Agonists and antagonists of protease activated receptors (PARs)

Protease activated receptors (PARs) are a category of G-protein coupled receptors (GPCRs) implicated in the progression of a wide range of diseases, including thrombosis, inflammatory disorders, and proliferative diseases. Signal transduction via PARs proceeds via an unusual activation mechanism. Instead of being activated through direct interaction with an extracellular signal like most GPCRs. they are self-activated following cleavage of their extracellular N-terminus by serine proteases to generate a new receptor N-terminus that acts as an intramolecular ligand by folding back onto itself and triggering receptor activation. Short synthetic peptides corresponding to this newly exposed N-terminal tethered ligand can activate three of the four known PARs in the absence of proteases. and such PAR activating peptides (PAR-APs) have served as templates for agonist/antagonist development. In fact much of the evidence for involvement of PARs in diseases has relied upon use of PAR-APs. often of low potency and uncertain selectivity. This review summarizes current structures of PAR agonists and antagonists, the need for more selective and more potent PAR ligands that activate or antagonize this intriguing class of receptors, and outlines the background relevant to PAR activation, assay methods, and physiological properties anticipated for PAR ligands.

Completion of the genome sequence of Lettuce necrotic yellows virus, type species of the genus Cytorhabdovirus

We completed the genome sequence of Lettuce necrotic yellows virus (LNYV) by determining the nucleotide sequences of the 4a (putative phosphoprotein), 4b, M (matrix protein), G (glycoprotein) and L (polymerase) genes. The genome consists of 12,807 nucleotides and encodes six genes in the order 3' leader-N-4a(P)-4b-M-G-L-5' trailer. Sequences were derived from clones of a cDNA library from LNYV genomic RNA and from fragments amplified using reverse transcription-polymerase chain reaction. The 4a protein has a low isoelectric point characteristic for rhabdovirus phosphoproteins. The 4b protein has significant sequence similarities with the movement proteins of capillo- and trichoviruses and may be involved in cell-to-cell movement. The putative G protein sequence contains a predicted 25 amino acids signal peptide and endopeptidase cleavage site, three predicted glycosylation sites and a putative transmembrane domain. The deduced L protein sequence shows similarities with the L proteins of other plant rhabdoviruses and contains polymerase module motifs characteristic for RNA-dependent RNA polymerases of negative-strand RNA viruses. Phylogenetic analysis of this motif among rhabdoviruses placed LNYV in a group with other sequenced cytorhabdoviruses, most closely related to Strawberry crinkle virus. (c) 2005 Elsevier B.V. All rights reserved.

Omega-conotoxins GVIA, MVIIA and CVID: SAR and clinical potential

Highly selective N-type voltage-gated calcium (Ca-V) channel inhibitors from cone snail venom (the omega-conotoxins) have emerged as a new class of therapeutics for the treatment of chronic and neuropathic pain. Earlier in 2005, Prialt ( Elan) or synthetic omega-conotoxin MVIIA, was the first omega-conotoxin to be approved by Food and Drug Administration for human use. This review compares the action of three omega-conotoxins, GVIA, MVIIA and CVID, describing their structure-activity relationships and potential as leads for the design of improved N-type therapeutics that are more useful in the treatment of chronic pain.

Solution structure and characterization of the LGR8 receptor binding surface of insulin-like peptide 3

Insulin-like peptide 3 (INSL3), a member of the relaxin peptide family, is produced in testicular Leydig cells and ovarian thecal cells. Gene knock-out experiments have identified a key biological role in initiating testes descent during fetal development. Additionally, INSL3 has an important function in mediating male and female germ cell function. These actions are elicited via its recently identified receptor, LGR8, a member of the leucine-rich repeat-containing G-protein- coupled receptor family. To identify the structural features that are responsible for the interaction of INSL3 with its receptor, its solution structure was determined by NMR spectroscopy together with in vitro assays of a series of B-chain alanine-substituted analogs. Synthetic human INSL3 was found to adopt a characteristic relaxin/ insulin-like fold in solution but is a highly dynamic molecule. The four termini of this two-chain peptide are disordered, and additional conformational exchange is evident in the molecular core. Alanine-substituted analogs were used to identify the key residues of INSL3 that are responsible for the interaction with the ectodomain of LGR8. These include Arg(B16) and Val(B19), with His(B12) and Arg(B20) playing a secondary role, as evident from the synergistic effect on the activity in double and triple mutants involving these residues. Together, these amino acids combine with the previously identified critical residue, Trp(B27), to form the receptor binding surface. The current results provide clear direction for the design of novel specific agonists and antagonists of this receptor.

Synaptic activation of transient receptor potential channels by metabotropic glutamate receptors in the lateral amygdala

Classical mammalian transient receptor potential channels form non-selective cation channels that open in response to activation of phospholipase C-coupled metabotropic receptors, and are thought to play a key role in calcium homeostasis in non-excitable cells. Within the nervous system transient receptor potential channels are widely distributed but their physiological roles are not well understood. Here we show that in the rat lateral amygdala transient receptor potential channels mediate an excitatory synaptic response to glutamate. Activation of group l etabotropic glutamate receptors on pyramidal neurons in the lateral amygdala with either exogenous or synaptically released glutamate evokes an inward current at negative potentials with a current voltage relationship showing a region of negative slope and steep outward rectification. This current is blocked by inhibiting G protein function with GTP-beta-S, by inhibiting phospholipase C or by infusing transient receptor potential antibodies into lateral amygdala pyramidal neurons. Using RT-PCR and Western blotting we show that transient receptor potential 1, transient receptor potential 4 and transient receptor potential 5 are present in the lateral amygdala. Single cell PCR confirms the presence of transient receptor potential 1 and transient receptor potential 5 in pyramidal neurons and we show by co-immunoprecipitation that transient receptor potential 1 and transient receptor potential 5 co-assemble as a heteromultimers in the amygdala. These results show that in lateral amygdala pyramidal neurons synaptically released glutamate activates transient receptor potential channels, which we propose are likely to be heteromultimeric channels containing transient receptor potential 1 and transient receptor potential 5/transient receptor potential 4. (c) 2005 Published by Elsevier Ltd on behalf of IBRO.

Endothelin A receptor localization in the internal mammary artery

Introduction: The vasoconstricting peptide endothelin-1 (ET-1) binds two G-protein-coupled receptor subtypes, the Endothelin A (ETA) and Endothelin B (ETB) receptors. The ETB receptor subtype has been predominantly localised to the arterial and venous endothelial cells both in-vivo and in culture. Stimulation of ET-1 through this receptor subtype can modulate the expression of endothelial nitric oxide and accelerate endothelial cell wound healing. In comparison the ETA receptor is abundantly expressed in medial vascular smooth muscle cells and mediates the vasoconstrictor action of ET-1 and is thought to play a key role in angiogenesis. Aims: To determine the levels of ETA receptor expression and localisation in the internal mammary artery (IMA). Methods: Twenty-four IMA sections were examined from patients undergoing coronary artery bypass (CABG) surgery (5F; 19M; mean age 67 years). And 14 organ donor IMA specimens were used as controls (7M; 7F; mean age 45 years. The tissue was fixed in formalin and processed for histology. Immunohistochemistry was performed on cross-sections of the left distal IMA to assess the areas of ETA receptor staining. The percentage are of ETA receptor staining in the media was calculated using image analysis software connected to an optical microscope and semiquantitative assessment was used to grade staining intensity, that is, mild (+), moderate (++) and strong (+++). Results: ETA receptor staining was significantly elevated in the media of the CABG specimens compared with the donor controls (46.88+/11.52% Vs 18.58+/7.65%, P = .0001). Interestingly, the endothelium (++) of the IMA, as well as the small microvessels in the adventitia (+++) stained positive for ETA receptor expression. Without using a haematoxylin counterstain, the nuclei of the cell stained more intensely (+++) with respect to the cytoplasm in both the medial smooth muscle (++) and endothelial cells (++). Fibroblasts in the medial adventitia junction were also positive for ETA receptor expression (+++). Further, this receptor subtype was also strongly expressed by inflammatory cells (monocytes and macrophages). Conclusions: These results demonstrate that the ETA receptor expression is increased in the medial SMC layer of the CABG IMA specimens and also present in the endothelium, vasa vasorum, fibroblasts and inflammatory cell types. Thus it is possible that in addition to affecting vascular tone, ET-1 may play an important role in IMA remodelling.

Microarray and proteomic analysis of the human alcoholic brain

The superior frontal cortex (SFC) is selectively damaged in chronic alcohol abuse, with localized neuronal loss and tissue atrophy. Regions such as motor cortex show little neuronal loss except in severe co-morbidity (liver cirrhosis or WKS). Altered gene expression was found in microarray comparisons of alcoholic and control SFC samples [1]. We used Western blots and proteomic analysis to identify the proteins that also show differential expression. Tissue was obtained at autopsy under informed, written consent from uncomplicated alcoholics and age- and sex-matched controls. Alcoholics had consumed 80 g ethanol/day chronically (often, 200 g/day for 20 y). Controls either abstained or were social drinkers ( 20 g/day). All subjects had pathological confirmation of liver and brain diagnosis; none had been polydrug abusers. Samples were homogenized in water and clarified by brief centrifugation (1000g, 3 min) before storage at –80°C. For proteomics the thawed suspensions were centrifuged (15000g, 50 min) to prepare soluble fractions. Aliquots were pooled from SFC samples from the 5 chronic alcoholics and 5 matched controls used in the previous microarray study [1]. 2-Dimensional electrophoresis was performed in triplicate using 18 cm format pH 4–7 and pH 6–11 immobilized pH gradients for firstdimension isoelectric focusing. Following second-dimension SDS-PAGE the proteins were fluorescently stained and the images collected by densitometry. 182 proteins differed by 2-fold between cases and controls. 141 showed lower expression in alcoholics, 33 higher, and 8 were new or had disappeared. To date 63 proteins have been identified using MALDI-MS and MS-MS. Western blots were performed on uncentrifuged individual samples from 76 subjects (controls, uncomplicated alcoholics and cirrhotic alcoholics). A common standard was run on every gel. After transfer, immunolabeling, and densitometry, the intensities of the unknown bands were compared to those of the standards. We focused on proteins from transcripts that showed clear differences in a series of microarray studies, classified into common sets including Regulators of G-protein Signaling and Myelin-associated proteins. The preponderantly lower level of differentially expressed proteins in alcoholics parallels the microarray mRNA analysis in the same samples. We found that mRNA and protein expression do not frequently correspond; this may help identify pathogenic processes acting at the level of transcription, translation, or post-translationally.