An ultrasensitive sorting mechanism for EGF Receptor Endocytosis
 Hannah SchmidtGlenewinkel^{1},
 Ivayla Vacheva^{1},
 Daniela Hoeller^{2},
 Ivan Dikic^{3} and
 Roland Eils^{1, 4}Email author
DOI: 10.1186/17520509232
© SchmidtGlenewinkel et al; licensee BioMed Central Ltd. 2008
Received: 28 June 2007
Accepted: 07 April 2008
Published: 07 April 2008
Abstract
Background
The Epidermal Growth Factor (EGF) receptor has been shown to internalize via clathrinindependent 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 predefined 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 EGFconcentration dependent response of CIE. The described mechanism involves a saturation effect of the dominant clathrindependent endocytosis pathway and implies distinct steadystates 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 steadystates 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 clearcut, 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 AP2 [6, 7]. Via direct or indirect binding, adaptors recruit the receptor to special membrane regions which are characterized by a particular composition of cageproteins 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].
Clathrindependent 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 caveolinrich membrane regions exist (Clathrinindependent 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].
From a modeling point of view, the behaviour of the clathrinindependent 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].
Other models of ultrasensitivity have been derived for MichaelisMenten 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 zeroorder 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 (monoUb) of the EGF Receptor could only be observed at high EGF concentrations, raising the question whether monoUb 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 internalization pathways are entered and explain how a switchlike response of CIE may result simply from a saturation effect of the CDE pathway. Together with the observation of EGFconcentration dependence of CIE, this analysis invites attention to an ultrasensitive regulatory mechanism for endocytic sorting. We give an analytical derivation of the switcheffect and derive regimes of reaction parameters and initial values for which the switch is preserved. Further, we describe its extensibility to more than two pathways. Importantly, the mechanism imposes only weak assumptions on the underlying interaction structure and parameter values. In summary, we give evidence for the hypothesis that the main purpose of postligand binding modifications of the EGF receptor such as ubiquitination does not lie in the discrimination between alternative endocytosis pathways.
Results
We built a system of ordinary differential equations (ODEs) that models the sorting of EGF receptor into clathrindependent or independent endocytosis pathways. The equations read:
d(EGF)/dt = k_{f} * EGF * R + k_{r} * R_EGF
d(CDE)/dt) = k_{cde} * R_EGF * CDE
d(CIE)/dt) = k_{cie} * R_EGF * CIE
d(R_{cde}) = k_{cde} * R_EGF * CDE
d(R_{cie}) = k_{cie} * R_EGF * CIE
The model contains the binding reaction of EGF to receptor R, which leads to one form of activated receptor (R_EGF), capable of entering clathrindependent or independent endocytosis.
In order to simulate the entry of ligandbound receptor into an endocytosis pathway, we introduced variables CDE and CIE, representing adaptors for clathrindependent and independent endocytosis which R_EGF can enter with rates k_{cde} and k_{cie}, respectively. The variables CDE and CIE represent the amount of the limiting factor in each pathway, which could be adaptor or cageproteins. We assume that the affinity of activated receptors R_EGF is significantly higher for the CDEpathway (k_{cde} ≫ k_{cie}). To quantify the fraction of receptor going either pathway, we introduced variables R_{i_cde} and R_{i_cie}. The steadystate values of these variables represent the amount of R_EGF internalized via CDE and CIE, respectively. The model equations were derived according to the law of massaction [50].
CIEinternalization depends on number of receptors and strength of EGFstimulation
The first class of initial values (Figure 2A) represents the case that either the initial number of unbound receptors (R_{0}) or EGFmolecules (EGF_{0}) is lower than the capacity of the CDEpathway (CDE_{0}) (Generally, X_{0} denotes the initial value of variable X). This corresponds to an experimental setting where cells are stimulated with low EGFconcentrations, i.e. EGF_{0} < CDE_{0}. Initial values from the second class are such that either R_{0} or EGF_{0} are below the capacity of both internalization pathways (CDE_{0} + CIE_{0}) (Figure 2B), whereas the third class reflects the case that both R_{0} and EGF_{0} exceed the capacity of both internalization pathways (Figure 2C).
It can be seen that in case A, R_{i_cie}production stays close to zero. In case B, internalization via CIE does occur, albeit to a lesser degree than CDE. For case C, receptors are equally partitioned between CDE and CIE.
Conditions on receptor number for switcheffect of CIEinternalization
The simulations shown in Figure 2 suggest conditions on the receptor number under which an EGFdependent switch of CIEinternalization will occur. If a cell possesses less receptors than CDEadaptors, CIEinternalization will be low independent of EGFstimulation (cf. Figure 2A). If the cell exhibits more receptors than adaptors for CDE, but less than for both pathways, then, for EGFstimulations exceeding CDE_{0}, a moderate fraction of receptor will internalize via CIE (cf. Figure 2B). Finally, if the amount of receptors is higher than the combined capacity of both pathways, CIEinternalization will be switched on equally strong as CDEinternalization for EGFstimulations that are higher than this combined capacity (cf. Figure 2C).
As predicted, for receptor levels lower than CDE_{0} (Figure 3A), CIEinternalization stays close to zero, independently of EGFstimulation. If the initial amount of receptors is greater than the capacity of CDE, CIEinternalization sets in abruptly, albeit to a moderate degree compared to CDEinternalization, for EGFstimulations greater than CDE_{0} (Figure 3B). Finally, if the initial number of receptors is greater than the capacity of both pathways, the CIE pathway switches on to an equal extent as CDEinternalization (Figure 3C).
We have thus derived an ultrasensitive response of CIEinternalization with respect to EGFstimulation, without assuming any discriminative receptor modifications. Rather, it is necessary and sufficient that the initial amount of receptors is higher than the capacity of the CDEpathway (CDE_{0} < R_{0}, moderate switch) or both pathways (CDE_{0} + CIE_{0} < R_{0}, maximal switch).
Correspondence to distinct classes of steadystate
For dynamical systems with multiple steadystates, a certain steadystate will be reached depending on whether the system starts in the corresponding basin of attraction [23, 51]. Thus, a switch between steadystates occurs for different vectors of initial values, provided that the separatrix, i.e. the hypersurface between neighboring basins of attraction, is crossed.
We investigated, whether the switcheffect of CIEinternalization corresponds to such a transition between distinct steadystates of the system. Analytically, one derives two classes of steadystates (see Methods for derivation of conditions):
where X* denotes the steadystate concentration of the respective component.
Note that classes of steadystates are used since not all variables are assigned specific values. For example, in both cases ${\text{R}}_{\text{i\_cie}}^{\ast}$ and ${\text{R}}_{\text{i\_cde}}^{\ast}$ are not uniquely determined and depend on the corresponding initial values. Steadystate class I reflects the case that either all available EGF (EGF* = 0) or all free receptors R (R* = 0) have been absorbed in the binding reaction and all activated receptors R_EGF have been internalized. In steadystate class II neither receptors nor ligand are limiting for the internalization process and have come to an equilibrium with R_EGF. Instead, the capacity of both internalization pathways has been depleted (CIE* = CDE* = 0).
The systematic scan of initial values and subsequent solving of the system until steadystate revealed initial conditions under which each steadystate class is reached. We found that if both EGFstimulation and initial receptor level are higher than the capacity of both internalization pathways (CDE_{0} + CIE_{0} < min{R_{0}, EGF_{0}}, initial value class C), the system tends towards steadystate class II. Otherwise, steadystate class I will be reached. In this case, if EGFstimulation is below the amount of receptors, all EGF will be depleted in the binding reaction (EGF* = 0), whereas if it is above, all receptors will be consumed (R* = 0).
Applying these derived conditions on the initial values, we can also show that the steadystates are stable. A steadystate is stable, if, for small perturbations, the system returns to this steadystate. Consider steadystate class I with R* = 0 and R_EGF* = 0. CIE* and CDE* are not clearly defined in this case, but according to the conditions on the initial values we derived, we know that R_{0} < CDE_{0} + CIE_{0}. This means that in steadystate, at least one of the two adaptor variables must be greater than zero, i.e. 0 = R* < CDE* + CIE*. If we apply a sufficiently small perturbation to the system, and set the obtained value as the new start vector, this last inequality will still hold due to the continuity of the functions. According to the conditions on initial values we derived in the previous paragraph, the system will tend back to R* = 0 and R_EGF* = 0. Hence we showed stability of steadystate class I. An analogous argument can be used to show stability of steadystate class II.
Conditions on initial values for steadystates. Assuming that the initial number of receptors is higher than the combined capacity of both pathways (R_{0} > CDE_{0} + CIE_{0}), low and high EGFstimulations lead to two different steadystates, respectively. In steadystate I, the receptor internalizes primarily via CDE, whereas in steadystate II it is equally partitioned between CDE and CIE.
Initial values  Steadystate  CIEinternalization 

EGF_{0} < CDE_{0}  I  low 
CDE_{0} + CIE_{0} < EGF_{0}  II  high 
Steepness of switch effect
An ultrasensitive response of a signaling system is characterized by a low, or damped response up to a certain threshold of stimulus, followed by an abrupt increase towards maximal response when this threshold is exceeded [23, 26, 27, 50]. It has been derived to result from positive feedback or multisitemodification [24, 25, 50, 52, 53]. Ultrasensitivity has also been shown to arise in (de)modification cycles if the enzymes operate near saturation [31], which makes the mechanism very sensitive to small parameter changes [26], if the abundance levels of unmodified substrate and enzyme are sufficiently high (ultrasensitization, [33]) or if the enzyme is inhibited [28].
Robustness of solution
It has been argued that biologically functional modules or pathways need to be robust against variations of reaction parameters and protein concentrations in order to ensure proper functioning [54, 55]. The concept of robustness refers to the 'purpose' of a certain module or pathway: it is expected that intracellular network structures have undergone an evolution that guarantees their proper functioning independently of precise parameter values [56].
To transfer this concept to the question of receptor sorting into alternative pathways, we asked to what extent the functioning of the here described module depends on exact parameter or initial values. As functioning we defined the clearcut sorting of the receptor into distinct routes, namely CDE at low, respectively CDE and CIE at high ligand concentrations.
Consider the curve for ${\text{R}}_{\text{i\_cie}}^{\ast}$ (blue). For initial adaptor values such that CDE_{0} + CIE_{0} < R_{0}, EGF_{0} (see arrow), the curve is largely independent of CDE_{0} and increases linearly as a function of CIE_{0} up to the threshold of 1.5. The independence of CDE_{0} reflects the fact that if neither receptors nor ligand are limiting for the internalization reaction (steadystate class II), the steadystate amount of receptor internalized via CIE is solely dependend on the initially available number of CIEadaptors. Outside of this range, ${\text{R}}_{\text{i\_cie}}^{\ast}$ decreases with increasing CDE_{0} and becomes zero for CDE_{0} > 1.5(cf. Figure 3A). ${\text{R}}_{\text{i\_cie}}^{\ast}$ (orange curve) is largely independent of CIE_{0} and increases linearly with CDE_{0} up to the threshold of 1.5 when ligand or receptor number become limiting.
The threshold of CIEinternalization (K_{ m }of the stimulusresponse curves) is independent of CIE_{0} (for CIE_{0} > 0) and is equal to CDE_{0} as shown in Figure 8B.
Role of Receptor modifications
It is wellknown that ligandinduced receptor modifications in the form of phosphorylation and/or ubiquitination play a functional role in signaling and contribute to the specificity of adaptorbinding. However, our analysis focused on the question, whether for a precise sorting of receptors into the two alternative endocytosis pathways discussed here a discriminative modification is necessary. In this light, R_EGF, which in our model indicates the activated receptor species capable of interacting with the endocytosis adaptors, could also represent an already modified form of the receptor. To illustrate this point, we extended the model as given in equations (1.1 – 1.7) to include the binding of the ubiquitinligase Cbl followed by ubiquitination of the receptor. The equations read
d(EGF)/dt = k_{f} * EGF * R + k_{r} * R_EGF
d(CDE)/dt =  k_{cde} * R_EGF_Ub * CDE
d(CIE)/dt =  k_{cde} * R_EGF_Ub * CIE
d(R_{i_cde})/dt = k_{cde} * R_EGF_Ub * CDE
d(R_{i_cie})/dt = k_{cie} * R_EGF_Ub * CIE
Here, k_{onCbl} and k_{offCbl} are the rate constants for the association and dissociation of Cbl to ligandbound receptor R_EGF, and k_{catCbl} is the rate of the ubiquitination step.
Model extension for more than two Pathways
For EGF receptor, evidence for the existence of more than just two independent endocytosis pathways has been given [19, 57]. To our knowledge, EGFconcentration dependence has not been shown. Here, we describe an extension of the above model to more than two pathways which might play a role in other contexts.
d(L)/dt = k_{f} * L * R + k_{r} * R_{L}
d(C_{i})/dt = k_{i} * R_{L} * C_{i}
d(R_{i})/dt = k_{i} * R_{L} * C_{i}
i = 1, 2, 3, 4
Discussion
Our analysis addresses the experimentally observed dependence of endocytosis on EGFconcentration [5, 21]. We propose an ultrasensitive sorting mechanism for EGF receptor internalization which does not require a discriminative receptor modification and give a systematic description of the parameter requirements to achieve proper sorting. We derive analytically the existence of alternative steadystates as well as conditions on the abundance levels of receptors, ligand and endocytic adaptors to reach these states.
Referring to previous models of sigmoidal responses based on cooperativity, for the EGF receptor in particular a cooperative binding effect of the ubiquitinligase Cbl during the ubiquitination reaction has been proposed to be necessary for the observed switcheffect of CIE internalization [44]. Our analysis explains how imposing weaker assumptions on the internalization machinery, namely that the two pathways are entered with distinct affinities, is sufficient to explain the observed switcheffect. Importantly, it is pointed out how by varying the abundance levels of active receptors or endocytic adaptors, cells may modulate their response to incoming EGFstimulations: depending on the initially available receptors or adaptors, the distribution of internalized receptors can be different for one and the same EGF concentration (cf. Figure 3, Figure 8A).
The lack of knowledge about the true parameter/initial values was accounted for by systematic variations over broad numerical ranges. The robustness of the switcheffect to exact parameter values argues for the plausibility of the introduced mechanism.
Mathematical models addressing the problem of receptor sorting into alternative endocytosis pathways do not currently exist. Previously proposed hypotheses based on experimental data only have not been able to give a satisfying answer to this question [9, 12, 18–21, 58]. Generally, the problem is considered at the 'singlemoleculelevel' : a single receptor is envisioned, which is thought to enter either the CDE or the CIEroute (see Figure 1). This picture misleadingly implies the necessity of a discriminative receptor modification.
Instead of thinking in terms of individual entities, we propose to consider the dynamical properties of a system of interacting molecule populations. Applying methods from the theory of dynamical systems, we were able to conceive that an increase in the ligand concentration above the capacity of the CDEpathway qualitatively alters the system behaviour by enforcing an alternative steadystate (Fig. 3, Table 1). Our model states that an abrupt, switchlike start of CIE occurs if the extracellular EGF concentration exceeds the capacity of the CDE machinery. This proposes an interesting implication of the regulation of receptor sorting: the cell achieves the switcheffect 'for free' since no extra cost has to be invested into a discriminative receptor modification. It can be assumed that cells have evolved to optimize energy efficiency [59]. Utilizing the kind of dynamics introduced here, where just one form of receptor is consumed by both pathways, could thus constitute an evolutionary advantage.
A second major observation we draw from the model is that the described mechanism provides a means for an individual cell to sense its surrounding medium: clathrinindependent endocytosis is switched on precisely when the extracellular ligand concentration exceeds the number of CDEadaptors. One might interpret this mechanism as a protein module, i.e. a small interaction network acting as a computational element, whose purpose is to store and process information [51, 60, 61].
One might argue that the simplicity of the model impairs its abilitiy to uncover unanticipated results. While an initiation of clathrinindependent endocytosis upon saturation of the clathrin pathway might have been proposed without mathematical modeling, the unexpected steepness of this switchingbehaviour as well as its robustness could not have been revealed by intuition alone [9, 18, 19]. Furthermore, we showed that extending the model by allowing a modification of the receptor does not increase the steepness of the response. Thus, we conclude that a modification of receptor is not required to discriminate between the pathways. This does notably not exclude the possibility that a modification of the receptor might have been chosen by nature to ensure proper endocytic sorting.
Finally, we want to emphasize that the generality of our model makes it applicable to ultrasensitivity in signaling processes other than the here discussed problem of receptor sorting. This is highlighted in the potential of the model to be extended to more than two overlapping signaling pathways (cf. Figure 9)
Conclusion
We describe the dynamical consequences of an interaction motif, whose molecular basis has already been well established experimentally and discuss its applicability to endocytic sorting of the EGF receptor. We give a simple, yet mathematically sound explanation how cell variation of endocytic sorting results from modulating abundance levels of involved cellular molecules. In the light of the difficulty to experimentally identify a discriminative receptor modification, our analysis points to the possibility of a systemslevel regulation of endocytic sorting. The natural extensibility of the model to more than two cases may prove applicable in other signaling contexts.
Methods
Numerical Simulations and SteadyState Analysis
To numerically solve the systems of equations (1.1 – 1.7) we used the MATLAB ODE15s function. The existence of distinct classes of steadystates was derived as follows: The system of ODEs exhibits four independent equations, namely equations (1.1), (1.3), (1.4) and (1.5). To obtain the values of the variables that represent a steadystate, we simultaneously set these equations equal to zero and solved for the variables.
Setting equations (1.4) and (1.5) equal to zero yields
k_{cde} * R_EGF *CDE = 0
k_{cie} * R_EGF *CDE = 0
Since all kinetic constants are assumed to be positive, one derives that either R_EGF* or both CDE* and CIE* have to be zero. Consider the case that R_EGF* = 0. Then, from setting equations (1.1) and (1.3) equal to zero, i.e.
which also leaves equation (1.1) equal to zero. This second set of values represents steadystate class II (see Results).
Approximability by Hillcurve
Here, V denotes reaction velocity, V_{max} the maximal velocity, x the substrate concentration, K_{m} the substrate concentration where halfmaximal velocity is reached and h, the Hillcoefficient, represents the steepness of the response.
In order to investigate the sensitivity of the switcheffect of CIEinternalization on specific parameter values, we adapted a procedure applied in [26]. We systematically varied reaction parameters and initial values of the model (equations 1.1 – 1.7) using equidistant sampling points. For each thus obtained set we computed the stimulusresponse curve and extracted the Hillcoefficient as $\text{h}=\frac{\mathrm{log}81}{\mathrm{log}({\text{x}}_{0.9})\mathrm{log}({\text{x}}_{0.1})}$[25]. Here, x_{0.1} and x_{0.9} are the EGFconcentrations where 10% or 90% of the maximal responseis reached, espectively.
To evaluate the approximability of the stimulusresponse curve by the Hillequation, we computed the mean deviation between the obtained stimulusresponse curves and a reference Hillcurve. In this way we formally determined a broad range of parameter values for which an ultrasensitive response of CIEinternalization occurs (Fig. 5 and Fig. 6).
List of Abbreviations
 The following abbreviations are used:

CDE – Clathrin dependent endocytosis, CIE – Clathrin independent endocytosis, EGF – Epidermal Growth Factor, EGFR – Epidermal Growth Factor Receptor, monoUb – monoubiquitination
Declarations
Acknowledgements
We would like to thank Frauke Melchior, Angela Stevens and Angel Alonso for helpful comments on the manuscript. We further acknowledge fruitful discussions on modelling aspects with Ivano Primi, Anna Marciniak, Sven Mesecke and Hauke Busch. This project was supported by the SMP Bioinformatics within the National Genome Research Network (NGFN) funded by the Federal Ministry of Education and Research (BMBF) and Endocyte (EU Foerderung).
Authors’ Affiliations
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