Interactive Fly, Drosophila



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Expression of TGF-alpha and its receptor

The responses of mouse blastocysts to TGF-alpha/EGF treatment are mediated by EGF receptors (EGFR) located on the apical surface of the trophectoderm (TE). Experiments using gold-labeled EGF confirm the presence of these apically located EGFRs. Immunoelectron microscopy (IEM) studies using anti-EGFR antibodies indicate that the receptor is preferentially distributed on the basolateral surface of the TE. The receptor is also present on the inner cell mass (ICM) and is likely to be functional, since treatment of isolated ICMs with TGF-alpha affects [35S]methionine uptake and incorporation into acid-insoluble material. IEM was also used to demonstrate that EGF, which is not synthesized by the mouse preimplantation embryo, is present in both the oviduct and the uterus. Maternally derived EGF is present in both ICM and TE cells in freshly isolated blastocysts, but is present in greatly reduced amounts following overnight culture of blastocysts in vitro. IEM was also used to demonstrate that TGF-alpha is preferentially localized to the ICM and polar TE. The co-localization of TGF-alpha and functional EGFRs to the ICM and polar TE suggests potential autocrine, juxtacrine, and paracrine roles for TGF-alpha in blastocyst development (Dardik, 1992).

The epidermal growth factor (EGF), the transforming growth factor alpha (TGFalpha) and the epidermal growth factor receptor (EGFr) have been immunolocalized at two developmental stages: (1) during the testicular postnatal development (i.e. at the perinatal, prepubertal and adult periods), and (2) during the seminiferous epithelium cycle in the different germ cell types. While TGFalpha is essentially observed in somatic cells, specifically in perinatal Leydig cells and in mature Sertoli cells, EGF is localized both in germ cells and in somatic cells with a preferential tubular expression. Identification of EGFr in different testicular cell types indicates that during postnatal development and spermatogenesis, testicular cells are potentially responsive to EGF because they express EGFr. Indeed, in the course of the gonadal development, the EGFr distribution is found both in somatic and germ cells with a specific germ cell pattern depending upon the seminiferous epithelium cycle. A predominant EGFr staining is found during the meiotic process and the spermiogenesis. This work suggests involvement of the TGFalpha/EGF system in the local control of testicular cells during development and particularly of the system's potential direct involvement in crucial steps of spermatogenesis such as meiosis and spermiogenesis (Caussanel, 1996).

The crystal structure, at 2.5 Å resolution, is reported of a truncated human EGFR ectodomain bound to TGFalpha. TGFalpha interacts with both L1 and L2 domains of EGFR, making many main chain contacts with L1 and interacting with L2 via key conserved residues. The results indicate how EGFR family members can bind a family of highly variable ligands. In the 2:2 TGFalpha:sEGFR501 complex, each ligand interacts with only one receptor molecule. There are two types of dimers in the asymmetric unit: a head-to-head dimer involving contacts between the L1 and L2 domains and a back-to-back dimer dominated by interactions between the CR1 domains of each receptor. Based on sequence conservation, buried surface area, and mutagenesis experiments, the back-to-back dimer is favored to be biologically relevant (Garrett, 2002).

TGF-alpha and EGF interaction with EGF receptor

Epidermal growth factor (EGF) and type alpha transforming growth factor (TGF-alpha) bind to a specific region in subdomain III of the extracellular portion of the EGF receptor (EGFR). Binding leads to receptor dimerization, auto-and transphosphorylation on intracellular tyrosine residues, and activation of signal transduction pathways. The binding and biological actions of EGF and TGF-alpha were compared in Chinese hamster ovary (CHO) cells expressing either wild-type human EGFR (HER497R) or a variant EGFR that has an arginine-to-lysine substitution in the extracellular domain at codon 497 (HER497K) within subdomain IV of EGFR. Both receptors exhibit two orders of binding sites with EGF. Similar results were obtained with TGF-alpha in cells expressing HER497R. In contrast, only one order of low-affinity binding sites was seen with TGF-alpha in the case of HER497K. Although EGF and TGF-alpha enhance tyrosine phosphorylation of both receptors, CHO cells expressing HER497K exhibit an attenuated growth response to EGF and TGF-alpha and a reduced induction of the protooncogenes FOS, JUN, and MYC. Moreover, high concentrations of TGF-alpha inhibit growth in these cells but not in cells expressing HER497R. These findings indicate that a region in subdomain IV of EGFR regulates signal transduction across the cell membrane and selectively modulates the binding characteristics of TGF-alpha (Moriai, 1994).

TGF-alpha and EGF are structurally related factors that bind to and induce tyrosine autophosphorylation of a common receptor. Proteolytic cleavage of the transmembrane TGF-alpha precursor's external domain releases several TGF-alpha species. However, membrane-bound TGF-alpha forms remain on the surface of TGF-alpha-expressing cell lines. To evaluate the biological activity of these forms, two cleavage sites were modified in the TGF-alpha precursor coding sequence, making impossible the precursor's processing into the 50 amino acid TGF-alpha. Overexpression of this cDNA in a receptor-negative cell line, as well as partial purification, and N-terminal sequence analysis indicate the existence of two transmembrane TGF-alpha forms. These solubilized precursors induce tyrosine autophosphorylation of the EGF/TGF-alpha receptor in intact receptor-overexpressing cells, and anchorage-independent growth of NRK fibroblasts. Cell-cell contact between TGF-alpha precursor-overexpressing cells and cells expressing high numbers of receptors also result in receptor activation. These findings suggest a role for transmembrane TGF-alpha forms in intercellular interactions in proliferating tissues (Brachmann, 1989).

The epidermal growth factor receptor plays crucial roles throughout the development of multicellular organisms, and inappropriate activation of the receptor is associated with neoplastic transformation of many cell types. The receptor is thought to be activated by ligand-induced homodimerization. However, in the absence of bound ligand the receptor has an ability to form a dimer and exists as a preformed dimer on the cell surface. The receptor dimerization has been analyzed by inserting cysteine residues at strategic positions about the putative alpha-helix axis of the extracellular juxtamembrane region. The mutant receptors spontaneously formed disulphide bridges and transformed NIH3T3 cells in the absence of ligand, depending upon the positions of the cysteine residue inserted. Kinetic analyses of the disulphide bonding indicate that EGF binding induces flexible rotation or twist of the juxtamembrane region of the receptor in the plane parallel with the lipid bilayer. The binding of an ATP competitor to the intracellular domain also induces similar flexible rotation of the juxtamembrane region. All the disulphide-bonded dimers have flexible ligand-binding domains with the same biphasic affinities for EGF as the wild-type. These results demonstrate that ligand binding to the flexible extracellular domains of the receptor dimer induce rotation or twist of the juxtamembrane regions (hence the transmembrane domains), and dissociate the dimeric, inactive form of the intracellular domains. The flexible rotation of the intracellular domains may be necessary for the intrinsic catalytic kinase to become accessible to the multiple tyrosine residues present in the regulatory domain and various substrates, and may be a common property of many cell-surface receptors, such as the insulin receptor (Moriki, 2001).

Two structurally related but different polypeptide growth factors, epidermal growth factor (EGF) and transforming growth factor-alpha (TGF-alpha), exert their activities after interaction with a common cell-surface EGF/TGF-alpha-receptor. Comparative studies of the effects of both ligands have established that TGF-alpha is more potent than EGF in a variety of biological systems. This observation is not explained by differences in the affinities of the ligands for the receptor, because the affinity-constants of both factors are very similar. The intracellular processing of ligand-receptor complexes using either EGF or TGF-alpha has been compared in two different cell systems. TGF-alpha dissociates from the EGF/TGF-alpha-receptor at much higher pH than EGF, which may reflect the substantial difference in the calculated isoelectric points. After internalization, the intracellular TGF-alpha is more rapidly cleared than EGF, and a substantial portion of the released TGF-alpha represents undegraded TGF-alpha, in contrast to the mostly degraded EGF. In addition, TGF-alpha does not induce a complete down-regulation of cell surface receptors, as observed with EGF, which,in the case of TGF-alpha, is at least in part responsible for a much sooner recovery of the ligand-binding ability after down-regulation. These differences in processing of the ligand-receptor complexes may explain why TGF-alpha exerts quantitatively higher activities than EGF (Ebner, 1991).

Many proteins contain so-called epidermal growth factor (EGF)-like domains that share the characteristic spacing of cysteines and glycines with members of the EGF family. They are, however, functionally unrelated, despite the fact that the three-dimensional structure of these EGF-like domains, is also often very similar to that of the EGF receptor agonists. In the present study, an EGF-like repeat from the Drosophila Notch protein was linked to the N- and C-terminal linear tail sequences of human EGF (hEGF). This chimera (E1N6E) is unable to bind or activate the hEGF receptor. This recombinant protein was then used as a basic construct for identifying the minimal requirements for high affinity EGF receptor binding and activation. A limited number of important hEGF-derived residues were selectively reintroduced, and by using this unique approach, hEGF/Notch chimeras could be made that, compared with wild type hEGF, show nearly 100% binding affinity and mitogenic activity on HER-14 cells expressing the hEGF receptor (van de Poll, 1998).

Proper spatial localization of EGFR signaling activated by autocrine ligands represents a critical factor in embryonic development as well as tissue organization and function, and ligand/receptor binding affinity is among the molecular and cellular properties suggested to play a role in governing this localization. A computational model has been used to predict how receptor-binding affinity affects local capture of autocrine ligand vis-a-vis escape to distal regions, and an experimental test was provided by constructing cell lines expressing EGFR along with either wild-type EGF or a low-affinity mutant, EGFL47M. The model predicts local capture of a lower affinity autocrine ligand to be less efficient when the ligand production rate is small relative to receptor appearance rate. The experimental data confirm this prediction, demonstrating that cells can use ligand/receptor binding affinity to regulate ligand spatial distribution when autocrine ligand production is limiting for receptor signaling (DeWitt, 2002).

Epidermal growth factor (EGF) regulates cell proliferation and differentiation by binding to the EGF receptor (EGFR) extracellular region, comprising domains I-IV, with the resultant dimerization of the receptor tyrosine kinase. In this study, the crystal structure of a 2:2 complex of human EGF and the EGFR extracellular region has been determined at 3.3 Å resolution. EGFR domains I-III are arranged in a C shape, and EGF is docked between domains I and III. The 1:1 EGF*EGFR complex dimerizes through a direct receptor*receptor interaction, in which a protruding beta-hairpin arm of each domain II holds the body of the other. The unique 'receptor-mediated dimerization' was verified by EGFR mutagenesis (Ogiso, 2002).

Epidermal growth factor (EGF) receptor is the prototype of the ErbB (HER) family receptor tyrosine kinases (RTKs), which regulate cell growth and differentiation and are implicated in many human cancers. EGF activates its receptor by inducing dimerization of the 621 amino acid EGF receptor extracellular region. The 2.8 Å resolution crystal structure of this entire extracellular region (sEGFR) in an unactivated state is described. The structure reveals an autoinhibited configuration, where the dimerization interface present in activated sEGFR structures is completely occluded by intramolecular interactions. To activate the receptor, EGF binding must promote a large domain rearrangement that exposes this dimerization interface. This contrasts starkly with other RTK activation mechanisms and suggests new approaches for designing ErbB receptor antagonists (Ferguson, 2003).

TGF-alpha mutation

Transforming growth factor alpha (TGF alpha) mutant mice are viable and fertile, but display pronounced waviness of the whiskers and fur, accompanied by abnormal curvature, disorientation, and misalignment of the hair follicles. Homozygous and, to a lesser extent, heterozygous mice display eye abnormalities of variable incidence and severity, including open eyelids at birth, reduced eyeball size, and superficial opacity. Histological examination reveals eyelid and anterior segment dysgenesis, corneal inflammation and scarring, and lens and retinal defects. Although TGF alpha deficiency affects skin and eyes, wound healing in these tissues is not impaired. Similar hair and eye defects have been previously associated with the recessive mutation waved-1 (wa-1), and there is reduced expression of TGF alpha in wa-1 mice. Crosses between wa-1 homozygotes and TGF alpha-targeted mice confirmed that wa-1 and TGF alpha are allelic (Luetteke, 1993).

TGF-alpha downstream signaling

Epidermal growth factor (EGF) has been shown to stimulate mouse placental lactogen I (mPL-I) secretion and inhibit mPL-II secretion. Does transforming growth factor alpha (TGF-alpha) regulate the production of mPL-I and mPL-II in a manner similar to that of EGF? In contrast to the activity of EGF, TGF-alpha inhibits secretion of mPL-I by placental cells isolated from mice on day 7 of pregnancy. Maximum inhibition of mPL-I secretion occurs on the third day of a 5-day culture period and ranges between 37% and 56%. Incubation of cells with hTGF-alpha and EGF is not followed by a change in the mPL-I concentration of the medium, suggesting the peptides antagonize each other's effects. TGF-alpha inhibits secretion of mPL-II; maximum inhibition ranged between 62% and 84% in multiple trials. EGF and TGF-alpha bind to the same receptors on placental cells, and both peptides stimulate receptor phosphorylation. There are three types of mPL-containing cells in placental cultures: cells that contain only mPL-I, cells that contain only mPL-II, and cells that contain both mPLs. TGF-alpha affects the differentiation of the subpopulations of PL-containing cells in a manner that differs from that of EGF. The data suggest that TGF-alpha and EGF do not regulate the production of mPL-I and mPL-II in a similar manner (Yamaguchi, 1995).

The glucocorticoid and transforming growth factor-alpha (TGF-alpha) regulation of growth and cell-cell contact was investigated in a mammary epithelial tumor cell line. In cell monolayers, dexamethasone coordinately suppresses DNA synthesis, stimulates monolayer transepithelial electrical resistance (TER), and decreases the paracellular leakage of inulin or mannitol across the monolayer. Constitutive production of TGF-alpha in transfected cells or exogenous treatment with TGF-alpha prevents the glucocorticoid growth suppression response and disrupts tight junction formation without affecting glucocorticoid responsiveness. DNA synthesis is not a requirement for the growth factor disruption of tight junctions. The ZO-1 tight junction protein is localized exclusively at the cell periphery in dexamethasone-treated cells; TGF-alpha causes ZO-1 to relocalize from the cell periphery back to a cytoplasmic compartment. Taken together, these results demonstrate that glucocorticoids can coordinately regulate growth inhibition and cell-cell contact of mammary tumor cells and that TGF-alpha can override both effects of glucocorticoids. These results have uncovered a novel functional "cross-talk" between glucocorticoids and TGF-alpha, which potentially regulates the proliferation and differentiation of mammary epithelial cells (Buse, 1995).

Transcriptional regulation of EGF

The hormone combination of insulin, dexamethasone and prolactin induce accumulation of preproepidermal growth factor (EGF) mRNA in HC11 mouse mammary epithelial cells 16-24 h after the hormones are added to the cultures. Individual hormones or combinations of two of the hormones have no effect on EGF mRNA concentrations. The same hormone combination is capable of inducing expression of a reporter gene construct containing -888 to +25 bp of the EGF gene fused with luciferase. Deletions of the promoter between -888 and -271 bp has no detectable effect on basal or hormone-induced reporter gene expression. However, further deletion from -270 to -74 bp increases baseline to approximately equal hormone-induced reporter gene expression. This deletion also abolishes the hormone-induced increase in reporter gene expression. Sequence analysis suggested that this region contains a binding site for Yin-Yang-1 (YY1: Drosophila homolog pleiohomeotic), which was confirmed by gelshift analysis. Mutation of the YY1 binding site increases baseline reporter gene expression to the same level as induced by insulin, dexamethasone and prolactin in the wild-type promoter. These results indicate that expression of the EGF gene in mammary epithelium is repressed by the YY1 site, and that removal of repression may play a part in regulating EGF gene expression in lactating mammary tissue (Fang, 1998).

Table of contents

spitz: Biological Overview | Regulation | Developmental Biology | Effects of Mutation | References

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