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. 2019 Apr 5;8:e42498. doi: 10.7554/eLife.42498

Figure 3. Binding half-life, not receptor occupancy, dominates CAR signaling.

(A) A cell exposed to a high LOV2 density but a short binding half-life can have the same receptor occupancy as a cell exposed to a low LOV2 density but a long binding half-life. (B) At constant receptor occupancy, an occupancy model predicts binding half-life should have no effect on signaling, while kinetic proofreading predicts that increasing binding half-life should increase signaling. (C) At constant receptor occupancy, increasing ligand binding half-life increases DAG signaling, as shown by both non-parametric kernel smoothing regression (green line) and Spearman’s correlation coefficient (ρ). Each black dot is a single cell measurement obtained from multiple experiments over a range of LOV2 concentrations. (D and E) During individual experiments, there is a fixed concentration of LOV2, meaning that both receptor occupancy and binding half-life change in response to blue-light. We measured Spearman’s correlation coefficient to ask whether DAG levels were best described as a function of receptor occupancy or binding half-life across different LOV2 concentrations. DAG levels are more correlated with binding half-life (E) than they are with CAR occupancy (D). This is reflected in the fact that the DAG response curves nearly overlap with each other when plotted as a function of binding half-life, indicating changing the concentration of LOV2 has little effect on downstream signaling. These data are consistent with binding half-life, not receptor occupancy, dominating CAR signaling. The mean is plotted with a 95% CI (two-sided Student’s t-test).

Figure 3.

Figure 3—figure supplement 1. Long LOV2 binding half-lives signal better than short binding half-lives, even at equal receptor occupancy.

Figure 3—figure supplement 1.

(A) Single cell measurements were binned together over narrow ranges of CAR occupancy, and DAG levels were plotted as a function of LOV2 binding half-life. Long LOV2 binding half-lives lead to higher DAG levels, despite nearly identical receptor occupancies. Each plot shows data from a different narrow range of CAR occupancy. (B) Single cell DAG measurement from A) are plotted over their narrow range of CAR occupancies. Grid positions of plots in A) correspond to those in B). Over these narrow ranges, there is no correlation between CAR occupancy and DAG levels, indicating that the increasing DAG levels observed in A) are due to increasing binding half-life and not small increases in receptor occupancy.
Figure 3—figure supplement 2. DAG levels are most strongly correlated with ligand binding half-life.

Figure 3—figure supplement 2.

DAG levels from cell stimulated with the same intensities of blue light but different LOV2 concentrations are plotted as a function of receptor occupancy or ligand binding half-life. Each black line connects measurements from the same cell. The mean response is plotted in red. DAG levels correlate better to ligand binding half-life than they do to receptor occupancy, indicating that binding half-life strongly influences downstream CAR signaling. ρ is Spearman’s correlation coefficient and p denotes the p-value.