidden Markov Model analyses. The amphioxus genome appears to express a number of D1-like dopamine receptor sequences, including one related to insect dopamine receptors. It also expresses a number of receptors that resemble invertebrate octopamine/tyramine receptors and others that resemble vertebrate alpha-adrenergic receptors. Amphioxus also expresses receptors that resemble vertebrate histamine receptors. Several of the novel receptor sequences have been identified in amphioxus cDNA libraries from a number of tissues.     

Platelet and vascular smooth muscle thromboxane A2/prostaglandin H2 receptors

Thromboxane A2 (TxA2) and prostaglandin H2 (PGH2) aggregate platelets and contract vascular smooth muscle. Inasmuch as both compounds produce the same effects and presumably through the same receptor, their receptors have been referred to as TxA2/PGH2 receptors. Pharmacological studies of stable agonists and antagonists of the TxA2/PGH2 receptors have shown different rank order potencies for these compounds in platelets compared with blood vessels. These studies have provided evidence to support the hypothesis that the platelet TxA2/PGH2 receptor is different from the one found in vascular tissue. The vascular receptor has been named [TxA2/PGH2]tau and the platelet receptor has been named [TxA2/PGH2]alpha. In the past few years several radiolabeled antagonists and agonists have been developed and used in radioligand-binding studies, primarily in platelets. One of these ligands, 125I-labeled PTA-OH, a TxA2/PGH2 receptor antagonist, has been extensively used to characterize the human platelet TxA2/PGH2-binding site. It has been found to have a Kd of approximately 20 nM and a Bmax of 2500 binding sites/platelet. Through the combination of pharmacological and biochemical approaches, it should be possible to characterize platelet and vascular TxA2/PGH2 receptors.     

GABA-benzodiazepine receptor complex and drug actions

This short review describes the benzodiazepine receptors, their interplay with GABAergic transmission and chloride ionophore, the search for endogenous ligands, and the drug responses that can be evoked through these receptors. Benzodiazepine receptors offer a unique pathway through which opposite drug actions e.g., anxiogenic and anxiolytic effects can be exerted, and these actions can be inhibited with competitive receptor antagonists. The most plausible endogenous ligand for benzodiazepine receptors discovered so far, a polypeptide DBI, exerts actions opposite to those of the benzodiazepines used in clinical therapy. This has been the stimulus for a new look at the physiological role for these receptors.     

Lipoprotein nanoplatform for targeted delivery of diagnostic and therapeutic agents

Low-density lipoprotein (LDL) provides a highly versatile natural nanoplatform for delivery of visible or near-infrared fluorescent optical and magnetic resonance imaging (MRI) contrast agents and photodynamic therapy and chemotherapeutic agents to normal and neoplastic cells that overexpress low-density lipoprotein receptors (LDLRs). Extension to other lipoproteins ranging in diameter from about 10 nm (high-density lipoprotein [HDL]) to over a micron (chylomicrons) is feasible. Loading of contrast or therapeutic agents onto or into these particles has been achieved by protein loading (covalent attachment to protein side chains), surface loading (intercalation into the phospholipid monolayer), and core loading (extraction and reconstitution of the triglyceride/cholesterol ester core). Core and surface loading of LDL have been used for delivery of optical imaging agents to tumor cells in vivo and in culture. Surface loading was used for delivery of gadolinium-bis-stearylamide contrast agents for in vivo MRI detection in tumor-bearing mice. Chlorin and phthalocyanine near-infrared photodynamic therapy agents (相似文献   

Solid Phase Synthesis and Application of Labeled Peptide Derivatives: Probes of Receptor-Opioid Peptide Interactions

Solid phase synthetic methodology has been developed in our laboratory to incorporate an affinity label (a reactive functionality such as isothiocyanate or bromoacetamide) into peptides (Leelasvatanakij and Aldrich J Peptide Res 56, 80, 2000), and we have used this synthetic strategy to prepare affinity label derivatives of a variety of opioid peptides. To date side reactions have been detected only in two cases, both involving intramolecular cyclization. We have identified several peptide-based affinity labels for δ opioid receptors that exhibit wash-resistant inhibition of binding to these receptors and are valuable pharmacological tools to study opioid receptors. Even in cases where the peptide derivatives do not bind covalently to their target receptor, studying their binding has revealed subtle differences in receptor interactions with particular opioid peptide residues, especially Phe residues in the N-terminal “message” sequences. Solid phase synthetic methodology for the incorporation of other labels (e.g. biotin) into the C-terminus of peptides has also been developed in our laboratory (Kumar and Aldrich Org Lett 5, 613, 2003). These two synthetic approaches have been combined to prepare peptides containing multiple labels that can be used as tools to study peptide ligand-receptor interactions. These solid phase synthetic methodologies are versatile strategies that are applicable to the preparation of labeled peptides for a variety of targets in addition to opioid receptors.     

Receptors for plant growth regulators: Recent advances

We have reviewed recent progress in research on plant growth regulator (PGR) receptors. For some growth regulators, no receptor protein has yet been identified, but promising new approaches are discussed. For other receptors, specific and sensitive probes have been developed and, in one case, the membrane-associated auxin-binding protein of maize, these have been used to study the function of the receptor. The maize receptor has been cloned and sequenced; cDNA probes will allow the expression of receptor genes in normal and transformed plants to be studied. PGR sensitivity mutants have been described and, in conjunction with biochemical probes, should prove valuable in elucidating the functions of receptors and the nature of subsequent signal transduction events.     

Nicotinic acetylcholine receptors: targets for commercially important insecticides

Nicotinic acetylcholine receptors (nAChRs) are major excitatory neurotransmitter receptors in both vertebrates and invertebrates. In insects, nAChRs are the target site for several naturally occurring and synthetic compounds that exhibit potent insecticidal activity. Several compounds isolated from plants are potent agonists or antagonists of nAChRs, suggesting that these may have evolved as a defence mechanism against insects and other herbivores. Nicotine, isolated from the tobacco plant, has insecticidal activity and has been used extensively as a commercial insecticide. Spinosad, a naturally occurring mixture of two macrocyclic lactones isolated from the microorganism Saccharopolyspora spinosa, acts upon nAChRs and has been developed as a commercial insecticide. Since the early 1990s, one of the most widely used and rapidly growing classes of insecticides has been the neonicotinoids. Neonicotinoid insecticides are potent selective agonists of insect nAChRs and are used extensively in both crop protection and animal health applications. As with other classes of insecticides, there is growing evidence for the evolution of resistance to insecticides that act on nAChRs.     

Structural Insights into the Interactions between Platelet Receptors and Fibrillar Collagen

Collagen peptides have been used to identify binding sites for several important collagen receptors, including integrin α₂β₁, glycoprotein VI, and von Willebrand factor. In parallel, the structures of these collagen receptors have been reported, and their interactions with collagen peptides have been studied. Recently, the three-dimensional structure of the intact type I collagen fiber from rat tail tendon has been resolved by fiber diffraction. It is now possible to map the binding sites of platelet collagen receptors onto the intact collagen fiber in three dimensions. This minireview will discuss these recent findings and their implications for platelet activation by collagen.     

Structural characteristics of two highly selective opioid peptides

The demonstration of opioid receptors by radioligand binding and the discovery of their endogenous peptide ligands has provided a new class of compounds that can be used for the development of novel opioids. The number of potential receptor targets for such opioids has been expanded by the identification of multiple opioid receptor types. The development of highly selective opioid peptides using the principles of conformational restriction permits the analysis of the structure-activity requirements of each receptor type, and is facilitating the elucidation of the functional properties of the different opioid receptors.     

Interaction of [3H](–)-SKF-10,047 with Brain σ Receptors: Characterization and Autoradiographic Visualization

The sigma opiates differ from other opiates in their stimulatory and psychotomimetic actions. The sigma opiate [3H](-)-SKF-10,047 has been used to characterize sigma receptors in rat nervous tissue. Binding of [3H](-)-SKF-10,047 to rat brain membranes was of high affinity, saturable, and reversible. Scatchard analysis revealed the apparent interaction of this drug with two distinct binding sites characterized by affinities of 0.03 and 75 nM (5 mM Tris-HCl buffer, pH 7.4, at 4 degrees C). Competition analyses involving rank order determinations for a series of opiates and other drugs indicate that the high-affinity binding site is the mu opiate receptor. The lower-affinity site (revealed after suppression of mu and delta receptor binding) has been identified as the sigma opiate/phencyclidine receptor. In vitro autoradiography has been used to visualize neuroanatomical patterns of receptors labeled using [3H](-)-SKF-10,047 in the presence of normorphine and [D-Ala2,D-Leu5]enkephalin to block mu and delta interactions, respectively. Labeling patterns differ markedly from those for mu, delta, or kappa receptors. The highest densities (determined by quantitative autoradiography) are found in the medial portion of the nucleus accumbens, amygdaloid nucleus, hippocampal formation, central gray, locus coeruleus, and the parabrachial nuclei. Receptors in these structures could account for the stimulatory, mood-altering, and analgesic properties of the sigma opiates. Although not the most selective sigma opiate ligand, [3H](-)-SKF-10,047 binds to sigma opiate receptors in brain, and this interaction can be readily distinguished from its interactions with other classes of brain opiate receptors.     

Studies on the role of iron binding ligands and the intestinal brush border receptors in iron absorption.

With a view to identifying ligands that could be used as promoters of iron absorption, the affinity of a number of iron chelating agents and the efficiency with which they can donate iron to the brush border receptors has been studied. A number of organic and inorganic compounds were found to chelate iron and keep it soluble at pH 7.5 of the intestinal lumen. This ligand-bound iron was taken up by the intestinal brush border receptors with varying degree of efficiency; ascorbic acid being the most effective and EDTA and citrate the least effective in donating the chelated iron to the receptors. Several polyphosphate compounds, used as food additives, chelated iron and kept it in solution but showed moderate potency for donating iron to the receptors.     

An ultrahigh-throughput screening assay for estrogen receptor ligands.

Members of the steroid and thyroid hormone receptor superfamily (nuclear receptors) play diverse roles in mammalian physiology, in both normal and pathological states. For this reason, and because nuclear receptors are natural receptors for lipophilic small molecules, they are important therapeutic targets for the pharmaceutical industry. Here we describe a method for screening for ligands for one of these receptors, the estrogen receptor. The method is rapid, robust, and reliable, and has been used in an ultrahigh-throughput robotic screen. The receptor is crosslinked to a scintillant-containing solid support (FlashPlate) via a receptor-specific antibody. Test compounds are assayed for competition with a radiolabeled estrogen for binding to the immobilized receptor. Receptor-ligand complexes are allowed to form and receptor-bound radioactivity is detected in a scintillation counter. The assay has been designed for both isoforms of the estrogen receptor, alpha and beta, using separate antibodies for each. Given a radioactive tracer and an appropriate antibody, many of which are now commercially available, the assay could be established for any nuclear receptor.     

A dual role for genetically modified lymphocytes in cancer immunotherapy

T cells as the ultimate effectors of adaptive immune responses are currently used to treat patients affected by infectious diseases and certain tumors. Recently, T cells have been manipulated ex vivo with viral vectors coding for chimeric antigen receptors, exogenous T cell receptors, or 'suicide' genes to potentiate their efficacy and minimize possible side effects. However, the introduction of exogenous genes into T lymphocytes, particularly bacterial or viral transgene products, has occasionally produced immune-mediated elimination of transduced lymphocytes. This immune effect has recently been exploited in a trial of active immunotherapy in melanoma patients. In this opinion article, we discuss the therapeutic possibilities presented by the dual aspects of genetically modified lymphocytes used to treat cancer patients.     

The quantitative patterns of the modified one-center bioreceptor model

A modified one site model of the bioreceptor have been used to estimate quantitatively the phenomenon of the full agonism. Threshold phenomenon, spare receptors and linear dependence of the biological effect on concentration of complex agonist-receptor has been determinate by the general correlation equations. The equations of one site model provides a good fit to the experimental curves "dose-response" for the full agonists and allows to calculate the value of space receptors. The model includes occupancy Clark's theory and law "all or nothing". The interaction of acetylcholine and aklyltrimethylammonium salts with muscarinic acetylholine receptors is analysed as an example of use of this equation.     

Orthosteric and allosteric binding sites of P2X receptors

P2X receptors for ATP comprise a distinct family of ligand gated ion channels with a range of properties. They have been shown to be involved in a variety of physiological processes including blood clotting, sensory perception, pain sensation, bone formation as well as inflammation and may provide a number of novel drug targets. In addition to the orthosteric site for ATP binding it has been suggested that there may be additional allosteric sites that regulate agonist action at the receptor. There is currently no crystal structure available for P2X receptors and the lack of sequence similarity to other ATP binding proteins has meant that a mutagenesis-based approach has been used primarily to investigate receptor structure-function. This review aims to provide an overview of recent work that gives an insight into residues involved in ATP action and allosteric regulation.