SUMMARY Many signaling systems show adaptation—the ability to reset themselves after responding to a stimulus. We computationally searched all possible three-node enzyme network topologies to identify those that could perform adaptation. Only two major core topologies emerge as robust solutions: a negative feedback loop with a buffering node and an incoherent feedforward loop with a proportioner node. Minimal circuits containing these topologies are, within proper regions of parameter space, sufficient to achieve adaptation. Morecomplex circuits that robustly performadaptation all contain at least one of these topologies at their core. This analysis yields a design table highlighting a finite set of adaptive circuits. Despite the diversity of possible biochemical networks, it may be common to find that only a finite set of core topologies can execute a particular function. These design rules provide a framework for functionally classifying complex natural networks and a manual for engineering networks. For a video summary of this article, see the PaperFlick file with the Supplemental Data available online.

A long-held tenet of molecular pharmacology is that canonical signal transduction mediated by G-protein-coupled receptor (GPCR) coupling to heterotrimeric G proteins is confined to the plasma membrane. Evidence supporting this traditional view is based on analytical methods that provide limited or non-subcellular resolution1. It has been subsequently proposed that signalling by internalized GPCR is restricted to G-protein-independent mechanisms such as scaffolding by arrestins2,3, or GPCR activation elicits a discrete form of persistent G protein activation4–9, or that internalized GPCR can indeed contribute to the acute G protein-mediated response10. Evidence supporting these various latter hypotheses is indirect or subject to alternate interpretation, and it remains unknown if endosome-localized GPCR are even present in an active form. Here we describe the application of conformation-specific single domain antibodies (nanobodies) to directly probe activation of the β2-adrenoceptor, a prototypical GPCR11, and its cognate G protein, Gs (ref. 12) in living mammalian cells. We show that the adrenergic agonist isoprenaline promotes receptor and G protein activation in the plasma membrane as expected, but also in the early endosome membrane; and that internalized receptors contribute to the overall cellular cyclic AMP response within several minutes after agonist application. These findings provide direct support for the hypothesis that canonical GPCR signalling occurs from endosomes as well as the plasma membrane, and suggest a versatile strategy for probing dynamic conformational change in vivo.

Computational modeling and experimentation in the unfolded protein response reveals a role for the ER-resident chaperone protein BiP in fine-tuning the system's response dynamics.

We show that difficulties in regulating cellular behavior with synthetic biological circuits may be circumvented using in silico feedback control. By tracking a circuit's output in Saccharomyces cerevisiae in real time, we precisely control its behavior using an in silico feedback algorithm to compute regulatory inputs implemented through a genetically encoded light-responsive module. Moving control functions outside the cell should enable more sophisticated manipulation of cellular processes whenever real-time measurements of cellular variables are possible.