A Particularly Discriminating Receptor.

A central challenge for systems biology is how to move beyond the characterization of processes as they occur in a given cell type under particular conditions to achieve generalizable insights that can be used to make accurate, quantitative predictions about how the system will function under different circumstances (Janes and Lauffenburger, 2013xJanes, K.A. and Lauffenburger, D.A. J. Cell Sci. 2013; 126: 1913–1921Crossref | PubMed | Scopus (17)See all ReferencesJanes and Lauffenburger, 2013). The problem arises because the behavior of signaling systems is strongly dependent on detailed features of the cellular context, including the expression levels of receptor components and other signaling proteins, and can also be influenced by crosstalk with other signaling processes occurring concurrently. Most critically, signaling is often regulated through dynamic modulation of the spatial co-localization of interacting species, meaning that behavior is driven by local rather than cell-wide conditions. Available experimental approaches often require the use of immortalized cell lines or isolated primary cells, cultured in vitro under highly artificial conditions. Thus, the development of knowledge that enables the extrapolation of quantitative systems properties from one cell type and experimental situation to another is critical to our ability to understand how these processes occur in vivo.An article in this issue of Cell Systems (Meyer et al., 2015xMeyer, A.S., Zweemer, A.J.M., and Lauffenburger, D.A. Cell Syst. 2015; 1: 25–36Abstract | Full Text | Full Text PDF | Scopus (4)See all ReferencesMeyer et al., 2015) illustrates how a combined experimental and modeling study can provide quantitative, generalizable insights into a complex cellular signaling process. In particular, the authors focus on how heterogeneity in the spatial distribution of ligand can influence the signaling response. Meyer et al. examine the receptor tyrosine kinase AXL, also known as UFO, and its primary ligand, growth-arrest-specific protein 6 (Gas6). AXL plays important roles in immune regulation, particularly the clearance of apoptotic cell fragments and other debris, and additionally has emerged as an important cancer target (Lemke, 2013xLemke, G. Cold Spring Harb. Perspect. Biol. 2013; 5: a009076Crossref | Scopus (32)See all ReferencesLemke, 2013).Each AXL molecule contains both a high-affinity ligand binding site, Ig1, and a low-affinity site, Ig2, which bind to distinct sites on Gas6. The key event in AXL activation is formation of a signaling-competent receptor dimer stabilized by two Gas6 molecules, each of which bridges two AXL molecules by binding to Ig1 on one receptor molecule and Ig2 on the other (Figure 1Figure 1A) (Sasaki et al., 2006xSasaki, T., Knyazev, P.G., Clout, N.J., Cheburkin, Y., Gohring, W., Ullrich, A., Timpl, R., and Hohenester, E. EMBO J. 2006; 25: 80–87Crossref | PubMed | Scopus (95)See all ReferencesSasaki et al., 2006). It has been known for some years that the lipid phosphatidylserine (PtdSer), a component of the inner leaflet of the plasma membrane, plays a role in AXL activation and function by binding to Gas6 through its N-terminal γ-carboxylglutamic acid-rich (GLA) domain (Graham et al., 2014xGraham, D.K., DeRyckere, D., Davies, K.D., and Earp, H.S. Nat. Rev. Cancer. 2014; 14: 769–785Crossref | PubMed | Scopus (37)See all ReferencesGraham et al., 2014). However, published studies disagree about precisely what role PtdSer plays in AXL activation, and whether it is an obligatory cofactor or is dispensable for some functions of the receptor.Figure 1Mechanism of AXL Engagement by Gas6 in the Absence and Presence of PtdSer(A) Molecular architecture of the complex between the Ig1 and Ig2 domains of AXL (green) and the C-terminal laminin G-like domains of Gas6 (blue) (PDB code 2C5D). Shown are a front view (left) and top view (right). The green arrows show the direction of AXL attachment to the membrane.(B) In the absence of PtdSer, dimerization of the 1:1 AXL-Gas6 complexes is weak, such that at moderate to high Gas6 concentrations the system adopts the non-signaling state shown at bottom.(C) When Gas6 is presented to the receptor arrayed on a PtdSer-coated particle (purple), the local density of the initially formed 1:1 AXL-Gas6 complexes is high compared to KD for dimerization, giving robust formation of activated receptor dimers.View Large Image | Download PowerPoint SlideUsing careful experimentation to disentangle the effects of exogenous Gas6 from those resulting from autocrine signaling, Meyer et al. were able to construct a detailed model of AXL activation. Strikingly, this model revealed exactly how interaction of Gas6 with PtdSer helps sensitize AXL to the presence of particulate species, such as apoptotic cell fragments, that contain exposed PtdSer-bearing membranes.This behavior can be understood by considering how Gas6 interacts with AXL in the absence of PtdSer. The initial 1:1 AXL-Gas6 complex, formed when Gas6 binds to Ig1, can dimerize through interaction of the bound Gas6 with the unoccupied Ig2 site in another AXL-Gas6 complex (Figure 1Figure 1B). However, as with all homodimeric receptors, a ligand that bridges the dimer will actually inhibit dimer formation at high ligand concentrations, as all binding sites on each receptor become fully occupied by separate ligand molecules, leaving no remaining vacant sites to be satisfied through receptor dimerization (Atanasova and Whitty, 2012xAtanasova, M. and Whitty, A. Crit. Rev. Biochem. Mol. Biol. 2012; 47: 502–530Crossref | PubMed | Scopus (14)See all ReferencesAtanasova and Whitty, 2012). Even at moderate ligand concentrations, significant dimer formation will occur only if the local receptor concentration approaches or exceeds the equilibrium dissociation constant (KD) for receptor dimerization (Whitty and Borysenko, 1999xWhitty, A. and Borysenko, C.W. Chem. Biol. 1999; 6: R107–R118Abstract | Full Text PDF | PubMed | Scopus (28)See all ReferencesWhitty and Borysenko, 1999).In the case of AXL, Meyer et al. showed that, because binding to Ig2 is weak, the receptor dimerization affinity of AXL-Gas6 is modest. Consequently, soluble Gas6 induces AXL dimerization only inefficiently, and can become inhibitory at relatively low concentrations (Figure 1Figure 1B). The situation changes dramatically, however, in the presence of a PtdSer-coated particle, which can bind multiple Gas6 molecules. The PtdSer-coated particle organizes the Gas6 and presents it to the AXL-expressing cell as a dense array, with limited mobility. As a result, in the presence of PtdSer, the initial monovalent AXL-Gas6 complexes are created at a high local concentration, effectively increasing the dimerization affinity and driving the equilibrium to favor activated AXL dimers (Figure 1Figure 1C).The model for AXL activation proposed by Meyer et al. explains AXL’s hypersensitivity to Gas6 in the presence of PtdSer-containing particulate species, such as apoptotic cell fragments. In doing so, the study provides a definitive and elegant example of how—to paraphrase Tip O’Neill—all signaling is local, and how heterogeneity in the spatial distribution of signaling molecules can therefore give rise to very large differences in the behavior of the system. The results also identify the low-affinity Ig2 site as a potential target site for inhibitor discovery.The notion that receptor clustering can enhance receptor engagement is, of course, by no means new. However, the new study from Meyer et al. combines experimentation and modeling to reveal quantitative details of how the observed signaling response relates to the individual molecular interaction events that drive receptor activation. Consequently, the model can potentially be applied to understand and predict the behavior of AXL in other cell types and situations and, with appropriate modifications, to the many other receptors that may use complex mechanisms to achieve sensitization to spatially distributed ligand.