The nuclear receptors are a group of ancient evolutionarily related transcription factors. Sequencing of the human genome has revealed 48 members of this class, of which about half appear to be orphans, i.e., no activity-modulating ligand has yet been identified for them. We will focus our attention on the ten separately encoded proteins whose activity is modulated by the hormones discussed in the following chapters.
The structural organization of the nuclear receptors for the classical steroid hormones, 1α,25(OH)2-vitamin D3, thyroid hormone, and retinoic acid is shown in Figure 1A. Each of these proteins functions as a DNA-binding protein, regulating, in a ligand dependent (and sometimes ligand-independent) way, the expression of genes related to the biological response of the hormone.
Fig1. Nuclear receptor structure. A. Primary structural organization. Shown are the structural features of nuclear receptors for thyroid hormone (TRα and TRβ), 1,25-dihydroxyvitamin D3 (VDR), the retinoic acid (RXR), estrogen (ERα and ERβ), cortisol (GR), aldosterone (MR), progesterone (PR B), and testosterone/dihydrotestosterone (AR). These receptors share a highly conserved DNA binding domain (C, green) and a short nonconserved region (D, blue), which serve as a hinge between the N-terminal and C-terminal portions of the molecule. The ligand binding domain (gold) is less conserved than the DNA binding domain, but is approximately the same size and adopts approximately the same three-dimensional structure in all the receptors. The difference in size between the receptor proteins is the highly variable N terminal A/B domain (pink). Two elements that are necessary for control of gene transcription, termed activation functions, exist, AF-1 in the A/B domain and AF-2 in the E/F domain. B. Three-dimensional structure of the DNA- and ligand-binding domains of the nuclear receptors. For the DNA binding domain (left), the interaction of the ER homodimer with DNA is shown. Each DNA binding site consists of two loops of DNA known as zinc fingers and described more fully in Figure 2. Recognition of the specific DNA sequence to be bound lies within the CI zinc finger (closest to the DNA) whereas CII is involved with receptor dimerization. The ligand-binding domains of the nuclear receptors are less conserved than the DNA-binding domain, but they share many common features. There are twelve α-helices arranged in three layers. The ligand binding pocket is within the more variable region. In addition to ligand binding, there are sites for a dimerization surface, a coregulator binding surface, and ligand-dependent transcriptional activation moiety, AF-2.
Fig2. Steroid hormone receptor zinc fingers. The amino acids in the DNA binding domain (see Figure 1) of a steroid hormone nuclear receptor are represented by circles. The coordination of a Zn2+ atom (blue) by four cysteines (pink) causes the formation of a loop. One of these, C1, which contains the P-box (light green), is involved in binding to the specific DNA binding site (hormone response element) and discriminating between closely related sites for different hormones. The D-box (dark green) in the second zinc finger, CII, plays a role in receptor dimerization.
The receptors for thyroid hormone (TR) and 1α,25(OH)2-vitamin D3 (VDR) are typically found in the nucleus of target cells where they (especially TR) may be bound to corepressor molecules which sup press DNA transcription (see Chapter 5). These receptors form heterodimers with RXR (also in the nucleus) to bind to specific DNA sequences. The receptors for cortisol (GR) and aldosterone (MR) are in the cytoplasm prior to ligand binding, where they are bound to chaperone proteins (heat shock proteins) that maintain them in an inactive state. Upon ligand binding they undergo nuclear translocation and homodimerization prior to binding to specific DNA sequences. The receptors for progesterone (PR), androgens (AR), and estro gens (ER) also form homodimers and may either be in the nucleus prior to ligand binding or travel between the two compartments.
As shown in Figure 1A, the nuclear receptors consist of a single polypeptide chain divided into six domains. The N-terminal sequence is highly variable in both sequence and length, accounting for the overall difference in size of the receptor proteins. It contains one DNA binding sequence, termed the AF-1 domain, which can regulate gene transcription independently of ligand binding, but can also be controlled by ligand binding. This section of the protein can undergo post-translational modification such as phosphorylation and may also interact with the C-terminal to affect the three-dimensional structure of the protein.
Functionally, the two most critical portions of the receptors are the DNA binding and ligand binding (E/F) domains. The three-dimensional organization of both is shown in Figure 1B. The C-domain, on the left, is a highly conserved sequence encoding a two zinc finger motif which is widely used in transcription factors. As seen in Figure 2 the “fingers” resulting from the coordination of a Zn2+ atom by four cysteine residues are the contact sites for DNA-binding, one of which carries the amino acid sequence for recognizing the correct specific DNA site (hormone response element, HRE; see section III.B) for binding.
The E/F domain is composed of twelve α-helices (Figure 1B) arranged in approximately the same three-dimensional structure for all the nuclear receptors. It is less highly conserved than the C-domain as it differs among receptors in order to accommodate different ligands in the ligand binding pocket. This region also contains the dimerization interface for the formation of both homodimers and heterodimers. Finally, the ligand-dependent transcription function, AF-2 resides in the E/F domain, specifically the C-terminal helix 12. This small helix has a great deal of ligand-dependent flexibility and its position determines the access of nuclear coregulators to the receptor protein.
