Author response: pYtags enable spatiotemporal measurements of receptor tyrosine kinase signaling in living cells

Abstract

Full text Figures and data Side by side Abstract Editor's evaluation Introduction Results Discussion Methods Appendix 1 Data availability References Decision letter Author response Article and author information Metrics Abstract Receptor tyrosine kinases (RTKs) are major signaling hubs in metazoans, playing crucial roles in cell proliferation, migration, and differentiation. However, few tools are available to measure the activity of a specific RTK in individual living cells. Here, we present pYtags, a modular approach for monitoring the activity of a user-defined RTK by live-cell microscopy. pYtags consist of an RTK modified with a tyrosine activation motif that, when phosphorylated, recruits a fluorescently labeled tandem SH2 domain with high specificity. We show that pYtags enable the monitoring of a specific RTK on seconds-to-minutes time scales and across subcellular and multicellular length scales. Using a pYtag biosensor for epidermal growth factor receptor (EGFR), we quantitatively characterize how signaling dynamics vary with the identity and dose of activating ligand. We show that orthogonal pYtags can be used to monitor the dynamics of EGFR and ErbB2 activity in the same cell, revealing distinct phases of activation for each RTK. The specificity and modularity of pYtags open the door to robust biosensors of multiple tyrosine kinases and may enable engineering of synthetic receptors with orthogonal response programs. Editor's evaluation Your study provides strong and convincing evidence that pYtags enable spatiotemporal measurements of receptor tyrosine kinase signaling in living cells. This is highly significant as it can be used to study in real-time receptor signaling in healthy and diseased cells. https://doi.org/10.7554/eLife.82863.sa0 Decision letter Reviews on Sciety eLife's review process Introduction Development and homeostasis of multicellular organisms require that cells sense and respond to diverse microenvironmental signals. Receptor tyrosine kinases (RTKs) are one widely expressed class of cell-surface receptors that play a key role in this information processing (Lemmon and Schlessinger, 2010). RTK activation is triggered by growth factors or hormones that bind to the extracellular domains of receptors, inducing conformational changes that lead to receptor dimerization and the autophosphorylation of tyrosine residues in the C-terminal tails. Phosphorylated RTKs, hereafter described as ‘activated’ RTKs, present phosphotyrosine-containing motifs that bind to downstream effectors that signal to multiple pathways. RTKs are thus positioned as the uppermost node of a complex network of intracellular signaling. RTK signaling provides a high-dimensional input space through which this class of receptors regulates a variety of cellular processes (Lemmon and Schlessinger, 2010). Human cells express at least 58 RTKs whose binding partners vary widely, so the pathways that are activated in a particular cell depend on the set of receptors expressed on its surface (Madhani, 2001; Salokas et al., 2022). Some classes of receptors can form homo- or heterodimers, further diversifying signaling responses (Kanakaraj et al., 1991; Harari and Yarden, 2000; Del Piccolo et al., 2017). Moreover, a single RTK can bind to many different ligands that are capable of inducing distinct conformational changes to tune downstream signaling dynamics (Freed et al., 2017; Hu et al., 2022). These observations underscore the substantial complexity present at even the uppermost node of the RTK signaling network: the interaction between receptors and their ligands. Recent advances in live-cell biosensors have enabled the study of intracellular signaling at unprecedented spatiotemporal resolution. Signaling responses can be measured with a temporal precision on the order of seconds, and recent studies have described approaches to multiplex biosensors of multiple pathways in a single cell (Regot et al., 2014) or to use barcoding strategies to monitor many biosensors within a population of cells (Kaufman et al., 2022; Yang et al., 2021). Yet the development of biosensors for specific RTKs has been somewhat limited. A fluorescent Grb2-based biosensor has been widely used to measure general RTK activity but does not distinguish between the many receptors that can recruit Grb2 (Reynolds et al., 2003). FRET-based biosensors of epidermal growth factor receptor (EGFR) (Komatsu et al., 2011; Sorkin et al., 2000; Itoh et al., 2005; Kurokawa et al., 2001) and platelet-derived growth factor receptor (PDGFR) (Seong et al., 2017) have been described, but rely on components of endogenous substrates that participate in interactions with multiple RTKs, and their specificity remains to be characterized. A more specific Src homology 2 (SH2)-based biosensor for EGFR was recently reported but required extensive engineering and may compete with other SH2 domain-containing proteins for binding to phosphotyrosine motifs required for signaling (Tiruthani et al., 2019). Consequently, the field still lacks modular live-cell biosensors to monitor the activity of any specific RTK of interest. Here, we describe pYtags, a versatile and modular RTK biosensing strategy. In the pYtag approach, the C-terminus of a user-defined RTK is labeled with a tyrosine activation motif that, when phosphorylated, binds selectively to a fluorescently labeled tandem SH2 (tSH2) reporter. This binding results in the depletion of the fluorescent reporter from the cytosol and local accumulation at membranes where the receptor is activated. We show that an EGFR pYtag biosensor quantitatively reports on the activity of EGFR with undetectable crosstalk from other unlabeled RTKs. We use pYtags to quantify EGFR activation in response to various ligands, revealing ligand- and dose-specific signaling dynamics. Mathematical modeling and experimental validation suggest that different ligands affect the signaling dynamics of EGFR by altering the dimerization affinity of ligand-bound receptors. We further demonstrate that pYtags can be applied to other receptors, including FGFR1, PDGFRβ, VEGFR3, and the ligand-less RTK ErbB2, a challenging case study due to its need to signal through receptor heterodimers. We develop a second, orthogonal pYtag that we use to simultaneously monitor EGFR and ErbB2 activity within the same cell. Finally, we demonstrate that pYtags can be inserted into the genome using CRISPR/Cas9-based editing, which enables reporting of endogenous receptors and eliminates confounding effects from ectopic expression. pYtags thus offer a modular strategy for measuring the activity of specific RTKs in individual living cells and may serve as a platform for engineering phosphotyrosine-based cargo that can be recruited to synthetic receptors. Results An orthogonal tag for monitoring tyrosine phosphorylation events Many biosensors of kinase activity are based on two components: a synthetic substrate that is predominantly phosphorylated by a single kinase of interest, and a domain that binds specifically to the phosphorylated substrate. To apply similar logic in the design of an RTK biosensor, it would be desirable to introduce a tyrosine-containing peptide that can be uniquely phosphorylated by an RTK of interest and a binding domain that interacts only with the phosphorylated peptide. Fulfilling these requirements is especially challenging in the case of tyrosine kinases, which typically lack one-to-one specificities between tyrosine kinase, tyrosine substrate, and SH2-binding domains (Miller, 2003). However, immune cells offer examples of highly specific phosphotyrosine signaling. During activation of the T-cell receptor (TCR), pairs of tyrosine residues located in immunoreceptor tyrosine-based activation motifs (ITAMs) are phosphorylated and serve as sites for the selective recruitment of the tandem SH2 domain of ZAP70 (ZtSH2), which binds 100-fold more tightly than individual ZAP70 SH2 domains with the same ITAMs and displays minimal crosstalk with other phosphotyrosine-containing peptides (Figure 1A; Isakov et al., 1995; Labadia et al., 1996; Wange et al., 1993). Because neither the TCR nor ZAP70 are expressed by nonimmune cells, we reasoned that an ITAM/ZtSH2 pair may be repurposed as an orthogonal interaction module to monitor the activity of a desired RTK in nonimmune contexts. If ITAMs appended to the C-terminal tail of an RTK are phosphorylated upon activation of the receptor, then this should create a selective binding site for a fluorescently labeled ZtSH2 (Figure 1B). At the cellular level, such a ZtSH2-based reporter should localize to the cytosol when RTK signaling is low and be recruited to the cell membrane when RTK signaling is high (Figure 1B). Figure 1 with 5 supplements see all Download asset Open asset pYtags: a biosensing strategy to monitor receptor tyrosine kinase (RTK) activity in living cells. (A) The T-cell receptor complex contains six immunoreceptor tyrosine-based activation motifs (ITAMs) from CD3 chains that, when phosphorylated, bind to the tSH2 domain of ZAP70 (ZtSH2). (B) Three repeats of CD3 ITAMs were appended to the C-terminus of epidermal growth factor receptor (EGFR) and clearance of ZtSH2 from the cytosol was assessed. (C) Timelapse images of NIH3T3 cells expressing EGFR pYtag (CD3ε variant), treated first with EGF (100 ng/mL) and then with gefitinib (10 µM). Scale bar, 20 µm. (D) Mean clearance of cytosolic ZtSH2 in cells co-expressing iRFP-ZtSH2 and EGFR C-terminally labeled with one of six CD3 ITAMs. EGF (100 ng/mL) and gefitinib (10 µM) were sequentially added at times denoted by arrows. n = 2 independent experiments. (E) Clearance of cytosolic ZtSH2 10 min post-EGF treatment and 40 min post-gefitinib treatment from (D). Lines denote mean values, boxes denote 25–75th percentiles, and whiskers denote minima and maxima. n ≥ 14 cells from two independent experiments. n.s., not significant, ***p<0.001 by Kolmogorov–Smirnov test with cells expressing no additional EGFR 10 min post-EGF. (F) Immunoblots of NIH3T3 cells expressing either WT EGFR or EGFR pYtag treated with EGF (100 ng/mL). (G) Mean ± SEM levels of EGFR, Akt, and Erk phosphorylation from (F). n = 3 independent experiments. (H) The EGFR pYtag was tested in SYF cells to determine whether SFKs are required for ITAM phosphorylation. (I) Representative images of NIH3T3 and SYF cells expressing EGFR pYtag, treated with EGF (100 ng/mL). Scale bars, 40 µm. (J) Mean clearance of cytosolic ZtSH2 in SYF and NIH3T3 cells 10 min after treatment with EGF. For each condition, n > 20 cells from three independent experiments. See also Figure 1—video 1. Figure 1—source data 1 Uncropped gels for Figure 1F. https://cdn.elifesciences.org/articles/82863/elife-82863-fig1-data1-v1.zip Download elife-82863-fig1-data1-v1.zip We first applied this biosensing approach to detect the activation of EGFR, the most well-characterized of the 58 known human RTKs (Salokas et al., 2022). In an initial screening experiment, we tested six ITAMs from the CD3γ, CD3δ, CD3ε, and CD3ζ chains of the TCR. In each case, we fused three identical repeats of the ITAM to the C-terminal tail of EGFR followed by the FusionRed fluorescent protein (EGFR-ITAM-FusionRed). We co-expressed each receptor with an iRFP-labeled ZtSH2 (iRFP-ZtSH2) in NIH3T3 fibroblasts (Figure 1B), which express very low levels of endogenous EGFR (Di Fiore et al., 1987; Livneh et al., 1986; Eierhoff et al., 2010). In cells expressing EGFR-ITAM-FusionRed, treatment with epidermal growth factor (EGF) resulted in rapid translocation of ZtSH2 from the cytosol to the cell membrane (Figure 1C, Figure 1—video 1), which we assessed by quantifying the percentage of ZtSH2 cleared from the cytosol (see ‘Methods’). Clearance of ZtSH2 from the cytosol was quickly reversed by treatment with gefitinib, an inhibitor of EGFR kinase activity, indicating that ZtSH2 can serve as a rapid, reversible reporter of EGFR activation (Figure 1C). Cells lacking EGFR or expressing an ITAM-less EGFR-FusionRed exhibited no change in ZtSH2 localization in response to treatment with EGF or gefitinib (Figure 1D and E), demonstrating that the ZtSH2 reporter responds specifically to the activation of an ITAM-labeled RTK. Although all six ITAMs that we tested appear capable of functioning as biosensors of RTK activity, we chose to focus on the CD3ε ITAM for all subsequent experiments due to its reported selectivity for ZtSH2 over other phosphotyrosine-binding domains (Osman et al., 1995; Ravichandran et al., 1993; Love and Hayes, 2010). We refer to the resulting two-component biosensor as a phosphotyrosine tag (pYtag): a tyrosine activation motif that recruits its complementary tSH2 reporter to report on signaling. Grb2-based biosensors have been used extensively to monitor receptor tyrosine kinase activity (Reynolds et al., 2003). However, Grb2 binds to many activated RTKs, making this biosensing strategy unable to specifically detect activity of a single RTK of interest. To compare our ITAM-labeling approach to Grb2-based biosensing, we simultaneously expressed a fluorescent Grb2 construct (Grb2-TagBFP) and iRFP-ZtSH2 in NIH3T3 cells expressing either EGFR-ITAM-FusionRed or an ITAM-less EGFR-FusionRed construct (Figure 1—figure supplement 1A). Stimulating these cells with EGF revealed that Grb2 localized to the membrane in both ITAM-tagged and ITAM-less EGFR cells, whereas ZtSH2 only showed membrane recruitment in the ITAM-tagged context (Figure 1—figure supplement 1B–E). These data further confirm that ZtSH2 is specific for ITAM-tagged RTKs, a feature that is not shared by Grb2-based reporters. Since pYtags introduce additional tyrosine residues and SH2-containing peptides to an RTK signaling complex, we asked whether this biosensing strategy interferes with signaling downstream of EGFR. We stimulated NIH3T3 cells expressing either EGFR-FusionRed or EGFR pYtag (EGFR-CD3ε-FusionRed; iRFP-ZtSH2) with EGF and measured signaling responses as a function of time by immunoblotting (Figure 1F). We found that EGFR, Erk, and Akt were phosphorylated at similar levels in the two cell lines (Figure 1G), suggesting that ITAM phosphorylation and ZtSH2 recruitment do not interfere with signaling downstream of EGFR. In T cells, CD3 ITAMs are typically phosphorylated by Src family kinases (SFKs) upon engagement of TCRs (Gaud et al., 2018). Because EGFR also activates SFKs, we reasoned that pYtags could be phosphorylated indirectly by EGFR-associated SFKs rather than by the kinase domain of EGFR itself (Figure 1H). To test whether SFK-mediated phosphorylation is required for pYtag function, we expressed EGFR pYtag in mouse embryonic fibroblasts lacking all three ubiquitously expressed SFKs: Src, Yes, and Fyn (SYF cells) (Figure 1H; Klinghoffer et al., 1999). As in NIH3T3 cells, SYF cells exhibited strong clearance of ZtSH2 from the cytosol after stimulation with EGF (Figure 1I and J). Combined with the rapid responses observed after EGFR stimulation and inhibition (Figure 1C–E), these results suggest that the EGFR pYtag acts as a direct biosensor of EGFR activity. Assessing reporter activity as a function of the expression levels of pYtag components Our pYtag biosensor relies on two components: an ITAM-tagged RTK and a fluorescent, cytosolic ZtSH2 probe. We reasoned that the measured output – the extent of ZtSH2 redistribution from cytoplasm to membrane – could depend on the expression level of both components, reminiscent of the responses observed for cellular optogenetics tools that re-localize between cytosol and membrane (Toettcher et al., 2011). To further investigate this dependency, we measured ZtSH2 membrane translocation while simultaneously quantifying the initial levels of membrane-associated EGFR and cytoplasmic ZtSH2 in individual cells by live imaging. These experiments were carried out in two cell lines used throughout our study: NIH3T3 cells expressing EGFR-CD3ε-FusionRed and iRFP-ZtSH2 (Figure 1—figure supplement 2) and HEK293T cells expressing EGFR-CD3ε-mNeonGreen and mScarlet-ZtSH2 (Figure 1—figure supplement 3). When comparing cells with similar levels of ZtSH2 but different levels of EGFR, we observed a greater degree of ZtSH2 redistribution in cells with higher EGFR expression (Figure 1—figure supplements 2A and 3A). Conversely, imaging cells with similar levels of EGFR but different levels of ZtSH2 revealed more complete ZtSH2 clearance in cells with lower ZtSH2 expression (Figure 1—figure supplements 2B and 3B). More broadly, quantifying cytosolic ZtSH2 clearance across all cells revealed consistently higher degrees of ZtSH2 clearance in cells with a high EGFR:ZtSH2 expression ratio, as measured by the ratio of their fluorescence intensities prior to stimulation (Figure 1—figure supplements 2C,D and 3C,D). This phenomenon was also matched by a simplified mathematical model of EGFR activation and ZtSH2 redistribution (see text for Figure 3 and ‘Methods’ for a detailed discussion of the model), where the percentage translocation of ZtSH2 varied linearly with the EGFR-to-ZtSH2 ratio until it saturated under conditions where the amount of ZtSH2 exceeded that of active EGFR (Figure 1—figure supplement 4). In sum, these results suggest that a simple ratio – the amount of ITAM-tagged EGFR relative to the amount of cytoplasmic ZtSH2 – sets the dynamic range of this translocation-based biosensor. We also tested whether the EGFR:ZtSH2 ratio might also affect the kinetics of translocation. We first quantified responses across all cells and sorted the resulting heatmaps in order of increasing EGFR:ZtSH2 ratio (Figure 1—figure supplements 2E and 3E), which again confirmed that ZtSH2 clearance depended on this ratio. We next min–max normalized each response, enabling us to compare response kinetics to be compared in an amplitude-independent manner (Figure 1—figure supplements 2F and 3F). While steady-state ZtSH2 cytosolic clearance depended on the EGFR:ZtSH2 ratio, we observed similarly rapid activation kinetics at all expression levels. These results suggest that the pYtag biosensor approach faithfully reports on the kinetics of RTK activation across a range of component expression levels, but that response amplitude should be compared only between cells at similar expression levels of both the ITAM-tagged RTK and fluorescent ZtSH2 probe. Our results also suggest guiding principles for future cell line engineering. Ideally, a translocation-based biosensor would exhibit measurable redistribution between cytosol and membrane, yet avoid completely clearing from the cytosol at low stimulus levels. Our results provide an intuitive way to achieve this response – matching the expression of the ZtSH2 cytosolic component to be proportional to the expression of the ITAM-tagged receptor. For example, if one desires to detect activity of RTKs that are expressed at low levels, it is important to engineer cells with correspondingly low ZtSH2 expression to observe potent redistribution of this biosensor. pYtags reveal the dynamics of EGFR signaling at high spatiotemporal resolution Because ZtSH2 can in principle be recruited rapidly on and off the membrane or other subcellular compartments where active receptors are found, we reasoned that pYtags could be used to monitor the subcellular activity of an RTK in individual cells over time. To test this possibility, we first performed rapid, high-resolution imaging of NIH3T3 cells treated with EGF (Figure 2A). We found that ZtSH2 was cleared from the cytosol in a biphasic manner, with an initial phase of rapid cytosolic clearance occurring within the first ~40 s, followed by a further gradual increase in cytosolic clearance over the subsequent ~20 min (Figure 2B). We observed sustained ZtSH2 translocation to the membrane for at least 30 min, indicating that high levels of membrane-localized, active EGFR are persistent at the membrane under these conditions (Figure 2A). Similar biphasic responses for EGFR were observed previously at the population-level using a split luciferase system (Macdonald-Obermann and Pike, 2014; Macdonald-Obermann et al., 2012) and in vitro for EGFR on synthetic vesicles (Kovacs et al., 2015), but have never been reported for individual cells. Figure 2 with 2 supplements see all Download asset Open asset Monitoring epidermal growth factor receptor (EGFR) signaling at subcellular and multicellular length scales. (A) Images of EGFR pYtag-expressing NIH3T3 cells treated with EGF (20 ng/mL). Scale bar, 20 µm. (B) Mean ± SD clearance of cytosolic ZtSH2 from (A). n = 3 independent experiments. (C) EGFR pYtag-expressing NIH3T3 cells treated with EGF (20 ng/mL) were monitored for internalized ZtSH2-positive vesicles, and then treated with gefitinib (10 µM). Scale bar, 20 µm. (D) Timelapse images from the region denoted by the blue dashed border from (C). Scale bar, 10 µm. (E) MCF10A human mammary epithelial cells cultured on soft substrata form round, multilayered clusters. EGFR pYtag and ErkKTR were used to spatiotemporally monitor both EGFR and Erk responses after stimulation with EGF. (F) Images of MCF10A cells cultured on soft substrata and treated with EGF (100 ng/mL). Scale bar, 25 µm. (G) Apical and lateral enrichment of ZtSH2 was quantified by line scans denoted by magenta and orange vectors, respectively. Scale bar, 10 µm. (H) Heatmaps of EGFR pYtag and ErkKTR responses from (F). Rows denote individual cells. For each cell, signaling responses of each biosensor were normalized to their respective minima and maxima. (I) Time to half maximal response for EGFR pYtag and ErkKTR for cells from (H). n = 19 (periphery) and n = 11 (interior) cells from three biological replicates. (J) Time to half maximal response for EGFR pYtag and ErkKTR. Responses of individual cells are denoted by points and connected by lines. n > 30 cells from three biological replicates. ***p<0.001 by Kolmogorov–Smirnov test. See also Figure 2—video 1. EGFR is well known to be heavily regulated by intracellular trafficking through processes such as internalization, degradation, and recycling back to the cell membrane (Sorkin and Goh, 2009). However, we observed continued ZtSH2 and EGFR membrane localization in NIH3T3 pYtag-expressing cells for at least 30 min after EGF stimulation (Figure 2A), indicating that endocytosis does not result in the rapid clearance of EGFR from the cell membrane in these cells, possibly due to saturation of receptor endocytosis at high expression levels (Lund et al., 1990). To gain more insight into trafficking in our system, we first validated that endocytosis of EGFR is not altered by expression of the novel pYtag components. We stimulated NIH3T3 cells expressing either EGFR-FusionRed or the pYtag system (EGFR-CD3ε-FR; iRFP-ZtSH2) and stained for the early endocytic marker EEA1 at various time points after stimulation with 100 ng/mL EGF (Figure 2—figure supplement 1A). Quantifying EGFR membrane intensity revealed that cells retain high levels of membrane-associated EGFR for at least 60 min after EGF stimulation, and that this response is independent of whether our pYtag components are present (Figure 2—figure supplement 1B and C). Despite sustained, high levels of membrane-localized EGFR, we also observed that pYtag-expressing cells stimulated with EGF contained puncta that were positive for both total EGFR and ZtSH2 (Figure 2C and D). Co-staining cells at various time points after EGF stimulation for the early endocytic marker EEA1 revealed colocalization between puncta containing EGFR, ZtSH2, and EEA1 in both NIH3T3 and HEK293T cells (Figure 2—figure supplement 1D–G). Furthermore, subsequent treatment with gefitinib rapidly eliminated ZtSH2 from EGFR-positive puncta, suggesting that the enrichment of ZtSH2 at puncta is indicative of signaling from endosomal compartments (Figure 2C and D). These results are consistent with prior reports that internalized EGFR remains bound to its ligand and can transmit signals from endosomal compartments (Haugh et al., 1999). Nevertheless, we find that EGFR-overexpressing NIH3T3 cells retain a high degree of membrane-associated receptor signaling for at least 1 hr after EGF stimulation. At the tissue scale, the spatiotemporal distribution of RTK signaling can also be regulated by several processes including paracrine signaling (De Simone et al., 2021), morphogen gradients (Casanova and Struhl, 1989; Sprenger and Nüsslein-Volhard, 1992), and the mechanical properties of the local microenvironment (Farahani et al., 2021). We therefore asked whether pYtags could be used to monitor RTK signaling in multicellular contexts. We previously found that MCF10A human mammary epithelial cells form multicellular clusters when cultured on soft substrata and exhibit a complex spatial pattern of EGF binding at cell membranes (Farahani et al., 2021). Measurements of fixed tissues revealed that EGF binds rapidly to the media-exposed membranes on the surface of the cluster but is excluded from lateral membranes and from cells located within the interior of the cluster (Farahani et al., 2021). To investigate the dynamics of this spatial pattern, we monitored EGFR signaling using two live-cell biosensors: EGFR pYtag and a kinase translocation reporter for downstream signaling through Erk (ErkKTR) (Regot et al., 2014). We treated MCF10A cells cultured on soft substrata with EGF and observed a radially directed wave of EGFR and Erk signaling: activation first appeared in cells at the periphery of the clusters before appearing in cells at the interior (Figure 2F, Figure 2—video 1). In cells at the periphery of clusters, we found that ZtSH2 was first enriched at the media-exposed membrane before localizing to lateral membranes (Figure 2G), highlighting the differences in receptor-level signaling between membrane subcompartments. Quantifying EGFR and Erk responses confirmed our qualitative observations, revealing a 2–4 min delay in EGFR and Erk signaling between the periphery and interior of clusters (Figure 2H and I). Notably, Erk responses also trailed those of EGFR by ~4 min, consistent with the delay in signal transmission previously observed from Ras to Erk (Figure 2J; Toettcher et al., 2013). These data support a model in which ligand–receptor interactions are limited at cell–cell contacts, producing an inward-traveling wave of RTK activation and downstream signaling. More broadly, our data demonstrate that pYtags can be used to reveal RTK signaling dynamics at high temporal resolution and over both subcellular and multicellular length scales. pYtags distinguish ligand- and dose-dependent signaling dynamics We next set out to apply pYtags to an unresolved question in RTK signaling. While it has long been known that different RTKs regulate cellular processes through the dynamics of downstream signaling (Freed et al., 2017; Marshall, 1995; Johnson and Toettcher, 2019; Santos et al., 2007), recent evidence suggests that signaling dynamics can also differ between ligands for the same receptor (Freed et al., 2017). For instance, high-affinity EGFR ligands such as EGF produce long-lived EGFR dimers that are internalized and degraded, whereas low-affinity ligands such as epiregulin (EREG) and epigen (EPGN) produce shorter-lived EGFR dimers that prolong signaling, leading to distinct cell-fate outcomes (Freed et al., 2017; Roepstorff et al., 2009). Yet how these different ligands might alter minutes-timescale EGFR signaling dynamics in single cells remains unknown. We reasoned that pYtags would be ideal for measuring whether different EGFR ligands elicit distinct fast-timescale signaling dynamics. We treated EGFR pYtag-expressing NIH3T3 cells with varying doses of either EGF or one of two low-affinity ligands, EREG or EPGN (see ‘Methods’). For all ligands, we found that ligand stimulation was able to activate our pYtag system, driving ZtSH2 clearance that persisted for at least 30 min (Figure 3—figure supplement 1). The spatial distributions of ZtSH2 and EGFR were similar across all ligands, with both components enriched at the cell membrane throughout the stimulation timecourse (Figure 3—figure supplement 1). However, quantification of single-cell responses revealed that the kinetics of EGFR activation varied substantially between doses and ligands (Figure 3A). For EGF, we observed a gradual rise in activity at lower doses (0.2–2 ng/mL). This response switched to a biphasic response at higher doses (5–100 ng/mL), with a rapid initial phase in first 1–2 min followed by a slower, continued rise (Figure 3A, left panel). In contrast, treatment with the low-affinity ligands EREG and EPGN produced a rapid rise in EGFR activity at all ligand doses, followed by a plateau (Figure 3A, middle and right panels). We also observed a small but reproducible transient peak of EGFR activation shortly after treatment with EREG and EPGN but not with EGF, indicative of a weakly adaptive response after stimulation with low-affinity ligands (Figure 3A, middle and right panels). These results demonstrate that different ligands produce markedly different signaling responses even in the first few minutes following stimulation. They also reveal an unusual property of the low-affinity RTK ligands EREG and EPGN: altering their concentrations has a strong effect on the amplitude, but not the kinetics, of EGFR activation. Figure 3 with 2 supplements see all Download asset Open asset Epidermal growth factor receptor (EGFR) pYtag reveals dose- and ligand-dependent signaling dynamics. (A) Mean ± SD responses of EGFR pYtag-expressing NIH3T3 cells to varying doses of EGF, epiregulin (EREG), and epigen (EPGN). The same 0 ng/mL control was used for each ligand. Data were collected from 475 cells across four indepen