Lateral and rotational diffusion of EGF-receptor complex: relationship to receptor-mediated endocytosis

The clustering and internalization of epidermal growth factor (EGF)–receptor complexes on human tumor cells are temperature-sensitive processes that are completely inhibited at 4°C. The rotational diffusion of EGF–receptor is limited by the viscosity of the lipid matrix, while additional constraints limit the lateral diffusion of the receptor molecule. Although the lateral and rotational diffusion of the occupied receptors are temperature sensitive, they do not provide a rate-determining factor for the clustering and subsequent internalization of the EGF–receptor. Even at 4°C the occupied receptors move quickly enough to allow the formation of EGF–receptor clusters. The lack of internalization at 4°C must be due to the inhibition of interactions other than a temperature effect on receptor mobility. EGF-induced receptor clustering involves the formation of microclusters composed of 10–50 receptor molecules.     

Direct visualization and quantitative analysis of epidermal growth factor-induced receptor clustering

Several observations have indicated that clustering of growth factor receptors plays an important role in the action of growth factors. In this investigation, we have used the label fracture method to study the effects of epidermal growth factor (EGF) on the lateral distribution of its receptors in A431 epidermoid carcinoma cells. This method allows a direct visualization of immunogold-labeled plasma membrane receptors on ultrastructural level and in addition permits an quantitative analysis of their lateral distribution. EGF receptors were immunogold-labeled according to standard procedures with the monoclonal anti-EGF receptor antibody 2E9 (IgG1), which binds to the EGF receptor in a 1:1 ratio. In the absence of EGF, EGF receptors located on the surface of A431 cells were found to be clustered, as deduced from Poisson variance analysis (p less than 0.001). Following treatment of A431 cells with EGF, receptor clustering increased rapidly, reaching the maximum within 10 min. Maximal clustering was maintained for 1 h, after which the lateral distribution of receptors returned to the control situation within another hour.     

Epidermal growth factor receptor of A431 cells. Characterization of a monoclonal anti-receptor antibody noncompetitive agonist of epidermal growth factor action

A monoclonal antibody to the epidermal growth factor (EGF) receptor of A431 cells, denoted 2D1-IgM, was generated after fusion of immunized BALB/c mouse spleen cells with SP2/0-Ag14 myeloma cells. Specific binding of 2D1-IgM to the A431 cell-surface receptor for EGF was demonstrated by indirect immunofluorescence, immunoprecipitation, and immunoblot analysis. Scatchard analysis of 125I-EGF binding to A431 cells demonstrated that 2D1-IgM treatment did not change the number of EGF receptors, but caused an increase in the affinity of EGF receptors from a population of low affinity to a uniform population of high affinity. Like EGF, 2D1-IgM induced phosphorylation of EGF receptors and EGF receptor clustering. As in the case of EGF, a biphasic growth response with stimulation of DNA synthesis at low and inhibition at high concentrations of 2D1-IgM was evident in A431 cells. The intrinsic "EGF-like" bioactivity of 2D1-IgM was enhanced by the presence of EGF. These results suggest that the binding of 2D1-IgM to the EGF receptor at a different site from that to which EGF binds can initiate an effective EGF-like biological response; and the EGF-like biological effects of 2D1-IgM may be mediated by a population of high affinity EGF receptors which may be involved in the control of cellular growth.     

Microaggregation of hormone-occupied epidermal growth factor receptors on plasma membrane preparations.

The rotational diffusion of the complexes of epidermal growth factor (EGF) with its specific receptor on plasma membrane vesicles prepared from human epidermoid carcinoma A431 cells was studied using the time-resolved polarization of phosphorescence of erythrosin-labeled hormone. The measured rotational correlation times of 16-20 microseconds at 4 degrees C are consistent with monomeric freely diffusing EGF receptor. Upon increasing the temperature to 37 degrees C, the rate of rotational diffusion slows down as evidenced by an increase in the correlation time to 75 microseconds. This finding suggests that small clusters of the occupied EGF receptor (microaggregation) form at the higher temperature, a property we have reported previously for occupied receptors on living A431 cells. Subsequent cooling of the membranes leads to a partial reversal of the microaggregation. We conclude that clustering of occupied EGF receptors can proceed at 37 degrees C in the absence of metabolic energy and external interactions, e.g. with components of the cytoskeleton, and thus reflects inherent properties of the receptor protein in its natural environment. A lag phase in the time course of microaggregation observed with the isolated membrane preparations may reflect cooperativity in the process of receptor association.     

Nanoscale Imaging of Epidermal Growth Factor Receptor Clustering: EFFECTS OF INHIBITORS*

The development of some solid tumors is associated with overexpression of the epidermal growth factor receptor (EGFR) and often correlates with poor prognosis. Near field scanning optical microscopy, a technique with subdiffraction-limited optical resolution, was used to examine the influence of two inhibitors (the chimeric 225 antibody and tyrosine phosphorylation inhibitor AG1478) on the nanoscale clustering of EGFR in HeLa cells. The EGFR is organized in small clusters, average diameter of 150 nm, on the plasma membrane for both control and EGF-treated cells. The numbers of receptors in individual clusters vary from as few as one or two proteins to greater than 100. Both inhibitors yield an increased cluster density and an increase in the fraction of clusters with smaller diameters and fewer receptors. Exposure to AG1478 also decreases the fraction of EGFR that colocalizes with both rafts and caveolae. EGF stimulation results in a significant loss of the full-length EGFR from the plasma membrane with the concomitant appearance of low molecular mass proteolytic products. By contrast, AG1478 reduces the level of EGFR degradation. Changes in receptor clustering provide one mechanism for regulating EGFR signaling and are relevant to the design of strategies for therapeutic interventions based on modulating EGFR signaling.     

Analysis of morphology and receptor metabolism in clonal variant A431 cells with differing growth responses to epidermal growth factor

Human epidermoid carcinoma A431 cell clones have been obtained whose growth is inhibited, stimulated, or unaffected by epidermal growth factor (EGF). In clones exhibiting each type of growth response, EGF induced similar morphologic changes consisting of aggregation of cells into dense clusters with baring of large areas of the culture dish. The similarity of the clones' morphologic responses, despite their differing growth responses, indicates that the effects of EGF on morphology are distinct from effects on growth. Cells whose growth was inhibited by EGF contained high numbers of EGF receptors, whereas the concentration of EGF receptors was reduced in cells whose growth was stimulated or unaffected by EGF. There were, however, no consistent differences in EGF receptor concentrations between stimulated or null clones. Cells that exhibited each type of growth response displayed similar rates of EGF binding to receptors, rates of internalization of EGF, and rates and extent of EGF-induced receptor down-regulation. Changes in EGF-stimulated tyrosine-specific protein kinase activity paralleled changes in EGF receptors, both between clones and upon down-regulation. These studies indicate that a reduction in the concentration of EGF receptors in A431 cells allows escape from the growth inhibitory effects of EGF, but suggest that the pattern of growth response depends on biochemical events subsequent to EGF-receptor metabolism and activation of tyrosine-specific protein kinase.     

Interaction of epidermal growth factor receptors with the cytoskeleton is related to receptor clustering

Recently it has been established that cytoskeleton-associated epidermal growth factor (EGF) receptors are predominantly of the high-affinity class and that EGF induces a recruitment of low-affinity receptors to the cytoskeleton. The nature of this EGF-induced receptor-cytoskeleton interaction, however, is still unknown. Therefore, we have studied the association of mutated EGF receptors with the cytoskeleton. Receptor deletion mutants lacking almost all intracellular amino acid residues displayed no interaction with the cytoskeleton, demonstrating that the cytoplasmic receptor domain is involved in this interaction. Further analysis revealed that receptor-cytoskeleton interaction is independent of receptor kinase activity and the C-terminal 126 amino acid residues, which include the auto-phosphorylation sites. Furthermore, it is shown that the high-affinity receptor subclass is not essential for association of low-affinity receptors to the cytoskeleton. EGF receptor-cytoskeleton interaction was increased, however, by treatment with sphingomyelinase, an enzyme known to induce membrane protein clustering, indicating that EGF receptor clustering may cause the association to the cytoskeleton.     

Cross-linking of epidermal growth factor receptors in intact cells: detection of initial stages of receptor clustering and determination of molecular weight of high-affinity receptors

A method was developed to label epidermal growth factor (EGF) receptors with 125I-EGF in whole cells using chemical cross-linking reagents. Polyacrylamide gel electrophoresis resolved an Mr approximately 180,000 EGF-receptor complex and larger Mr greater than or equal to 360,000 aggregates. The formation of the larger complexes was time and temperature dependent and appeared to represent the initial events of EGF receptor clustering. Alteration of the ratio of 125I-EGF-labeled high- (Kd approximately 0.16 nM) and low- (Kd approximately 1.5 nM) affinity complexes by competition with unlabeled EGF or by induction of additional high-affinity sites with dexamethasone suggested that both sites were represented by the Mr approximately 180,000 125I-EGF-receptor complexes. Digestion of cells before cross-linking detected a small population of trypsin-resistant Mr approximately 180,000 receptors, which could represent previously described cryptic and/or high-affinity receptors. Few of the Mr approximately 360,000 receptors were trypsin resistant. Glucocorticoid induction of high-affinity EGF receptors failed to induce detectable changes in the microclustering of EGF receptors but did result in a 50% increase in EGF-induced receptor phosphorylation in HeLa S3 cell membranes at 4 degrees C. Thus, glucocorticoids increase high-affinity EGF binding sites, EGF-induced receptor phosphorylation, and cell growth.     

Properties of a monoclonal antibody to epidermal growth factor receptor with implications for the mechanism of action of EGF.

A monoclonal antibody, EGR/ G49 , raised against the receptor for epidermal growth factor (EGF) present in A431 cells inhibits EGF binding by decreasing the affinity of the major population of low affinity receptors while leaving the minor high affinity population relatively unperturbed. The antibody, which binds to a carbohydrate determinant at a site distinct from the EGF binding site, induces clustering and internalisation of the receptor without stimulating the EGF receptor-kinase or affecting its ability to undergo stimulation by EGF. It is toxic to A431 cells and induces morphological changes similar to those seen when these cells are challenged with EGF in the concentration range 1-10 nM. These results suggest that high and low affinity EGF receptors can be distinguished and that they may serve different functions.     

Alteration in growth, cell morphology, and cytoskeletal structures of KB cells induced by epidermal growth factor and transforming growth factor-beta

Long-term biological effects of epidermal growth factor (EGF), insulin, insulin-like growth factor-I (IGF-I), and transforming growth factor-beta (TGF-beta) were examined with human epidermoid carcinoma KB cells. EGF inhibited the growth of KB cells in both serum-containing and serum-free synthetic media by reducing the growth rate and by lowering the saturation density. The cells cultured with EGF showed relatively high motility and grew dispersely as single cells, whereas the cells cultured in the absence of EGF grew in clusters. Although TGF-beta itself did not inhibit the growth of KB cells, it augmented the growth inhibition by EGF. TGF-beta also affected the cell morphology. In the presence of TGF-beta, the cells became flattened and actin stress fibers were well developed compared to those cultured in its absence. The effects of EGF on growth, cell motility, and cell morphology were reversible. Tyrosine phosphorylation of EGF receptors was continuously observed for at least 50 h in the presence of EGF. TGF-beta did not increase the phosphorylation induced by EGF. These results suggested that signals continuously transmitted through EGF receptors caused the changes in cell growth and morphology and that TGF-beta did not act on the cells by modulating binding of EGF to its receptors or activation of the receptor kinase. In contrast to EGF and TGF-beta, neither insulin nor IGF-I affected cell morphology or growth, although KB cells express their receptors and the receptor kinases were also continuously activated during exposure of the cells to insulin or IGF-I.     

Sequestration of epidermal growth factor receptors in non-caveolar lipid rafts inhibits ligand binding

Cholesterol depletion has been shown to increase mitogen-activated protein kinase activation in response to stimulation with epidermal growth factor (EGF) (Furuchi, T., and Anderson, R. G. W. (1998) J. Biol. Chem. 273, 21099-21104). However, the underlying mechanisms are unknown. We show that cholesterol depletion increases EGF binding, whereas cholesterol loading lowers EGF binding. Based on binding analyses, we demonstrate that the observed changes in EGF binding are caused by alterations in the number of EGF receptors available for ligand binding, whereas the affinity of the receptor for EGF remains unaltered. We also show by immunofluorescence that in unstimulated cells the EGF receptor is localized in non-caveolar lipid rafts containing the ganglioside GM1 and that patching of these rafts by cholera toxin B-chain causes co-patching of EGF receptors. Experiments with solubilization in different detergents at 4 degrees C show that the association of the EGF receptor with these rafts is sensitive to Triton X-100 extraction but insensitive to extraction with another non-ionic detergent, Brij 58. Furthermore, experiments with cholesterol-depleted cells show that the association is cholesterol-dependent. We propose that non-caveolar lipid rafts function as negative regulators of EGF receptor signaling by sequestering a fraction of the EGF receptors in a state inaccessible for ligand binding.     

Mobility of epidermal growth factor surface receptors and modulation by cytoplasmic actin.

Epidermal growth factor (EGF) receptors were demonstrated in cultured bovine odontogenic epithelial and ectomesenchymal cells and in rat embryonic and foetal lung fibroblasts by immunofluorescent reactivity with EGF and anti-EGF traced with a fluorescein-isothiocyanate-labelled goat anti-rabbit globulin. Mobility of EGF receptors, either alone or cross linked by anti-EGF, was manifest as sequential changes in staining patterns progressing from a random pattern to clusters, supranuclear caps and large perinuclear globules. Double fluorochrome studies showed that supranuclear caps of EGF receptors occurred in sites closely related to those occupied by cytoplasmic actin; the latter was demonstrated by anti-actin antibody traced with a rhodamine-labelled goat anti-human globulin. Prior treatment of cells with 2 x 10-5 M cytochalasin B for 10-120 min promoted capping of EGF receptors, while its presence throughout the staining procedure inhibited capping. The dual opposing effects of cytochalasin B on capping suggest that actin may have roles in the opposing functions of EGF receptor anchorage and mobility.     

Electric field-induced redistribution and postfield relaxation of epidermal growth factor receptors on A431 cells

The lateral mobility of the epidermal growth factor (EGF) receptor in the plane of the plasma membrane of cultured A431 cells was investigated using direct and indirect fluorescent probes to measure the generation and relaxation of electric field-induced receptor asymmetry. A steady electric field of 15 V/cm for 30 min at 23 degrees C induced a redistribution of the unoccupied EGF receptor such that there was approximately a three-fold higher concentration of receptors at the cathode-facing pole. After termination of the field, the unoccupied receptors back diffused at 37 degrees C with a rate corresponding to a diffusion coefficient of 2.6-3.5 X 10(-10) cm2/s. No diffusion was detected at 4 degrees C. Formation of the hormone-receptor complex is known to induce receptor clustering and internalization. By inhibiting internalization with metabolic poisons, we were able to study the cell surface mobility of clusters of the hormone-receptor complex. The same degree of asymmetry was induced when the occupied receptor was exposed to an electric field and the rate of back diffusion of clusters of the hormone-receptor complex corresponded to a diffusion coefficient of 0.68-0.95 X 10(-10) cm2/s. Although the unoccupied receptor is somewhat more mobile than the hormone-receptor complex, it was still far less mobile than one would predict for an unconstrained protein imbedded in a phospholipid bilayer.     

Cholesterol depletion induces dynamic confinement of the G-protein coupled serotonin1A receptor in the plasma membrane of living cells

Cholesterol is an essential constituent of eukaryotic membranes and plays a crucial role in membrane organization, dynamics, function, and sorting. It is often found distributed non-randomly in domains or pools in biological and model membranes and is thought to contribute to a segregated distribution of membrane constituents. Signal transduction events mediated by seven transmembrane domain G-protein coupled receptors (GPCRs) are the primary means by which cells communicate with and respond to their external environment. We analyzed the role of cholesterol in the plasma membrane organization of the G-protein coupled serotonin_1A receptor by fluorescence recovery after photobleaching (FRAP) measurements with varying bleach spot sizes. Our results show that lateral diffusion parameters of serotonin_1A receptors in normal cells are consistent with models describing diffusion of molecules in a homogenous membrane. Interestingly, these characteristics are altered in cholesterol-depleted cells in a manner that is consistent with dynamic confinement of serotonin_1A receptors in the plasma membrane. Importantly, analysis of ligand binding and downstream signaling of the serotonin_1A receptor suggests that receptor function is affected in a significantly different manner when intact cells or isolated membranes are depleted of cholesterol. These results assume significance in the context of interpreting effects of cholesterol depletion on diffusion characteristics of membrane proteins in particular, and cholesterol-dependent cellular processes in general.     

Dimerization of internalized epidermal growth factor receptors.

Binding of epidermal growth factor (EGF) to cell surface EGF receptors initiates the formation of the receptor homodimers that can be detected by covalent cross-linking in intact cells or in detergent-solubilized cell extracts. Low pH dissociation of EGF from surface receptors results in immediate monomerization of receptor dimers. Using chemical cross-linking during mild permeabilization or cell solubilization, we have detected dimers of internalized EGF receptors in human carcinoma A-431 cells and transfected NIH 3T3 cells that express human EGF receptors. The percentage of internalized cross-linked receptor dimers was similar to that observed for surface EGF receptors. Furthermore, at the time of maximal accumulation of EGF-receptor complexes within the endosomal compartment (10-15 min of incubation at 37 degrees C), both the dimeric and monomeric forms of the EGF receptor are tyrosine-phosphorylated to the same extent as surface dimer and monomer species. In transfected NIH 3T3 cells, the level of dimerized and internalized kinase-negative EGF receptors was not different from that observed for wild-type receptors. These data suggest that for some time after internalization EGF does not dissociate from its receptor and indicate that a receptor conformation is preserved intracellularly that allows maintenance of receptor-receptor interactions and tyrosine kinase activity.     

Separate endocytic pathways of kinase-defective and -active EGF receptor mutants expressed in same cells

Ligand binding to the membrane receptor for EGF induces its clustering and internalization. Both receptor and ligand are then degraded by lysosomal enzymes. A kinase defective point mutant (K721A) of EGF receptor undergoes internalization similarly to the wild-type receptor. However, while internalized EGF molecules bound to either the wild-type or mutant receptors are degraded, the K721A mutant receptor molecules recycle to the cell surface for reutilization. To investigate the mechanism of receptor trafficking, we have established transfected NIH-3T3 cells coexpressing the kinase-negative mutant (K721A) together with a mutant EGF receptor (CD63) with active kinase. CD63 was chosen because it behaves like wild-type EGF receptor with respect to biological responsiveness and cellular routing but afforded immunological distinction between kinase active and inactive mutants. Although expressed in the same cells, the two receptor mutants followed their separate endocytic itineraries. Like wild-type receptor, the CD63 mutant was downregulated and degraded in response to EFG while the kinase-negative mutant K721A returned to the cell surface for reutilization. Intracellular trafficking of EGF receptor must be determined by a sorting mechanism that specifically recognizes EGF receptor molecules according to their intrinsic kinase activity.     

The physical and functional thermal sensitivity of bacterial chemoreceptors

The bacterium Escherichia coli exhibits chemotactic behavior at temperatures ranging from approximately 20 °C to at least 42 °C. This behavior is controlled by clusters of transmembrane chemoreceptors made from trimers of dimers that are linked together by cross-binding to cytoplasmic components. By detecting fluorescence energy transfer between various components of this system, we studied the underlying molecular behavior of these receptors in vivo and throughout their operating temperature range. We reveal a sharp modulation in the conformation of unclustered and clustered receptor trimers and, consequently, in kinase activity output. These modulations occurred at a characteristic temperature that depended on clustering and were lower for receptors at lower adaptational states. However, in the presence of dynamic adaptation, the response of kinase activity to a stimulus was sustained up to 45 °C, but sensitivity notably decreased. Thus, this molecular system exhibits a clear thermal sensitivity that emerges at the level of receptor trimers, but both receptor clustering and adaptation support the overall robust operation of the system at elevated temperatures.     

Reply to Claudio Cobelli and Gianna Toffolo,identifiability from parameter bounds. Structural and numerical aspects. Math. Biosci. 71:237–243 (1984)

We present an analysis of receptor mediated endocytosis which includes the following elements: ligand binding to receptors, interaction of the ligand-receptor complex with coated pits, internalization of coated pit contents, recycling of receptors, and degradation of ligand. The model accounts quantitatively for epidermal growth factor binding and clustering in coated pits at 4°C, for its internalization and degradation at 37°C, and for EGF receptor down-regulation. Steady state analysis of the model indicates that the slope and intercept of a Scatchard plot are functions of the kinetic parameters of the endocytic loop and do not necessarily reflect the affinity and number of receptors in metabolically active cells. Moreover, the model predicts that for homogeneous receptors, a Scatchard plot can be either linear or nonlinear, depending on the concentration of proteins in coated pits which interact with ligand-receptor complexes. A slight generalization of the model in which phorbol ester-receptor complexes compete with EGF-receptor complexes for the same coated pit proteins provides a quantitative explanation for the loss of the high affinity portion of the EGF Scatchard plot subsequent to preincubation with phorbol esters. This explanation leads to the prediction of a local homology between a portion of the phorbol ester receptor sequence and a portion of the EGF receptor sequence.     

EGF signalling amplification induced by dynamic clustering of EGFR

Lateral interaction is an important feature of various types of cell surface receptors including the receptor tyrosine kinases (RTKs). Here we report that dynamic lateral interaction produces amplification and variation in signalling of the EGF receptor, a member of RTKs. Binding of EGF is known to induce transphosphorylation inside EGFR dimers. Using single-molecule techniques, the relationship between EGF binding and EGFR phosphorylation has been determined. The number of phosphorylated EGFR molecules became larger than that of EGF binding as unliganded EGFR was phosphorylated, meaning an amplification of EGF signalling. EGFR formed clusters continuously exchanging their elements through thermal diffusion, and direct and/or indirect lateral interactions. As a result, various types of activation sites differing in number of activated receptors were generated. Amplification required no cytoplasmic factors and was observed on semi-intact cells for a wide range of number of EGFR molecules (10(4)-10(6) per cell) suggesting generality of this process.     

Cholesterol levels modulate EGF receptor-mediated signaling by altering receptor function and trafficking

A variety of signal transduction pathways including PI turnover, MAP kinase activation, and PI 3-kinase activation have been shown to be affected by changes in cellular cholesterol content. However, no information is available regarding the locus (or loci) in the pathways that are susceptible to modulation by cholesterol. We report here that depletion of cholesterol with methyl-beta-cyclodextrin increases cell surface (125)I-EGF binding by approximately 40% via a mechanism that does not involve externalization of receptors from an internal pool. Cholesterol depletion also enhances in vivo EGF receptor autophosphorylation 2-5-fold without altering the rate of receptor dephosphorylation. In vitro kinase assays, which are done under conditions where phosphotyrosine phosphatases are inhibited and receptor trafficking cannot occur, demonstrate that treatment with methyl-beta-cyclodextrin leads to an increase in intrinsic EGF receptor tyrosine kinase activity. EGF receptors are localized in cholesterol-enriched lipid rafts but are released from this compartment upon treatment with methyl-beta-cyclodextrin. These data are consistent with the interpretation that localization to lipid rafts partially suppresses the binding and kinase functions of the EGF receptor and that depletion of cholesterol releases the receptor from lipid rafts, relieving the functional inhibition of the receptor. Cholesterol depletion also inhibits EGF internalization and down-regulation of the EGF receptor, and this likely contributes to the enhanced ability of EGF to stimulate downstream signaling pathways such as the activation of MAP kinase.