An ultrasensitive sorting mechanism for EGF Receptor Endocytosis

Hannah Schmidt-Glenewinkel 2 Ivayla Vacheva 2 Daniela Hoeller 1 Ivan Dikic 0 Roland Eils 2 3 0 Institute for Biochemistry II, Goethe University Medical School , 60590 Frankfurt , Germany 1 Innsbruck Medical University , Biocenter , Medical Biochemistry , A-6020 Innsbruck , Austria 2 Division Theoretical Bioinformatics, German Cancer Research Center (DKFZ) , 69120 Heidelberg , Germany 3 Institute for Pharmacy and Molecular Biotechnology (IPMB) , 69120 Heidelberg , Germany Background: The Epidermal Growth Factor (EGF) receptor has been shown to internalize via clathrin-independent endocytosis (CIE) in a ligand concentration dependent manner. From a modeling point of view, this resembles an ultrasensitive response, which is the ability of signaling networks to suppress a response for low input values and to increase to a pre-defined level for inputs exceeding a certain threshold. Several mechanisms to generate this behaviour have been described theoretically, the underlying assumptions of which, however, have not been experimentally demonstrated for the EGF receptor internalization network. Results: Here, we present a mathematical model of receptor sorting into alternative pathways that explains the EGF-concentration dependent response of CIE. The described mechanism involves a saturation effect of the dominant clathrin-dependent endocytosis pathway and implies distinct steady-states into which the system is forced for low vs high EGF stimulations. The model is minimal since no experimentally unjustified reactions or parameter assumptions are imposed. We demonstrate the robustness of the sorting effect for large parameter variations and give an analytic derivation for alternative steady-states that are reached. Further, we describe extensibility of the model to more than two pathways which might play a role in contexts other than receptor internalization. Conclusion: Our main result is that a scenario where different endocytosis routes consume the same form of receptor corroborates the observation of a clear-cut, stimulus dependent sorting. This is especially important since a receptor modification discriminating between the pathways has not been found experimentally. The model is not restricted to EGF receptor internalization and might account for ultrasensitivity in other cellular contexts. - Background Endocytosis is the process by which activated transmembrane receptors are directed into the endosomal system from the plasma membrane [1-4]. In the past years, it has emerged as a powerful mechanism for the cell to temporally and spatially control its signaling response [5]. Ligand induced phosphorylation of EGF receptor creates docking sites for adaptor proteins, such as EPS15, epsin and AP-2 [6,7]. Via direct or indirect binding, adaptors recruit the receptor to special membrane regions which are characterized by a particular composition of cage-proteins and/or -lipids [8,9]. The forming vesicles pinch off the membrane and carry their cargo to distinct intracellular locations, which might account for the specificity of the invoked signal [1,10]. Endocytosis may direct the receptors for lysosomal degradation or recycle them back to the membrane [10-12]. Proper sorting of the EGF receptor into the correct endocytosis route is crucial for cell functioning as indicated by the fact that corruption of the sorting e.g. by viral proteins [13,14] may result in impaired receptor downregulation and increased mitogenic activity [15]. Clathrin-dependent endocytosis (CDE) was the first receptor internalization mechanism to be discovered and is generally considered the major route for EGF receptor (reviewed in [1,5,6,9]). Nevertheless, receptor internalization mechanisms that do not employ the structural protein clathrin, but arise from lipid rafts and caveolin-rich membrane regions exist (Clathrin-independent endocytosis, CIE) [8,9,16,17]. The important question which molecular events govern the sorting of the EGF receptor into the different endocytosis pathways remains unanswered [5,8,9,18-20]. A study addressing the sorting between Clathrin- vs lipid raft/Caveolae-mediated Endocytosis in mammalian cells suggested an interesting mechanism for the sorting process [21]: the distribution of receptors into the two pathways was shown to be EGF-concentration dependent. In the presence of low concentrations of EGF, the receptor was exclusively internalized via CDE, whereas at high concentrations, receptors were equally distributed between CDE and CIE (Figure 1). From a modeling point of view, the behaviour of the clathrin-independent pathway resembles an ultrasensitive response: activation of the pathway is suppressed for low input EGF values, to reach the same level as the clathrindependent pathway for high input levels. Theoretically, several different mechanisms can explain ultrasensitive behaviour. Multisite modifications lead to a sigmoidal response of the modified molecule [22-24], an effect that can be enhanced by consecutive arrangement in the form of cascades [25-29] which has also been validated experimentally [30]. FCiDguEraend1 CIE pathways of EGF receptor CDE and CIE pathways of EGF receptor. An illustration of CDE and CIE pathways of EGF receptor. High EGF concentrations induce CIE, whereas CDE is observed at low and high EGF concentrations. The adaptors for the respective endocytosis pathways are referred to as CDE- or CIE-adaptors, respectively. See list of abbreviations. Other models of ultrasensitivity have been derived for Michaelis-Menten type enzyme reactions: the presence of a stoichiometric inhibitor of an enzyme can suppress a reaction up to a certain threshold [28]. In (de-)modification cycles ultrasensitivity occurs when the opposing enzymes work in the zero-order regime [31], a mechanism which has been shown to work during morphogen directed pattern formation [32], or if the abundance levels of unmodified substrate and enzyme are sufficiently high [33]. Mathematical modeling has previously played a significant role in elucidating the mechanisms of EGF receptor signaling and endocytosis [34-42]. In a series of quantitative studies the interaction between receptors and endocytosis machinery was evaluated [34,35,38,43]. Here, the existence of at least two distinct internalization pathways with different affinities for the EGF receptor was discovered [35,43]. In [21] it was reported that monoubiquitination (mono-Ub) of the EGF Receptor could only be observed at high EGF concentrations, raising the question whether mono-Ub might serve as a discriminative feature, which, when appended to the receptor, selectively targets the receptor to CIE [19,44]. This, however, conflicts with reports on the involvement of ubiquitinbinding adaptor proteins such as epsin and EPS15 during CDE [19,20,45-49]. To address this controversy, we built a mathematical model of the sorting process. We address the functional consequences of different affinities with which internal (...truncated)