M A R C B L

F. Nuclear Receptors

Members of the NR superfamily regulate gene expression by binding to cis-active elements in target genes and either activating or repressing transcription. Ligand binding to NRs modifies the DNA-binding and transcriptional properties of these receptors resulting in the activation or repression of target genes. Ligand binding induces conformational change in NRs and promotes association with diverse nuclear proteins that may function as coactivators of transcription through a conserved sequence motif present in the coactivators (Fig. 21). The coactivator proteins that associate with NRs in a ligand-dependent manner varied from 2 for estrogen receptor (ERAP) to 12 for vitamin D receptor (DRIPS). In addition to the receptor binding assays, transcription, DNA binding, and coactivator binding assays are in use for functional screens of NRs.

1. Transcription Assays

The ligands for NR, on entering the cells, bind to their cognate receptors with high affinity and induce a conformational change that activates the receptor. In some cases, the receptor may dissociate from heat shock proteins and conforma-tional changes that allow the receptor to bind with other proteins involved in transcriptional regulation. Activated receptors bind to specific DNA sequences called hormone response elements (HRE) and increase transcription of the linked downstream gene [121]. High-throughput transcription assays have been developed in which the NR is expressed in a cell line under a certain selection of antibiotic, and in the same cell a plasmid-containing reporter gene like luciferase under the control of a promoter containing appropriate HRE is expressed under selection of a second antibiotic. Incubation of the cell with a ligand activates the expressed NR receptor, dimerizes and translocated into nucleus where it binds to the HREs in the reporter plasmid and induces the expression of the reporter gene (Fig. 21). The agonist-induced expression of the reporter gene results in the increased production of the reporter gene-product, which can be quantitatively assayed. The common reporters used for this purpose include firefly luciferase

Figure 21 Schematic illustration of the regulation of gene expression by a nuclear receptor (NR). Upon binding of ligand, NR undergoes conformational changes and is translocated into the nucleus, where it binds to HRE as a dimer and activates gene expression.

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