Model of influenza A virus infection: dynamics of viral antagonism and innate immune response.

Viral antagonism of host responses is an essential component of virus pathogenicity. The study of the interplay between immune response and viral antagonism is challenging due to the involvement of many processes acting at multiple time scales. Here we develop an ordinary differential equation model to investigate the early, experimentally measured, responses of human monocyte-derived dendritic cells to infection by two H1N1 influenza A viruses of different clinical outcomes: pandemic A/California/4/2009 and seasonal A/New Caledonia/20/1999. Our results reveal how the strength of virus antagonism, and the time scale over which it acts to thwart the innate immune response, differs significantly between the two viruses, as is made clear by their impact on the temporal behavior of a number of measured genes. The model thus sheds light on the mechanisms that underlie the variability of innate immune responses to different H1N1 viruses.

Functional selectivity in GPCR heterocomplexes.

G protein-coupled receptors (GPCRs) can couple to more than one signaling pathway. Biophysical studies and pharmacological theory indicate that they exist in different active conformations that differ in their capacity to activate specific signaling pathways. Individual agonists stabilize particular active conformations and thereby can differ in their relative activation of different signaling pathways coupled to the same receptor, a phenomenon referred to as functional selectivity. Many pairs of GPCRs have been shown to interact and form heterocomplexes in vitro and in vivo. Recent studies implicate these complexes in the responses to some therapeutic drugs and drugs of abuse, and raise the possibility that they may be involved in mediating functional selectivity.

Power-laws in interferon-B mRNA distribution in virus-infected dendritic cells.

Interferon-beta (IFNB1) mRNA shows very large cell-to-cell variability in primary human dendritic cells infected by Newcastle disease virus, with copy numbers varying from a few to several thousands. Analysis of data from the direct measurement of the expression of this gene in its natural chromatin environment in primary human cells shows that the distribution of mRNA across cells follows a power law with an exponent close to -1, and thus encompasses a range of variation much more extensive than a Gaussian. We also investigate the single cell levels of IFNB1 mRNA induced by infection with Texas influenza A mutant viruses, which vary in their capacity to inhibit the signaling pathways responsible for activation of this gene. Here as well we observe power-law behavior for the distribution of IFNB1 mRNA, albeit over a truncated range of values, with exponents similar to the one for cells infected by Newcastle disease virus. We propose a model of stochastic enhanceosome and preinitiation complex formation that incorporates transcriptional pulsing. Analytical and numerical results show good agreement with the observed power laws, and thus support the existence of transcriptional pulsing of an unmodified, intact gene regulated by a natural stimulus.

Shared kinase fluctuations between two enzymatic reactions.

Kinases serve crucial roles in many cellular signaling pathways that process and transfer information. When signaling kinases phosphorylate two targets, these can serve as branch points that distribute information among two pathways. Responses to stimuli transmitted by activated kinases show high levels of cell-to-cell variation that influence cellular function. We ask how fluctuations around a steady state, due to kinase fluctuations and intrinsic noise, are distributed between two reactions with substrates phosphorylated by a shared kinase. We develop the formalism to answer this question and, for a realistic set of biological constants, we illustrate various features of fluctuations and relaxation times to a steady state. We find that the steady-state response determines the size and range in enzyme concentration of phosphorylated substrate fluctuations, and that the choice of an operating point can have a large impact on how shared kinase noise is distributed among two available pathways.

BAC-mediated transgenic expression of fluorescent autophagic protein Beclin 1 reveals a role for Beclin 1 in lymphocyte development.

Beclin 1/Atg6 is an essential component of the evolutionary conserved PtdIns(3)-kinase (Vps34) protein complex that regulates macroautophagy (autophagy) in eukaryotic cells and also interacts with antiapoptotic Bcl-2 family members, Bcl-2, and Bcl-x(L). To elucidate the physiological function of Beclin 1, we generated transgenic mice producing a green fluorescent Beclin 1 protein (Beclin 1-GFP) under Beclin 1 endogenous regulation. The beclin 1-GFP transgene is functional because it completely rescues early embryonic lethality in beclin 1-deficient mice. The transgenic mice appear normal, with undetected change in basal autophagy levels in different tissues, despite the additional expression of functional Beclin 1-GFP. Staining of Beclin 1-GFP shows mostly diffuse cytoplasmic distribution in various tissues. Detailed analysis of the transgene expression by flow cytometry reveals a Bcl-2-like biphasic expression pattern in developing T and B cells, as well as differential regulation of expression in mature versus immature thymocytes following in vitro stimulation. Moreover, thymocytes expressing high Beclin 1-GFP levels appear increasingly sensitive to glucocorticoid-induced apoptosis in vitro. Our results, therefore, support a role for Beclin 1 in lymphocyte development involving cross talk between autophagy and apoptosis.

Effective use of microarrays in neuroendocrine research.

The development of microarray technology makes it possible to simultaneously assay the expression level of hundreds to tens of thousands of mRNA transcripts in one experiment. Genome-wide transcriptional analysis has increasing importance for many areas of neuroendocrinology research. The expense and technical complexity of microarray experiments can make it difficult to navigate the terrain of rival platforms and technologies. In this review, we provide a practical view and comparison of various microarray technologies. Affymetrix arrays, high-density cDNA arrays, membrane arrays and experimental design and data analysis are all discussed by researchers currently using these techniques to study gene regulation in neuroendocrine tissues.

D1 dopamine receptor regulation of microtubule-associated protein-2 phosphorylation in developing cerebral cortical neurons.

This study addresses the hypothesis that the previously described capacity of D1 dopamine receptors (D1Rs) to regulate dendritic growth in developing cortical neurons may involve alterations in the phosphorylation state of microtubule-associated protein-2 (MAP2). The changes in phosphorylation of this protein are known to affect its ability to stabilize the dendritic cytoskeleton. The study involved two systems: primary cultures of mouse cortical neurons grown in the presence of the D1R agonists, SKF82958 or A77636, and the cortex of neonatal transgenic mice overexpressing the D1A subtype of D1R. In both models, a decrease in dendritic extension corresponded with an elevation in MAP2 phosphorylation. This phosphorylation occurred on all three amino acid residues examined in this study: serine, threonine, and tyrosine. In cultured cortical neurons, D1R stimulation-induced increase in MAP2 phosphorylation was blocked by the protein kinase A (PKA) inhibitor, H-89, and mimicked by the PKA activator, S(p)-cAMPS. This indicates that D1Rs modulate MAP2 phosphorylation through PKA-associated intracellular signaling pathways. We also observed that the elevations in MAP2 phosphorylation in neuronal cultures in the presence of D1R agonists (or S(p)-cAMPS) were maintained for a prolonged time (up to at least 96 hr). Moreover, MAP2 phosphorylation underwent a substantial increase between 24 and 72 hr of exposure to these drugs. Our findings are consistent with the idea that D1Rs can modulate growth and maintenance of dendrites in developing cortical cells by regulating the phosphorylation of MAP2. In addition, our observations suggest that MAP2 phosphorylation by long-term activation of D1Rs (and PKA) can be divided into two phases: the initial approximately 24-hr-long phase of a relatively weak elevation in phosphorylation and the delayed phase of a much more robust phosphorylation increase taking place during the next approximately 48 hr.

Gonadotropin-releasing hormone receptor-coupled gene network organization.

An early gene cDNA microarray was developed to study genes that are regulated immediately following gonadotropin-releasing hormone (GnRH) receptor activation. 956 selected candidate genes were printed in triplicate, a t statistic-based regulation algorithm was used for data analysis, and the response to GnRH in a time course from 1 to 6 h was determined. Measurements were highly reproducible within arrays, between arrays, and between experiments. Accuracy and algorithm reliability were established by real-time polymerase chain reaction assays of 60 genes. Gene changes ranging from 1.3- to 31-fold on the microarray were confirmed by real-time polymerase chain reaction. Many of the genes were found to be highly regulated. The regulated genes identified were all elevated at 1 h of treatment and returned nearly or completely to baseline levels of expression by 3 h of treatment. This broad, robust, and transient transcriptional response to constant GnRH exposure includes modulators of signal transduction (e.g. Rgs2 and IkappaB), cytoskeletal proteins (e.g. gamma-actin), and transcription factors (e.g. c-Fos, Egr1, and LRG21). The interplay of the activators, repressors, and feedback inhibitors identified embodies a combinatorial code to direct the activity of specific downstream secondary genes.

Insertional mutagenesis of the arginine cage domain of the gonadotropin-releasing hormone receptor.

The pattern of side-chain conservation at the cytoplasmic side of the third transmembrane domain of rhodopsin family G protein-coupled receptors, Asp/Glu-Arg-Tyr/X-X-X-Ile/Val, defines a structural "arginine cage" domain. Previous computational and mutagenesis studies of the GnRH receptor indicated an important contribution of local interactions to the function of this domain. We have investigated the functional importance of the intrahelical position and orientation of the arginine cage using insertional mutagenesis. Introduction of a single Ala proximal to the conserved Asp-Arg of this domain caused loss of detectable ligand binding. Inserting a second Ala, however, restored high-affinity agonist binding. Further insertion of three or four Ala residues at this site generated receptors that bound agonist with an affinity 3- to 10-fold higher than that of the wild-type receptor. Loss of detectable coupling to inositol phosphate turnover in all these mutant receptors confirms that the structure required in this region for efficient signaling is highly constrained. In contrast, the recovery of agonist binding with the progressive insertion of two to four Ala residues indicates that specific orientations of this segment can stabilize high-affinity receptor conformations that are uncoupled from signal transduction.

A chicken gonadotropin-releasing hormone receptor that confers agonist activity to mammalian antagonists. Identification of D-Lys(6) in the ligand and extracellular loop two of the receptor as determinants.

Mammalian receptors for gonadotropin-releasing hormone (GnRH) have over 85% sequence homology and similar ligand selectivity. Biological studies indicated that the chicken GnRH receptor has a distinct pharmacology, and certain antagonists of mammalian GnRH receptors function as agonists. To explore the structural determinants of this, we have cloned a chicken pituitary GnRH receptor and demonstrated that it has marked differences in primary amino acid sequence (59% homology) and in its interactions with GnRH analogs. The chicken GnRH receptor had high affinity for mammalian GnRH (K(i) 4.1 +/- 1.2 nM), similar to the human receptor (K(i) 4.8 +/- 1.2 nM). But, in contrast to the human receptor, it also had high affinity for chicken GnRH ([Gln(8)]GnRH) and GnRH II ([His(5),Trp(7),Tyr(8)]GnRH) (K(i) 5.3 +/- 0.5 and 0.6 +/- 0.01 nM). Three mammalian receptor antagonists were also pure antagonists in the chicken GnRH receptor. Another three, characterized by D-Lys(6) or D-isopropyl-Lys(6) moieties, functioned as pure antagonists in the human receptor but were full or partial agonists in the chicken receptor. This suggests that the Lys side chain interacts with functional groups of the chicken GnRH receptor to stabilize it in the active conformation and that these groups are not available in the activated human GnRH receptor. Substitution of the human receptor extracellular loop two with the chicken extracellular loop two identified this domain as capable of conferring agonist activity to mammalian antagonists. Although functioning of antagonists as agonists has been shown to be species-dependent for several GPCRs, the dependence of this on an extracellular domain has not been described.

Conserved helix 7 tyrosine functions as an activation relay in the serotonin 5HT(2C) receptor.

The function of the helix VII Tyr in the conserved Asn-Pro-X-X-Tyr segment of rhodopsin-like G protein coupled receptors has been investigated in many receptors. Various effects of site-directed mutation of this locus have been found, including altered coupling, sequestration and agonist affinity. We report the first constitutively active mutations of this Tyr. In the serotonin 5HT(2C) receptor, substituting Ala or Cys for Tyr resulted in a marked increase in the basal level of inositol phosphate accumulation in transfected COS-1 cells. This constitutive signaling was abolished by the inverse agonist SB206553. Introducing Phe at this locus eliminated both basal and agonist-stimulated signaling. All three mutant receptors showed an increase in binding affinity for the structurally dissimilar agonists 5-hydroxytryptamine (5HT), (+/-)-1-(2,5-dimethoxy-4-iodophenyl)-2-aminopropane (DOI), and quipazine, suggesting that both the activating and inactivating mutations stabilize a high affinity state. These results implicate the conserved Tyr in the conformational rearrangements that occur during agonist complexing and receptor activation.

Dopamine receptors: from structure to behavior.

The responses obtained with drugs that act at dopamine receptors depend on the spectrum of receptors stimulated, the pattern of stimulation and the neuronal signal-transduction pathways that are activated. In the absence of drugs that reliably discriminate between the various cloned receptors, elucidating the role of these receptors has largely relied on molecular genetic approaches that include expression of genes for receptors in cell lines and manipulation of this expression in animal models. Connecting molecular events that occur consequent to receptor stimulation with the resulting physiological effects entails bridging a complex network of interactions. This article reviews the current understanding of the molecular, cellular and systemic consequences of activation of the different dopamine receptors.

Multiple interactions of the Asp(2.61(98)) side chain of the gonadotropin-releasing hormone receptor contribute differentially to ligand interaction.

Mutation of Asp(2.61(98)) at the extracellular boundary of transmembrane helix 2 of the gonadotropin-releasing hormone (GnRH) receptor decreased the affinity for GnRH. Using site-directed mutagenesis, ligand modification, and computational modeling, different side chain interactions of Asp(2.61(98)) that contribute to high-affinity binding were investigated. The conservative Asp(2. 61(98))Glu mutation markedly decreased the affinity for a series of GnRH analogues containing the native His(2) residue. This mutant showed smaller decreases in affinity for His(2)-substituted ligands. The loss of preference for His(2)-containing ligands in the mutant receptor shows that Asp(2.61(98)) determines the specificity for His(2). Analysis of the affinities of a series of position 2-substituted ligands suggests that a hydrogen bond forms between Asp(2.61(98)) and the delta NH group of His(2) and that Asp(2. 61(98)) forms a second hydrogen bond with the ligand. Substitution of Asp(2.61(98)) with an uncharged residue further decreased the affinity for all ligands and also decreased receptor expression. Computational modeling indicates an intramolecular ionic interaction of Asp(2.61(98)) with Lys(3.32(121)) in transmembrane helix 3. The uncharged, Lys(3.32(121))Gln mutation also markedly decreased agonist affinity. The modeling and the similar phenotypes of mutants with uncharged substitutions for Asp(2.61(98)) or Lys(3.32(121)) are consistent with the presence of this helix 2-helix 3 interaction. These studies support a dual role for Asp(2.61(98)): formation of an interhelical interaction with Lys(3.32(121)) that contributes to the structure of the agonist binding pocket and an interaction with His(2) of GnRH that helps stabilize agonist complexing.

Dopamine receptors and locomotor responses: molecular aspects.

The responses obtained with drugs acting as dopamine receptors depend on the spectrum of receptors activated and the neuronal signal transduction pathways that are activated. Drugs that reliably discriminate among the various cloned dopamine receptors are not yet available. Therefore, elucidating the role of these receptors has largely relied on molecular genetic approaches, including heterologous receptor expression in cell lines and manipulation of receptor expression in animal models. The current understanding of the molecular, cellular and systemic consequences of dopaminergic activation of the different cloned dopamine receptors is reviewed.

Evaluation of the role of the D2 dopamine receptor in myoclonus dystonia.

A novel Val154-->Ile mutation in the D2 dopamine receptor (DRD2) on chromosome 11q23 has recently been shown to be associated with myoclonus dystonia (M-D) in one large family. Sequence analysis of the DRD2 gene in 5 M-D patients from different families did not reveal any mutations, nor was there evidence of linkage to the 11q23 region in the DRD2 gene in four other families. Receptor binding and signal transduction assays of the DRD2 mutant and wild-type receptors revealed identical agonist and antagonist affinities and functional responses. These studies suggest that M-D is genetically heterogeneous. The molecular mechanisms through which the Val-->Ile mutation may contribute to M-D remain to be determined.

The functional microdomain in transmembrane helices 2 and 7 regulates expression, activation, and coupling pathways of the gonadotropin-releasing hormone receptor.

Structural microdomains of G protein-coupled receptors (GPCRs) consist of spatially related side chains that mediate discrete functions. The conserved helix 2/helix 7 microdomain was identified because the gonadotropin-releasing hormone (GnRH) receptor appears to have interchanged the Asp(2.50) and Asn(7.49) residues which are conserved in transmembrane helices 2 and 7 of rhodopsin-like GPCRs. We now demonstrate that different side chains of this microdomain contribute specifically to receptor expression, heterotrimeric G protein-, and small G protein-mediated signaling. An Asn residue is required in position 2.50(87) for expression of the GnRH receptor at the cell surface, most likely through an interaction with the conserved Asn(1.50(53)) residue, which we also find is required for receptor expression. Most GPCRs require an Asp side chain at either the helix 2 or helix 7 locus of the microdomain for coupling to heterotrimeric G proteins, but the GnRH receptor has transferred the requirement for an acidic residue from helix 2 to 7. However, the presence of Asp at the helix 7 locus precludes small G protein-dependent coupling to phospholipase D. These results implicate specific components of the helix 2/helix 7 microdomain in receptor expression and in determining the ability of the receptor to adopt distinct activated conformations that are optimal for interaction with heterotrimeric and small G proteins.

A novel human GnRH receptor homolog gene: abundant and wide tissue distribution of the antisense transcript.

Gonadotropin releasing hormone (GnRH) regulates the reproductive system through a specific G-protein-coupled receptor (GPCR) in pituitary gonadotropes. The existence of two (or more) forms of GnRH in most vertebrates suggested the existence of GnRH receptor subtypes (I and II). Using sequence information for extracellular loop 3 of a putative Type II GnRH receptor from a reptile species, we have looked for a Type II GnRH receptor gene in the human genome EST (expressed sequence tag) database. A homolog was identified which has 45% and 41% amino acid identity with exons 2 and 3 of the known human GnRH pituitary receptor (designated Type I) and much lower homology with all other GPCRs. A total of 27 contiguous ESTs was found and comprised a continuous sequence of 1642 nucleotides. The EST sequences were confirmed in the cloned human gene and in PCR products of cDNA from several tissues. All EST transcripts detected were in the antisense orientation with respect to the novel GnRH receptor sequence and were highly expressed in a wide range of human brain and peripheral tissues. PCR of cDNA from a wide range of tissues revealed that intronic sequence equivalent to intron 2 of the Type I GnRH receptor was retained. The failure to splice out putative intron sequences in transcripts which spanned exon-intron boundaries is expected in antisense transcripts, as candidate donor and acceptor sites were only present in the gene when transcribed in the orientation encoding the GnRH receptor homolog. No transcripts extended 5' to the sequence corresponding to intron 2 of the Type I GnRH as the antisense transcripts terminated in poly A due to the presence of a polyadenylation signal sequence in the putative intron 2 when transcribed in the antisense orientation. These findings suggest that a Type II GnRH receptor gene has arisen during vertebrate evolution and is also present in the human. However, the receptor may have become vestigial in the human, possibly due to the abundant and universal tissue transcription of the opposite DNA strand to produce antisense RNA.

Nuclear translocation of GAPDH-GFP fusion protein during apoptosis.

Increased expression and nuclear accumulation of glyceraldehyde-3-phosphate dehydrogenase (GAPDH) are early, critical events in several forms of apoptosis. In order to investigate the subcellular trafficking of GAPDH in vivo, the localization of a GAPDH-green fluorescent protein (GFP) fusion was studied in PC12, HEK 293 and COS-1 cells. In control cells, fusion protein autofluorescence was largely restricted to the cytoplasm, rather than the nuclear concentration favored by GFP alone. In contrast, as early as 30 min after an insult, nuclear fluorescence increased in all cell lines studied. The fusion protein redistribution paralleled the dynamics of endogenous GAPDH. These data suggest that some nuclear GAPDH observed during apoptosis represents protein previously resident in the cytosol. This construct provides an in vivo monitor for an early change in apoptosis.

Paradoxical locomotor behavior of dopamine D1 receptor transgenic mice.

The behavioral effects of augmenting dopamine D1 receptor expression in the brain were investigated in mice incorporating additional copies of the mouse D1 receptor gene. Two transgenic lines showed increases in brain D1 receptor binding sites, which were greatest in extrastriatal regions. The full D1 agonist SKF 81297, when administered systemically to control animals, stimulated a dose-dependent increase in locomotor activity. In contrast, in D1 receptor overexpressing transgenic mice, this drug caused a marked suppression of locomotion due to a decrease in the frequency of movement initiation. Amphetamine and cocaine induced comparable locomotor activation in both transgenic animals and their control littermates. In the transgenic animals, D1 agonist-induced rearing and climbing behaviors were suppressed. However, on rotarod testing, the agonist-treated transgenic and control mice performed comparably, indicating that sensorimotor coordination was unaffected. These studies demonstrate that altering the levels of D1 receptor expression reverses the effects of D1 agonism on locomotor initiation and rearing.