Immunoaffinity purification of the epidermal growth factor receptor. Stoichiometry of binding and kinetics of self-phosphorylation

Epidermal growth factor (EGF) receptor protein has been purified in a single high-yield step by immunoaffinity chromatography of extracts of A431 cells. A monoclonal antibody directed against the EGF binding site of the receptor was immobilized to Sepharose 4B as a specific immune absorbent and competitive elution with EGF was used to obtain purified EGF receptor protein with tyrosine kinase activity. The stoichiometry of EGF binding was determined by comparing 125I-EGF binding to A431 cells with the mass of EGF receptor protein in those cells as measured by immunoaffinity chromatography, radioimmunoassay, and immune precipitation. Each measurement indicated one EGF binding site/EGF receptor protein molecule. Study of the kinetics of autophosphorylation revealed rapid incorporation of 1 mol of phosphate/mol of enzyme followed by slower incorporation of additional phosphate groups. The autophosphorylation reaction has a Km for ATP (0.2 microM) which is about 10-fold lower than that for phosphorylation of exogenous substrates. The kinetically preferred autophosphorylation is an intramolecular reaction.     

Epidermal growth factor receptor. Characterization of a monoclonal antibody specific for the receptor of A431 cells

A monoclonal antibody to the epidermal growth factor (EGF) receptor of A431 cells was obtained after fusion of immunized BALB/c mouse spleen cells with NS-1 myeloma cells. Specific binding of the antibody to the plasma membrane of A431 cells was demonstrated by indirect immunofluorescence and electron microscopy. The antibody did not react with human KB cells, normal rat kidney cells, or Swiss 3T3 cells. The antibody is an IgG3K; it specifically immunoprecipitated a Mr approximately 170,000 protein from radiolabeled A431 cell extracts. This protein is phosphorylated in a EGF-dependent manner in intact A431 cells and in Triton X-100-solubilized plasma membranes. The specificity of the interaction of the antibody with the Mr = 170,000 protein was confirmed by electrophoretic transfer of A431 cell proteins to nitrocellulose followed by incubation with the antibody and 125I-protein A. When 125I-EGF was covalently cross-linked to its receptor, the 125I-EGF-receptor complex was specifically precipitated by the antibody. The monoclonal antibody did not inhibit the binding of 125I-EGF to its receptor in intact A431 cells and also failed to stimulate the phosphorylation of the Triton X-100-solubilized EGF receptor. The results indicate that the antibody and EGF bind to different sites on the EGF receptor. The antibody will be useful for isolating the EGF receptor in an unactivated form.     

An antibody that blocks insulin-like growth factor (IGF) binding to the type II IGF receptor is neither an agonist nor an inhibitor of IGF-stimulated biologic responses in L6 myoblasts

To better define the biologic function of the type II insulin-like growth factor (IGF) receptor, we raised a blocking antiserum in a rabbit by immunizing with highly purified rat type II IGF receptor. On immunoblots of crude type II receptor preparations, only bands corresponding to the type II IGF receptor were seen with IgG 3637, indicating that the antiserum was specific for the type II receptor. Competitive binding and chemical cross-linking experiments showed that IgG 3637 blocked binding of 125I-IGF-II to the rat type II IGF receptor, but did not block binding of 125I-IGF-I to the type I IGF receptor, nor did IgG 3637 block binding of 125I-insulin to the insulin receptor. In addition, IgG 3637 did not inhibit the binding of 125I-IGF-II to partially purified 150- and 40-kDa IGF carrier proteins from adult and fetal rat serum. L6 myoblasts have both type I and type II IGF receptors. IGF-I was more potent than IGF-II in stimulating N-methyl-alpha-[14C]aminoisobutyric acid uptake, 2-[3H]deoxyglucose uptake, and [3H]leucine incorporation into cellular proteins. IgG 3637 did not stimulate either 2-[3H]deoxyglucose uptake, N-methyl-alpha-[14C]aminoisobutyric acid uptake, or [3H]leucine incorporation into protein when tested alone. Furthermore, IgG 3637 at concentrations sufficient to block type II receptors under conditions of the uptake and incorporation experiments did not cause a shift to the right of the dose-response curve for stimulation of these biologic functions by IGF-II. We conclude that the type II IGF receptor does not mediate IGF stimulation of N-methyl-alpha-[14C]aminoisobutyric acid and 2-[3H]deoxyglucose uptake and protein synthesis in L6 myoblasts; presumably, the type I receptor mediates these biologic responses. The anti-type II receptor antibody inhibited IGF-II degradation in the media by greater than 90%, suggesting that the major degradative pathway for IGF-II in L6 myoblasts utilizes the type II IGF receptor.     

Epidermal growth factor and transforming growth factor alpha bind differently to the epidermal growth factor receptor

Epidermal growth factor (EGF) and transforming growth factor alpha (TGF alpha) compete with each other for binding to the EGF receptor. These two growth factors have similar actions, but there are distinguishable differences in their biological activities. It has never been clear how this one receptor can mediate different responses. A monoclonal antibody to the EGF receptor (13A9) has been identified which has only small effects on the binding of EGF to the EGF receptor, but which has very large effects on the binding of TGF alpha to the EGF receptor; 5 micrograms/mL antibody has been shown to totally block 0.87 microM TGF alpha from binding to purified EGF receptor and to lower both the high- and low-affinity binding constants of TGF alpha binding to EGF receptor on A431 cells by about 10-fold. The 13A9 antibody causes a 2.5-fold stimulation of the tyrosine kinase activity of partially purified EGF receptor, compared to a 4.0-fold stimulation of the tyrosine kinase activity by EGF under the same conditions. The data suggest either that the antibody stabilizes a conformation of the EGF receptor which is not favorable for TGF alpha binding or that it blocks a part of the surface of the receptor which is necessary for TGF alpha binding but not EGF binding.     

Photoaffinity labelling of the ATP-binding site of the epidermal growth factor-dependent protein kinase.

Epidermal growth factor (EGF), after binding to its receptor, activates a tyrosine-specific protein kinase which phosphorylates several substrates, including the EGF receptor itself. The effects of a photoaffinity analogue of ATP, 3'-O-(3-[N-(4-azido-2-nitrophenyl)amino]propionyl)adenosine 5'-triphosphate (arylazido-beta-alanyl-ATP) on the EGF-dependent protein kinase in A431 human tumour cell plasma membrane vesicles was investigated. This analogue was capable of inactivating the EGF-receptor kinase in a photodependent manner. Partial inactivation occurred at an analogue concentration of 1 microM and complete inactivation occurred at 10 microM when a 2 min light exposure was used. Arylazido-beta-alanine at 100 microM and ATP at 100 microM were incapable of inactivating the enzyme with 2 min of light exposure. The photodependent inactivation of the enzyme by the analogue could be partially blocked by 20 mM-ATP and more effectively blocked by either 20 mM-adenosine 5'-[beta gamma-imido]triphosphate or 20 mM-guanosine 5'-[beta gamma-imido]triphosphate, indicating nucleotide-binding site specificity. Arylazido-beta-alanyl-[alpha-32P]ATP was capable of labelling membrane proteins in a photodependent manner. Numerous proteins were labelled, the most prominent of which ran with an apparent Mr of 53000 on polyacrylamide-gel electrophoresis. A band of minor intensity was seen of Mr corresponding to the EGF receptor (170000). Immunoprecipitation of affinity-labelled and solubilized membranes with an anti-(EGF receptor) monoclonal antibody demonstrated that the Mr 170000 receptor protein was photoaffinity labelled by the analogue. The Mr 53000 peptide was not specifically bound by the anti-receptor antibody. The affinity labelling of the receptor was not enhanced by EGF, suggesting that EGF stimulation of the kinase activity does not result from changes in the affinity of the kinase for ATP. These studies demonstrate that arylazido-beta-alanyl-ATP interacts with the ATP-binding site of the EGF-receptor kinase with apparent high affinity and that this analogue is an effective photoaffinity label for the kinase. Furthermore, these studies demonstrate that the EGF receptor, identified by using monoclonal antibodies, contains an ATP-binding site, providing further confirmation that the EGF receptor and EGF-dependent protein kinase are domains of the Mr 170000 protein.     

An insulin epidermal growth factor-binding protein from Drosophila has insulin-degrading activity

We have recently described the purification and characterization of an insulin-degrading enzyme (IDE) from Drosophila melanogaster that can cleave porcine insulin, is highly conserved through evolution and is developmentally regulated. We now report that the IDE is, in fact, an insulin EGF-binding protein (dp100) that we had isolated previously from Drosophila using an antihuman EGF receptor antiserum. This conclusion is based upon the following evidence. (a) dp100, identified by its ability to cross-link to labeled insulin, EGF, and transforming growth factor-alpha (TGF-alpha), and to be immunoprecipitated by anti-EGF receptor antisera, copurifies with the IDE activity. Thus, the purified IDE can be affinity labeled with either 125I-insulin, 125I-EGF, or 125I-TGF-alpha, and this labeling is specifically inhibited with unlabeled insulin, EGF, and the insulin B chain. (b) The antiserum to the human EGF receptor, which recognizes dp100, is able to specifically immunoprecipitate the insulin-degrading activity. (c) The purified IDE preparation contains a single protein of 110 kD which is recognized by both the anti-EGF receptor antiserum and anti-Drosophila IDE antiserum. (d) Polyclonal antiserum to the purified IDE, which specifically recognized only the 110-kD band in Drosophila Kc cells, immunoprecipitates dp100 cross-linked to 125I-TGF-alpha and dp100 cross-linked to 125I-insulin from the purified IDE preparation. (e) EGF, which competes with insulin for binding to dp100, also inhibits the degradation of insulin by the purified IDE. These results raise the possibility that a functional interaction between the insulin and EGF growth factor families can occur which is mediated by the insulin-degrading enzyme.     

Binding of human C-reactive protein to mouse macrophages is mediated by distinct receptors

Human C-reactive protein (CRP) is known to activate mouse macrophages (M phi) to a tumoricidal state and to serve as an opsonin for M phi. Therefore, cell surface receptors for CRP on mouse M phi were characterized and their relationship to the IgG FcR determined. The specific binding of 125I-CRP to resident or elicited mouse M phi was saturable, reversible, and involved both a high and a low affinity receptor population. Binding of CRP to the mouse M phi cell lines PU5 1.8 and J774 was nearly identical to that observed with peritoneal M phi. The high affinity receptor population had a calculated K of 10 nM and a receptor density of approximately 10(5) sites per cell. Mouse Ig of the IgG2a, IgG2b, or IgG1 isotypes inhibited binding of 125I-CRP to PU5 1.8 cells at concentrations five-fold greater than that of the homologous ligand. In the converse experiment, unlabeled CRP failed to inhibit specific binding of 125I-labeled IgG2a, IgG2b or IgG1. Isolation of CRP binding proteins from surface iodinated PU5 1.8 cells by ligand-affinity chromatography or chemical cross-linking yielded a major protein band of 57 to 60 kDa which appeared to be distinct from the IgG1/IgG2b FcR (FcR-II) membrane proteins. Removal of radiolabeled IgG2b/IgG1 binding membrane proteins by affinity chromatography did not remove CRP-binding proteins. The rat mAb 2.4G2 which inhibits binding of radiolabeled mouse IgG2b, did not inhibit the binding of CRP. A rat polyclonal antiserum to CRP-binding membrane proteins of PU5 1.8 cells inhibited 125I-CRP binding, but not 125IgG2b binding. The rat polyclonal antibody reacted with two 57 to 60 kDa membrane proteins from PU5 1.8 cells that appear to be of a similar size on Western blots. The 125I-CRP was internalized via endosomes and intact CRP subunits could be detected intracellularly. The findings suggest that binding of CRP occurs through a receptor that is distinct from the IgG FcRs, but that CRP-R activity may be influenced by an association with an IgG FcR.     

Thyrotropin releasing hormone action in pituitary cells. Protein kinase C-mediated effects on the epidermal growth factor receptor

Thyrotropin releasing hormone (TRH) causes phosphatidylinositol bisphosphate hydrolysis to form inositol trisphosphate and diacylglycerol. Since diacylglycerol activates protein kinase C (Ca2+/phospholipid-dependent enzyme), this enzyme may be involved in mediating the physiological response to TRH. Activation of protein kinase C leads to phosphorylation of receptors for epidermal growth factor (EGF) and decreased EGF affinity. The present study examined the effect of TRH on EGF binding to intact GH4C1 rat pituitary tumor cells to test whether TRH activates protein kinase C. Cells were incubated with TRH at 37 degrees C and specific 125I-EGF binding was then measured at 4 degrees C. 125I-EGF binding was decreased by a 10-min treatment with 0.1-100 nM TRH to 30-40% of control in a dose-dependent manner. 125I-EGF binding was not altered if cells were incubated at 4 degrees C, although TRH receptors were saturated or in a variant pituitary cell line without TRH receptors. TRH (10 min at 37 degrees C) decreased EGF receptor affinity but caused little change in receptor density, 125I-EGF internalization, or degradation. When cells were incubated continuously with TRH, there was a recovery of 125I-EGF binding after 24 h. Incubation with the protein kinase C activating phorbol ester TPA caused an immediate (less than 10 min) profound (greater than 85%) decrease in 125I-EGF binding followed by partial recovery at 24 h. Maximally effective doses of TRH and TPA decreased EGF receptor affinity with half-times of 3 min. EGF treatment (5 min) caused an increase in the tyrosine phosphate content of several proteins; prior incubation with TRH resulted in a small decline in the EGF response. GH4C1 cells were incubated with 500 nM TPA for 24 h in order to down-regulate protein kinase C. Protein kinase C depletion was confirmed by immunoblots and the effects of TRH and TPA on 125I-EGF binding were tested. TRH and TPA were both much less effective in cells pretreated with phorbol esters. TRH increased cytoplasmic pH measured with an intracellularly trapped pH sensitive dye after mild acidification with nigericin. This TRH response is presumed to be the result of protein kinase C-mediated activation of the amiloride-sensitive Na+/H+ exchanger and was blunted in protein kinase C-depleted cells. All of these results are consistent with the view that TRH acts rapidly in the intact cell to activate protein kinase C and that a consequence of this activation is EGF receptor phosphorylation and Na+/H+ exchanger activation.     

Dissociation of cellular responses to epidermal growth factor using anti-receptor monoclonal antibodies.

Three biologically active monoclonal antibodies against the human epidermal growth factor (EGF) receptor (2E9, 2D11 and 2G5) have been used to analyse the interrelationship between various cellular responses to EGF. Antibody 2E9 (IgG1) is directed against the protein core of the receptor, close to or at the EGF binding site, while 2D11 (IgG3) and 2G5 (IgG2a) recognize blood-group A-related carbohydrate determinants of the receptor. These antibodies have EGF-like effects in that they can activate the receptor tyrosine kinase both in vitro and in vivo. Cross-linking of the receptor-bound antibodies by a second antibody mimics EGF in inducing a rapid aggregation of receptors on the cell surface. However, all three antibodies fail to mimic EGF in raising cytoplasmic pH and free Ca2+ and do not stimulate DNA synthesis in quiescent fibroblasts, even after external cross-linking of the occupied receptors. It is concluded that EGF-R tyrosine kinase activity as well as substrate specificity can be modulated by ligands other than EGF, even if they bind to sites distinct from the EGF binding domain; activation of the receptor tyrosine kinase, receptor clustering and induction of the ionic signals are causally unrelated events; and tyrosine kinase activation and receptor cross-linking are not sufficient for stimulation of DNA synthesis.     

Regulation of epidermal growth factor receptor number and phosphorylation by fasting in rat liver

The binding of 125I-epidermal growth factor (EGF) to microsomal membrane preparations from the livers of rats fasted for 72 h or fed control or high carbohydrate diets was examined to determine whether alterations in nutrient intake could affect the EGF receptor system. Fasted rats had 40-50% less membrane binding than did control or carbohydrate-fed rats. Scatchard analysis of the binding data indicated that the decrease in EGF binding in fasted rats was due to a decrease in receptor number with no change in receptor affinity. Cross-linking of 125I-EGF to EGF receptors with disuccinimidyl suberate revealed specific binding of a Mr 170,000 protein, which was diminished by approximately 75% in fasting, and a Mr = 150,000 protein, which accounted for 40-50% of the total labeling in the control and carbohydrate-fed rats and which was relatively unchanged by fasting. The sum of the labeling of the 2 bands was reduced by approximately 40% in fasting and is consistent with the reduction in EGF binding detected by Scatchard analysis. EGF stimulated a 1.5-3-fold increase in 32P incorporation into one major protein of 170 kDa in all 3 groups. Basal and EGF-stimulated autophosphorylation of 170 kDa, when normalized for protein, was 75% lower in membranes from fasted animals, compared to those from control or carbohydrate-fed rats. The comparable reduction of 125I-EGF binding to, and 32P incorporation into, the 170-kDa EGF receptor protein suggested that kinase activity/receptor was unaffected by fasting. Moreover, EGF receptor kinase activity in the 3 groups was comparable for an exogenous substrate, as judged by equal basal and EGF-stimulated phosphorylation of Val5-angiotensin II, when normalized for total EGF-binding capacity. These results suggest that fasting regulates EGF receptor kinase activity primarily by regulation of the number of hepatic EGF receptors. The possibility exists that some in vivo effects of fasting may be mediated by a reduction in EGF receptor levels.     

Inability of anti-epidermal growth factor receptor monoclonal antibody to block "autocrine" growth stimulation in transforming growth factor-secreting melanoma cells

Mouse monoclonal antibodies to the human epidermal growth factor (EGF) receptor were raised by immunizing with plasma membrane vesicles prepared from A431 cells. This paper describes the characterization of one of the IgG anti-receptor monoclonal antibodies generated and its use to probe the role of transforming growth factor (TGF) in the autonomous growth of a melanoma cell line in culture. This antibody blocks: 1) the binding of 125I-EGF to the A431 EGF receptor; 2) the EGF stimulation of the EGF-dependent protein kinase in vitro; and 3) human fibroblast DNA synthesis and proliferation in culture. It can precipitate the EGF receptor from metabolically labeled A431 cells and human fibroblasts and these receptors have indistinguishable peptide maps. No EGF receptor could be detected by immunoprecipitation after fibroblasts were treated with EGF or conditioned medium from the melanoma cells which secrete EGF-like TGF (alpha TGF). The antibody itself did not down-regulate the receptor but could block down-regulation caused by EGF and alpha TGF. Despite its ability to block EGF-stimulated growth and down-regulation in fibroblasts, the antibody was unable to block the growth and soft agar colony formation of alpha TGF-secreting melanoma cells, nor could the antibody detect EGF receptor in these cells under the conditions developed to prevent down-regulation and lysosomal degradation of the EGF receptor. These studies suggest that these melanoma cells do not have the intact EGF receptor and that the secretion of alpha TGF by these cells plays no role in their growth in culture. The absence of receptor cannot be explained by down-regulation by secreted alpha TGF.     

Monoclonal antibody that immunoreacts with a subclass of human receptors for epidermal growth factor

Spleen cells from BALBc mice immunized with human epidermoid carcinoma A431 cells were fused with mouse myeloma P3NP cells. One of the isolated hybridoma lines, B4G7, secreted a monoclonal antibody of the IgG class which inhibited the binding of [125I]-EGF to A431 cells and human fibroblasts, but not to mouse 3T3 cells. This inhibition was partial (65-70%) and Scatchard analysis of the EGF binding data suggested that the B4G7 antibody interacts preferentially with a low-affinity class of EGF receptors. This monoclonal antibody specifically precipitated EGF receptors (Mr = 170,000 and 155,000) of A431 cells which were directly crosslinked with [125I]-EGF. It also precipitated EGF receptors from cells whose surface proteins were labeled with 125I, from cells grown in the presence of [35S]-methionine or [32P]-orthophosphate, and from membrane fractions phosphorylated in vitro with [32P]-gamma-ATP. Receptors subjected to EGF-induced phosphorylation, both in vivo and in vitro, were also precipitated. The B4G7 antibody blocked approximately 70% of the EGF receptors in human fibroblasts, but did not stimulate DNA synthesis in these cells. However, in the presence of this antibody, cells showed the full mitogenic response to EGF, presumably through the unblocked receptors that are likely to be of the high-affinity type.     

Transactivation of the epidermal growth factor receptor is involved in 12-O-tetradecanoylphorbol-13-acetate-induced signal transduction

The mechanism of 12-O-tetradecanoylphorbol-13-acetate (TPA)-induced tumor promotion is still not well understood even though it is thought to be related to the protein kinase C/mitogen-activated protein kinase/AP-1 pathway. Recently, TPA was also found to induce epidermal growth factor receptor (EGFR) activity. Here, we investigated whether the EGFR is a necessary component for TPA-induced signal transduction associated with tumor promotion. We demonstrated that potent inhibitors of the EGFR, PD153035 and AG1478, blocked TPA-induced phosphorylation of extracellular signal-regulated kinases (ERKs), AP-1 activity, and cell transformation. Egfr gene deficiency blocked TPA-induced ERK activity and AP-1 binding activity. The blocking of the ectodomain of the EGFR by a monoclonal antibody depressed TPA-induced ERK activity and AP-1 DNA binding activity. The use of a neutralizing antibody for heparin-binding EGF, one of the ligands of EGFR, blocked TPA-induced phosphorylation of ERKs. BB-94, a potent inhibitor of matrix metalloproteinases, which are activators of ectodomain shedding of EGFR ligands, also blocked TPA-induced ERK activity, AP-1 DNA binding, and cell transformation but had no effect on EGF-induced signal transduction. Anti-EGFR, anti-heparin-binding EGF, and BB-94 each blocked TPA-induced EGFR phosphorylation, but only anti-EGFR could block EGF-induced EGFR phosphorylation. Based on these results, we conclude that the EGFR is required for mediating TPA-induced signal transduction. EGFR transactivation induced by TPA is a mechanism by which the EGFR mediates TPA-induced tumor promotion-related signal transduction.     

Identification of müllerian inhibiting substance specific binding in human cell lines.

The receptor for Müllerian Inhibiting Substance (MIS), a gonadal glycoprotein hormone, has not been previously identified. Plasma membranes from MIS-sensitive human tumor cell lines (HTB-111, endometrial carcinoma; and A-431, vulvar squamous carcinoma) were detergent extracted and incubated with 125I-labeled MIS anti-idiotypic antibody, or radioiodinated human recombinant MIS (125I rhMIS), with and without unlabeled competitors. 125I anti-idiotypic MIS antibody bound to HTB-111 membrane extracts was displaceable by unlabeled anti-idiotypic antibody, but not by anti-isotypic antibody prior to cross-linking. Specific binding of the anti-idiotypic MIS antibody to endometrial carcinoma cells was verified using fluorescence activated cell analysis and fluoresceinated antibody. Furthermore, unlabeled anti-idiotypic MIS antibody competed for 125I rhMIS binding to A-431 vulvar carcinoma membranes. The labeled anti-idiotypic MIS antibody binding complex could be separated from 32P labeled EGF receptor in the A-431 membranes, indicating that EGF, a natural inhibitor of MIS activity, and MIS itself bind to different receptors. These studies demonstrate a specific, displaceable binder for MIS in the plasmalemmae of two human tumor lines. Purification of this cell surface receptor protein will be greatly aided by using the MIS anti-idiotypic antibody.     

Angiotensin II stimulates ERK via two pathways in epithelial cells: protein kinase C suppresses a G-protein coupled receptor-EGF receptor transactivation pathway.

In GN4 rat liver epithelial cells, angiotensin II (Ang II) produces intracellular calcium and protein kinase C (PKC) signals and stimulates ERK and JNK activity. JNK activation appears to be mediated by a calcium-dependent tyrosine kinase (CADTK). To define the ERK pathway, we established GN4 cells expressing an inhibitory Ras(N17). Induction of Ras(N17) blocked EGF- but not Ang II- or phorbol ester (TPA)-dependent ERK activation. In control cells, Ang II and TPA produced minimal increases in Ras-GTP level and Raf kinase activity. PKC depletion by chronic TPA exposure abolished TPA-dependent ERK activation but failed to diminish the effect of Ang II. In PKC-depleted cells, Ang II increased Ras-GTP level and activated Raf and ERK in a Ras-dependent manner. In PKC depleted cells, Ang II stimulated Shc and Cbl tyrosine phosphorylation, suggesting that without PKC, Ang II activates another tyrosine kinase. PKC-depletion did not alter Ang II-dependent tyrosine phosphorylation or activity of p125(FAK), CADTK, Fyn or Src, but PKC depletion or incubation with GF109203X resulted in Ang II-dependent EGF receptor tyrosine phosphorylation. In PKC-depleted cells, EGF receptor-specific tyrosine kinase inhibitors blocked Ang II-dependent EGF receptor and Cbl tyrosine phosphorylation, and ERK activation. In summary, Ang II can activate ERK via two pathways; the latent EGF receptor, Ras-dependent pathway is equipotent to the Ras-independent pathway, but is masked by PKC action. The prominence of this G-protein coupled receptor to EGF receptor pathway may vary between cell types depending upon modifiers such as PKC.     

The membrane-proximal intracellular domain of the epidermal growth factor receptor underlies negative cooperativity in ligand binding

The binding of EGF induces dimerization of its receptor, leading to the stimulation of its intracellular tyrosine kinase activity. Kinase activation occurs within the context of an asymmetric dimer in which one kinase domain serves as the activator for the other kinase domain but is not itself activated. How ligand binding is related to the formation and dynamics of this asymmetric dimer is not known. The binding of EGF to its receptor is negatively cooperative--that is, EGF binds with lower affinity to the second site on the dimer than to the first site on the dimer. In this study, we analyzed the binding of (125)I-EGF to a series of EGF receptor mutants in the intracellular juxtamembrane domain and demonstrate that the most membrane-proximal portion of this region plays a significant role in the genesis of negative cooperativity in the EGF receptor. The data are consistent with a model in which the binding of EGF to the first site on the dimer induces the formation of one asymmetric kinase dimer. The binding of EGF to the second site is required to disrupt the initial asymmetric dimer and allow the formation of the reciprocal asymmetric dimer. Thus, some of the energy of binding to the second site is used to reorient the first asymmetric dimer, leading to a lower binding affinity and the observed negative cooperativity.     

Epidermal growth factor receptor kinase translocation and activation in vivo

The rat liver epidermal growth factor (EGF) receptor was assessed for EGF-dependent autophosphorylation as well as phosphorylation of a defined exogenous substrate in purified plasmalemma and Golgiendosome fractions isolated from rat liver homogenates. While EGF-dependent kinase activity was readily detected in plasmalemma the corresponding activity in Golgi-endosome fractions required detergent. Consequent to the systemic injection of EGF in vivo, the majority (approximately 60%) of receptor as evaluated by 125I-EGF binding was rapidly lost (T 1/2 approximately 8 min) from the plasmalemma and correspondingly accumulated in the Golgi-endosome fraction in a dose-dependent manner. Electron microscope radioautography of 125I-EGF uptake into Golgi-endosome fractions identified internalization into lipoprotein-filled vesicles of heterogenous size and shape but not into stacked saccules of the Golgi apparatus. Evaluation of receptor kinase activity in plasmalemma fractions isolated at various times after EGF injection in vivo showed more rapid loss of EGF-dependent autophosphorylation activity (T 1/2 approximately 10 s) than of receptor content (T 1/2 approximately 8 min). In contrast to the EGF receptor kinase of the plasmalemma fraction, kinase activity accumulating in endosomes was activated, i.e. maximally stimulated, in the absence of EGF or Triton X-100 in vitro. Furthermore, following the peak time of accumulation of EGF receptor kinase in endosomes (5-15 min) EGF-dependent autophosphorylation activity and EGF receptor content were lost more slowly (T 1/2 approximately 27 and 87 min for the loss of autophosphorylation activity and receptor content, respectively). The rapidity of translocation of activated EGF receptor into endosomes (30 s) and the dose response to low levels (1 microgram) of EGF injected are consistent with a physiological role for internalized EGF receptor kinase activity.     

Perinuclear location and recycling of epidermal growth factor receptor kinase: immunofluorescent visualization using antibodies directed to kinase and extracellular domains

This paper describes studies on the migratory behavior of epidermal growth factor (EGF) receptor kinase using antibodies that are specific for either the kinase domain or the extracellular domain of the receptor. Antiserum was raised to a 42,000-D subfragment of EGF receptor, which was shown earlier to carry the kinase catalytic site but not the EGF-binding site. Another antiserum was raised to the pure intact 170,000-D EGF receptor. The specificities of these antibodies were established by immunoprecipitation and immunoblotting experiments. The domain specificity was examined by indirect immunofluorescent staining of fixed cells. The anti-42-kD peptide antibody could bind specifically to EGF receptors of both human and murine origin and was found to be directed to the cytoplasmic part of the molecule. It did not bind to EGF receptor-negative cells, which contained other types of tyrosine kinases. The antibodies raised against the intact receptor recognized only EGF receptor-specific epitopes and were directed to the extracellular part of the molecule. The anti-receptor antibodies described above were used to visualize the cyclic locomotory behavior of EGF receptor kinase under various conditions of EGF stimulation and withdrawal. The receptor was examined in fixed and permeabilized cells by indirect immunofluorescent staining. The results demonstrate the following: (a) the receptor kinase domain migrates to the perinuclear region upon challenge with EGF; (b) both extracellular and cytoplasmic domains of the receptor are involved in migration as a unit; (c) withdrawal of EGF results in rapid recycling of the perinuclear receptors to the plasma membrane; (d) this return to the cell surface is inhibited by methylamine, chloroquine, and monensin; and (e) neither the internal migration nor the recycling process is blocked by inhibitors of protein biosynthesis.