Research
Basics of PDGF and PDGF receptor signaling
PDGF receptor signaling: kinetics, mechanisms, and crosstalk
Spatial gradient sensing in directed fibroblast migration
Signaling in the immune system: IL-2/IL-4 receptor signaling in T cells
Signal transduction reactions in cell membranes
Regulation of protein tyrosine phosphatases (PTPs)
Model of human growth hormone-stimulated cell proliferation
Compartmentalized signaling by internalized receptors
Basics of PDGF and PDGF receptor signaling
First, a little historical context. The observation that fibroblasts proliferate robustly in the presence of serum (fluid derived from blood after clotting), but not plasma (fluid derived from blood without clotting), led to the discovery that a specific growth factor is secreted by activated platelets. The aptly named platelet-derived growth factor (PDGF) was later found to exhibit broad specificity, targeting not only fibroblasts but also smooth muscle cells and other connective tissue cells; it was also shown to be synthesized by numerous cell types in addition to platelets. Beyond proliferation signaling, PDGF is a strong chemoattractant for dermal fibroblasts, directing migration to sites of platelet-mediated clotting during wound healing, and it can prolong survival of cells under stress. As in most cases, there are actually multiple forms of PDGF (five have been characterized so far).
As with the vast majority of compounds that elicit cell responses, PDGF exerts its effects by engaging a specific receptor protein on the cell surface (actually, there are two structurally related PDGF receptors). Only cells that express PDGF receptors can respond to PDGF (the ligand), and each such cell would have thousands of receptor copies, allowing the magnitude of a response to vary with the extracellular ligand concentration (the dose-response relationship).
PDGF receptors belong to the well-studied class of signal transducers collectively known as receptor tyrosine kinases, which also include receptors for epidermal, insulin-like, fibroblast, and nerve growth factors. Ligand-induced dimerization of PDGF receptors potentiates their intrinsic enzymatic activity, which catalyzes the phosphorylation of specific tyrosine residues in certain proteins; this is how the signal is transduced inside the cell. The dimerized receptors themselves are actually the first proteins to be phosphorylated. The phosphotyrosine-decorated receptors then serve as scaffolds to which certain intracellular signaling proteins reversibly and specifically attach. This recruitment, mediated by modular domains in those signaling proteins, can foster signal transduction in a multitude of ways: 1) direct activation of an enzymatic activity through an allosteric mechanism (conformational change in the protein); 2) facilitating, by induced proximity, the phosphorylation of the protein by the receptor; and 3) localization at the cell membrane, offering enhanced access to membrane-associated substrates and/or other binding partners.
We have held a long-time fascination with, and focused much of our efforts on, the latter mechanism. While it is no secret that protein localization is huge in signal transduction, recruitment to membranes introduces complexity in the mathematical modeling of intracellular signaling, which must be approached as a combination of solution as well as surface chemistries.
Of the signaling pathways activated by PDGF and other growth factors, the Ras-Erk (extracellular signal-regulated kinase) cascade is arguably the best studied, with its strong connection to fibroblast proliferation. The phospholipase C (PLC) pathway is also activated by PDGF receptors and plays a role in cell migration and chemotaxis. However, of particular importance in PDGF signaling of fibroblasts is the phosphoinositide (PI) 3-kinase pathway, given its particularly strong activation by the PDGF receptor and requisite roles in multiple functional responses. PI 3-kinases are composed of regulatory and catalytic subunits and phosphorylate phosphatidylinositol lipids on the D-3 position, producing specific lipid second messengers in cell membranes that recruit and facilitate activation of other signaling enzymes. One such enzyme is the serine-threonine kinase Akt, which controls multiple cell survival pathways.
Specificity of the three best characterized PDGF ligands (AA, AB, and BB) for dimerization and activation of the two PDGF receptors (alpha and beta).
Recruitment of signaling proteins to specific receptor phosphorylation sites (tyrosine residue numbers are for the human PDGF beta-receptor). TK=tyrosine kinase. Protein sizes not to scale.
Partial interaction map of PDGF receptor-mediated intracellular signaling. Broken arrows signify multiple steps.
PDGF receptor signaling: kinetics, mechanisms, and crosstalk
Analysis of kinetic data is a well-established method for quantitatively assessing the mechanisms and relative rates of molecular processes, yet this approach is not routinely used in cell biological research. It requires the collection of quantitative data sets and the ability to model the underlying processes in mathematical terms. Hence, our first goal was to analyze the early, pre-steady state activation kinetics of PDGF receptor and of Akt, a strictly PI 3-kinase-dependent kinase that promotes cell survival and proliferation. Quantitative, high-throughput biochemical assays were developed for these measurements, and the trends were analyzed for general quantitative features as well as the ability of various kinetic models to fit these data.
The results of our kinetic analyses were as follows. We found that PDGF receptor phosphorylation exhibits positive cooperativity with respect to PDGF concentration, consistent with a mechanism in which receptor dimerization is initially mediated by association of two 1:1 PDGF/PDGF receptor complexes. Receptor phosphorylation is transient at high PDGF concentrations, which we attributed to receptor endocytosis. By comparison, Akt activation responds to lower PDGF concentrations, and with more sustained kinetics. Further analysis indicated that the pathway is saturated at the level of PI 3-kinase, i.e., a fraction of the maximum receptor activation level is sufficient to recruit all of the PI 3-kinase from the cytosol, with Akt activation closely following the level of 3' phosphoinositides. Mathematical modeling, when closely integrated with quantitative cell measurements, can be used to evaluate molecular mechanisms in signaling and their sensitivities to input(s). In this case, these sensitivities were found to impact the kinetics as well as the magnitude of PI 3-kinase signaling.
One of the current challenges in engineering cell function is the analysis of crosstalk interactions among signaling pathways, which imply the existence of integrated signaling networks. A notable example is the regulatory switch governing proliferation and programmed cell death, arguably the most widely studied signaling system in cell physiology. Not surprisingly, mitogens such as PDGF trigger proliferation, through the Ras/Erk pathway, as well as protection from cell death, through the phosphoinositide 3-kinase (PI3K)/Akt pathway. The well-documented positive and negative crosstalk interactions between them suggest that cell life and death are co-regulated.
Building on the kinetic analysis described above, we are characterizing multiple points of branching and convergence in this network through genetic and pharmacological perturbations, beginning with the direct interaction between Ras-GTP and PI3K downstream of PDGF receptors. Receptors and Ras-GTP are stably membrane-associated and engage distinct subunits of PI3K, suggesting that these molecules might cooperate to form a stable, ternary complex at the plasma membrane. Indeed, through expression of dominant-negative or constitutively active Ras and analysis of a simple equilibrium-binding model, we have shown that modulation of Ras-GTP levels affects PDGF-stimulated PI3K/Akt activation in a manner that is uniquely consistent with cooperative assembly of receptor/PI3K/Ras complexes.
Kaur, H.*, Park, C.S.*, Lewis, J.M., and Haugh, J.M. (2006). (*co-first authors).
Quantitative model of Ras/phosphoinositide 3-kinase signalling cross-talk
based on co-operative molecular assembly.
Biochemical Journal, 393: 235-243. (doi:10.1042/BJ20051022)Park, C.S., Schneider, I.C., and Haugh, J.M. (2003).
Kinetic analysis of platelet-derived growth factor receptor/phosphoinositide 3-kinase/Akt
signaling in fibroblasts.
Journal of Biological Chemistry, 278: 37064-37072. (link)
Top panels: PDGF receptor phosphorylation (left; quantitative ELISA) and activation of Akt (right; in vitro kinase assay) were measured as a function of time and PDGF concentration. The solid curves are from a best global fit to a mechanistic model of the pathway. The dose responses of receptor phosphorylation and Akt activation were confirmed by quantitative immunoblotting (not shown). Bottom panels: Mechanistic models of receptor, PI 3-kinase, and Akt activation steps. Our approach from here is to "whittle" such models down, through appropriate kinetic assumptions, as prescribed by the number of features in the data set. We are thus assured that the model output will be sensitive to the values of all parameters, and the lumped parameters can always be related back to elementary rate steps. Adapted from Park et al., 2003.
Top: Receptor signaling rarely proceeds in a linear, sequential fashion. Each receptor typically activates multiple pathways (pleiotropy), and these pathways often interact with each other downstream of the receptor (crosstalk). Other sources of complexity include cooperation of distinct signaling pathways in determining functional outcomes and the integration of multiple extracellular inputs. Bottom panels: It's not just the topology of the network that matters; one must also consider the specific mechanisms. In our equilibrium-binding model of Ras/PI 3-kinase crosstalk, cytosolic PI 3-kinase (P) is recruited to the plasma membrane by activated receptors R* or Ras-GTP S*. These interactions produce RP and PS complexes, with equilibrium dissociation constants KD,R and KD,S, respectively. The ternary RPS complex may then form, and it is postulated that the second binding step is greatly enhanced by induced proximity at the plasma membrane (χ >> 1). Alternatively, the two interactions may exhibit no cooperativity (χ = 1), negative cooperativity (0 < χ < 1), or purely competitive binding (χ = 0). Such simple models are ideal for interpretation of experimental results. Indeed, we found that Ras apparently enhances the affinity of PI 3-kinase for receptors in PDGF-stimulated fibroblasts; in the context of the model, this suggests that a ternary complex forms, and that the second step is greatly enhanced through membrane localization and possibly allosteric effects. Adapted from Kaur, Park, et al., 2006.
Spatial gradient sensing in directed fibroblast migration
During wound healing, PDGF and other factors released by platelets (and later by macrophages) diffuse into the surrounding tissue, where they stimulate cell responses. Fibroblasts are the "construction workers" in wound repair and closure, and these cells proliferate in response to PDGF stimulation. Importantly, fibroblasts also migrate in a directed fashion towards the wound site by following the gradient of PDGF that forms in the tissue (chemotaxis). Eukaryotic cells sense gradients spatially; that is, the spatial pattern of extracellular cues yields, through activation of receptors, a pattern of intracellular signaling that locally alters cell motility processes. Spatial sensing mechanisms are actively being studied in neutrophils and the slime mold Dictyostelium discoideum, but to our knowledge we are the only group doing quantitative analysis and modeling of fibroblast chemotaxis towards PDGF. The systems are similar in that spatial sensing is apparently controlled by 3' phosphoinositides (PIs), products of the aforementioned PI 3-kinases. Our experimental system involves the real-time imaging of a fluorescent, 3' PI-specific probe in living cells using total internal reflection fluorescence (TIRF) microscopy. In many cases, we directly compare the spatiotemporal fluorescence patterns with reaction-diffusion models, using the actual geometry of each cell's contact area. Thus, we extract useful parameter values for each individual cell (apparent 3' PI production rate, diffusion coefficient and turnover rate constant), and more importantly we are studying how these parameters are regulated spatially in response to various cues.
Our work in this area, together with the kinetic analysis of PDGF receptor/PI 3-kinase activation described above, has culminated in an experimentally validated, mechanistic model of PDGF gradient sensing (a comprehensive version of this model is publicly available through the Virtual Cell modeling software). Compared with the chemotactic responses of Dictyostelium discoideum and neutrophils, PDGF gradient sensing in fibroblasts exhibits less sensitivity in general and a greater dependence on the midpoint concentration of the gradient. Optimal gradient sensing is observed in a relatively narrow range of PDGF concentrations that yield near maximal PI 3-kinase recruitment without saturating PDGF receptor occupancy. Most recently, this model has been integrated in a mathematical description of PDGF-mediated fibroblast invasion; this coarse-grain, population-level model explains how robust fibroblast chemotaxis might be maintained over relatively large length scales, across which the PDGF concentration profile might span several logs, given the characterized sensitivity of PDGF gradient sensing.
Schneider, I.C. and Haugh, J.M. (2006).
Mechanisms of gradient sensing and chemotaxis: conserved pathways, diverse regulation.
Cell Cycle, 5: 1130-1134 (Extra View). (link)Haugh, J.M. and Schneider, I.C. (2006).
Effectiveness factor for spatial gradient sensing in living cells.
Chemical Engineering Science, 61: 5603-5611. (doi:10.1016/j.ces.2006.04.041)Haugh, J.M. (2006).
Deterministic model of dermal wound invasion incorporating receptor-mediated
signal transduction and spatial gradient sensing.
Biophysical Journal, 90: 2297-2308. (doi:10.1529/biophysj.105.077610)Schneider, I.C. and Haugh, J.M. (2005).
Quantitative elucidation of a distinct spatial gradient-sensing mechanism in fibroblasts.
Journal of Cell Biology, 171: 883-892. (doi:10.1083/jcb.200509028)Schneider, I.C., Parrish, E.M., and Haugh, J.M. (2005).
Spatial analysis of 3' phosphoinositide signaling in living fibroblasts, III:
Influence of cell morphology and morphological polarity.
Biophysical Journal, 89: 1420-1430. (doi:10.1529/biophysj.105.061218)Schneider, I.C. and Haugh, J.M. (2004).
Spatial analysis of 3' phosphoinositide signaling in living fibroblasts: II.
Parameter estimates for individual cells from experiments.
Biophysical Journal, 86: 599-608. (link)Haugh, J.M. and Schneider, I.C. (2004).
Spatial analysis of 3' phosphoinositide signaling in living fibroblasts: I.
Uniform stimulation model and bounds on dimensionless groups.
Biophysical Journal, 86: 589-598. (link)Haugh, J.M., Codazzi, F., Teruel, M., and Meyer, T. (2000).
Spatial sensing in fibroblasts mediated by 3' phosphoinositides.
Journal of Cell Biology, 151: 1269-1279. (link)
Live-cell fluorescence imaging and spatial analyses of PI 3-kinase signaling in fibroblasts. Total internal reflection fluorescence (TIRF) microscopy is used to image the contact area "footprint" of living NIH 3T3 fibroblasts transfected with the 3' PI-specific fluorescent probe, GFP-AktPH. a) Representative cell response to uniform PDGF stimulation; a PI 3-kinase inhibitor was added shortly after panel 4. The radial gradient is caused by restriction of PI 3-kinase activation to the nonadherent plasma membrane. b&c) Direct comparison and best fits of a quantitative reaction-diffusion model to individual cell fluorescence tracks (a-c are adapted from Schneider & Haugh, 2004). d) TIRF image of a fluorescent dextran gradient emanating from a micropipette (upper left corner), as a marker for gradients of extracellular ligand. e) TIRF image, as in a, of a cell responding to a PDGF gradient (orientation indicated by the arrow); a finite-element simulation of the reaction-diffusion problem, with realistic cell geometry, is shown for comparison. f) The same cell as in e was then uniformly saturated with PDGF (the geometries of the simulated cells shown in e&f were smoothed significantly; this is not a necessary step in general).
Top panels: A simple, quasi-steady state version of our PDGF gradient sensing model yields three testable predictions: 1) The gradient in PI 3-kinase signaling, Δe, is sensitive to both the relative PDGF gradient, δ, and its midpoint concentration, with the greatest sensitivity at intermediate midpoint concentrations; 2) Given parameter values consistent with the measured dose responses of PDGF receptor and PI 3-kinase activation, the effective PDGF concentration range spans roughly two logs; and 3) Near the optimal concentration of PDGF, PI 3-kinase activation at the front of the cell exceeds the level observed at receptor saturation, and so the subsequent addition of a high PDGF dose will force the 3' PI level at the front to decrease. Bottom panels: Experiments using fluorescent AktPH-transfected mouse fibroblasts and TIRF microscopy are in qualitative and quantitative agreement with the model predictions. Adapted from Schneider & Haugh, JCB, 2005.
Signaling in the immune system: IL-2/IL-4 receptor signaling in T cells
We have begun to explore molecular aspects of signaling in T lymphocytes, agents of the immune system that orchestrate the defense against infectious disease. Aside from their obvious importance in human health, the highly specialized and tightly controlled activities of T cells offer a unique test case for the fundamental understanding of cell regulation. Three aspects of T cell signaling are currently being investigated: 1) crosstalk in the signaling network stimulated by the receptor for interleukin (IL)-2, a soluble cytokine that explicitly stimulates proliferation of T cells as the immune system mounts its defense against a specific pathogen; 2) cooperation of these interconnected pathways in triggering T cell proliferation and survival; and 3) interactions between the signaling networks of IL-2 and IL-4 receptors. IL-4 elicits differentiation and expansion of a specialized subset of T cells, and IL-4 synergizes with IL-2 in stimulating the proliferation and/or survival of this population.
These cytokine receptors are relatively complex, in that they are composed of distinct subunits and must activate separate, nonreceptor tyrosine kinases in order to be phosphorylated (see below), but thereafter the signal transduction is very similar to that of receptor tyrosine kinases. We are studying the roles of their shared and unique signaling pathways in bringing about the synergy in cell responsiveness.
Signal transduction reactions in cell membranes
As outlined in the sections above, phosphorylated receptors recruit and activate intracellular enzymes in the first steps of signal transduction. The fact that nearly all of these enzymes act upon membrane-associated molecules such as lipids or lipid-tethered proteins (as seen in each of the phospholipase C, PI 3-kinase, and Ras pathways) suggests a general theme in intracellular signaling. It has long been speculated that the relatively slow diffusion of membrane constituents could give rise to rates of reaction that are limited by the frequency of intermolecular collisions, i.e., diffusion-limited kinetics. In such cases, nanometer-scale gradients of active signaling components would transiently arise in the proximity of a receptor-recruited enzyme; significantly, such gradients currently cannot be detected through direct measurements.
We and others have therefore examined this problem theoretically, following a rich tradition of reaction-diffusion problems in planar geometries. Obviously, our focus has uniquely centered on intracellular signaling (in particular the specific pathways mentioned above), with consideration of the mobility, consumption, and in some cases the regulated insertion of membrane substrates as well as the dynamics of receptor-mediated enzyme recruitment.
Most recently, we have developed Brownian dynamics algorithms for simulating potentially complex interactions/reactions in cell membranes. For relatively simple systems, we have confirmed that simulation results are in excellent agreement with the continuum theory.
Monine, M.I. and Haugh, J.M. (2005).
Reactions on cell membranes: Comparison of continuum theory and Brownian dynamics simulations.
Journal of Chemical Physics, 123: 074908 (6 pages). (doi:10.1063/1.2000236)Haugh, J.M. (2002).
A unified model for signal transduction reactions in cellular membranes.
Biophysical Journal, 82: 591-604. (link)Haugh, J.M., Wells, A., and Lauffenburger, D.A. (2000).
Mathematical modeling of epidermal growth factor receptor signaling through the phospholipase C pathway: mechanistic insights and predictions for molecular interventions.
Biotechnology & Bioengineering, 70: 225-238. (link)Haugh, J.M. and Lauffenburger, D.A. (1997).
Physical modulation of intracellular signaling processes by locational regulation.
Biophysical Journal, 72: 2014-2031. (link)
Regulation of protein tyrosine phosphatases (PTPs)
Signaling pathways are generally modulated by kinase-mediated phosphorylation, yet equally important in determining the magnitude and kinetics of such a response is protein dephosphorylation, catalyzed by phosphatase enzymes. A growing body of evidence indicates that certain protein tyrosine phosphatases (PTPs), like tyrosine kinases, are affected by intermolecular interactions that alter the specific activity or localization of their catalytic domains. Using a detailed kinetic modeling framework, we have begun to theoretically explore the regulation of PTPs through their association with receptor tyrosine kinases, as noted for the Src homology 2-domain-containing PTPs, Shp-1 and -2. Receptor-PTP binding, in turn, is expected to influence the phosphorylation pattern of those receptors and proteins they associate with, and in our initial efforts we have shown how PTPs might serve to co- or counter-regulate parallel pathways in a signaling network.
In collaboration with Jim Faeder and the Cell Signaling Group at Los Alamos National Laboratory, we have recently built a rule-based computational model of Shp2-receptor interaction and Shp2 regulation that incorporates the modular domain structure of the Shp2 protein. The two SH2 domains of Shp2 differentially regulate the enzymatic activity by a well-characterized mechanism, but they also affect the targeting of Shp2 to signaling receptors in cells. Our kinetic model integrates these potentially competing effects by considering the intra- and intermolecular interactions of the Shp2 SH2 domains and catalytic site as well as the effect of Shp2 phosphorylation. Even for the isolated Shp2/receptor system, which may seem simple by certain standards, we find that the network of possible binding and phosphorylation states is comprised of over one thousand members. To our knowledge, this is the first kinetic model to fully consider the modular, multifunctional structure of a signaling protein, and the computational approach should be generally applicable to other complex intermolecular interactions.
Barua, D., Faeder, J.R., and Haugh, J.M. (2007).
Structure-based kinetic models of modular signaling protein function: focus on Shp2.
Biophysical Journal, 92: 2290-2300. (doi:10.1529/biophysj.106.093484)Haugh, J.M., Schneider, I.C., and Lewis, J.M. (2004).
On the cross-regulation of protein tyrosine phosphatases and receptor tyrosine kinases
in intracellular signaling.
Journal of Theoretical Biology, 230: 119-132. (doi:10.1016/j.jtbi.2004.04.023)
Model of human growth hormone-stimulated cell proliferation
Human growth hormone (hGH) is a classical pituitary hormone that controls numerous physiological processes, most notably skeletal growth. Structurally and functionally, the interactions of hGH with its receptor have been resolved in fine detail, such that hGH and hGH receptor variants can be practically engineered by either random or rational approaches to achieve significant changes in the free energies of binding. A somewhat unique feature of hGH action is its homodimerization of two hGH receptors, which is required for intracellular signaling and stimulation of cell proliferation. It is well known that such an activation mechanism naturally gives rise to a bell-shaped dose response curve, as higher ligand concentrations progressively decrease the number of unoccupied receptors available for dimerization.
Given the extensive experimental work on this system, it has been used as a model example of receptor dynamics in Prof. Haugh's specialty course, Molecular Cell Engineering. As a direct outcome of in-class discussions, it became clear that certain observations - namely the efficacies of hGH receptor agonists and antagonists in cell-based assays - could not be adequately explained by kinetic models that only considered receptor-ligand interactions. It was subsequently found that a model considering hGH receptor internalization, which imposes a limit on the lifetime of an active receptor complex at the cell surface, is uniformly consistent with the numerous published observations regarding hGH receptor agonism and antagonism.
Haugh, J.M. (2004).
A mathematical model of human growth hormone (hGH)-stimulated cell proliferation explains
the efficacy of hGH variants as receptor agonists or antagonists.
Biotechnology Progress, 20: 1337-1344. (doi:10.1021/bp0499101)
Compartmentalized signaling by internalized receptors
For his graduate thesis, Prof. Haugh studied the effects of epidermal growth factor (EGF) receptor internalization on the magnitude and specificity of intracellular signaling. The Lauffenburger lab had previously analyzed, along with Steven Wiley's group (then at Univ. of Utah), the dyamics of EGF receptor trafficking and the resulting effects on cell proliferation, assuming a phenomenological relationship between receptor occupancy and response. The subsequent analyses of the underlying signaling pathways were undertaken in collaboration with Alan Wells and his group (then at UAB), who had previously elucidated the role of the phospholipase C pathway in EGF-stimulated cell motility. This topic is currently not a major focus of our laboratory, but the interested reader is referred to the following publications.
Haugh, J.M. (2002).
Localization of receptor-mediated signal transduction pathways: the inside story.
Molecular Interventions, 2: 292-307 (Review). (link)Haugh, J.M. and Meyer, T. (2002).
Active EGF receptors have limited access to PI(4,5)P2 in endosomes:
implications for phospholipase C and PI 3-kinase signaling.
Journal of Cell Science, 115: 303-310. (link)Haugh, J.M., Huang, A.C., Wiley, H.S., Wells, A., and Lauffenburger, D.A. (1999).
Internalized epidermal growth factor receptors participate in the activation of p21(ras) in fibroblasts.
Journal of Biological Chemistry, 274: 34350-34360. (link)Haugh, J.M., Schooler, K., Wells, A., Wiley, H.S., and Lauffenburger, D.A. (1999).
Effect of epidermal growth factor receptor internalization on regulation of the
phospholipase C-γ1 signaling pathway.
Journal of Biological Chemistry, 274: 8958-8965. (link)Haugh, J.M. and Lauffenburger, D.A. (1998).
Analysis of receptor internalization as a mechanism for modulating signal transduction.
Journal of Theoretical Biology, 195: 187-218. (doi:10.1006/jtbi.1998.0791)Lauffenburger, D.A., Fallon, E.F. and Haugh, J.M. (1998).
Scratching the (cell) surface: cytokine engineering for improved ligand/receptor trafficking dynamics.
Chemistry & Biology, 5: R257-R263 (Review). (doi:10.1016/S1074-5521(98)90110-7)
Cartoon of compartmentalized receptor signaling, most resembling the epidermal growth factor (EGF) receptor system. Hydrolysis and phosphorylation of phosphatidylinositol (4,5)-bisphosphate (PIP2) by phospholipase C (PLC) and phosphoinositide 3-kinase (PI3-K), respectively, are restricted to the plasma membrane. Internalized receptors, insofar as they remain ligated, retain the ability to recruit those enzymes, but PIP2 is not accessible to them in endosomal membranes. On the other hand, surface and internalized receptor-ligand complexes contribute equally to the production of Ras-GTP. Adapted from the Molecular Interventions review.
