Nuclear receptors often function in the cytoplasm.A triple conveyor belt pumps ligand (signal) into the nucleus and onto the DNA.The active export of importins enhances signaling to the nucleus.Sharing a single nuclear pore may reduce rather than increase crosstalk.
Nuclear receptors (NRs) derive their family name from the early observation that they are located in the nucleus, despite responding to extracellular signals such as hormones (e.g., cortisol) (Fanestil and Edelman, 1966). According to the ‘classical' paradigm of NR signaling, the NR resides in the nucleus, attached to a DNA response element, waiting for its ligand to bind. The actual systems have multiple additional features (reviewed in Cutress et al, 2008; Cao et al, 2009; Levin, 2009a; Bunce and Campbell, 2010), such as that NRs shuttle between the nucleus and the cytoplasm (Von Knethen et al, 2010) and ligand addition changes receptor location dynamically (Pratt et al, 1989; Liu and DeFranco, 2000; Kumar et al, 2004, 2006; Tanaka et al, 2005; Heitzer et al, 2007; Prüfer and Boudreaux, 2007; Ricketson et al, 2007; Cutress et al, 2008): Figure 1 summarizes the current understanding of the topology of the reaction networks involved in NR signaling, in systems biological graphical notation (SBGN), with NR activation, importin-α and -β binding, nuclear pore complex (NPC)-mediated import, recycling of importins, NR binding to target promoter sequences, exportin-mediated nuclear export of the NR, exportin cycling and free energy-driven Ran recycling. This topology is surprisingly complex when compared with the ‘classical' paradigm. To address to what extent this extra complexity is just detail or contributes essential functionality, we have simulated the dynamics of the NR transcriptional response in maximally realistic mathematical models of increasingly complex designs.
The calculations revealed significant disadvantages of the classical and simplest mechanism for endocrine NR-mediated signaling, i.e., the one with localization of the NR exclusively on the DNA (design 1 in Figure 2A): the transcriptional response was very low (Figure 2B). A high concentration of free NR in the nucleus (design 2) improved sensitivity, but made the responsiveness much slower (Figure 2B). If the NR was equally distributed between the nucleus and the cytoplasm without the NR being able to traverse the nucleocytoplasmic membrane (design 3), then, although the NR diffuses more slowly than the much smaller ligand molecule, the higher concentration of the NR increased flux from the plasma membrane to the nuclear membrane; the steady state was reached faster (Figure 2B and C; compare design 3 relative to design 2). Enabling the NR to traverse the nucleocytoplasmic membrane (design 4), further accelerated the response (Figure 2B and C).
Designs 1–4 considered the permeation of the NR through the nuclear membrane to be passive, implying an import/export activity ratio of 1. When varying the import to export activity ratio (design 5), a trade-off between the fast responsiveness of design 4 and the high sensitivity of design 2 was calculated (Figure 2B). In order to maximize responsiveness, core-NR should be concentrated in the cytoplasm, whereas to gain sensitivity, liganded NR should be concentrated in the nucleus. This suggested that performance could be improved by making nuclear import and export selective for liganded over unliganded NR (design 6; Figure 2A). Indeed, retention of core-NR in the cytoplasm provided high influx of ligand into the nucleus (Figure 2D), and also the highest concentration of ligand in the nucleus (Figure 2C): Apart from its classical receptor role in transcription regulation, the NR may function as (part of) an active pump for its ligand, resembling a triple conveyor belt: importins and exportins cycle as conveyor belts and drive the cycling of the third conveyor belt consisting of the NR that pumps ligand into the nucleus.
Two other striking features of the NR signaling network (Figure 1) are related to the facts that the energy of GTP hydrolysis is coupled to an active export of importins rather than to direct active import of NR and that the same NPC is used for all transport processes. At first sight, the former may waste free energy and the latter might cause fragility due to interferences between different NRs and other signaling pathways. However, our models show that active nuclear export of importins is a design preventing NR sequestration in the nucleus by nuclear importins and, equally paradoxically, the transport of all cargo through the same NPC makes the transport of any particular cargo robust with respect to perturbations in the availability of any other cargo.
Our calculations also predict that there is an optimal ratio of nuclear to cytoplasmic fractions of the NR (Figure 2G) that depends on the specific properties of the ligand and on the transcription activation requirements. This may help to explain the observation that different NRs have different predominant intracellular localizations. Our model calculations are thereby in line with many experimental observations, but specific cases of NR signaling may only exhibit a subset of all features. Our models can aid in identifying which subsets are important in any particular case of NR signaling, as we demonstrate for an example.
In this study, we have shown that complex networks of biochemical and signaling reactions can harbor subtle design principles that can be understood rationally in terms of simplified but not simple models (which are available to the reader).
The topology of nuclear receptor (NR) signaling is captured in a systems biological graphical notation. This enables us to identify a number of ‘design' aspects of the topology of these networks that might appear unnecessarily complex or even functionally paradoxical. In realistic kinetic models of increasing complexity, calculations show how these features correspond to potentially important design principles, e.g.: (i) cytosolic ‘nuclear' receptor may shuttle signal molecules to the nucleus, (ii) the active export of NRs may ensure that there is sufficient receptor protein to capture ligand at the cytoplasmic membrane, (iii) a three conveyor belts design dissipating GTP-free energy, greatly aids response, (iv) the active export of importins may prevent sequestration of NRs by importins in the nucleus and (v) the unspecific nature of the nuclear pore may ensure signal-flux robustness. In addition, the models developed are suitable for implementation in specific cases of NR-mediated signaling, to predict individual receptor functions and differential sensitivity toward physiological and pharmacological ligands.