two hormones 20E and JH, the molecular basis of 20E action is much better understood. The pioneering work of Clever and Karlson (1960) and Ashburner (1973) on the action of 20E to induce/ repress ''puffs'' in the polytene chromosomes of Chironomus and Drosophila led Ashburner and co-workers to propose a model for ecdysteroid action (Ashburner et al., 1974). According to this model, 20E binds to an ecdysone receptor to differentially regulate several classes of''early'' and ''late'' genes. While the ''early'' genes are activated by the 20E-receptor complex, the '' late'' genes are repressed. The protein products of the ''early'' genes such as E75 and CHR3 derepress the expression of the ''late'' genes such as DOPA decarboxylase
(DDC) and at the same time repress their own expression.
18.104.22.168.2. Ecdysone receptors The ecdysone receptor complex is a heterodimer of two proteins, ecdysone receptor (EcR) and ultraspiracle (USP), which is a homolog of the mammalian retinoic acid receptor (RXR) (Yao etal, 1992,1995; Thomas et al., 1993). In several insects, both EcR and USP exist in several transcriptional and splice variants, presumably for use in a stage- and tissue-specific way (review: Riddiford et al., 2001). Both EcR and USP are members of the steroid receptor superfamily that have characteristic DNA and ligand binding domains. Ecdysteroids have been shown to bind to EcR only when EcR and USP exist as heterodimers (Yao et al., 1993), although additional transcrip-tional factors are required for ecdysteroid dependent gene regulation (Arbeitman and Hogness, 2000; Tran et al., 2000). Moreover, EcR can hetero-dimerize with RXR to form a functional ecdysteroid receptor complex in transfected cells (Yao et al., 1992; Tran et al., 2000). cDNAs encoding both EcR and USPs from a number of dipteran (Koelle et al., 1991; Imhof et al., 1993; Cho et al., 1995; Kapitskaya et al., 1996; Hannan and Hill, 1997, 2001; Veras et al., 1999), lepidopteran (Kothapalli et al., 1995; Swevers et al., 1995), coleopteran (Mouillet et al., 1997; Dhadialla and Tzertzinis,
1997), homopteran (Zhang et al., 2003; Dhadialla et al., unpublished data; Ronald Hill, personal communication), and orthopteran (Saleh et al., 1998; Hayward et al., 1999, 2003) insects, tick (Guo et al., 1997) and crab (Chung et al., 1998) have been cloned. Some of the EcRs and USPs have been characterized in ligand binding (Kothapalli et al., 1995; Kapitskaya et al., 1996; Dhadialla et al.,
1998) and cell transfection assays (Kumar et al., 2002; Toya et al., 2002). In all cases, the DNA binding domains (DBDs) of EcRs show a very high degree of homology and identity. However, homol-ogy between the ligand binding domains (LBDs) of EcRs varies from 70% to 90%, although all EcRs studied so far bind 20E and other active ecdyster-oids. The DBDs of USPs are also highly conserved. The USP LBDs, however, show very interesting evolutionary dichotomy: the LBDs from the locust, Locusta migratoria, the mealworm beetle, Tenebrio molitor, the hard tick, Amblyoma americanum, and the fiddler crab, Uca puglitor, show about 70% identity with their vertebrate homolog, but the same sequences from dipteran and lepidopteran USPs show only about 45% identity with those from other arthropods and vertebrates (Guo et al., 1997; Hayward et al., 1999; Riddiford et al., 2001).
The functional significance of RXR-like LBDs in USPs of primitive arthropods is not well understood, because EcRs from the same insects still bind ecdys-teroids (Guo et al., 1997; Chung et al., 1998; Hayward et al., 2003; Dhadialla, unpublished data for Tenebrio molitor EcR and USP (TmEcR/TmUSP)).
The crystal structures of USPs from both Heliothis virescens and Drosophila melanogaster have been elucidated by two groups (Billas et al., 2001; Clayton et al., 2001). The crystal structure of USP is similar to its mammalian homolog RXR, except that USP structures show a long helix-1 to helix-3 loop and an insert between helices 5 and 6. These variations seem to lock USP in an inactive conformation by displacing helix 12 from the agonist conformation. Both groups found that crystal structures of the two USPs had large hydrophobic cavities, which contained phospholipid ligands.
Finally, the crystal structures of Heliothis viresens EcR/USP (HvEcR/HuUSP) heterodimers liganded with an ecdysteroid or a nonsteroidal ecdysone agonist have been determined (Billas et al., 2003; see Section 22.214.171.124.2 for more details). The crystal structure of liganded EcR/USP from the silverleaf whitefly, Bemesia tabaci, has also been determined and awaits publication (Ronald Hill, personal communication).
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