Cytoplasmic Receptors

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Cytoplasmic receptors are ligand binding transcription factors. All except the aryl-hydrocarbon receptor (AhR, a basic helix-loop-helix-PAS [bHLH-PAS] family member) are members of the classical steroid family, which is now part of the nuclear receptor (NR) superfamily (for recent reviews see Refs. 26-29). Because AhR was originally characterized in biochemical studies as a steroid receptor, other putative steroid receptors may actually be bHLH-PAS structural family members. The biochemical features of cytoplasmic receptor ligands are tailor made for drug discovery efforts: they are small lipophilic compounds that average 350 daltons, show exquisite specificity and efficacy, and mediate physiological processes that are vital in arthropods and vertebrates. Steroids and synthetic mimics have medical utility for many conditions including osteoporosis, cancer, and a variety of inflammatory disorders, as well as agricultural utility to enhance production in livestock. The majority of these compounds was discovered by traditional screening approaches. With increasing knowledge of the three-dimensional structure of these receptors and the auxiliary proteins involved in receptor mediated transcription, it is now possible rationally to select drug targets and synthesize ligands (for reviews see Refs. 30-32).

The exploitation of steroid receptor targets in agricultural chemical and veterinary therapeutic discovery has resembled medical discovery strategies, fa voring a mechanism-based drug discovery approach. Environmentally favored commercial insecticides, whose mode of action is to interfere with vital biological pathways unique to insects, are referred to as insect growth regulators (IGR). The discoveries of the IGRs RH5849 (subsequently optimized to tebufenozide) and methoprene were serendipitous. Subsequently, it was shown that both are nonsteroid analogues of insect specific steroid hormones: tebufenozide is a bis-acylhydrazine nonsteroid agonist of the ecdysone receptor, and methoprene is a terpenoid, similar in structure to juvenile hormones [33,34]. The ecdysone receptor (EcR) is a member of the steroid receptor family [26]. The Drosophila melanogaster methoprene resistance gene (Met), which remains after a decade of research as the most likely candidate for the putative nuclear receptor-like component of the juvenile hormone receptor, was recently identified as a bHLH-PAS family member [35,36]. D. melanogaster has 18 nuclear receptors [37], six bHLH-PAS family members [36,38-40], and the size of these families may increase several fold with the enlargement of expressed-sequence tag (EST) data bases and complete sequencing of the Drosophila genome. In addition, there are a variety of related receptors in other insect species.

The structural features of cytoplasmic receptors (the four classes of steroid/ nuclear receptors plus AhR) determine the specific requirements and strategies for screen design. On the primary amino acid level, cytoplasmic receptors have a tripartite structure of independent functional domains. Nuclear receptors (NR) have an N-terminal transactivation domain (TA), a middle DNA binding domain (DBD) and C-terminal ligand binding domain (LBD). AhRs have a similar topology except the TA is moved from the N-to the C-terminus. The DBD of both NR and AhR binds a cis-DNA element referred to as a hormone response element (HRE), for which consensus sequences have been identified. The NR superfamily is divided into four groups based on their DNA binding and dimerization properties. Families I and II are ligand binding receptors and families III and IV are orphan receptors. Family I includes the steroid receptors: glucocorticoid receptor (GR), mineralocorticoid receptor (MR), estrogen receptor (ER a and P), progesterone receptor (PR a and P and androgen receptor (AR). In the absence of ligand, these receptors are located in the cytoplasm in association with heat shock proteins (HSPs). The presence of ligand causes the release of the HSP, translocation into the nucleus, binding as homodimers to the HRE, and activation of transcription.

Family II includes the receptors that heterodimerize with the 9-cis-retinoid X receptor (RXR) and that bind their cognate HRE in the absence of ligand. Based on their HRE consensus sequence, this family was recently divided into two subfamilies: the original family members, peroxisome proliferator activated receptor (PPAR a, P, and y), RXR (a, P, and y), retinoic acid receptor (RAR a, P, and y), and vitamin D3 receptor, thyroxine receptor (TR a and P), and the newer members whose ligands were recently identified, constitutively active receptor P (CARP), benzoate X receptor (BXR), pregnane X receptor (PXR), and steroid and xenobiotic receptor (SXR). The classification into families serves only as a guideline, since ER functions as a hybrid of family I and family II receptors.

In family III, the orphan receptors bind the HRE as monomers (e.g., nerve factor induced orphan receptor), and in family IV as dimers (e.g., chicken ovalbumin upstream promoter transcription factor) [26,41,42]. The ability to hetero-dimerize with RXR is a signature feature that an orphan receptor may have a ligand [27]. Novel ligands for orphan receptors are intracrine rather than being endocrine, which may explain why they have been more difficult to identify. The ligand mediated transcriptional activity of some receptors is regulated by a growing list of cofactors that act as corepressors or coactivators. A number of coactiva-tors are histone acetyltransferases (HATs) and members of the bHLH-PAS family. These cofactors function by recruiting multicomponent complexes that promote chromatin remodeling: corepressors are associated with multiple histone HATS and coactivators with histone deacetylases (HDACs) [31]. Recently, a novel cofactor that is both a corepressor and a coactivator called NR-binding SET-domain containing protein has been identified [43].

AhR shares properties of both families I and II ligand activated NRs. Like family I members, unligated AhR is in the cytoplasm associated with HSP. Upon ligand binding, the HSP dissociate and aryl hydrocarbon receptor nuclear trans-locator (ARNT) associates and transports the complex to the nucleus, where it binds to the xenobiotic response elements (XRE) and activates transcription [29]. ARNT was the first bHLH-PAS member identified and like RXR and the NR family II members, functions as a heterodimeric partner to other bHLH-PAS members. The extent to which NRs and bHLH-PASs interact with each other and share common regulator pathways is being intensely investigated [30,44].

Since yeast has no endogenous cytoplasmic receptors and has a limited metabolic capability, it is a model system for reconstituting ligand mediated cyto-plasmic receptor function. The ligand inducible transactivation activity of all the known mammalian steroid receptors and a growing list of RXR receptors and their heterodimeric partners has been successfully reconstituted in yeast by the classic cis-trans assay (Table 1) [45-53] (for reviews see Refs. 42, 54-58). Yeast is transformed with a receptor expression plasmid(s) and a reporter plasmid driven by hormone-responsive promoter fused to a tractable marker gene. The approaches we routinely use are based on the standard strategies for expression of steroid receptors in yeast [45,54,59] (see Ref. 58 for alternative approaches). The salient feature of the yeast expression vector is that it codes for a ubiquitin-receptor fusion protein, which is cleaved by endogenous yeast protease at the junction to generate an authentic receptor. In addition, either a copper inducible metalothionein promoter is used to regulate the expression level of the receptor or the triosephosphate dehydrogenase promoter is used to obtain constitutive expression. With the AhR system, an expression vector for yeast (pBEVY) that has

Table 1 Cytoplasmic Receptors Expressed in cis/trans Assay in Yeast

Ligand inducible nuclear receptors Reference

Estrogen receptor alpha 46,47,51-53,57,58

Glucocorticoid receptor 62 Mineralocorticoid receptor

Androgen receptor 53

Progesterone receptor 45,51,53

Retinoic X receptors (RXR) 164 Vitamin D receptor

Retinoic acid receptors 164 Oncogenic thyroid hormone receptor (c-erbA)

Thyroid hormone receptor (c-erbA) and RXR 46 Vitamin D receptor and RXR

Retinoic acid receptors and RXR 54,164

Peroxisome proliferator-activated receptor and RXR 48,49

Constitutively active nuclear receptors

Chicken ovalubumin upstream promoter transcription factor 46 (COUP)

Hepatic nuclear factor 4 165

Ecdysone receptor (EcR) 59,61

Androgen receptor/EcR chimera and ultraspiracle 61

bHLH-PAS receptors

Aryl hydrocarbon nuclear receptor 60

a bidirectional galactose inducible promoter was used [60], and the recombinant protein was not a fusion product. Expression vectors with bidirectional promoters are becoming more important because several proteins must be expressed to reconstitute or optimize the activity of some receptors. One note of caution for the use of galactose inducible promoters is that some galactose media are contaminated with bioactive steroid (i.e., estrogen and progesterone [51]).

A frequently used yeast reporter plasmid, YRpC2, has an Xhol cloning site in which to insert the HRE upstream of the yeast cytochrome C promoter and lacZ reporter gene. The HRE, which typically consists of a duplicate copy of the binding sequence, is critical and may need to be optimized for specific needs. Although lacZ is the most frequently used reporter and its expression is quantifiable, it is not a selectable maker. The auxiotrophic markers URA3 [52] and HIS3 [46,57] were both used as reporter genes with the ER. The advantage of URA3 is that its activity is quantifiable, and it can be used as a counter-selectable marker:

the gene product orotidine-5'monophosphate decarboxylase can be measured in a liquid OMPdecase assay, and kills yeast by conferring sensitivity to the toxic antimetabolite 5-fluoro-orotic acid (5-FOA). In a Usp antagonist screen, we use the canavanine permease gene (CAN1) as a counter-selectable marker [61] (see Table 2). The protease-deficient yeast strain BJ2168 is frequently used for cytoplasmic receptor expression. While in our experience the EcRs and Usp showed comparable activity in BJ2168 and a strain expressing the normal complement of proteases [61], others have observed that the high levels of receptors produced in strain BJ2168 were not always desirable and perhaps were even detrimental for particular assays [54]. Typically, yeast is grown for 4 to 24 h in the presence of ligand to detect reporter gene expression, and P-galactosidase assays are performed in a 96-well format [53,54]. In Usp screen, which employs a CAN1 reporter, cells are seeded in agar media and test compounds are applied on the surface, typically 144 natural products or up to 576 chemicals per plate.

Whether the cis-trans assay in yeast will work for a particular receptor is not predictable. Basic receptor research has demonstrated that ''adapter proteins'' are sometimes required for proper expression. For example, TR, RAR, VDR, ER, and RXR, which all function in yeast, require TR interacting protein (TRIP1) for their ligand activating abilities. However, the yeast SUG1 gene (suppressor of gal4D lesions) is the homologue of mammalian TRIP1 and is able functionally to replace TRIP1 [42]. In screens to evaluate endocrine modulators, RSP5 was overexpressed with PR and SPT3 with AR. The rationale for this approach is that the overexpression of the human homologues RPF1 and TAF18 in mammalian systems enhanced transcriptional efficiency without altering potency or specificity [53]. It is speculated that SUG1 and RSP5 act by affecting the turnover of receptors rather than mediating agonist [52]. RXR, VDR, and the steroid receptors are functional in yeast without the additional coexpression of corepressors like TR and the RAR associated corepressor (TRAC), which interact with TR and RAR and do not appear to have a yeast counterpart. Compounds that act as antagonists in mammalian cells function as partial agonists in yeast (tamoxifen

Table 2 S. cerevisiae Counterselectable Markers

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