Kaposi's sarcoma associated herpesvirus G-protein coupled receptor activation of cyclooxygenase-2 in vascular endothelial cells

Background

Taking as a pattern, the T4 and lambda viruses interacting with each other and with their Gram-negative host, Escherichia coli, a general model is constructed for the evolution of 'gentle' or temperate pathogens. This model is not simply either pure group or kin selection, but probably is common in a variety of host-parasite pairs in various taxonomic groups. The proposed mechanism is that for its own benefit the pathogen evolved ways to protect its host from attack by other pathogens and this has incidentally protected the host. Although appropriate mechanisms would have been developed and excluded related viral species and also other quite different pathogens, the important advance would have been when other individuals of the same species that arrive at the host subsequent to the first infecting one were excluded.

Results

Such a class of mechanisms would not compete one genotype with another, but simply would be of benefit to the first pathogen that had attacked a host organism.

Conclusion

This would tend to protect and extend the life of the host against the detrimental effects of a secondarily infecting pathogen. This leads to the pathogens becoming more temperate via the now favorable co-evolution with its host, which basically protects both host and virus against other pathogens but may cause slowing of the growth of the primary infecting pathogen. Evolution by a 'gentle' strategy would be favored as long as the increased wellbeing of the host also favored the eventual transmission of the early infecting pathogen to other hosts.     

Using genetic algorithms for the construction of phylogenetic trees: application to G-protein coupled receptor sequences

Many different phylogenetic clustering techniques are used currently. One approach is to first determine the topology with a common clustering method and then calculate the branch lengths of the tree. If the resulting tree is not optimal exchanging tree branches can make some local changes in the tree topology. The whole process can be iterated until a satisfactory result has been obtained. The efficiency of this method fully depends on the initially generated tree. Although local changes are made, the optimal tree will never be found if the initial tree is poorly chosen. In this article, genetic algorithms are applied such that the optimal tree can be found even with a bad initial tree topology. This tree generating method is tested by comparing its results with the results of the FITCH program in the PHYLIP software package. Two simulated data sets and a real data set are used.     

Sequence alignment of the G-protein coupled receptor superfamily.

The multitude of G-protein coupled receptor (GPR) superfamily cDNAs recently isolated has exceeded the number of receptor subtypes anticipated by pharmacological studies. Analysis of the sequence similarities and unique features of the members of this family is valuable for designing strategies to isolate related cDNAs, for developing hypotheses concerning substrate-ligand and receptor-effector interactions, and for understanding the evolution of these genes. We have compiled and aligned the 74 unique amino acid sequences published to date and review the present understanding of the structural motifs contributing to ligand binding and G-protein coupling.     

Acute internalization of gap junctions in vascular endothelial cells in response to inflammatory mediator-induced G-protein coupled receptor activation

During the inflammatory response, activation of G-protein coupled receptors (GPCRs) by inflammatory mediators rapidly leads to inhibition of gap junction intercellular communication (GJIC); however, the steps that lead to this inhibition are not known. Combining high-resolution fluorescence microscopy and functional assays, we found that activation of the GPCRs PAR-1 and ET_A/B by their natural inflammatory mediator agonists, thrombin and endothelin-1, resulted in rapid and acute internalization of gap junctions (GJs) that coincided with the inhibition of GJIC followed by increased vascular permeability. The endocytosis protein clathrin and the scaffold protein ZO-1 appeared to be involved in GJ internalization, and ZO-1 was partially displaced from GJs during the internalization process. These findings demonstrate that GJ internalization is an efficient mechanism for modulating GJIC in inflammatory response.     

VIGR--a novel inducible adhesion family G-protein coupled receptor in endothelial cells

Using a signal sequence trap for selection of differentially expressed secretory and membrane proteins, we identified a novel member of the adhesion family of G-protein coupled receptors (GPCRs), termed vascular inducible GPCR (VIGR). VIGR contains C1r-C1s, Uegf and Bmp1 (CUB) and pentraxin (PTX)-like modules and a mucin-like spacer, followed by seven transmembrane domains. By surface biotinylation as well as by immunofluorescence analysis we demonstrate that endogenous, highly glycosylated VIGR is expressed on the cell surface of endothelial cells (ECs) upon LPS or thrombin treatment, and inducible expression is mediated by MAP kinases, but not NF-kappaB. We show that VIGR is selectively expressed in ECs derived from larger vessels, but not from microvessels. In summary, VIGR represents a novel GPCR of the adhesion family, which is unique in its long extra-cellular domain comprising CUB and PTX-like modules and in its inducibility by LPS and thrombin in a subset of ECs, suggesting an important function in cell-adhesion and potentially links inflammation and coagulation.     

Modelling of the activation of G-protein coupled receptors: drug free constitutive receptor activity

G-protein coupled receptors (GPCRs) form a crucial component of approximately 80% of hormone pathways. In this paper, the most popular mechanism for activation of GPCRs—the shuttling mechanism—is modelled mathematically. An asymptotic analysis of this model clarifies the dynamics of the system in the absence of drug, in particular which reactions dominate during the different timescales. Equilibrium analysis of the model demonstrates the model’s ability to predict constitutive receptor activity.     

Applications of BRET to study dynamic G-protein coupled receptor interactions in living cells

Summary Protein-protein interactions are fundamental processes for many biological systems including those involving the superfamily of G-protein coupled receptors (GPCRs). When addressing key questions concerning the regulation of GPCR-protein complexes and their functional significance, the development and refinement of non-invasive techniques to study these interactions will be of great value. One such technique, bioluminescence resonance energy transfer (BRET), is a recently described biophysical method that represents a powerful tool with which to measure protein-protein interactions in live cells, in real time. This minireview highlights the impact that evolving techniques such as BRET have had on the study of dynamic protein interactions involving GPCRs. In particular, the application of BRET to the study of protein interactions involving the receptors for hypothalamic peptide hormones, thyrotropin-releasing hormone (TRH) and gonadotropin-releasing hormone (GnRH), will be discussed. Using these receptors, BRET has successfully been used to demonstrate formation of both agonist-dependent and independent GPCR-GPCR complexes (oligomerization) and the agonist-dependent interaction of GPCRs with their intracellular adaptor protein partners, the arrestins. In summary, BRET is a highly snnsitive method that will not only aid in advancing our understanding of GPCR signalling and trafficking bout coud also potentially lead to the development of novel therapeutics that target these GPCR-protein complexes.     

Picosecond dynamics of G-protein coupled receptor activation in rhodopsin from time-resolved UV resonance Raman spectroscopy

The protein response to retinal chromophore isomerization in the visual pigment rhodopsin is studied using picosecond time-resolved UV resonance Raman spectroscopy. High signal-to-noise Raman spectra are obtained using a 1 kHz Ti:Sapphire laser apparatus that provides <3 ps visible (466 nm) pump and UV (233 nm) probe pulses. When there is no time delay between the pump and probe events, tryptophan modes W18, W16, and W3 exhibit decreased Raman scattering intensity. At longer pump-probe time delays of +5 and +20 ps, both tryptophan (W18, W16, W3, and W1) and tyrosine (Y1 + 2xY16a, Y7a, Y8a) peak intensities drop by up to 3%. These intensity changes are attributed to decreased hydrophobicity in the microenvironment near at least one tryptophan and one tyrosine residue that likely arise from weakened interaction with the beta-ionone ring of the chromophore following cis-to-trans isomerization. Examination of the crystal structure suggests that W265 and Y268 are responsible for these signals. These UV Raman spectral changes are nearly identical to those observed for the rhodopsin-to-Meta I transition, implying that impulsively driven protein motion by the isomerizing chromophore during the 200 fs primary transition drives key structural changes that lead to protein activation.     

Applications of BRET to study dynamic G-protein coupled receptor interactions in living cells

Protein-protein interactions are fundamental processes for manybiological systems including those involving the superfamily ofG-protein coupled receptors (GPCRs). When addressing keyquestions concerning the regulation of GPCR-protein complexes andtheir functional significance, the development and refinement ofnon-invasive techniques to study these interactions will be ofgreat value. One such technique, bioluminescence resonanceenergy transfer (BRET), is a recently described biophysicalmethod that represents a powerful tool with which to measureprotein-protein interactions in live cells, in real time. Thisminireview highlights the impact that evolving techniques such asBRET have had on the study of dynamic protein interactionsinvolving GPCRs. In particular, the application of BRET to thestudy of protein interactions involving the receptors forhypothalamic peptide hormones, thyrotropin-releasing hormone(TRH) and gonadotropin-releasing hormone (GnRH), will bediscussed. Using these receptors, BRET has successfully beenused to demonstrate formation of both agonist-dependent andindependent GPCR-GPCR complexes (oligomerization) and theagonist-dependent interaction of GPCRs with their intracellularadaptor protein partners, the arrestins. In summary, BRET is ahighly sensitive method that will not only aid in advancing ourunderstanding of GPCR signalling and trafficking but could alsopotentially lead to the development of novel therapeutics thattarget these GPCR-protein complexes.     

Structures of the transmembrane helices of the G-protein coupled receptor, rhodopsin.

An hypothesis is tested that individual peptides corresponding to the transmembrane helices of the membrane protein, rhodopsin, would form helices in solution similar to those in the native protein. Peptides containing the sequences of helices 1, 4 and 5 of rhodopsin were synthesized. Two peptides, with overlapping sequences at their termini, were synthesized to cover each of the helices. The peptides from helix 1 and helix 4 were helical throughout most of their length. The N- and C-termini of all the peptides were disordered and proline caused opening of the helical structure in both helix 1 and helix 4. The peptides from helix 5 were helical in the middle segment of each peptide, with larger disordered regions in the N- and C-termini than for helices 1 and 4. These observations show that there is a strong helical propensity in the amino acid sequences corresponding to the transmembrane domain of this G-protein coupled receptor. In the case of the peptides from helix 4, it was possible to superimpose the structures of the overlapping sequences to produce a construct covering the whole of the sequence of helix 4 of rhodopsin. As similar superposition for the peptides from helix 1 also produced a construct, but somewhat less successfully because of the disordering in the region of sequence overlap. This latter problem was more severe for helix 5 and therefore a single peptide was synthesized for the entire sequence of this helix, and its structure determined. It proved to be helical throughout. Comparison of all these structures with the recent crystal structure of rhodopsin revealed that the peptide structures mimicked the structures seen in the whole protein. Thus similar studies of peptides may provide useful information on the secondary structure of other transmembrane proteins built around helical bundles.     

Using random forests for assistance in the curation of G-protein coupled receptor databases

Background

Biology is experiencing a gradual but fast transformation from a laboratory-centred science towards a data-centred one. As such, it requires robust data engineering and the use of quantitative data analysis methods as part of database curation. This paper focuses on G protein-coupled receptors, a large and heterogeneous super-family of cell membrane proteins of interest to biology in general. One of its families, Class C, is of particular interest to pharmacology and drug design. This family is quite heterogeneous on its own, and the discrimination of its several sub-families is a challenging problem. In the absence of known crystal structure, such discrimination must rely on their primary amino acid sequences.

Methods

We are interested not as much in achieving maximum sub-family discrimination accuracy using quantitative methods, but in exploring sequence misclassification behavior. Specifically, we are interested in isolating those sequences showing consistent misclassification, that is, sequences that are very often misclassified and almost always to the same wrong sub-family. Random forests are used for this analysis due to their ensemble nature, which makes them naturally suited to gauge the consistency of misclassification. This consistency is here defined through the voting scheme of their base tree classifiers.

Results

Detailed consistency results for the random forest ensemble classification were obtained for all receptors and for all data transformations of their unaligned primary sequences. Shortlists of the most consistently misclassified receptors for each subfamily and transformation, as well as an overall shortlist including those cases that were consistently misclassified across transformations, were obtained. The latter should be referred to experts for further investigation as a data curation task.

Conclusion

The automatic discrimination of the Class C sub-families of G protein-coupled receptors from their unaligned primary sequences shows clear limits. This study has investigated in some detail the consistency of their misclassification using random forest ensemble classifiers. Different sub-families have been shown to display very different discrimination consistency behaviors. The individual identification of consistently misclassified sequences should provide a tool for quality control to GPCR database curators.

     

Reorganization of the actin cytoskeleton upon G-protein coupled receptor signaling

The actin cytoskeleton is involved in a multitude of cellular responses besides providing structural support. While the role of the actin cytoskeleton in cellular processes such as trafficking and motility has been extensively studied, reorganization of the actin cytoskeleton upon signaling by G-protein coupled receptors (GPCRs) represents a relatively unexplored area. The G-protein coupled receptor superfamily is an important protein family in mammals, involved in signal transduction across membranes. G-protein coupled receptors act as major signaling hubs and drug targets. The serotonin(1A) receptor is a representative member of the G-protein coupled receptor superfamily and plays a crucial role in the generation and modulation of various cognitive, developmental and behavioral functions. In order to monitor the changes in the actin cytoskeleton upon serotonin(1A) receptor signaling in a quantitative manner, we developed an approach based on high magnification imaging of F-actin in cells, followed by image reconstruction. Our results suggest that the actin cytoskeleton is reorganized in response to serotonin(1A) receptor signaling. In addition, we show that reorganization of the actin cytoskeleton is strongly dependent on adenosine 3',5'-cyclic monophosphate level, and is mediated by the activation of protein kinase A. Our results are consistent with the possibility of a feedback mechanism involving the actin cytoskeleton, adenosine 3',5'-cyclic monophosphate level and the serotonin(1A) receptor.     

Stability study of the human G-protein coupled receptor, Smoothened

Smoothened is a member of the G-protein coupled receptor (GPCR) family responsible for the transduction of the Hedgehog signal to the intracellular effectors of the Hedgehog signaling pathway. Aberrant regulation of this receptor is implicated in many cancers but also in neurodegenerative disorders. Despite the pharmacological relevance of this receptor, very little is known about its functional mechanism and its physiological ligand. In order to characterize this receptor for basic and pharmacological interests, we developed the expression of human Smoothened in the yeast Saccharomyces cerevisiae and Smoothened was then purified. Using Surface Plasmon Resonance technology, we showed that human Smoothened was in a native conformational state and able to interact with its antagonist, the cyclopamine, both at the yeast plasma membrane and after purification. Thermostability assays on purified human Smoothened showed that this GPCR is relatively stable in the classical detergent dodecyl-β-d-maltoside (DDM). The fluorinated surfactant C₈F₁₇TAC, which has been proposed to be less aggressive towards membrane proteins than classical detergents, increased Smoothened thermostability in solution. Moreover, the replacement of a glycine by an arginine in the third intracellular loop of Smoothened coupled to the use of the fluorinated surfactant C₈F₁₇TAC during the mutant purification increased Smoothened thermostability even more. These data will be very useful for future crystallization assays and structural characterization of the human receptor Smoothened.     

An algebra of dimerization and its implications for G-protein coupled receptor signaling

Many species of receptors form dimers, but how can we use this information to make predictions about signal transduction? This problem is particularly difficult when receptors dimerize with many different species, leading to a combinatoric increase in the possible number of dimer pairs. As an example system, we focus on receptors in the G-protein coupled receptor (GPCR) family. GPCRs have been shown to reversibly form dimers, but this dimerization does not directly affect signal transduction. Here we present a new theoretical framework called a dimerization algebra. This algebra provides a systematic and rational way to represent, manipulate, and in some cases simplify large and often complicated networks of dimerization interactions. To compliment this algebra, Monte Carlo simulations are used to predict dimerization's effect on receptor organization on the membrane, signal transduction, and internalization. These simulation results are directly comparable to various experimental measures such as fluorescence resonance energy transfer (FRET), and as such provide a link between the dimerization algebra and experimental data. As an example, we show how the algebra and computational results can be used to predict the effects of dimerization on the dopamine D2 and somatastatin SSTR1 receptors. When these predictions were compared to experimental findings from the literature, good agreement was found, demonstrating the utility of our approach. Applications of this work to the development of a novel class of dimerization-modulating drugs are also discussed.     

Modelling the activation of G-protein coupled receptors by a single drug

In this paper, the most popular proposed mechanism for activation of G-protein coupled receptors (GPCRs) - the shuttling mechanism - is modelled mathematically. An asymptotic analysis of this model clarifies the dynamics of the system in the presence of a drug, in particular identifying which reactions dominate during the different timescales. The modelling also reveals challenging behaviour in the form of a peak response. This new mechanism gives simple explanations for complex, possibly misunderstood, behaviour.     

Mechanisms of regulation and function of G-protein coupled receptor kinases

G protein-coupled receptor kinases (GRKs) are key modulators of G protein-coupled receptor (GPCR) signaling. They constitute a family of seven mammalian serine-threonine protein kinases that phosphorylate agonist-bound receptor. GRKs-mediated receptor phosphorylation rapidly initiates profound impairment of receptor signaling and desensitization. Activity of GRKs and subcellular targeting is tightly regulated by interaction with receptor domains, G protein subunits, lipids, anchoring proteins and calcium sensitive proteins. Moreover, GRK phosphorylation by several other kinases and autophosphorylation have recently been shown to modulate its functionality. This review summarize our current knowledge of GRKs regulatory mechanisms and GRKs physiological function.     

Structural models for dimerization of G-protein coupled receptors: the opioid receptor homodimers

Among the most exciting functional features of G-protein coupled receptors (GPCRs) that are coming into focus lately are those relating to the role and structural characteristics of their oligomerization (mostly homo- and heterodimers). The structural underpinnings of these novel functional insights are still not clear, as current experimental techniques have not yet succeeded in identifying the dimerization interfaces between GPCR monomers. Two computational approaches have recently been designed in our lab to provide reasonable three-dimensional (3D) molecular models of the transmembrane (TM) regions of GPCR dimers based on a combination of the structural information of receptor monomers and analyses of correlated mutations in receptor families. The modeling of GPCR heterodimers has been described recently. We present here a related approach for modeling of GPCR homodimers that identifies the interfaces in the most likely configurations of the complexes. The approach is illustrated for the three cloned opioid receptor subtypes (OPRD, OPRM, and OPRK).     

Modeling activation and desensitization of G-protein coupled receptors provides insight into ligand efficacy.

Signaling through G-protein coupled receptors is one of the most prevalent and important methods of transmitting information to the inside of cells. Many mathematical models have been proposed to describe this type of signal transduction, and the ternary complex (ligand/receptor/G-protein) model and its derivatives are among the most widely accepted. Current versions of these equilibrium models include both active (i.e. signaling) and inactive conformations of the receptor, but do not include the dynamics of G-protein activation or receptor desensitization. Yet understanding how these dynamic events effect response behavior is crucial to determining ligand efficacy. We developed a mathematical model for G-protein coupled receptor signaling that includes G-protein activation and receptor desensitization, and used it to predict how activation and desensitization would change if either the conformational selectivity (the effect of ligand binding on the distribution of active and inactive receptor states) or the desensitization rate constant was ligand-specific. In addition, the model was used to explore the implications of measuring responses far downstream from G-protein activation. By comparing the experimental data from the beta(2)-adrenergic, micro-opioid, D(1)dopamine, and neutrophil N -formyl peptide receptors with the predictions of our model, we found that the conformational selectivity is the predominant factor in determining the amounts of activation and desensitization caused by a particular ligand.