Regulation of cell proliferation by epidermal growth factor

Epidermal Growth Factor (EGF) is a 6045 dalton polypeptide which stimulates the proliferation of various cell types in vitro and in vivo. EGF binds to diffusely distributed membrane receptors which rapidly cluster primarily on coated pits areas on the plasma membrane. Subsequently, the EGF-receptor complexes are endocytosed and degraded by lysosomal enzymes. The lateral diffusion coefficient (D) of EGF-receptor complexes on cultured cells increases gradually from D = 2.8 X 10(-10) cm2/sec at 5 degrees C to 8.5 X 10(-10) cm2/sec at 37 degrees C. In the same range of temperature the rotational correlation times change from 25 to 50 microseconds to approximately 350 microseconds. Hence, at 4 degrees C, the occupied EGF receptors translate and rotate rapidly in the plane of the membrane. At 37 degrees C, EGF receptors form microclusters composed of 10 to 50 molecules. Moreover, it is concluded that both at 4 degrees C and 37 degrees C lateral diffusion of the occupied receptors is not the rate determining step for either receptor clustering or internalization. EGF receptor is a 150,000 to 170,000 dalton glycoprotein. The receptor is in close proximity to an EGF-sensitive, cAMP-independent, tyrosine-specific protein kinase which also phosphorylates the receptor molecules itself. The EGF sensitive kinase is similar to the kinase activity which is associated with certain RNA tumor viruses. The fact that the non-mitogenic cyanogen-bromide cleaved EGF is as potent as native EGF in stimulating phosphorylation suggests that EGF-induced, protein phosphorylation is a necessary but insufficient signal for the induction of DNA synthesis by EGF. EGF receptor serves also as the binding site for Transforming Growth Factors (TGF) which compete with EGF and induce anchorage-independent growth of normal cells in soft agar. Tumor promoters such as phorbol ester effect the binding of EGF to its membrane receptors and its ability to stimulate DNA synthesis. EGF itself has also some tumor promoting activity. Hence, the membrane receptor for EGF seems to participate in the regulation of normal and neoplastic growth. Monoclonal antibodies against EGF receptor (IgM) induce various early and delayed effects of EGF, while their monovalent Fab' fragments are devoid of biological activity. These observations support the notions that EGF receptor rather than EGF itself is the active moiety and that the role of the hormone is to perturb the receptor in the appropriate way, probably by inducing the microaggregation of EGF receptors.     

Regulation of the epidermal growth factor receptor by phosphorylation

The receptor for epidermal growth factor (EGF) is a glycosylated transmembrane phosphoprotein that exhibits EGF-stimulable protein tyrosine kinase activity. On EGF stimulation, the receptor undergoes a self-phosphorylation reaction at tyrosine residues located primarily in the extreme carboxyl-terminal region of the protein. Using enzymatically active EGF receptor purified by immunoaffinity chromatography from A431 human epidermoid carcinoma cells, the self-phosphorylation reaction has been characterized as a rapid, intramolecular process which is maximal at 30-37 degrees C and exhibits a very low Km for ATP (0.2 microM). When phosphorylation of exogenous peptide substrates was measured as a function of receptor self-phosphorylation, tyrosine kinase activity was found to be enhanced two to threefold at 1-2 mol of phosphate per mol of receptor. Analysis of the dependence of the tyrosine kinase activity on ATP concentration yielded hyperbolic kinetics when plotted in double-reciprocal fashion, indicating that ATP can serve as an activator of the enzyme. Higher concentrations of peptide substrates were found to inhibit both the self- and peptide phosphorylation, but this inhibition could be overcome by first self-phosphorylating the enzyme. These results suggest that self-phosphorylation can remove a competitive/inhibitory constraint so that certain exogenous substrates can have greater access to the enzyme active site. In addition to self-phosphorylation, the EGF receptor can be phosphorylated on threonine residues by the calcium- and phospholipid-dependent protein kinase C. The sites on the EGF receptor phosphorylated in vitro by protein kinase C are identical to the sites phosphorylated on the receptor isolated from A431 cells exposed to the tumor promoters 12-O-tetradecanoylphorbol 13-acetate or teleocidin. This phosphorylation of the EGF receptor results in a suppression of its tyrosine kinase and EGF binding activities both in vivo and in vitro. The EGF receptor can thus be variably regulated by phosphorylation: self-phosphorylation can enhance tyrosine kinase activity whereas protein kinase C-catalyzed phosphorylation can depress enzyme activity. Because these two phosphorylations account for only a fraction of the phosphate present in the EGF receptor in vivo, other protein kinases can apparently phosphorylate the receptor and these may exert additional controls on EGF receptor/kinase function.     

Control of cell metabolism by the epidermal growth factor receptor

The epidermal growth factor receptor (EGFR) triggers the activation of many intracellular signals that control cell proliferation, growth, survival, migration, and differentiation. Given its wide expression, EGFR has many functions in development and tissue homeostasis. Some of the cellular outcomes of EGFR signaling involve alterations of specific aspects of cellular metabolism, and alterations of cell metabolism are emerging as driving influences in many physiological and pathophysiological contexts. Here we review the mechanisms by which EGFR regulates cell metabolism, including by modulation of gene expression and protein function leading to control of glucose uptake, glycolysis, biosynthetic pathways branching from glucose metabolism, amino acid metabolism, lipogenesis, and mitochondrial function. We further examine how this regulation of cell metabolism by EGFR may contribute to cell proliferation and differentiation and how EGFR-driven control of metabolism can impact certain diseases and therapy outcomes.     

Regulation of epidermal growth factor receptor gene expression

Synthesis and metabolism of the epidermal growth factor (EGF) receptor are extensively regulated to modulate cellular responses to ligand. To study regulation of EGF receptor gene expression, the 5' region of the gene was isolated from a human placental genomic library. A 5' proximal 1.1-kilobase fragment (-1100 to -19 relative to the ATG translation start site) and subfragments of this were subcloned in both forward and reverse orientations into the luciferase expression vector pSVOAL delta 5' and transfected into human cell lines. Luciferase activity was stimulated by treatment of transfected HeLa cells with EGF, 12-O-tetradecanoylphorbol 13-acetate (TPA), (Bu)2 cAMP, retinoic acid, and dexamethasone. Deletion analysis indicated full retention of activity after removal of the -1100 to -485 region (-485 to -19 fragment), but a 5-fold reduction in activity on removal of the -485 to -153 region (-153 to -19 fragment). Despite a reduction in basal activity, the proximal 134-basepair fragment retained responses to all inducers. Additivity was observed in response to maximal concentrations of TPA plus retinoic acid and of TPA plus (Bu)2 cAMP; the response to a combination of four inducers exceeded that to the RSV-LTR strong promoter. Differences in stimulated responses were observed in various recipients, with hepatoma HepG2 cells lacking responses to (Bu)2 cAMP and glioblastoma T98G cells lacking responses to EGF and TPA. These results indicate that a 134-basepair DNA fragment closely adjacent to the translation start site contains elements responsible for directing basal and stimulated expression of the EGF receptor gene.     

Regulation of epidermal growth factor receptor internalization by G protein-coupled receptors

The epidermal growth factor (EGF) receptor (EGFR) plays a central role in regulating cell proliferation, differentiation, and migration. Cellular responses to EGF are dependent upon the amount of EGFR present on the cell surface. Stimulation with EGF induces sequestration of the receptor from the plasma membrane and its subsequent downregulation. Recently, internalization of the EGFR was also shown to be required for mitogenic signaling via the activation of MAP kinases. Therefore, mechanisms regulating internalization of the EGFR represent an important facet for the control of cellular response. Here, we demonstrate that EGFR is removed from the cell surface not only following stimulation with EGF, but also in response to stimulation of G protein-coupled lysophosphatidic acid (LPA) and beta2 adrenergic (beta2AR) receptors. Using a FLAG epitope-tagged EGFR to quantitate receptor internalization, we show that incubation with EGF, LPA, or isoproterenol (ISO) causes the time-dependent loss of cell surface EGFR. Internalization of EGFR by these ligands involves the tyrosine kinase activity of the receptor itself and c-Src, as well as the GTPase activity of dynamin. Unexpectedly, we find that internalization of the EGFR by EGF is dependent upon Gbetagamma and beta-arrestin proteins; expression of minigenes encoding the carboxyl terminii of the G protein-coupled receptor kinase 2, or beta-arrestin1, attenuates LPA-, ISO-, and EGF-mediated internalization of EGFR. Thus, G protein-coupled receptors can control the function of the EGFR by regulating its endocytosis.     

Regulation of high- and low-affinity epidermal growth factor receptors by glucocorticoids

It is reported that receptors for epidermal growth factor (EGF) in HeLa S₃ cells exist in two forms, which differ in both affinity and capacity. Both the number of receptors and their distribution into low- and high-affinity forms are modulated by glucocorticoids. Scatchard analysis of saturation binding assays performed at 0 °C indicates that there is a low-affinity class of receptors ($\text{K}{}_{\mathrm{d}}\text{? 1.5}\text{nm}$), which contains approximately 6 × 10⁴ binding sites per cell, and a second, high-affinity class of receptors ($\text{K}{}_{\mathrm{d}}\text{? 0.16}\text{nm}$) containing approximately 5 × 10³ binding sites per cell. Exposure of HeLa S₃ cells to 10^?7m dexamethasone for 24 h increased EGF binding to whole cells by increasing the numbers of low- and high-affinity receptors by 20 and 114%, respectively. The increase in EGF binding depends upon the dose of dexamethasone, being raised from 10^?11 to 10^?6m. EGF binding is half-maximal near 2–4 × 10^?9m, a concentration equal to the K_d of dexamethasone for the glucocorticoid receptor in these cells. The increase in EGF binding is specific for glucocorticoids, occurring when the HeLa S₃ cells are exposed to 10^?7m cortisol or dexamethasone for 24 h, but not when the cells are similarly treated with testosterone, 5α-dihydroxytestosterone, 17β-estradiol, or progesterone. The effect on EGF binding appears to be biphasic; the initial rapid increase occurs between 8 and 12 h, is blocked by both 10^?6m cyclohexamide and 0.1 μg/ml actinomycin D, and is followed by a more gradual increase thereafter. These data indicate that glucocorticoids are able to regulate both the number of EGF receptors and their distribution into high- and low-affinity components. Press, Inc.     

Regulation of epidermal growth factor receptor degradation by heterotrimeric Galphas protein

Heterotrimeric G proteins have been implicated in the regulation of membrane trafficking, but the mechanisms involved are not well understood. Here, we report that overexpression of the stimulatory G protein subunit (Galphas) promotes ligand-dependent degradation of epidermal growth factor (EGF) receptors and Texas Red EGF, and knock-down of Galphas expression by RNA interference (RNAi) delays receptor degradation. We also show that Galphas and its GTPase activating protein (GAP), RGS-PX1, interact with hepatocyte growth factor-regulated tyrosine kinase substrate (Hrs), a critical component of the endosomal sorting machinery. Galphas coimmunoprecipitates with Hrs and binds Hrs in pull-down assays. By immunofluorescence, exogenously expressed Galphas colocalizes with myc-Hrs and GFP-RGS-PX1 on early endosomes, and expression of either Hrs or RGS-PX1 increases the localization of Galphas on endosomes. Furthermore, knock-down of both Hrs and Galphas by double RNAi causes greater inhibition of EGF receptor degradation than knock-down of either protein alone, suggesting that Galphas and Hrs have cooperative effects on regulating EGF receptor degradation. These observations define a novel regulatory role for Galphas in EGF receptor degradation and provide mechanistic insights into the function of Galphas in endocytic sorting.     

Regulation of protein breakdown by epidermal growth factor in A431 cells

Addition of epidermal growth factor (EGF) to cultures of A431 human epidermoid carcinoma cells produces an increase in the rate of intracellular protein breakdown that cannot be accounted for by increased proteolysis in lysates from EGF-treated cells. In support of this observation, inhibition of protein synthesis with cycloheximide does not reduce the EGF response in cell monolayers. On the other hand, inhibitors of lysosomal proteolytic function such as leupeptin, vinblastine and especially the weak base, ammonia, are able to block the ability of EGF to increase protein breakdown. Additional results suggest that the EGF effect is mediated via a stimulation of autophagy. First, the autophagocytosis inhibitor, 3-methyladenine, reduces the EGF response, and second, the ability of insulin to inhibit protein breakdown by preventing the formation of autophagic vacuoles is overcome by EGF. Moreover, the actions of inhibitors and competing hormones are similar to those reported for glucagon, a hormone known to increase autophagy. The EGF response on protein breakdown persists for at least 6 h after thorough washing of the A431 monolayers. This result contrasts with the rapid reversal of EGF effects in other cell lines. Examination of the fate of bound EGF in cells washed and incubated for 2 h at 37 degrees C shows that some 500-fold more EGF per mg protein is retained on the surface of A431 cells compared to AG2804-transformed fibroblasts, a difference which probably explains the unusual persistence of the EGF effect on protein breakdown.     

Regulation of epidermal growth factor receptor down-regulation by UBPY-mediated deubiquitination at endosomes

Ligand-activated receptor tyrosine kinases undergo endocytosis and are transported via endosomes to lysosomes for degradation. This "receptor down-regulation" process is crucial to terminate the cell proliferation signals produced by activated receptors. During the process, ubiquitination of the receptors serves as a sorting signal for their trafficking from endosomes to lysosomes. Here, we describe the role of a deubiquitinating enzyme UBPY/USP8 in the down-regulation of epidermal growth factor (EGF) receptor (EGFR). Overexpression of UBPY reduced the ubiquitination level of EGFR and delayed its degradation in EGF-stimulated cells. Immunopurified UBPY deubiquitinated EGFR in vitro. In EGF-stimulated cells, UBPY underwent ubiquitination and bound to EGFR. Overexpression of Hrs or a dominant-negative mutant of SKD1, proteins that play roles in the endosomal sorting of ubiquitinated receptors, caused the accumulation of endogenous UBPY on exaggerated endosomes. A catalytically inactive UBPY mutant clearly localized on endosomes, where it overlapped with EGFR when cells were stimulated with EGF. Finally, depletion of endogenous UBPY by RNA interference resulted in elevated ubiquitination and accelerated degradation of EGF-activated EGFR. We conclude that UBPY negatively regulates the rate of EGFR down-regulation by deubiquitinating EGFR on endosomes.     

Regulation of growth hormone and epidermal growth factor receptors by progestins in breast cancer cells

A 24 hr incubation of T-47D human breast cancer cells with R5020, a synthetic progestin, resulted in a 200-250% increase in the specific binding of human growth hormone (hGH) and epidermal growth factor (EGF) by these cells. This effect was specific for progestins in that similar responses were observed with progesterone, medroxyprogesterone acetate and ORG 2058 but no significant increases in hGH or EGF binding were observed in cells incubated with testosterone, estradiol or hydrocortisone. Increased binding was due to an increase in the concentration of receptors (hGH, control = 6,490 +/- 500, progestin treated = 13,180 +/- 3,270 sites/cell; EGF, control = 33,380 +/- 7,410, progestin treated = 67,460 +/- 20,330 sites/cell) while the affinity constants for the hormone-receptor interactions were unchanged by progestin treatment. The specific binding of insulin, calcitonin, transferrin and concanavalin A was unaffected by these treatments. It is concluded that expression of hGH and EGF receptors in this breast cancer cell line is regulated by progestins.     

Regulation of the ligand-dependent activation of the epidermal growth factor receptor by calmodulin

Calmodulin (CaM) is the major component of calcium signaling pathways mediating the action of various effectors. Transient increases in the intracellular calcium level triggered by a variety of stimuli lead to the formation of Ca(2+)/CaM complexes, which interact with and activate target proteins. In the present study the role of Ca(2+)/CaM in the regulation of the ligand-dependent activation of the epidermal growth factor receptor (EGFR) has been examined in living cells. We show that addition of different cell permeable CaM antagonists to cultured cells or loading cells with a Ca(2+) chelator inhibited ligand-dependent EGFR auto(trans)phosphorylation. This occurred also in the presence of inhibitors of protein kinase C, CaM-dependent protein kinase II and calcineurin, which are known Ca(2+)- and/or Ca(2+)/CaM-dependent EGFR regulators, pointing to a direct effect of Ca(2+)/CaM on the receptor. Furthermore, we demonstrate that down-regulation of CaM in conditional CaM knock out cells stably transfected with the human EGFR decreased its ligand-dependent phosphorylation. Substitution of six basic amino acid residues within the CaM-binding domain (CaM-BD) of the EGFR by alanine resulted in a decreased phosphorylation of the receptor and of its downstream substrate phospholipase Cγ1. These results support the hypothesis that Ca(2+)/CaM regulates the EGFR activity by directly interacting with the CaM-BD of the receptor located at its cytosolic juxtamembrane region.     

Regulation of epidermal growth factor receptor traffic by the small GTPase rhoB.

Members of the Rho family of small GTPases control cell adhesion and motility through dynamic regulation of the actin cytoskeleton. Although twelve family members have been identified, only three of these - RhoA, Rac and Cdc42 - have been studied in detail. RhoA regulates the formation of focal adhesions and the bundling of actin filaments into stress fibres. It is also involved in other cell signalling pathways including the regulation of gene expression and the generation of lipid second messengers [1] [2]. RhoA is very closely related to two other small GTPases about which much less is known: RhoB and RhoC (which are approximately 83% identical). Perhaps the most intriguing of these is RhoB. RhoA is largely cytosolic but translocates to the plasma membrane on activation. RhoB, however, is entirely localised to the cytosolic face of endocytic vesicles [3] [4]. This suggests a potential role for RhoB in regulating endocytic traffic; however, no evidence has been presented to support this. RhoA has been shown to act at the plasma membrane to regulate the clathrin-mediated internalisation of transferrin receptor [5] and of the muscarinic acetylcholine receptor [6]. We have recently demonstrated that RhoB binds the RhoA effector, PRK1 and targets it to the endosomal compartment [7]. We show here that RhoB acts through PRK1 to regulate the kinetics of epidermal growth factor receptor traffic.     

A nonmitogenic analogue of epidermal growth factor induces early responses mediated by epidermal growth factor

Cyanogen bromide-cleaved epidermal growth factor (CNBr-EGF) binds to EGF receptors with reduced affinity compared to the native hormone but fails to induce DNA synthesis. However, at similar receptor occupancy, CNBr-EGF is as potent as EGF in activating early cell responses to the hormone. The phosphorylation of membrane proteins, the stimulation of Na+-K+-ATPase as reflected by the ouabain-sensitive uptake of 86Rb of fibroblasts, changes in the organization of microfilaments and in cell-morphology, and the activation of the enzyme ornithine-decarboxylase are all induced by CNBr-EGF as well as EGF Our results are consistent with the notion that EGF-induced phosphorylation could act as a "second messenger" for the action of various EGF-induced responses such as activation of Na+-K+-ATPase, changes in the cytoskeleton and cell morphology, and the activation of the enzyme ornithine decarboxylase. However, the stimulation of phosphorylation of membrane proteins and other early responses are either not required or necessary but insufficient for the induction of DNA synthesis. Suboptimal concentrations of EGF together with CNBr-EGF stimulate DNA synthesis in human fibroblasts. Other growth factors such as insulin, fibroblast growth factor, and prostaglandin F2 alpha, which potentiate the mitogenic response of EGF, do not effect the response to CNBr-EGF. This suggests that the restoration of the mitogenic properties of CNBr-EGF by suboptimal doses of EGF occurs at the level of EGF receptors or during their processing.     

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.