Signal Transduction

competence does not persist and a cyclic disassembly and re-assembly of the complex ensues until ligand binding occurs. Hormone-bound receptors are transported along microtubules to nuclear pores, where the entire complex is shuttled into the nuclear space. Inside the nucleus the complex dissociates, allowing the dimerized receptor to bind to its response element (in the promoter region of genes). They then engage a different set of proteins to become part of a large transcription complex. Subsequently, used or ‘experienced’ receptors lose their hormone and again associate with the chaperones as they are released from their DNA binding sites. They may then either be degraded, exported back into the cytosol or reused within the nucleus, where they may bind hormone once again.

This sequence of events has been demonstrated most clearly for GRs, which in the absence of ligand are principally cytosolic, but there is evidence

that a similar mechanism operates for the other steroid receptors. Even ER, predominantly resident in the nucleus, shows a dependence on hsp90. Other receptors that are exclusively nuclear appear to be associated with DNA in the absence of ligand, but bind to specific sites in its presence.

DNA binding

Nuclear receptors bind to DNA as dimers at specific loci within the regulatory region of target genes. These sites, known as response elements, consist

of two half-sites, each of six base pairs. For steroid receptors the consensus sequences of the sites are palindromic, inverted repeats separated by a 3 bp spacer (termed IR3). GR, PR, MR, and AR bind as homodimers to a response element having the consensus sequence 5’-AGAACA-3’ (Figure 10.7). ER also binds to an IR3 inverted repeat, but the consensus sequence is different, 5’- AGGTCA-3’. Most non-steroid receptors bind to half-sites that are direct repeats of the AGGTCA hexad (termed DRn, where n is the number of spacers). They may bind either as homodimers (TR, VDR) or commonly with higher affinity, as heterodimers with RXR, as in the case of TR, VDR, RAR, LXR, FXR, PXR, CAR, and PPAR. The binding of heterodimers provides a combinatorial mechanism in which the two different receptors can coregulate transcription at a single response element.

Since the sites at which the different heterodimers bind have the same consensus sequence, how is specificity achieved? A measure of discrimination is provided by differences in binding site geometry determined by the number of spacers (DR1–DR5) between the half-sites (see Figure 10.7). Each additional base-pair displaces the half-sites by 3.4 Å and introduces a relative rotation of 36°. Note that RXR is always situated upstream (5’) of its binding partner, except in the case of the RAR-RXR heterodimer that binds at DR1 sites. When the linker length is 2 or 5, the dimer assembles in the order RXR-RAR.

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FIG 10.7  Dimerization and binding to DNA half-sites.

Steroid hormones bind to inverted repeats as homodimers. Other receptors bind to direct repeats as homodimers or as heterodimers with RXR. Binding site geometry is affected by the number of base pairs in the linker sequence. (H hormone, 9 9-cis retinoic acid).

For many receptors, further specificity comes from interactions of other residues in the DBD with flanking regions of DNA. For example, the C-terminal extension of the DBD may bind to sites upstream of the hexad sequences. Finally, it should also be stressed that the actual sequences of the response element half-sites that are recognized by nuclear receptors commonly differ from the consensus sequences at one or more positions.

Recognizing response elements

Nuclear receptor DBDs contain two C4-type zinc fingers in which four

cysteine residues are linked to a tetrahedrally chelated Zn2 ion (see page 781). These structures nucleate the protein fold. A schematic representation

of the glucocorticoid DBD is shown in Figure 10.8a and the three-dimensional structure of the homodimer bound to DNA half-sites with the idealized AGAACA sequence is shown in Figure 10.8b. An N-terminal recognition helix inserts into the major groove of the target DNA, where it contacts specific

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FIG 10.8  The zinc finger DNA-binding domain of the glucocorticoid receptor.

(a) Amino acid sequence of a single DBD. (b) Structure of receptor DBD dimers binding to DNA. P-boxes are coloured red and D-boxes are yellow.36,37 (1r4).38

bases by means of a motif called the P-box. For the non-oestrogenic steroids, such as GR, the P-box sequence is GSCKV. For ER it is EGCKA and for receptors that form heterodimers with RXR, it is EGCKG. A mutation of the GR P-box, G→E, produces a receptor that can bind either GR or ER response elements. Complete swapping of the GR P-box for that of ER confers an ability to bind and activate at oestrogen response elements, eliminating glucocorticoid responsiveness.

For receptors binding to inverted repeats, dimerization occurs upon binding, through interactions between a D-box motif on each monomer, as illustrated in Figure 10.8. For receptor dimers that bind direct repeats, the monomers are oriented head to tail and there is no D-box interface. Instead, a more diffuse set of interactions between the DBDs ensures heterodimer stability. Dimerization may also be reinforced by interactions between sites on the two LBDs. Such interactions enable some receptors to bind as dimers to DNA without a bound ligand. In these circumstances they may act to repress transcription.

In summary, nuclear receptors may be divided into two main classes: those that bind as homodimers to inverted repeat DNA half-sites, such

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