Receptor-Based Mechanism of Cell Memory and Relative Sensing in Mammalian Signaling Networks

Detecting relative rather than absolute changes in external signals enables cells to make decisions in fluctuating environments and diverse biological contexts. However, how mammalian signaling networks store the memories of past stimuli and use them to compute relative signals is not well understood. Using the growth factor-activated PI3K-Akt signaling pathway, we develop computational and analytical models, and experimentally validate a novel mechanism of relative sensing in mammalian cells. This non-transcriptional mechanism relies on a new form of cellular memory, where cells effectively encode past stimulation levels in the abundance of cognate receptors on the cell surface. We show the robustness and specificity of the relative sensing for two physiologically important ligands, epidermal growth factor (EGF) and hepatocyte growth factor (HGF), and across wide ranges of background stimuli. The described memory and sensing mechanism could play a role in multiple other sensory cascades where stimulation leads to a proportional reduction in the abundance of cell surface receptors.

Detecting relative rather than absolute changes in external signals enables cells to make decisions in fluctuating environments and diverse biological contexts. However, how mammalian signaling networks store the memories of past stimuli and use them to compute relative signals is not well understood. Using the growth factor-activated PI3K-Akt signaling pathway, we develop computational and analytical models, and experimentally validate a novel mechanism of relative sensing in mammalian cells. This non-transcriptional mechanism relies on a new form of cellular memory, where cells effectively encode past stimulation levels in the abundance of cognate receptors on the cell surface. We show the robustness and specificity of the relative sensing for two physiologically important ligands, epidermal growth factor (EGF) and hepatocyte growth factor (HGF), and across wide ranges of background stimuli. The described memory and sensing mechanism could play a role in multiple other sensory cascades where stimulation leads to a proportional reduction in the abundance of cell surface receptors.

Main text
Stimulation of mammalian cells with growth factors elicits a variety of contextdependent phenotypic responses, including cell migration, proliferation, and cell survival (1). Akt serves as a central hub in many signaling cascades activated by growth factors (2). Naturally, Akt phosphorylation-dependent (pAkt) pathways are implicated in multiple human diseases, such as many types of cancers (2,3), diabetes (4) and psychiatric disorders (5,6).  (Fig. 1d). Thus, the pAkt response to EGF stimulation is strongly affected by background EGF levels and this effect is mediated by the removal of activated EGFRs from cell surface (7). We constrained the ranges of model parameters based on literature-derived estimates (SM Table 1), and fitted the model using pAkt time courses and steady state sEGFR levels at different doses of EGF stimulations. We used simulated annealing to optimize model parameters (SM section III), and considered multiple distinct parameter sets from the optimization runs for further computational analysis (SM Fig. 9a, b).
Using the fitted dynamical model, we explored the ability of the Akt pathway to respond to relative, rather than absolute, changes in EGF levels. We simulated the pAkt response by exposing the model in silico to a range of background EGF levels. We then simulated different fold change increases in EGF concentration (Fig. 2b). The model predicted that the maximal pAkt response indeed depends primarily on the EGF fold change relative to the background stimulation (Fig. 2c). Relative sensing occurred over an order of magnitude of background EGF concentrations and the resulting pAkt response was approximately proportional to the logarithm of the EGF fold change (Fig.   2d). Notably, the model predicted relative sensing in the range of EGF background concentrations where endocytosis was sensitive to background ligand stimulation. At low EGF background concentrations (< 0.01 ng/ml), no substantial sEGFR removal was predicted at steady state (SM Fig. 9b ). Error bars represent the standard deviation of top 10 model fits.
We next experimentally tested the model-predicted relative sensing behavior in MCF10A cells. Cells were first treated with various background EGF concentrations for three hours to ensure steady state sEGFR levels (Fig. 1d), and that pAkt had decayed after a transient increase (Fig. 1c). As in the computational analysis (Fig. 2b), cells were then exposed to different fold changes in EGF levels. pAkt levels were measured at 2.5, 5, 10, 15, 30 and 45 minutes after the step increase in EGF stimulation (SM Fig. 1a, b); similar results were obtained in two independent biological replicates (SM Figures 3 and   4a, b). The experiments confirmed the predictions of the computational model that maximum pAkt response depends primarily on the fold change in EGF levels and not its absolute concentration (Fig. 3a, SM Fig. 2). Specifically, across more than an order of magnitude of EGF background concentrations (0.03 -0.5 ng/ml) the same EGF fold change (lines of the same colors in Figure 3a) elicited similar pAkt responses. The concentration range in which relative sensing was observed is consistent with recent estimations of in vivo EGF levels (9). Finally, in good agreement with model predictions, the maximum pAkt response was approximately proportional to the logarithm of EGF fold change (Fig. 3b). ). Error bars represent the standard deviation of technical replicates.
To better understand the mechanism responsible for the observed relative sensing of extracellular EGF concentration, we constructed a simplified analytical model and that the maximal receptor phosphorylation response [LR where a and b are numerical constants (SM section IV). As a result of these relationships, the phosphorylation response [LR  In addition to EGF, Akt phosphorylation can be induced by multiple other ligands, including hepatocyte growth factor (HGF) (10) which binds to its cognate receptor cMet (11). To investigate the specificity of the receptor-based cell memory to past ligand exposures, we used the two ligands, EGF and HGF, which share most of their signaling components downstream of their cognate receptors (12). We stimulated cells with background doses of either HGF or EGF for three hours, and then treated cells using either the same or the other growth factor to elicit pAkt response (Fig. 5a, b). Preexposure with HGF did not substantially downregulate EGF-induced pAkt responses, but substantially decreased HGF-induced responses (Fig. 5a). Similarly, there was a relatively small desensitization of HGF-induced responses due to pre-exposure with EGF, while a significant desensitization of EGF-induced pAkt responses was observed (Fig. 5b). We further confirmed that exposure of MCF10A cells to various concentrations of HGF leads to pronounced HGF-dependent removal of cMet from the cell surface, without significant removal of sEGFR (SM Fig. 5a). Similarly, the pre-exposure of cells to EGF leads to EGF-dependent removal of sEGFR without a significant change in surface cMet abundance (SM Fig. 5b). These observations support the mechanism in which the relative sensing of extracellular ligands relies on the memory of their past exposures encoded primarily in the abundances of their cognate cell-surface receptors.

Fig. 5: Desensitization and cell memory for EGF-and HGF-induced pAkt response.
MCF10A cells were exposed to various background doses of either HGF or EGF for three hours, and then stimulated using either the same or the other growth factor. pAkt levels were then measured 10 minutes after the addition of the second stimulus. (a) EGF (blue, 2.5 ng/ml) or HGF (red, 4 ng/ml) induced pAkt response in cells pre-exposed with various doses of HGF (shown on the x axis) for three hours. (b) EGF (blue, 2.5 ng/ml) or HGF (red, 4 ng/ml) induced pAkt response in cells pre-exposed with various doses of EGF for three hours. Error bars represent the standard deviation of technical replicates. (c) The maximum pAkt response in MCF10A cells exposed to different background doses of HGF (x axis) for 3 hours followed by 2-, 4-, and 8-fold increase (different colors) of HGF. Inset shows experimental pAkt response over a wider range of background HGF levels. (d) The maximum pAkt responses to HGF fold changes depended approximately logarithmically on the fold change. Maximum pAkt responses from experiments with various HGF background levels (indicated by data points with the same shape and color) were combined and plotted as a function of the fold change in HGF dose (x axis). Dashed line represents log-linear fit to data (Pearson's r 2 = 0.88, p < 10 -6 ). Error bars represent the standard deviation of technical replicates.
Given the observed HGF-dependent removal of cell surface cMet receptors and resulting pAkt desensitization, we investigated whether the maximum pAkt response depends, similarly to EGF, on the relative fold changes in the level of extracellular HGF.
To that end, we exposed cells to a range of different background levels of HGF, and then stimulated cells with different fold changes in HGF concentrations (Fig. 5c,d and   SM Fig. 6). These experiments demonstrated that HGF-induced phosphorylation of Akt also depends primarily on the fold change in extracellular HGF concentration across almost an order of magnitude of background HGF exposures (between 0.1 and 1 ng/ml HGF) and can distinguish up to 8 fold changes in HGF concentration (Fig. 5c).
Moreover, similar to EGF, the maximum pAkt levels depends approximately log-linearly on the fold change in HGF (Fig. 5d).
Relative sensing of extracellular ligands should affect important biological targets of the PI3K-Akt pathway. The FoxO3 transcription factor is a key effector of the pathway, and is involved in multiple cellular processes including apoptosis, proliferation, and metabolism (13). Akt phosphorylation of FoxO3 leads to its translocation from the nucleus to cytoplasm and subsequent transcriptional deactivation (13). To investigate FoxO3 activity following EGF stimulation, we used quantitative immunofluorescence to measure its nuclear-to-cytoplasm ratio (14). We exposed cells to two different background EGF levels for three hours, and then treated them with two different fold changes in EGF concentrations. Consistent with relative sensing by pAkt, the nuclear-tocytoplasmic ratio of FoxO3 reflected the relative, but not the absolute changes in EGF stimulation ( Fig. 6 and SM Fig. 8). Thus, relative sensing of the signal is faithfully transmitted to at least some of the physiologically important effectors of the PI3K-Akt pathway. Fig. 6: Relative sensing of EGF concentrations by FoxO3. MCF10A cells were exposed to two background doses of EGF for three hours, and then treated with 3-and 6-fold increase in EGF concentrations. The ratio of nuclear-to-cytoplasmic FoxO3 levels (y-axis) was measured using quantitative immunofluorescence (see SM section II) after 15 minutes of the EGF fold changes. Following the patterns observed for pAkt activation, the ratio of nuclear-to-cytoplasmic FoxO3 level depended on the relative but not absolute change in EGF concentration. Statistical significance was calculated using the Wilcoxon rank sum test (n = 5); * corresponds to p < 0.01, and n. s. corresponds to p > 0.1. Error bars represent the standard deviation of technical replicates.