PXR (pregnane X receptor) and CAR (constitutive androstane receptor) are expressed in liver and gut. They coordinate protection against potentially toxic compounds by inducing the expression of drug transporters and drugmetabolizing enzymes of the cytochrome P450 family.

PXR is a xenobiotic receptor that has a particularly large ligand binding cavity and demonstrates low affinity binding for a wide range of ligands.

CAR is a xenobiotic receptor that also regulates the metabolism of bilirubin and thyroid hormone. It has a high basal activity in vivo, which can be increased by ligand. The steroid androstanol is an inverse agonist.

feedback mechanism causes the repression of the CYP7A1 gene in response to bile acid synthesis, but this is indirect. It involves three different nuclear receptors FXR (page 281), SHP (NR0B2, an atypical receptor that lacks a DBD), and LHR-1 (NR5A2, responsible for the basal transcription of CYP7A1). Firstly, FXR is activated by bile acids to induce expression of SHP, which in turn, represses LHR-1 by forming a heterodimeric complex. In the opposite sense, LXR acts to increase throughput in the catabolic pathway by inducing the expression of reverse cholesterol transporters in the tissues. In mouse and rat it increases throughput through the expression of CYP7A1.

Bile acids are hepatotoxic and a further layer of control is provided to avoid excessive accumulation in the liver (cholestasis). This involves CAR and PXR. These adopted orphans were initially identified as detectors of a range of lipophilic xenobiotics (including many drugs). They induce expression of CYP3A4, which brings about hydroxylation and leads to detoxification. It later emerged that they have physiological activators that include precursors and products of cholesterol metabolism. Thus these receptors also act to repress transcription of CYP7A1 in response to bile acids, but at a 10–fold lower affinity than FXR and by a different mechanism. They also activate expression of transporters which promote

export of bile acids from the liver and of CYP3A enzymes that increase bile acid metabolism and lead to excretion of the products. Even this is a very simplified picture of the network regulating bile acid metabolism. There are many other components and interactions. In general, although the circuitry of such networks may be complicated, they are often accessible to pharmacological intervention and, less fortunately, to environmental influence.

Interaction with other signalling pathways

Nuclear receptor signalling does not exist in isolation from other forms of cellular signal transduction. Not only is there cross-talk between different ligandactivated nuclear receptor pathways, there are also interactions with other signalling mechanisms. The effects can range from modulation of transcriptional activation to its activation in the absence of nuclear receptor ligand.

Phosphorylation

Although nuclear receptors exist as phosphoproteins in the absence of their ligands, their function may be modified by hyperphosphorylation.37 The target sites are concentrated on the N-terminal A/B domain, but they may also be present on each of the other major domains. The number of sites and their locations vary with receptor type; for example, PR has 13 possible phosphorylation sites on its N-terminal domain, others have just one or two

and VDR, exceptionally, is not phosphorylated in this region. Target residues in the A/B domain are mostly serines in the vicinity of prolines.

Nuclear receptors are phosphorylated by a wide range of kinases. The cyclindependent kinases, which play a role in regulation of the cell division cycle, are responsible for the ligand-independent, basal phosphorylation and also hyperphosphorylation in response to nuclear receptor ligand binding. This can cause the level of phosphorylation to vary throughout the cell cycle. For example, in S phase the basal level of GR phosphorylation is low and it is high in G2/M, while its hyperphosphorylation is the reverse.

Nuclear receptors are also targets of the downstream kinases of the major signalling pathways, namely PKC and PKA (activated by G-protein-coupled receptors: see Chapter 9), ERK and PKB/Akt (activated by growth factor receptors via Ras and PI 3-kinase respectively: Chapters 12 and 18), JNK/SAPK and p38-MAP kinase (activated by stress and cytokine receptors: Chapter 16).

Phosphorylation may upor down-regulate transcription

Phosphorylation at a given site on a given receptor may facilitate or inhibit transcription. Given the number of potential sites and the variety of kinases and receptors, the picture is complicated. In general, ligand-independent phosphorylation by cyclin-dependent kinases on a receptor AF-1 region promotes transcription. Phosphorylation in the N-terminal region of receptors such as PR, ER, AR, and PPAR by members of the MAP kinase family (ERK, JNK/SAPK, p38-MAP kinase) also enhances transcription by helping the recruitment of coactivators, HAT activity, and chromatin remodelling proteins. Furthermore, ER and AR may be phosphorylated in their N-terminal domains by PKB/Akt, again enhancing transcription. Phosphorylation of the AF-2 region of ER by the tyrosine kinase Src also up-regulates transcriptional activity. Coregulators may be also be phosphorylated by MAP kinases (and other kinases). This has the effect of promoting the binding of coactivators

to nuclear receptors in the presence of ligand and inhibiting the binding of corepressors. The general trend of all this is positive regulation, ensuring the formation of an efficient transcription initiation complex.

Transcriptional activity can be down-regulated by phosphorylation of residues in the DBD by PKC or PKA, by preventing recognition of response element half-sites or by modifying receptor dimerization surfaces. MAP kinase phosphorylation of the LBD of RXR inhibits transcription by RAR/RXR and VDR/RXR heterodimers through conformational changes that disrupt coregulator interactions.

Ligand-independent activation

Growth factors such as EGF (see page 318) or IGF (see page 554) can activate transcription by ER apparently in the absence of oestrogens. This appears to

CYP3A4, the most prominent P450 protein in human liver, is thought to be responsible for the metabolism of some 60% of all clinically used drugs (MIM 124010).