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1.
I Lax A K Mitra C Ravera D R Hurwitz M Rubinstein A Ullrich R M Stroud J Schlessinger 《The Journal of biological chemistry》1991,266(21):13828-13833
Ligand-induced oligomerization is a universal phenomenon among growth factor receptors. Although the mechanism involved is yet to be defined, much evidence indicates that receptor oligomerization plays a crucial role in receptor activation and signal transduction. Here we show that epidermal growth factor (EGF) is able to stimulate the oligomerization of a recombinant, soluble, extracellular ligand-binding domain of EGF receptor. Covalent cross-linking experiments, analysis by sodium dodecyl sulfate-gel electrophoresis, size exclusion chromatography, and electron microscopy demonstrate that receptor dimers, trimers and larger multimers are formed in response to EGF. This establishes that receptor oligomerization is an intrinsic property of the extracellular ligand-binding domain of EGF receptor. Ligand-induced conformational change in the extracellular domain will stimulate receptor-receptor interactions. This may bring about the allosteric change involved in signal transduction from the extracellular domain across the plasma membrane, resulting in the activation of the cytoplasmic kinase domain. Electron microscopic images of individual extracellular ligand-binding domains appear as clusters of four similarly-sized stain-excluding areas arranged around a central, relatively less stain-excluded area. This suggests that the extracellular ligand-binding domain is structurally composed of four separate domains. 相似文献
2.
Hofman EG Bader AN Voortman J van den Heuvel DJ Sigismund S Verkleij AJ Gerritsen HC van Bergen en Henegouwen PM 《The Journal of biological chemistry》2010,285(50):39481-39489
The current activation model of the EGF receptor (EGFR) predicts that binding of EGF results in dimerization and oligomerization of the EGFR, leading to the allosteric activation of the intracellular tyrosine kinase. Little is known about the regulatory mechanism of receptor oligomerization. In this study, we have employed FRET between identical fluorophores (homo-FRET) to monitor the dimerization and oligomerization state of the EGFR before and after receptor activation. Our data show that, in the absence of ligand, ~40% of the EGFR molecules were present as inactive dimers or predimers. The monomer/predimer ratio was not affected by deletion of the intracellular domain. Ligand binding induced the formation of receptor oligomers, which were found in both the plasma membrane and intracellular structures. Ligand-induced oligomerization required tyrosine kinase activity and nine different tyrosine kinase substrate residues. This indicates that the binding of signaling molecules to activated EGFRs results in EGFR oligomerization. Induction of EGFR predimers or pre-oligomers using the EGFR fused to the FK506-binding protein did not affect signaling but was found to enhance EGF-induced receptor internalization. Our data show that EGFR oligomerization is the result of EGFR signaling and enhances EGFR internalization. 相似文献
3.
Two EGF molecules contribute additively to stabilization of the EGFR dimer. 总被引:10,自引:3,他引:7
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M A Lemmon Z Bu J E Ladbury M Zhou D Pinchasi I Lax D M Engelman J Schlessinger 《The EMBO journal》1997,16(2):281-294
Receptor dimerization is generally considered to be the primary signaling event upon binding of a growth factor to its receptor at the cell surface. Little, however, is known about the precise molecular details of ligand-induced receptor dimerization, except for studies of the human growth hormone (hGH) receptor. We have analyzed the binding of epidermal growth factor (EGF) to the extracellular domain of its receptor (sEGFR) using titration calorimetry, and the resulting dimerization of sEGFR using small-angle X-ray scattering. EGF induces the quantitative formation of sEGFR dimers that contain two EGF molecules. The data obtained from the two approaches suggest a model in which one EGF monomer binds to one sEGFR monomer, and that receptor dimerization involves subsequent association of two monomeric (1:1) EGF-sEGFR complexes. Dimerization may result from bivalent binding of both EGF molecules in the dimer and/or receptor-receptor interactions. The requirement for two (possibly bivalent) EGF monomers distinguishes EGF-induced sEGFR dimerization from the hGH and interferon-gamma receptors, where multivalent binding of a single ligand species (either monomeric or dimeric) drives receptor oligomerization. The proposed model of EGF-induced sEGFR dimerization suggests possible mechanisms for both ligand-induced homo- and heterodimerization of the EGFR (or erbB) family of receptors. 相似文献
4.
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 homodimerisation. Here, however, we show by chemical cross-linking and sucrose density-gradient centrifugation that 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. We also analysed the receptor dimerisation 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 induced similar flexible rotation of the juxtamembrane region. All the disulphide-bonded dimers had 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. 相似文献
5.
The EGF receptor is a transmembrane receptor tyrosine kinase that is enriched in lipid rafts. Subdomains I, II and III of the extracellular domain of the EGF receptor participate in ligand binding and dimer formation. However, the function of the cysteine-rich subdomain IV has not been elucidated. In this study, we analyzed the role of the membrane-proximal portion of subdomain IV in EGF binding and signal transduction. A double Cys-->Ala mutation that breaks the most membrane-proximal disulfide bond (Cys600 to Cys612), ablated high affinity ligand binding and substantially reduced signal transduction. A similar mutation that breaks the overlapping Cys596 to Cys604 disulfide had little effect on receptor function. Mutation of residues within the Cys600 to Cys612 disulfide loop did not alter the ligand binding or signal transducing activities of the receptor. Despite the fact that the C600,612A EGF receptor was significantly impaired functionally, this receptor as well as all of the other receptors with mutations in the region of residues 596 to 612 localized normally to lipid rafts. These data suggest that the disulfide-bonded structure of the membrane-proximal portion of the EGF receptor, rather than its primary sequence, is important for EGF binding and signaling but is not involved in localizing the receptor to lipid rafts. 相似文献
6.
Self-phosphorylation of epidermal growth factor receptor: evidence for a model of intermolecular allosteric activation 总被引:43,自引:0,他引:43
The membrane receptor for epidermal growth factor (EGF) is a 170,000-dalton glycoprotein composed of an extracellular EGF-binding domain and a cytoplasmic kinase domain connected by a stretch of 23 amino acids traversing the plasma membrane. The binding of EGF to the extracellular domain activates the cytoplasmic kinase function even in highly purified preparations of EGF receptor, suggesting that the activation occurs exclusively within the EGF receptor moiety. Conceivably, kinase activation may require the transfer of a conformational change through the single transmembrane region from the ligand binding domain to the cytoplasmic kinase region. Alternatively, ligand-induced receptor-receptor interactions may activate the kinase and thus bypass this requirement. Both mechanisms were contrasted by employing independent experimental approaches. The following lines of evidence support an intermolecular mechanism for the activation of the detergent-solubilized receptor: the EGF-induced receptor self-phosphorylation has a parabolic dependence on the concentration of EGF receptor, cross-linking of EGF receptors by antibodies or lectins stimulates receptor self-phosphorylation, immobilization of EGF receptor on various solid matrices prevents EGF from activating the kinase function, and cross-linking of EGF receptors increases their affinity toward EGF. On the basis of these results, an allosteric aggregation model is formulated for the activation of the cytoplasmic kinase function of the receptor by EGF. This model may be relevant to the mechanism by which the mitogenic signal of EGF is transferred across the membrane. 相似文献
7.
The binding of epidermal growth factor (EGF) to its plasma membrane receptor results in the stimulation of a tyrosyl residue-specific protein kinase, which has been shown to be part of the receptor. The mechanism by which EGF binding give rise to the stimulation of kinase activity is not understood in detail; however, a number of recent studies have implicated receptor dimerization or oligomerization in this process. We prepared Triton X-100 extracts of A431 cells in which the concentration of EGF receptors was on the order of 10(-7) M. When samples of the extracts were incubated with or without EGF and then treated with the high-yield cross-linking reagent bis(sulfosuccinimidyl)suberate (BS3), covalent receptor dimers could be detected in high yield in samples that had been treated with both EGF and BS3, whereas only monomeric receptor was detected in untreated samples or in samples that had been treated with either EGF or BS3. The yield of receptor dimers trapped by cross-linking correlated with the stimulation of autophosphorylation by EGF and with the concentration of EGF present. EGF-induced receptor dimers were also efficiently cross-linked in highly purified receptor preparations, suggesting that EGF-induced dimerization is a process intrinsic to the receptor, requiring no additional accessory proteins. 相似文献
8.
Chafen Lu Li-Zhi Mi Thomas Schürpf Thomas Walz Timothy A. Springer 《The Journal of biological chemistry》2012,287(45):38244-38253
We study a mechanism by which dimerization of the EGF receptor (EGFR) cytoplasmic domain is transmitted to the ectodomain. Therapeutic and other small molecule antagonists to the kinase domain that stabilize its active conformation, but not those that stabilize an inactive conformation, stabilize ectodomain dimerization. Inhibitor-induced dimerization requires an asymmetric kinase domain interface associated with activation. EGF and kinase inhibitors stimulate formation of identical dimer interfaces in the EGFR transmembrane domain, as shown by disulfide cross-linking. Disulfide cross-linking at an interface in domain IV in the ectodomain was also stimulated similarly; however, EGF but not inhibitors stimulated cross-linking in domain II. Inhibitors similarly induced noncovalent dimerization in nearly full-length, detergent-solubilized EGFR as shown by gel filtration. EGFR ectodomain deletion resulted in spontaneous dimerization, whereas deletion of exons 2–7, in which extracellular domains III and IV are retained, did not. In EM, kinase inhibitor-induced dimers lacked any well defined orientation between the ectodomain monomers. Fab of the therapeutic antibody cetuximab to domain III confirmed a variable position and orientation of this domain in inhibitor-induced dimers but suggested that the C termini of domain IV of the two monomers were in close proximity, consistent with dimerization in the transmembrane domains. The results provide insights into the relative energetics of intracellular and extracellular dimerization in EGFR and have significance for physiologic dimerization through the asymmetric kinase interface, bidirectional signal transmission in EGFR, and mechanism of action of therapeutics. 相似文献
9.
Treatment of A431 human epidermoid carcinoma cells with 4-phorbol 12-myristate 13-acetate (PMA) causes an inhibition of the high affinity binding of epidermal growth factor (EGF) to cell surface receptors and an inhibition of the EGF receptor tyrosine protein kinase activity. The hypothesis that PMA controls EGF receptor function by regulating the oligomeric state of the receptor was tested. Dimeric EGF receptors bound to 125I-EGF were identified by covalent cross-linking analysis using disuccinimidyl suberimidate. Treatment of cells with PMA in the presence of 20 nM 125I-EGF caused no significant change in the level of labeled cross-linked monomeric and dimeric receptor species. Investigation of the in vitro autophosphorylation of receptor monomers and dimers cross-linked with 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide demonstrated that the treatment of cells with PMA caused an inhibition of the tyrosine phosphorylation of both monomeric and dimeric EGF receptors. We conclude that the inhibition of the EGF receptor tyrosine protein kinase activity caused by PMA is not associated with the regulation of the oligomeric state of the EGF receptor. 相似文献
10.
Jennifer L. Macdonald-Obermann Linda J. Pike 《The Journal of biological chemistry》2009,284(20):13570-13576
We have previously shown that the binding of epidermal growth factor (EGF)
to its receptor can best be described by a model that involves negative
cooperativity in an aggregating system (Macdonald, J. L., and Pike, L. J.
(2008) Proc. Natl. Acad. Sci. U. S. A. 105, 112–117). However,
despite the fact that biochemical analyses indicate that EGF induces
dimerization of its receptor, the binding data provided no evidence for
positive linkage between EGF binding and dimer assembly. By analyzing the
binding of EGF to a number of receptor mutants, we now report that in naive,
unphosphorylated EGF receptors, ligand binding is positively linked to
receptor dimerization but the linkage is abolished upon autophosphorylation of
the receptor. Both phosphorylated and unphosphorylated EGF receptors exhibit
negative cooperativity, indicating that mechanistically, cooperativity is
distinct from the phenomenon of linkage. Nonetheless, both the positive
linkage and the negative cooperativity observed in EGF binding require the
presence of the intracellular juxtamembrane domain. This indicates the
existence of inside-out signaling in the EGF receptor system. The
intracellular juxtamembrane domain has previously been shown to be required
for the activation of the EGF receptor tyrosine kinase (Thiel, K. W., and
Carpenter, G. (2007) Proc. Natl. Acad. Sci. U. S. A. 104,
19238–19243). Our experiments expand the role of this domain to include
the allosteric control of ligand binding by the extracellular domain.The EGF2 receptor
is a tyrosine kinase composed of an ∼620-amino-acid extracellular domain
that recognizes and binds EGF, a single pass α-helical transmembrane
domain, and an intracellular tyrosine kinase domain, encompassing roughly
residues 685–950 (1). In
addition, the receptor contains an ∼230-amino-acid-long C-terminal tail
that contains the bulk of the sites of receptor autophosphorylation
(2–4).
An intracellular juxtamembrane domain of about 40 residues connects the
transmembrane domain to the kinase domain and has been shown to be crucial in
the allosteric activation of the EGF receptor kinase
(5,
6).In the membrane, the EGF receptor exists as a monomer, but a wealth of data
indicate that the binding of EGF induces the formation of EGF receptor dimers
(7–10).
Dimerization appears to be mediated in large part by the extracellular domain
of the receptor, which is comprised of four subdomains, designated I through
IV. X-ray crystallography data suggest that in the absence of ligand, the
extracellular domain is held in a closed configuration through the interaction
of loops or arms that extend from the backs of subdomains II and IV
(11). Upon binding of EGF,
this intramolecular tether is released, allowing the receptor to adopt an open
conformation in which EGF is tightly bound between subdomains I and III. In
this configuration, the “dimerization arm” that was previously
involved in tethering the receptor closed mediates the formation of a
back-to-back EGF receptor dimer
(12,
13).Analyses of the binding of 125I-EGF to its receptor have
invariably resulted in concave up Scatchard plots that have been interpreted
as indicating the presence of two classes of EGF binding sites. However, we
have recently used global analysis of the binding of 125I-EGF to
cells expressing increasing levels of EGF receptors to show that EGF binding
is best described by a model involving negative cooperativity in an
aggregating system (14) (see
Fig. 6). Ligand binding is
negatively cooperative if the binding of ligand to the first site on a dimer
reduces the affinity of the ligand for binding to the second site on the
dimer.Open in a separate windowFIGURE 6.Model for the binding of EGF to its receptor. Circles
represent receptor subunits. E represents a molecule of EGF. The
equilibrium association constants are written above or beside the reaction to
which they apply.The concept of cooperativity only applies to existing dimers. It does not
relate to the effect of ligand on the assembly or disassembly of those dimers.
The effect of ligand on the formation of receptor dimers is captured in the
concept of linkage (15,
16). If ligand binding is
positively linked to dimer formation, then ligand promotes the assembly of
receptor dimers. In a monomer-dimer equilibrium, positive linkage arises when
a ligand binds with higher affinity to the first site on the dimer than to the
monomer. Under these circumstances, the ligand will preferentially bind to the
dimer, shifting the equilibrium in favor of the dimeric species. In the case
of the EGF receptor, biochemical data suggest that EGF induces receptor
dimerization; however, evidence for positive linkage in binding studies has
been lacking.By analyzing the binding of 125I-EGF to cells expressing various
EGF receptor mutants, we now report that in naive, unphosphorylated EGF
receptors, ligand binding is, in fact, positively linked to receptor
dimerization. Autophosphorylation of the EGF receptor abolishes the positive
linkage that is present during the initial phase of the ligand binding
reaction. Negative cooperativity is present in both the phosphorylated and the
non-phosphorylated states of the receptor. Structure-function analyses
demonstrate that both cooperativity and linkage are lost when the EGF receptor
is truncated immediately after the transmembrane domain. However, both forms
of regulation are restored in receptors that include the additional 40 amino
acids that correspond to the intracellular juxtamembrane domain. These data
expand the role of the intracellular juxtamembrane domain to include the
allosteric regulation of EGF binding by the extracellular domain and
demonstrate the presence of inside-out signaling in the EGF receptor
system. 相似文献
11.
Preformed oligomeric epidermal growth factor receptors undergo an ectodomain structure change during signaling
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Fluorescence resonance energy transfer (FRET) was used to reveal aspects of the mechanism of signal transduction by epidermal growth factor receptors (EGFR). The superpositions of epidermal growth factor (EGF), transforming growth factor-alpha (TGFalpha) and an antibody fragment (29.1) to the carbohydrate extremity of the receptor's ectodomain as measured by FRET, show that 14% of EGFRs in A431 cells are oligomerized before growth factor binding. After binding growth factor and signaling, these oligomers dissociate before releasing growth factor. Time courses of the FRET-derived distances between constitutively oligomerized EGFRs during signal transduction show a transient structural change in the extracellular domain, which occurs simultaneously with the production of intracellular Ca2+ signals. The FRET measurements also show a slow increase in oligomerization of EGFR monomers after growth factor binding. The structural change found in the extracellular domain of oligomeric EGFRs is similar to that shown by others for EPO, Neu, Fas, and tumor necrosis factor receptors, and may therefore be a common property of the transduction of the receptor-mediated signals. 相似文献
12.
Cellular signaling mediated by the epidermal growth factor receptor (EGFR or ErbB) family of receptor tyrosine kinases plays an important role in regulating normal and oncogenic cellular physiology. While structures of isolated EGFR extracellular domains and intracellular protein tyrosine kinase domains have suggested mechanisms for growth factor-mediated receptor dimerization and allosteric kinase domain activation, understanding how the transmembrane and juxtamembrane domains contribute to transmembrane signaling requires structural studies on intact receptor molecules. In this report, recombinant EGFR constructs containing the extracellular, transmembrane, juxtamembrane, and kinase domains are overexpressed and purified from human embryonic kidney 293 cell cultures. The oligomerization state, overall structure, and functional stability of the purified EGF-bound receptor are characterized in detergent micelles and phospholipid bilayers. In the presence of EGF, catalytically active EGFR dimers can be isolated by gel filtration in dodecyl maltoside. Visualization of the dimeric species by negative stain electron microscopy and single particle averaging reveals an overall structure of the extracellular domain that is similar to previously published crystal structures and is consistent with the C-termini of domain IV being juxtaposed against one another as they enter the transmembrane domain. Although detergent-soluble preparations of EGFR are stable as dimers in the presence of EGF, they exhibit differential functional stability in Triton X-100 versus dodecyl maltoside. Furthermore, the kinase activity can be significantly stabilized by reconstituting purified EGF-bound EGFR dimers in phospholipid nanodiscs or vesicles, suggesting that the environment around the hydrophobic transmembrane and amphipathic juxtamembrane domains is important for stabilizing the tyrosine kinase activity in vitro. 相似文献
13.
Mutations in the cytoplasmic domain of EGF receptor affect EGF binding and receptor internalization. 总被引:39,自引:6,他引:33
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Binding of epidermal growth factor (EGF) to its receptor results in a cascade of events that culminate in cell division. The receptor is present on the cell surface in two forms of high and low affinity binding for EGF. EGF binding activates the receptor's intracellular tyrosine kinase activity and subsequently causes the receptor to be rapidly internalized into the cell via clathrin-coated pits. We have cloned the EGF receptor cDNA into a retroviral expression vector and made mutations in vitro to investigate the function of different receptor domains. Deletion of cytoplasmic sequences abolishes high but not low affinity sites as well as impairing the ability of the protein to internalize into cells. Thus, cytoplasmic sequences must be involved in the regulation of high affinity sites and are required for EGF-induced receptor internalization. A four amino acid insertion mutation at residue 708 abolishes the protein-tyrosine kinase activity of the immunoprecipitated receptor. However, this receptor mutant exhibits both the high and low affinity states, internalizes efficiently and is able to cause cells to undergo DNA synthesis in response to EGF. Another four amino acid insertion mutation (residue 888) abolishes protein-tyrosine kinase activity, high affinity binding, internalization and mitogenic responsiveness. Finally, a chimaeric receptor composed of the extracellular EGF binding domain and the cytoplasmic v-abl kinase region transforms Rat-I cells. This chimaeric receptor possesses intrinsic protein tyrosine kinase activity which cannot be regulated by EGF. Moreover, EGF fails to induce the internalization of the chimaeric receptor. 相似文献
14.
Conformational stability of the epidermal growth factor (EGF) receptor as influenced by glycosylation,dimerization and EGF hormone binding 总被引:1,自引:0,他引:1
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The epidermal growth factor receptor (EGFR) is an important transmembrane glycoprotein kinase involved the initiation or perpetuation of signal transduction cascades within cells. These processes occur after EGFR binds to a ligand [epidermal growth factor (EGF)], thus inducing its dimerization and tyrosine autophosphorylation. Previous publications have highlighted the importance of glycosylation and dimerization for promoting proper function of the receptor and conformation in membranes; however, the effects of these associations on the protein conformational stability have not yet been described. Molecular dynamics simulations were performed to characterize the conformational preferences of the monomeric and dimeric forms of the EGFR extracellular domain upon binding to EGF in the presence and absence of N‐glycan moieties. Structural stability analyses revealed that EGF provides the most conformational stability to EGFR, followed by glycosylation and dimerization, respectively. The findings also support that EGF–EGFR binding takes place through a large‐scale induced‐fitting mechanism. Proteins 2017; 85:561–570. © 2016 Wiley Periodicals, Inc. 相似文献
15.
M K Collins J Downward A Miyajima K Maruyama K Arai R C Mulligan 《Journal of cellular physiology》1988,137(2):293-298
Epidermal growth factor (EGF) is a small protein that acts as a mitogen for various epidermal, epithelial, and fibroblastic cells that bear specific EGF receptors. The molecule that binds EGF is a 175-kD transmembrane protein, with an extracellular ligand binding domain and an intracellular domain that possesses tyrosine kinase activity, thought to be involved in the mitogenic signalling process. Here we have constructed a recombinant murine retrovirus that transduces a human cDNA encoding the 175-kD protein and used this retrovirus to infect BAF3, a murine, bone marrow-derived cell line, which is dependent on the haematopoietic factor interleukin-3 (IL3) for its growth in culture. The EGF receptors expressed in the infected cells exhibit two affinity states, as well as EGF-stimulated autophosphorylation. Furthermore, EGF can replace IL3 in supporting short-term proliferation of these cells. These data identify functional properties of the EGF receptor upon expression of the 175-kD EGF binding protein in a haemotopoietic cell that does not express endogenous receptors. They also suggest that gene transfer of growth factor receptors to heterologous cells may allow novel growth stimuli to be exploited. 相似文献
16.
Trebak M Begg GE Chong JM Kanazireva EV Herlyn D Speicher DW 《The Journal of biological chemistry》2001,276(3):2299-2309
The GA733-2 antigen (GA733) is a homotypic calcium-independent cell adhesion molecule (CAM) present in most normal human epithelial cells and gastrointestinal carcinomas. Because oligomerization of some CAMs regulates cell adhesion and signal transduction, the correlation between GA733 oligomeric state and cell-cell adhesion was investigated. Sedimentation equilibrium studies showed that full-length (-FL) GA733 exists as dimers and tetramers in solution, whereas the GA733 extracellular domain (-EC) is a monomer. The Kd of GA733-FL is less than 10 nm for the monomer-dimer association, whereas the dimer-tetramer association is about 1000-fold weaker (Kd approximately 10 microm). Chemical cross-linking of purified GA733-FL in solution resulted in a major product corresponding to GA733 dimers, and minor amounts of trimers and tetramers. However, GA733-EC cross-linked under the same conditions was consistently a monomer. Chemical cross-linking of dissociated colon carcinoma cells produced predominantly GA733 dimers, whereas cross-linking of cells in monolayers yielded some tetramers as well. GA733-FL retained its cell-cell adhesion function as shown by inhibition of cell aggregation, whereas monomeric GA733-EC was inactive. These data show that GA733 exists predominantly as high affinity noncovalent cis-dimers in solution and on dissociated colon carcinoma cells. The lower affinity association of dimers to form tetramers is most likely the head-to-head interaction between GA733 cis-dimers on opposing cells that represents its cell-cell adhesion activity. 相似文献
17.
Marcel A. G. Van der Heyden Mirjam Nievers Arie J. Verkleij Johannes Boonstra Paul M. P. Van Bergen en Henegouwen 《FEBS letters》1997,410(2-3)
Although all EGF receptors in EGF receptor-expressing cells are molecularly identical, they can be subdivided in two different classes that have either a high or a low affinity for EGF. Specifically the high-affinity class is associated with filamentous actin. To determine whether the interaction of the EGF receptor with actin induces its high-affinity state, we studied EGF-binding properties of an EGF receptor mutant that lacks the actin-binding site. Interestingly, we found that cells expressing this mutant receptor still display both high- and low-affinity classes of EGF receptors, indicating that the actin-binding domain does not determine the high-affinity binding state. By further mutational analysis we identified a receptor domain, within the tyrosine kinase domain, that regulates the affinity for EGF. 相似文献
18.
Jennifer L. Macdonald-Obermann Linda J. Pike 《The Journal of biological chemistry》2014,289(38):26178-26188
The EGF receptor has seven different cognate ligands. Previous work has shown that these different ligands are capable of inducing different biological effects, even in the same cell. To begin to understand the molecular basis for this variation, we used luciferase fragment complementation to measure ligand-induced dimer formation and radioligand binding to study the effect of the ligands on subunit-subunit interactions in EGF receptor (EGFR) homodimers and EGFR/ErbB2 heterodimers. In luciferase fragment complementation imaging studies, amphiregulin (AREG) functioned as a partial agonist, inducing only about half as much total dimerization as the other three ligands. However, unlike the other ligands, AREG showed biphasic kinetics for dimer formation, suggesting that its path for EGF receptor activation involves binding to both monomers and preformed dimers. EGF, TGFα, and betacellulin (BTC) appear to mainly stimulate receptor activation through binding to and dimerization of receptor monomers. In radioligand binding assays, EGF and TGFα exhibited increased affinity for EGFR/ErbB2 heterodimers compared with EGFR homodimers. By contrast, BTC and AREG showed a similar affinity for both dimers. Thus, EGF and TGFα are biased agonists, whereas BTC and AREG are balanced agonists with respect to selectivity of dimer formation. These data suggest that the differences in biological response to different EGF receptor ligands may result from partial agonism for dimer formation, differences in the kinetic pathway utilized to generate activated receptor dimers, and biases in the formation of heterodimers versus homodimers. 相似文献
19.
Pike LJ 《Biochemical Society transactions》2012,40(1):15-19
Scatchard analyses of the binding of EGF (epidermal growth factor) to its receptor (EGFR) yield concave up Scatchard plots, indicative of some type of heterogenity in ligand-binding affinity. This was typically interpreted as being due to the presence of two independent binding sites: one of high affinity representing ≤10% of the receptor population, and one of low affinity making up the bulk of the receptors. However, the concept of two independent binding sites is difficult to reconcile with the X-ray structures of the dimerized EGFR that show symmetrical binding of the two ligands. A new approach to the analysis of 125I-EGF-binding data combined with the structure of the singly-occupied Drosophila EGFR have now shown that this heterogeneity is due to the presence of negative co-operativity in the EGFR. Concerns that negative co-operativity precludes ligand-induced dimerization of the EGFR confuse the concepts of linkage and co-operativity. Linkage refers to the effect of ligand on the assembly of dimers, whereas co-operativity refers to the effect of ligand binding to one subunit on ligand binding to the other subunit within a preassembled dimer. Binding of EGF to its receptor is positively linked with dimer assembly, but shows negative co-operativity within the dimer. 相似文献
20.
Localization of a major receptor-binding domain for epidermal growth factor by affinity labeling. 总被引:19,自引:2,他引:17
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I Lax W H Burgess F Bellot A Ullrich J Schlessinger D Givol 《Molecular and cellular biology》1988,8(4):1831-1834
Epidermal growth factor (EGF) receptor was affinity labeled with 125I-labeled EGF, using bifunctional covalent cross-linking agents. The affinity-labeled receptor was isolated and cleaved with CNBr to yield a single-labeled fragment, which was unequivocally identified by site-specific antibodies and other methods to encompass residues 294 to 543 of the EGF receptor. On the basis of amino acid sequence conservation, the extracellular portion of EGF receptor can be divided into four domains. The labeled CNBr fragment contains the entire sequence which is flanked by the two cysteine-rich domains of extracellular portion of the EGF receptor denoted as domain III. On the basis of these and other results, we propose that domain III contributes most of the interactions that define ligand-binding specificity of the EGF receptor. 相似文献