System theoretical investigation of human epidermal growth factor receptor-mediated signalling.

The partitioning of biological networks into coupled-functional modules is being increasingly applied for developing predictive models of biological systems. This approach has the advantage that predicting a system-level response does not require a mechanistic description of the internal dynamics of each module. Identification of the input-output characteristics of the network modules and the connectivity between the modules provide the necessary quantitative representation of system dynamics. However, the determination of the input-output relationships of the modules is not trivial; it requires the controlled perturbation of module inputs and systematic analysis of experimental data. In this report, the authors apply a system theoretical analysis approach to derive the time-dependent input-output relationships of the functional module for the human epidermal growth factor receptor (HER) mediated Erk and Akt signalling pathways. Using a library of cell lines expressing endogenous levels of epidermal growth factor receptor (EGFR) and varying levels of HER2, the authors show that a transfer function-based representation can be successfully applied to quantitatively characterise information transfer in this system.

A model of cytokine shedding induced by low doses of gamma radiation.

A model for sustained shedding of epidermal growth factor (EGF) in response to low doses of gamma radiation was developed based on a time delay in the feedback from mitogen-activated protein kinase (MAPK) activation to metalloprotease activity in an autocrine signaling process. We determined the kinetic parameters of our model using the data available for MAPK activation by gamma irradiation in the 1-2-Gy dose range and then showed that predictions of the model were consistent with experimental results for the kinetics of EGF shedding into the growth medium after exposure of human mammary epithelial cells to 1-5 cGy of gamma radiation in the presence of antibodies that block ligand binding to EGF receptors. The model allowed us to estimate the rate of radiation-induced cytokine release per cell from measurements of EGF concentration in the growth medium and to assess the effectiveness of EGF shedding and subsequent diffusion through the medium as a mechanism for signal transmission between hit cells and bystanders.

Metalloprotease-mediated ligand release regulates autocrine signaling through the epidermal growth factor receptor.

Ligands that activate the epidermal growth factor receptor (EGFR) are synthesized as membrane-anchored precursors that appear to be proteolytically released by members of the ADAM family of metalloproteases. Because membrane-anchored EGFR ligands are thought to be biologically active, the role of ligand release in the regulation of EGFR signaling is unclear. To investigate this question, we used metalloprotease inhibitors to block EGFR ligand release from human mammary epithelial cells. These cells express both transforming growth factor alpha and amphiregulin and require autocrine signaling through the EGFR for proliferation and migration. We found that metalloprotease inhibitors reduced cell proliferation in direct proportion to their effect on transforming growth factor alpha release. Metalloprotease inhibitors also reduced growth of EGF-responsive tumorigenic cell lines and were synergistic with the inhibitory effects of antagonistic EGFR antibodies. Blocking release of EGFR ligands also strongly inhibited autocrine activation of the EGFR and reduced both the rate and persistence of cell migration. The effects of metalloprotease inhibitors could be reversed by either adding exogenous EGF or by expressing an artificial gene for EGF that lacked a membrane-anchoring domain. Our results indicate that soluble rather than membrane-anchored forms of the ligands mediate most of the biological effects of EGFR ligands. Metalloprotease inhibitors have shown promise in preventing spread of metastatic disease. Many of their antimetastatic effects could be the result of their ability to inhibit autocrine signaling through the EGFR.

ErbB-2 amplification inhibits down-regulation and induces constitutive activation of both ErbB-2 and epidermal growth factor receptors.

ErbB-2/HER2 is an important signaling partner for the epidermal growth factor receptor (EGFR). Overexpression of erbB-2 is also associated with poor prognosis in breast cancer. To investigate how erbB-2 amplification affects its interactions with the EGFR, we used a human mammary epithelial cell system in which erbB-2 expression was increased 7-20-fold by gene transfection. We found that amplification of erbB-2 caused constitutive activation of erbB-2 as well as ligand-independent activation of the EGFR. Overexpression of erbB-2 strongly inhibited erbB-2 down-regulation following transactivation by EGFR. Significantly, down-regulation of activated EGFR was also inhibited by erbB-2 amplification, resulting in enhanced ligand-dependent activation of the EGFR. The rate of EGFR endocytosis was not affected by erbB-2 overexpression, but the rate of lysosomal targeting was significantly reduced. In addition, erbB-2 overexpression promoted rapid recycling of activated EGFR back to the cell surface and decreased ligand dissociation from the EGFR. Our data suggest that overexpression of erbB-2 inhibits both its down-regulation and that of the EGFR. The net effect is increased signaling through the EGFR system.

Quantitative assessment of autocrine cell loops.

Regeneration of functioning tissue essentially involves recapitulating relevant aspects of organogenesis, so that the starting composite of cells, matrix, and molecular factors develops into the desired structure and physiology. A crucial aspect of development is local cell-cell communication; that is, molecular regulatory factors are more typically paracrine and autocrine than endocrine in nature. Autocrine loops were originally thought of predominantly as being involved in pathological behavior, but it is becoming increasingly clear that a large portion of normal physiological behavior-and a tremendous portion of development-is strongly regulated by autocrine factors (1). Thus, continuing progress of the field of tissue engineering will require increased understanding of how autocrine loops operate, so that they can be designed or manipulated systematically. We have made an effort in this direction, and some early experimental and modeling results can be found in the literature (2-5). In this chapter, we describe the methods we have used for creating autocrine cell loops and quantitatively assessing their operation.

Differential signaling and regulation of apical vs. basolateral EGFR in polarized epithelial cells.

Overexpression of the epidermal growth factor receptors (EGFR) in polarized kidney epithelial cells caused them to appear in high numbers at both the basolateral and apical cell surfaces. We utilized these cells to look for differences in the regulation and signaling of apical vs. basolateral EGFR. Apical and basolateral EGFR were biologically active and mediated EGF-induced cell proliferation to similar degrees. Receptor downregulation and endocytosis were less efficient at the apical surface, resulting in prolonged EGF-induced tyrosine kinase activity at the apical cell membrane. Tyrosine phosphorylation of EGFR substrates known to mediate cell proliferation, Src-homologous and collagen protein (SHC), extracellularly regulated kinase 1 (ERK1), and ERK2 could be induced similarly by activation of apical or basolateral EGFR. Focal adhesion kinase was tyrosine phosphorylated more by basolateral than by apical EGFR; however, beta-catenin was tyrosine phosphorylated to a much greater degree following the activation of mislocalized apical EGFR. Thus EGFR regulation and EGFR-mediated phosphorylation of certain substrates differ at the apical and basolateral cell membrane domains. This suggests that EGFR mislocalization could result in abnormal signal transduction and aberrant cell behavior.

Removal of the membrane-anchoring domain of epidermal growth factor leads to intracrine signaling and disruption of mammary epithelial cell organization.

Autocrine EGF-receptor (EGFR) ligands are normally made as membrane-anchored precursors that are proteolytically processed to yield mature, soluble peptides. To explore the function of the membrane-anchoring domain of EGF, we expressed artificial EGF genes either with or without this structure in human mammary epithelial cells (HMEC). These cells require activation of the EGFR for cell proliferation. We found that HMEC expressing high levels of membrane- anchored EGF grew at a maximal rate that was not increased by exogenous EGF, but could be inhibited by anti-EGFR antibodies. In contrast, when cells expressed EGF lacking the membrane-anchoring domain (sEGF), their proliferation rate, growth at clonal densities, and receptor substrate phosphorylation were not affected by anti-EGFR antibodies. The sEGF was found to be colocalized with the EGFR within small cytoplasmic vesicles. It thus appears that removal of the membrane-anchoring domain converts autocrine to intracrine signaling. Significantly, sEGF inhibited the organization of HMEC on Matrigel, suggesting that spatial restriction of EGF access to its receptor is necessary for organization. Our results indicate that an important role of the membrane-anchoring domain of EGFR ligands is to restrict the cellular compartments in which the receptor is activated.

Endocytosis and lysosomal targeting of epidermal growth factor receptors are mediated by distinct sequences independent of the tyrosine kinase domain.

Ligand-induced internalization of the epidermal growth factor receptor (EGFR) leads to accelerated receptor degradation. Two models have been proposed to explain this. In the first model, induced internalization expands the intracellular pool of receptors, leading to enhanced lysosomal targeting. The second model proposes that activation of intrinsic receptor kinase activity induces inward vesiculation of endosomes, thus interrupting receptor recycling. To test these models, we created EGFR mutants that lack the conserved tyrosine kinase domain, but retain different parts of the distal carboxyl terminus regulatory region. Mutants lacking all distal regulatory sequences underwent slow internalization (0.02 min-1) and turnover (t1/2 approximately 24 h), similar to unoccupied, holo-EGFR. Mutant receptors that lacked the kinase domain, but retained the entire distal regulatory domain, were constitutively internalized and targeted to lysosomes, even in the absence of EGF. The turnover of these receptors (t1/2 approximately 11 h) was similar to that of occupied, kinase-active holo-EGFR (t1/2 approximately 9.5 h). These results show that receptor tyrosine kinase activity is not required for the targeting of EGFR to lysosomes. Receptor mutants which expressed previously identified endocytic sequences underwent rapid internalization. Unexpectedly, enhanced turnover of EGFR mutants required additional sequences located between residues 945 and 991 in the holo-EGFR. Thus, internalization and lysosomal targeting of EGFR are separate processes mediated by distinct sequences. Our results indicate that induced internalization is necessary, but not sufficient, for enhanced EGFR degradation. Instead, down-regulation requires exposure of previously cryptic internalization and lysosomal targeting sequences. Occupied EGFR thus appear to be handled by the endocytic machinery in the same fashion as other constitutively internalized or lysosomally targeted receptors.

Regulation of postendocytic trafficking of the epidermal growth factor receptor through endosomal retention.

Little is known about the regulation of EGF receptor (EGF-R) trafficking following endocytosis. We investigated this by using a series of EGF-R with altered cytoplasmic tails and comparing their ability to undergo recycling and lysosomal targeting in both the occupied and empty state. We found that 2-3% of empty EGF-R are internalized each minute, but rapidly recycle (t1/2 approximately 5 min). This constitutive internalization and recycling of empty receptors was independent of cytoplasmic receptor sequences. Occupied EGF-R, in contrast, displayed a much slower rate of recycling (t1/2 between 10-23 min) due to retention within recycling endosomes. Endosomal retention of different EGF-R correlated with lysosomal targeting of EGF. Intrinsic receptor tyrosine kinase activity had no discernible effect on postendocytic trafficking of EGF. Although sequences within the cytoplasmic tail of the EGF-R appear to be required for occupancy-dependent endosomal retention, they are distinct from those required for ligand-induced endocytosis. Our studies indicate that intracellular trafficking of the EGF-R is regulated by endosomal components that preferentially recognize occupied receptors. Down-regulation of the EGF-R thus involves two distinct regulatory processes: one at the level of internalization and one at the level of recycling.

Highly polarized EGF receptor tyrosine kinase activity initiates egg activation in Xenopus.

Progesterone-matured Xenopus oocytes are arrested at second metaphase but resume meiosis following fertilization. To explore the role of tyrosine kinase activity and phosphatidylinositol turnover in this activation process, we caused oocytes to express three types of human epidermal growth factor receptor (EGF-R), which differ in their ability to stimulate these biochemical processes. Following mRNA injection we found that receptor expression was highly polarized, with most receptors located on the animal hemisphere. Occupancy of the wild-type EGF-R in progesterone-matured oocytes resulted in full egg activation as indicated by an activation potential, increased intracellular-free Ca2+ ([Ca2+]i), fertilization envelope liftoff, and cortical contraction. Fura-2 imaging showed that the wave of EGF-mediated Ca2+ release started in the animal hemisphere and progressed completely around the cell. These responses required receptor tyrosine kinase activity. Matured oocytes expressing the c'973 EGF-R, which possesses kinase activity but only weakly stimulates phosphatidylinositol turnover, responded differently to EGF addition. Cortical contraction and fertilization envelope liftoff appeared normal, but there was no activation potential. Significantly, [Ca2+]i was only slightly elevated and was topologically restricted to the regions expressing receptors. Our results suggest that some aspects of egg activation can occur through a tyrosine kinase pathway. However, phosphatidylinositol hydrolysis appears necessary for both amplification and propagation of signals generated locally by activated EGF-R.

Functional reconstitutional of the human epidermal growth factor receptor system in Xenopus oocytes.

We have expressed the human EGF receptor (hEGF-R) in Xenopus oocytes by injecting mRNA synthesized in vitro using SP6 vectors containing receptor cDNAs. Each oocyte could express over 1 x 10(10) receptors of a single affinity class and these were able to bind and rapidly internalize EGF. Occupancy resulted in receptor tyrosine autophosphorylation, downregulation, and release of intracellular calcium. Occupied receptors also rapidly induced meiotic maturation in stage VI oocytes. Receptors lacking tyrosine kinase activity bound EGF normally, but did not downregulate or induce any biological responses. The rate of oocyte maturation was proportional to hEGF-R occupancy and was significantly faster than progesterone-induced maturation at nanomolar EGF concentrations. Mutant hEGF-R truncated at residue 973 displayed identical phenotypes in both mammalian cells and oocytes in that they were defective in their ability to release intracellular calcium, undergo ligand induced internalization and receptor downregulation. However, these receptors were fully capable of inducing oocyte maturation. The remarkable retention of specific biological activities of different hEGF-R in the context of oocytes suggests that this receptor system interacts with generally available cellular components that have been conserved during evolution. In addition, it suggests that cell surface tyrosine kinase activity may play an important role in regulating resumption of the cell cycle.

Quantitative analysis of the endocytic system involved in hormone-induced receptor internalization.

We have developed a quantitative method to evaluate the interaction between cell surface receptors and the endocytic apparatus. This method exploits occupancy-dependent changes in internalization rates that occur in cells expressing high numbers of receptors. We found that constitutive internalization of the transferrin receptor behaves as a simple, first order process that is unaltered by ligand. Internalization of the epidermal growth factor (EGF) receptor, however, behaves as a saturable, second order process that is induced by receptor occupancy. Internalization of EGF receptors occurs through at least two distinct pathways: a low capacity pathway that has a relatively high affinity for occupied receptors, and a low affinity pathway that has a much higher capacity. The high affinity pathway was observed in all cells having receptors with intrinsic tyrosine kinase activity. Mutant EGF receptors lacking kinase activity could not utilize the high affinity pathway and were internalized only through the low affinity one. Mutated receptors with decreased affinity for kinase substrates were also internalized at decreased rates through the high affinity, inducible pathway. In the case of vitellogenin receptors in Xenopus oocytes, occupied receptors competed more efficiently for internalization than empty ones. Insulin increased the endocytic capacity of oocytes for vitellogenin receptors. Similarly, serum increased the capacity of the inducible pathway for EGF receptors in mammalian cells. These data are consistent with a model of internalization in which occupied receptors bind to specific cellular components that mediate rapid internalization. Ligand-induced internalization results from an increase in the affinity of occupied receptors for the endocytic apparatus. Hormones can also indirectly regulate endocytosis by increasing the number of coated pits or their rate of internalization. The ability to dissect receptor-specific effects from cell-specific ones should be very useful in investigating the molecular mechanisms of receptor mediated endocytosis.

Specific proteolysis regulates fusion between endocytic compartments in Xenopus oocytes.

We examined the role of proteolytic ligand modification in endosomal targeting using vitellogenin (VTG) uptake by Xenopus oocytes as a model system. Non-cleavable VTG is internalized, but does not appear in yolk platelets. We identified two inhibitors of VTG processing into the yolk proteins: the ionophore monensin and pepstatin A, a specific inhibitor of cathepsin D. Pepstatin neither affected ligand binding and internalization, nor inhibited the degradation of nonspecifically incorporated proteins, whereas monensin inhibited all of these processes. Inhibiting VTG processing prevented its deposition into yolk platelets by strongly interfering with endosome-yolk platelet fusion. Monensin treatment resulted in morphologically abnormal endosomes, while pepstatin only inhibited VTG cleavage and the subsequent fusion of endosomes with yolk platelets. Since VTG cleavage is initiated prior to its deposition in platelets, we postulate that ligand proteolysis could be necessary for normal endosomal targeting.

Receptor-mediated endocytosis in Xenopus oocytes. I. Characterization of the vitellogenin receptor system.

We have investigated the vitellogenin (VTG) receptor system in Xenopus oocytes since these cells are specialized for endocytosis. Oocytes have between 0.2 and 3 X 10(11) receptors per 1-mm cell. There is only a single class of receptors of low affinity (1.3 X 10(-6) M at 22 degrees C and 2-4 X 10(-6) M at 0 degree C), but high specificity (less than 5% nonspecific binding at 2 X 10(-6) M). The specific internalization rate of the VTG receptor (around 2 X 10(-3) s-1) is first order, highly variable, and at the upper end of the range of values reported for mammalian cells. The receptor association rate constant (9.6 X 10(2) M-1 s-1) is extremely low although the dissociation rate constant was immeasurable. Calcium is required for VTG binding, and low pH does not dissociate the VTG-receptor complex. Monensin treatment at 100 microM caused the loss of surface receptors with a t1/2 of 3 h and the accumulation of internalized ligand in a "pre-lysosomal" endocytic compartment. Conversely, the recovery of surface VTG receptors that were removed with trypsin occurred with a t1/2 of about 2 h. These observations indicate that oocytes have very large intracellular pools of receptors and that although surface receptors are internalized on the time scale of minutes, the intracellular pool is recycled on the time scale of hours.

Receptor-mediated endocytosis in Xenopus oocytes. II. Evidence for two novel mechanisms of hormonal regulation.

Xenopus oocytes exhibit enhanced rates of vitellogenin (VTG) endocytosis following exposure to insulin in vitro and human chorionic gonadotropin in vivo. We investigated this phenomenon using kinetic and steady state analyses and found that the stimulation of VTG uptake was not due to an increase in surface VTG receptors. Instead, both hormones acted by stimulating the specific internalization rate (ke) of the VTG receptor, although by apparently different mechanisms. The stimulation of ke by insulin was most obvious at low levels of receptor occupancy. Insulin also increased the affinity of the VTG receptor for its ligand. Both hormones increased the rate of fluid phase endocytosis, but the magnitude of stimulation was not sufficient to account for the observed increase in VTG uptake. The steady state binding of VTG was biphasic with respect to increasing ligand concentration, primarily due to a nonlinear receptor internalization rate as a function of occupancy. The nonlinear VTG binding was abolished by incubating the oocytes at 0 degree C. Our data are consistent with a model in which there is a cell surface regulatory component that facilitates the internalization of the occupied VTG receptor. Although both human chorionic gonadotropin and insulin stimulate VTG uptake by increasing the net rate of endocytosis, insulin also increases the association of the occupied VTG receptor with this regulatory component.

An enzymatic method for radiolabeling vertebrate vitellogenin.

Phosphoprotein kinases from Xenopus and chicken liver have been purified and these enzymes have been used to label Xenopus vitellogenin, a phosphoprotein, to high specific activity with [gamma-32P]ATP. The enzymes were isolated by (NH4)2SO4 fractionation followed by chromatography on DE-52 cellulose and phosphocellulose. This procedure resulted in greater than 20,000-fold enrichment for the enzymes. Both enzyme preparations were used to selectively label vitellogenin in the serum of estrogen-treated animals. Thus, isolation of the vitellogenin prior to radiolabeling was not necessary. The [32P]vitellogenin labeled in situ was incorporated by oocytes at a rate similar to [32P]vitellogenin labeled in vivo, was translocated to the yolk platelets, and was correctly processed into the yolk proteins.

The oocyte as an endocytic cell.

Oocytes of Xenopus laevis grow primarily by sequestering vitellogenin (VTG) selectively from the maternal bloodstream. Morphological observations have demonstrated that an endocytic system is responsible for VTG uptake. Binding studies indicate the presence of 2-28 X 10(10) surface VTG receptors per oocyte. These are continuously internalized into endosomes whether or not they are occupied by VTG, and other macromolecules may become trapped in the process. VTG-containing endosomes give rise to dense transitional yolk bodies; these fuse with yolk platelets only after the cleavage of vitellogenin. In the absence of VTG, endosomes appear to fuse directly with yolk platelets. From these observations it is postulated that receptor occupancy can act as a transmembrane signal which directs the postendocytic compartmentation of proteins. Yolk platelet proteins do not undergo subsequent turnover, whereas adventitiously incorporated protein is gradually lost from the oocyte by a dual mechanism which may involve both lysosomal proteolysis and secretion from the oocyte as a consequence of membrane recycling. Although these observations may not apply to all growing oocytes, the X. laevis oocyte nevertheless appears to be a particularly attractive experimental system for studies of endocytic compartmentation and membrane receptor recycling.

Differential postendocytotic compartmentation in Xenopus oocytes is mediated by a specifically bound ligand.

Xenopus laevis oocytes were used as a model system to study the intracellular fate of proteins incorporated by endocytosis. We found that the intracellular stability and compartmentation of proteins incorporated by receptor-mediated endocytosis differed substantially from that of proteins incorporated by nonspecific endocytosis. After its uptake, the specifically sequestered yolk precursor protein vitellogenin was converted to the yolk proteins lipovitellin and phosvitin which were stable with time (up to 13 days in culture). In contrast, nonspecifically incorporated albumin (125I-labeled or 3H-labeled bovine serum albumin) was rapidly degraded. To determine whether the differential stability of these proteins was related to their entry into different postendocytotic compartments, we examined the intracellular transfer pathways taken by these proteins. The transfer of vitellogenin from coated vesicles to the yolk platelets was found to involve a secondary compartment formed by the fusion of the incoming endosomes. This compartment, termed transitional yolk bodies (TYB), underwent a progressive condensation until it attained its terminal density (1.21 g/cm3) after approximately 1.5 hr. The fusion of the TYB with the yolk platelets then occurred coincidentally with the time at which vitellogenin was proteolytically processed into the yolk proteins within the TYB. When the proteolytic cleavage of vitellogenin was blocked there was no fusion of the two compartments. In contrast, we found that albumin incorporated in the absence of vitellogenin was directly transferred from endosomes to yolk platelets without the formation of, or fusion with a secondary compartment. However, when oocytes were exposed simultaneously to albumin and vitellogenin both proteins followed identical routes of compartmentation (that of vitellogenin) with no evidence of direct transfer of either protein to the yolk platelets. These results suggest that the incorporation of a specifically bound ligand can result in the formation of a unique intracellular compartment. Moreover, since yolk platelets were able to fuse only with vesicles lacking occupied receptors (in the case of albumin alone) or with a compartment in which the specific ligand had been proteolytically cleaved and presumably released from its receptor (in the case of VTG), we suggest that occupied receptors can act as a transmembrane signal which directs the postendocytotic compartmentation of proteins.