spineless

EVOLUTIONARY HOMOLOGS

Juvenile hormone analog (JHA) insecticides are relatively nontoxic to vertebrates and offer effective control of certain insect pests. Recent reports of resistance in whiteflies and mosquitoes demonstrate the need to identify and understand genes for resistance to this class of insect growth regulators. Mutants of the Methoprene-tolerant (Met) gene in Drosophila melanogaster show resistance to both JHAs and JH, and previous biochemical studies have demonstrated a mechanism of resistance involving an intracellular JH binding-protein that has reduced ligand affinity in Met flies. Met flies are resistant to the toxic and morphogenetic effects of JH and several JHAs, but not to other classes of insecticide. Biochemical studies reveal a target-site resistance mechanism, that of reduced JH binding in cytosolic extracts from either of two JH target tissues in Met flies. This property of reduced JH binding was cytogenetically localized to the Met region on the X chromosome and can account for the resistance. Possible identities for this binding protein include either an accessory JH-binding protein in the cytoplasm, similar to the cellular retinoic acid-binding protein in vertebrates, or a JH receptor protein involved in the action of JH (Ashok, 1998).

The Met+ gene has been cloned by transposable P-element tagging and reduced transcript level has been found in several mutant alleles, showing that underproduction of the normal gene product can lead to insecticide resistance. Transformation of Met flies with a Met+ cDNA results in susceptibility to methoprene, indicating that the cDNA encodes a functional Met+ protein. Met shows homology to the basic helix-loop-helix (bHLH)-PAS family of transcriptional regulators, implicating Met in the action of JH at the gene level in insects. This family also includes the vertebrate dioxin receptor, a transcriptional regulator known to bind a variety of environmental toxicants. Met shows three regions of homology to members of a family of transcriptional activators known as bHLH-PAS proteins. Met generally has higher homology to the vertebrate bHLH-PAS proteins than to those identified in D. melanogaster. A D. melanogaster ARNT-like gene has recently been cloned, and DARNT has higher homology to vertebrate ARNT than does Met, suggesting that DARNT, not Met, may function like ARNT in flies. Met homology to these proteins includes the bHLH region that is involved in DNA binding (30-38% identity), the PAS-A region (28-40%), and the PAS-B region (22-35%). The arrangement of these domains in the Met gene is the same as for other bHLH-PAS genes (Ashok, 1998).

The aryl hydrocarbon receptor (AHR) is a ligand-activated transcription factor, until now described only in vertebrates, that mediates many of the carcinogenic and teratogenic effects of certain environmental pollutants. Orthologs of AHR and its dimerization partner AHR nuclear translocator (ARNT) in the nematode Caenorhabditis elegans are encoded by the genes ahr-1 and aha-1, respectively. The corresponding proteins, AHR-1 and AHA-1, share biochemical properties with their mammalian cognates. Specifically, AHR-1 forms a tight association with HSP90, and AHR-1 and AHA-1 interact to bind DNA fragments containing the mammalian xenobiotic response element with sequence specificity. Yeast expression studies indicate that C. elegans AHR-1, like vertebrate AHR, requires some form of post-translational activation. This requirement depends on the presence of the domains predicted to mediate binding of HSP90 and ligand. Preliminary experiments suggest that if AHR-1 is ligand-activated, its spectrum of ligands is different from that of the mammalian receptor: C. elegans AHR-1 is not photoaffinity labeled by a dioxin analog, and it is not activated by beta-naphthoflavone in the yeast system. The discovery of these genes in a simple, genetically tractable invertebrate should allow elucidation of AHR-1 function and identification of its endogenous regulators (Powell-Coffman, 1998).

Evolution of aryl hydrocarbon receptor

The aryl hydrocarbon receptor (AHR) is a ligand-activated transcription factor through which halogenated aromatic hydrocarbons such as 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) cause altered gene expression and toxicity. The AHR belongs to the basic helix-loop-helix/Per-ARNT-Sim (bHLH-PAS) family of transcriptional regulatory proteins, whose members play key roles in development, circadian rhythmicity, and environmental homeostasis; however, the normal cellular function of the AHR is not yet known. As part of a phylogenetic approach to understanding the function and evolutionary origin of the AHR, the PAS homology domain of AHRs from several species of early vertebrates have been sequenced and phylogenetic analyses of these AHR amino acid sequences have been performed in relation to mammalian AHRs and 24 other members of the PAS family. AHR sequences have been identified in a teleost (the killifish Fundulus heteroclitus), two elasmobranch species (the skate Raja erinacea and the dogfish Mustelus canis), and a jawless fish (the lamprey Petromyzon marinus). Two putative AHR genes, designated AHR1 and AHR2, are found both in Fundulus and Mustelus. Phylogenetic analyses indicate that the AHR2 genes in these two species are orthologous, suggesting that an AHR gene duplication occurred early in vertebrate evolution and that multiple AHR genes may be present in other vertebrates. Database searches and phylogenetic analyses identified four putative PAS proteins in the nematode Caenorhabditis elegans, including possible AHR and ARNT homologs. Phylogenetic analysis of the PAS gene family reveals distinct clades containing both invertebrate and vertebrate PAS family members; the latter include paralogous sequences that are proposed have arisen by gene duplication early in vertebrate evolution. Overall, these analyses indicate that the AHR is a phylogenetically ancient protein present in all living vertebrate groups (with a possible invertebrate homolog), thus providing an evolutionary perspective to the study of dioxin toxicity and AHR function (Hahn, 1997).

Expression of aryl hydrocarbon receptor

The basic helix-loop-helix-PAS (bHLH-PAS) protein ARNT is a dimeric partner of the Ah receptor (AHR) and hypoxia inducible factor 1alpha (HIF1alpha). These dimers mediate biological responses to xenobiotic exposure and low oxygen tension. The recent cloning of ARNT and HIF1 (homologs (ARNT2 and HIF2alpha) indicates that at least six distinct bHLH-PAS heterodimeric combinations can occur in response to a number of environmental stimuli. In an effort to understand the biological relevance of this combinatorial complexity, their relative expression at a number of developmental time points was characterized by parallel in situ hybridization of adjacent tissue sections. In general, there is limited redundancy in the expression of these six transcription factors and each of these bHLH-PAS members displays a unique pattern of developmental expression emerging as early as embryonic day 9.5 (Jain, 1998).

Mutation of aryl hydrocarbon receptor

The Ah receptor (AHR) is a ligand-activated transcription factor that mediates a pleiotropic response to environmental contaminants such as benzo[a]pyrene and 2,3,7,8-tetrachlorodibenzo-p-dioxin. In an effort to gain insight into the physiological role of the AHR and to develop models useful in risk assessment, gene targeting was used to inactivate the murine Ahr gene by homologous recombination. Ahr-/- mice are viable and fertile but show a spectrum of hepatic defects that indicate a role for the AHR in normal liver growth and development. The Ahr-/- phenotype is most severe between 0-3 weeks of age and involves slowed early growth and hepatic defects, including reduced liver weight, transient microvesicular fatty metamorphosis, prolonged extramedullary hematopoiesis, and portal hypercellularity with thickening and fibrosis (Schmidt, 1996).

The aryl hydrocarbon (Ah) receptor (AHR) mediates many carcinogenic and teratogenic effects of environmentally toxic chemicals such as dioxin. An AHR-deficient (Ahr-/-) mouse line was constructed by homologous recombination in embryonic stem cells. Almost half of the mice die shortly after birth, whereas survivors reached maturity and are fertile. The Ahr-/- mice show decreased accumulation of lymphocytes in the spleen and lymph nodes, but not in the thymus. The livers of Ahr-/- mice are reduced in size by 50 percent and showed bile duct fibrosis. Ahr-/- mice are also nonresponsive with regard to dioxin-mediated induction of genes encoding enzymes that catalyze the metabolism of foreign compounds. Thus, the AHR plays an important role in the development of the liver and the immune system (Fernandez-Salguero, 1995).

Ah receptor/Arnt heterodimers

The human aryl hydrocarbon receptor (AhR) and aryl hydrocarbon receptor nuclear translocator protein (Arnt - Drosophila homolog: Tango) were coexpressed in the yeast Saccharomyces cerevisiae to create a system for the study of the Ahr/Arnt heterodimeric transcription factor. Specific transcriptional activation mediated by AhR/Arnt heterodimer (which is a functional indicator of receptor expression) was assessed by beta-galactosidase activity produced from a reporter plasmid. Yeast expressing AhR and Arnt display constitutive transcriptional activity that is not augmented by the addition of AhR agonists in strains that required exogenous tryptophan for viability. In contrast, strains with an intact pathway for tryptophan biosynthesis do respond to AhR agonists and have lower levels of background beta-galactosidase activity. In the yeast system, hexachlorobenzene, benzo(a)pyrene, and beta-naphthoflavone are effective AhR agonists for beta-galactosidase activity induction. Tryptophan, indole, indole acetic acid, and tryptamine activate transcription in yeast coexpressing AhR and Arnt. Indole-3-carbinol is an exceptionally potent AhR agonist in yeast. This yeast system is useful for the study of AhR/Arnt protein complexes, and may prove to be generally applicable to the investigation of other multiprotein complexes (Miller, 1997).

In mouse hepatoma cells, the environmental contaminant 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD, or dioxin) induces Cyp1A1 gene transcription, a process that requires two basic helix-loop-helix regulatory proteins: the aromatic hydrocarbon receptor (AhR) and the aromatic hydrocarbon receptor nuclear translocator (Arnt). Ligation-mediated PCR technique was used to analyze dioxin-induced changes in protein-DNA interactions and chromatin structure of the Cyp1A1 enhancer-promoter in its native chromosomal setting. Dioxin-induced binding of the AhR/Arnt heterodimer to enhancer chromatin is associated with a localized (about 200 bp) alteration in chromatin structure that is manifested by increased accessibility of the DNA; these changes probably reflect direct disruption of a nucleosome by AhR/Arnt. Dioxin induces analogous AhR/Arnt-dependent changes in chromatin structure and accessibility at the Cyp1A1 promoter. However, the changes at the promoter must occur by a different, more indirect mechanism, because they are induced from a distance and do not reflect a local effect of AhR/Arnt binding. Dose-response experiments indicate that the changes in chromatin structure at the enhancer and promoter are graded, mirroring the graded induction of Cyp1A1 transcription by dioxin (Okino, 1995).

Function of Ah receptor/Arnt heterodimers: a role for phosphorylation

The Ah receptor binds aryl hydrocarbons such as 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) with high affinity. After binding aryl hydrocarbons, the receptor releases the 90-kDa heat shock protein and forms a heterodimer with the Arnt protein capable of binding at xenobiotic-responsive elements (XREs) and stimulating the transcription of genes involved in the metabolism of aryl hydrocarbons. The activity of the Ah receptor/Arnt dimer can be decreased by treatments that cause the down-regulation of protein kinase C and decrease the nuclear accumulation of the receptor. Incubation with acid phosphatase or with alkaline phosphatase has been reported to block XRE binding. Thus the literature suggests that phosphorylation regulates Ah receptor activity by affecting DNA binding and/or nuclear transport. A reporter plasmid containing two XREs was used to investigate the effects of phosphatase inhibitors on TCDD-dependent transcription carried out by the Hepa-1 mouse liver cell line. The inhibitors calyculin A and okadaic acid cause two- to threefold increases in TCDD-dependent transcription, at concentrations capable of selectively inhibiting protein phosphatase 1 and protein phosphatase 2A. The inhibitor cyclosporin A doubles TCDD-dependent transcription at a concentration capable of selectively inhibiting protein phosphatase 2B. All three of the phosphatase inhibitors increase TCDD-dependent transcription without affecting transcription in the absence of TCDD. Nuclear extracts were prepared from cells treated with concentrations of either okadaic acid or cyclosporin A, both of which substantially stimulate TCDD-dependent transcription. Neither of the inhibitors significantly increase the level of TCDD-dependent XRE binding in the extracts. GAL4-Arnt fusion proteins were used to further investigate whether the phosphatase inhibitors affected a step other than DNA binding. Okadaic acid treatment specifically increases the ability of a GAL4 fusion protein containing the Arnt PAS and transactivation domains to stimulate transcription. These results suggest that serine/threonine-specific protein phosphatases can act at a level subsequent to XRE binding to inhibit the ability of the Ah receptor/Arnt dimer to stimulate transcription (Li, 1997).

Interaction of Ahr with HSP90

Functional domains of the mouse aryl hydrocarbon receptor (Ahr) were investigated by deletion analysis. Ligand binding is localized to a region encompassing the PAS B repeat. The ligand-mediated dissociation of Ahr from the 90-kDa heat shock protein (HSP90) does not require the aryl hydrocarbon receptor nuclear translocator (Arnt), but it is slightly enhanced by this protein. One HSP90 molecule appears to bind within the PAS region. The other molecule of HSP90 appears to require interaction at two sites: one over the basic helix-loop-helix region, and the other located within the PAS region. Each mutant was analyzed for dimerization with full-length mouse Arnt and subsequent binding of the dimer to the xenobiotic responsive element (XRE). In order to minimize any artificial steric hindrances to dimerization and XRE binding, each Ahr mutant was also tested with an equivalently deleted Arnt mutant. The basic region of Ahr is required for XRE binding but not for dimerization. Both the first and second helices of the basic helix-loop-helix motif and the PAS region are required for dimerization. These last results are analogous to those previously obtained for Arnt, compatible with the notion that equivalent regions of Ahr and Arnt associate with each other. Deletion of the carboxyl-terminal half of Ahr does not affect dimerization or XRE binding but, in contrast to an equivalent deletion of Arnt, eliminates biological activity, as assessed by an in vivo transcriptional activation assay, suggesting that this region of Ahr plays a more prominent role in transcriptional activation of the cyp1a1 gene than does the corresponding region of Arnt (Fukunaga, 1995).

Expression of a series of Ah receptor (AhR) deletion mutants in an in vitro translation system has been previously used to map several functional domains of the murine AhR. In this report, quantitative immunoprecipitation of 90-kDa heat shock protein (hsp90) from reticulocyte lysate allowed a measurement of expression levels for the AhR and AhR deletion mutants, complexed with hsp90. After translation of a series of deletion mutants it was determined that there are two distinct domains important in forming a stable AhR/hsp90 complex, corresponding to amino acid sequences 1-166 and 289-347 of the AhR. Neither Arnt, nor Per are able to stably interact with hsp90. Thus, the AhR appears to be a unique member of the PAS domain family of proteins that binds a known ligand and stably interacts with hsp90 (Perdew, 1996).

Neural function of ahr-1 in C. elegans

The aryl hydrocarbon receptors (AHR) are bHLH-PAS domain containing transcription factors. In mammals, they mediate responses to environmental toxins such as 2, 3, 7, 8-tetrachlorodibenzo-p-dioxin (TCDD). Such functions of AHRs require a cofactor, the aryl hydrocarbon receptor nuclear translocator (ARNT), and the cytoplasmic chaperonins HSP90 and XAP2. AHR homologs have been identified throughout the animal kingdom. The C. elegans orthologs of AHR and ARNT, ahr-1 and aha-1, regulate GABAergic motor neuron fate specification. Four C. elegans neurons known as RMED, RMEV, RMEL and RMER express the neurotransmitter GABA and control head muscle movements. ahr-1 is expressed in RMEL and RMER neurons. Loss of function in ahr-1 causes RMEL and RMER neurons to adopt a RMED/RMEV-like fate, whereas the ectopic expression of ahr-1 in RMED and RMEV neurons can transform them into RMEL/RMER-like neurons. This function of ahr-1 requires aha-1, but not daf-21/hsp90. These results demonstrate that C. elegans ahr-1 functions as a cell-type specific determinant. This study further supports the notion that the ancestral role of the AHR proteins is in regulating cellular differentiation in animal development (Huang, 2004).

The mammalian aryl hydrocarbon receptor (AHR) is a ligand-activated transcription factor that mediates the toxic effects of dioxins and related compounds. Dioxins have been shown to cause a range of neurological defects, but the role of AHR during normal neuronal development is not known. This study investigated the developmental functions of ahr-1, the Caenorhabditis elegans aryl hydrocarbon receptor homolog. ahr-1:GFP is expressed in a subset of neurons, and animals lacking ahr-1 function have specific defects in neuronal differentiation, as evidenced by changes in gene expression, aberrant cell migration, axon branching, or supernumerary neuronal processes. In ahr-1-deficient animals, the touch receptor neuron AVM and its sister cell, the interneuron SDQR, exhibit cell and axonal migration defects. Dorsal migration of SDQR is mediated by UNC-6/Netrin, SAX-3/Robo, and UNC-129/TGFbeta, and this process requires the functions of both ahr-1 and its transcription factor dimerization partner aha-1. A role for ahr-1 during the differentiation of the neurons that contact the pseudocoelomic fluid has also been document. In ahr-1-deficient animals, these neurons are born but they do not express the cell-type-specific markers gcy-32:GFP and npr-1:GFP at appropriate levels. Additionally, it was shown that ahr-1 expression is regulated by the UNC-86 transcription factor. It is proposed that the AHR-1 transcriptional complex acts in combination with other intrinsic and extracellular factors to direct the differentiation of distinct neuronal subtypes. These data, when considered with the neurotoxic effects of AHR-activating pollutants, support the hypothesis that AHR has an evolutionarily conserved role in neuronal development (Qin, 2004).

C. elegans ahr-1 is orthologous to the mammalian aryl hydrocarbon receptor, and it functions as a transcription factor to regulate the development of certain neurons. This study describes the role of ahr-1 in a specific behavior: the aggregation of C. elegans on lawns of bacterial food. This behavior is modulated by nutritional cues and ambient oxygen levels, and aggregation is inhibited by the npr-1 G protein-coupled neuropeptide receptor gene. Loss-of-function mutations in ahr-1 or its transcription partner aha-1 (ARNT) suppress aggregation behavior in npr-1-deficient animals. This behavioral defect is not irreparable. Aggregation behavior can be restored to ahr-1-deficient animals by heat-shock induction of ahr-1 transcription several hours after ahr-1-expressing neurons have normally differentiated. ahr-1 and aha-1 promote cell-type-specific expression of soluble guanylate cyclase genes that have key roles in aggregation behavior and hyperoxia avoidance. Aggregation behavior can be partially restored to ahr-1 mutant animals by expression of ahr-1 in only 4 neurons, including URXR and URXL. It is concluded that the AHR-1:AHA-1 transcription complex regulates the expression of soluble guanylate cyclase genes and other unidentified genes that are essential for acute regulation of aggregation behavior (Qin, 2006).

Aryl hydrocarbon receptor activation by cAMP vs. dioxin: Divergent signaling pathways

Even before the first vertebrates appeared on earth, the aryl hydrocarbon receptor (AHR) gene was present to carry out one or more critical life functions. The vertebrate AHR then evolved to take on functions of detecting and responding to certain classes of environmental toxicants. These environmental pollutants include polycyclic aromatic hydrocarbons (e.g., benzo[a]pyrene), polyhalogenated hydrocarbons, dibenzofurans, and the most potent small-molecular-weight toxicant known, 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD or dioxin). After binding of these ligands, the activated AHR translocates rapidly from the cytosol to the nucleus, where it forms a heterodimer with aryl hydrocarbon nuclear translocator, causing cellular responses that lead to toxicity, carcinogenesis, and teratogenesis. The nuclear form of the activated AHR/aryl hydrocarbon nuclear translocator complex is responsible for alterations in immune, endocrine, reproductive, developmental, cardiovascular, and central nervous system functions whose mechanisms remain poorly understood. Here, it is shown that the second messenger, cAMP (an endogenous mediator of hormones, neurotransmitters, and prostaglandins), activates the AHR, moving the receptor to the nucleus in some ways that are similar to and in other ways fundamentally different from AHR activation by dioxin. It is suggested that this cAMP-mediated activation may reflect the true endogenous function of AHR; disruption of the cAMP-mediated activation by dioxin, binding chronically to the AHR for days, weeks, or months, might be pivotal in the mechanism of dioxin toxicity. Understanding this endogenous activation of the AHR by cAMP may help in developing methods to counteract the toxicity caused by numerous environmental and food-borne toxic chemicals that act via the AHR (Oesch-Bartlomowicz, 2005).

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