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 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.     

Trans-signaling by cytokine and growth factor receptors

Although ligand-induced dimerization or oligomerization of receptors is a well established mechanism of growth factor signaling, increasing evidence indicates that biological responses are often mediated by receptor trans-signaling mechanisms involving two or more receptor systems. These include G protein-coupled receptors, cytokine, growth factor and trophic factor receptors. Greater responsiveness and inhibitory signaling responses are provided when different signaling pathways merge through receptor trans-signaling.     

Cellular signaling by fibroblast growth factor receptors

The 22 members of the fibroblast growth factor (FGF) family of growth factors mediate their cellular responses by binding to and activating the different isoforms encoded by the four receptor tyrosine kinases (RTKs) designated FGFR1, FGFR2, FGFR3 and FGFR4. Unlike other growth factors, FGFs act in concert with heparin or heparan sulfate proteoglycan (HSPG) to activate FGFRs and to induce the pleiotropic responses that lead to the variety of cellular responses induced by this large family of growth factors. A variety of human skeletal dysplasias have been linked to specific point mutations in FGFR1, FGFR2 and FGFR3 leading to severe impairment in cranial, digital and skeletal development. Gain of function mutations in FGFRs were also identified in a variety of human cancers such as myeloproliferative syndromes, lymphomas, prostate and breast cancers as well as other malignant diseases. The binding of FGF and HSPG to the extracellular ligand domain of FGFR induces receptor dimerization, activation and autophosphorylation of multiple tyrosine residues in the cytoplasmic domain of the receptor molecule. A variety of signaling proteins are phosphorylated in response to FGF stimulation including Shc, phospholipase-Cgamma, STAT1, Gab1 and FRS2alpha leading to stimulation of intracellular signaling pathways that control cell proliferation, cell differentiation, cell migration, cell survival and cell shape. The docking proteins FRS2alpha and FRS2beta are major mediators of the Ras/MAPK and PI-3 kinase/Akt signaling pathways as well as negative feedback mechanisms that fine-tune the signal that is initiated at the cell surface following FGFR stimulation.     

Regulation of hepatic growth hormone receptors by insulin.

Induction of diabetes in the rat with streptozotocin caused a decrease in the specific binding of human growth hormone to liver receptors. The decrease was due to a loss of binding sites, with no change in the affinity constant for growth hormone (5.6 × 10⁹M^?1). A highly significant correlation was seen between serum insulin levels and hepatic growth hormone binding. Specific insulin binding to hepatic receptors was increased in diabetes, with a highly significant negative correlation between serum insulin levels and insulin binding. The loss of growth hormone receptors was reversed by treating diabetic rats with insulin. Since hormones which bind to “lactogenic” binding sites in the liver are reported to regulate somatomedin levels, the insulin dependence of human growth hormone receptors might account for the decrease in serum somatomedin in diabetes.     

Regulation of microtubule assembly and disassembly by nucleotides

The role of nucleotides in microtubular assembly and disassembly has been reviewed. Two possible functions of GTP hydrolysis during assembly are discussed: (1) hydrolysis renders sensitivity to factor(s) regulating microtubule depolymerization; (2) the energy of GTP hydrolysis is utilized for the subunit flow from one end of the microtubule to the other. In the second part of the review, experiments are considered showing that microtubular disassembly takes place in the cells only in the presence of ATP, and, therefore, this process is regulated via some ATP-dependent mechanism (most probably, phosphorylation of microtubule-associated proteins).     

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 lymphocyte tumor necrosis factor receptors by IL-2

Activated lymphocytes are known to express TNF receptors. The precise stimuli involved in induction and regulation of these receptors have not been elucidated. Our findings demonstrate that IL-2, alone and in serum-free conditions, can trigger and regulate TNF receptor expression on normal lymphocytes. Flow cytometric analyses demonstrated that the receptor was rapidly induced on CD4, CD8, and CD16+ cells after IL-2 stimulation. Receptors increased with culture duration, became maximal between days 5 and 9, and were maintained for 18 to 20 days in the presence of IL-2. By using 125I-TNF and FITC-TNF binding, we present evidence that IL-2 concentration determines the magnitude of lymphoid TNF receptor expression--influencing both the percentage of TNF-positive cells within the population and the number of receptors/cell. Collectively, our results are persuasive for consideration of IL-2 as a central mediator in the regulation of lymphocyte TNF receptors.     

Epidermal growth factor receptors

EGF-Rs are cell membrane glycoproteins of wide distribution. They have not yet been fully characterized or purified but are probably molecules of 170-190,000 mol. wt. in most cells. The growth factor EGF binds and will saturate cell surface receptors with a KA of about 5 X 10(9) M-1 although a receptor class with an affinity in excess of 10(10) M-1 has been detected in some cells. The number of receptors on a cell does not determine the level of its response. Some cell types have receptors which bind EGF, but with no mitogenic response. The ways in which receptor affinity and/or number is modulated are described. This and other evidence is reviewed in a search for a suitable model of a mechanism of action on the cell, which best fits the current data. There is ample evidence that EGF binds to the receptor; that ligand-receptor complexes cluster or aggregate; and then are internalized and degraded, but evidence for a direct connection between internalization and the subsequent mitogenic response is lacking. Good correlations between internalization and mitogenic responses have been observed and developed into a theory of endocytic activation, but there is a body of evidence which cannot be accommodated by this theory. Instead, an alternative model is suggested.     

Hematopoietic growth factor receptors

The formation of the cellular constituents of the blood is regulated by a series of endogenous polypeptides with largely paracrine function. A number of these hematopoietic growth factors (HGF's), which include colony stimulating factors, interleukins, and erythropoietin, have been purified to homogeneity and cloned, which in turn has led to extensive investigations of their biochemical properties and biological effects and functions. The HGF's act on target cells by binding to receptors. The kinetics and, to an even larger extent, dynamics of the factor/receptor associations display several intriguing characteristics, most of which are still poorly understood. Herein, the biochemical characteristics of HGF's receptors as well as the binding properties, post-receptor binding events and receptor modulation resulting from the association of HGF's and their target cells are reviewed.Abbreviations BMDM Bone Marrow-Derived Macrophages - CSF Colony-Stimulating Factor - cAMP cyclic 3,5-adenosine monophosphate - EPO Erythropoietin - fMLP formyl-methionylleucyl-phenylalanine - IFN Interferon - IL Interleukin - LPS Lipopolysaccharide - PEM Peritoneal E Exudate Macrophages - PKC Protein Kinase C - TNF Tumor Necrosis Factor - TPA 12-O-tetradecanoylphorbol-13-acetate - PtdIns(4,5)P₂ Phosphatidylinositol-4,5 bisphosphate     

Cyclic AMP potentiates down regulation of epidermal growth factor receptors by platelet-derived growth factor

Pretreatment of $\text{Balb}\text{c}\text{-3T3}$ cells with platelet-derived growth factor (PDGF) has been shown to decrease binding sites for ¹²⁵I-labelled epidermal growth factor (EGF) (1,2,3). Agents which elevate cellular cyclic AMP concentrations enhance this ability, and the change in EGF binding is inversely proportional to the elevation of cyclic AMP. In quiescent density arrested cells, the sensitivity of cells to down regulation of EGF receptors by PDGF is proportional to the cyclic AMP content of the cultures in three different cell lines. Agents which elevate cyclic AMP and which potentiate PDGF mediated heterologous down regulation of EGF receptors are able, like cholera toxin (3), to stimulate cells to synthesize DNA in defined medium in the absence of EGF. Down regulation of EGF receptors by PDGF in combination with agents elevating cyclic AMP effectively mimics the action of EGF.     

Signal transduction by vascular endothelial growth factor receptors

VEGFs (vascular endothelial growth factors) control vascular development during embryogenesis and the function of blood vessels and lymphatic vessels in the adult. There are five related mammalian ligands, which act through three receptor tyrosine kinases. Signalling is modulated through neuropilins, which act as VEGF co-receptors. Heparan sulfate and integrins are also important modulators of VEGF signalling. Therapeutic agents that interfere with VEGF signalling have been developed with the aim of decreasing angiogenesis in diseases that involve tissue growth and inflammation, such as cancer. The present review will outline the current understanding and consequent biology of VEGF receptor signalling.     

Endocytosis of growth factor receptors

Binding of a growth factor (GF) to its specific receptor on the cell surface causes the initiation of a signal transduction cascade which eventually results in mitosis. GF:receptor complexes are removed from the cell surface via receptor-mediated endocytosis, a process which involves clathrin-coated pits. After internalization into the endosomal compartment, a significant pool of GFs and GF receptors escape recycling to the cell surface and are sorted to the degradation pathway. The ligandinduced internalization and lysosomal degradation of GF receptors result in the dramatic loss of surface receptors, a phenomenon termed receptor down-regulation. In this review, we discuss relevant biochemical, morphological and kinetic studies of the mechanism of GF endocytosis, and the possible role of this process in mitogenic signaling by growth factor receptors.     

Regulation of hepatic energy metabolism by epidermal growth factor

Employing the non-recirculating perfused rat liver preparation, we have investigated the regulation of hepatic gluconeogenesis, and metabolic fluxes through the tricarboxylic acid cycle and 2-oxoglutarate dehydrogenase reaction by epidermal growth factor (EGF) which mimics the actions of both insulin and Ca(2+)-mobilizing hormones (e.g. vasopressin). As monitored by the rate of 14CO2 production from [2-14C]pyruvate (0.5 mM), EGF (10 nM) transiently stimulated the activity of the tricarboxylic acid cycle. EGF also transiently stimulated hepatic gluconeogenesis from pyruvate. The transient stimulation of tricarboxylic acid cycle activity and gluconeogenesis were accompanied by an increase in perfusate Ca2+ content indicating that EGF also altered hepatic Ca2+ fluxes. EGF-elicited stimulation of gluconeogenesis was, at least in part, the result of a transient (50%) inhibition of pyruvate kinase activity. Likewise, EGF-mediated stimulation of tricarboxylic acid cycle activity can, in part, be attributed to EGF-elicited stimulation of metabolic flux through the mitochondrial, Ca(2+)-sensitive, 2-oxoglutarate dehydrogenase reaction. The regulation of hepatic metabolism by EGF appears to be the manifestation of alteration in cellular Ca2+ content since in experiments performed under conditions known to abolish the ability of EGF to alter cytosolic free-Ca2+ concentrations, i.e. in livers of pertussis-toxin-treated rats, EGF did not alter either perfusate Ca2+ content or any of the metabolic parameters monitored. Additionally, experiments involving pulsatile infusion of either EGF or phenylephrine into livers demonstrated that, unlike the alpha 1-adrenergic receptor, homologous desensitization of the EGF receptor occurs. Such a homologous desensitization of the EGF receptor can explain the transient nature of EGF-elicited stimulation of various metabolic processes. Since protein kinase C activation by EGF can lead to receptor desensitization, experiments were performed with phorbol esters which either activate or do not alter protein kinase C activity. While the inactive phorbol ester 4 alpha-phorbol 12,13-didecanoate did not modulate the hepatic actions of EGF, activation of protein kinase C by 4 beta-phorbol 12-myristate 13-acetate (70 nM) abolished the ability of EGF to stimulate gluconeogenesis, tricarboxylic acid cycle activity and metabolic flux through the 2-oxoglutarate dehydrogenase complex.     

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.