Internalization and processing of the EGF receptor in the induction of DNA synthesis in cultured fibroblasts: the endocytic activation hypothesis.

The addition of EGF to cultured murine 3T3 cells produces a decrease in EGF binding activity with concomitant internalization and degradation of the initially bound EGF. When the EGF receptor on cultured 3T3 cells is affinity labeled with high specific activity 125I-EGF, and the fate of the affinity labeled EGF-receptor complex determined, the loss in binding activity was accounted for by receptor internalization and subsequent proteolytic processing of the EGF receptor molecules in the lysosomes. Studies of the effects of EGF concentration on EGF binding by cells, EGF-induced receptor internalization and EGF-induced stimulation of 3H-thymidine uptake into cellular DNA show that there is a direct correlation between EGF-induced receptor internalization and EGF-induced stimulation of DNA synthesis, but not between EGF binding and EGF-induced stimulation of DNA synthesis. This correlation is lost at high EGF concentrations, where stimulation of DNA synthesis is suboptimal. Optimal stimulation of DNA synthesis requires a minimum of 6 h of incubation of EGF with cells, and the suboptimal stimulation of DNA synthesis at high EGF concentration is intensified when the period of incubation of EGF with cells is less than 6 h. These data are consistent with a model of hormone signal transmission by Endocytic Activation, wherein the activation of EGF-induced processes requires constant EGF-induced internalization of receptor for a requisite 6-8 h period as an obligatory step in production of "second messenger" in the action of this hormone.     

Mitogenic response to epidermal growth factor: relationship to number, affinity, and down-regulation of EGF receptors in three murine embryo cell lines.

Swiss 3T3 and C3H-M2 cells have a greater mitogenic response to epidermal growth factor (EGF) than do C3H10T 1/2 cells. The latter cell line, however, has a number of EGF receptors per cell intermediate between the two cell lines that have a more vigorous response to EGF. Scatchard analysis of binding data indicate that all three cell lines have one class of EGF receptor, with indistinguishable affinity for the ligand. When exposed to 10-nM EGF all three cell lines "down-regulate" their EGF receptors with the same time course, and to the same percentage of initial receptors.     

New strategies in drug discovery for GPCRs: high throughput detection of cellular ERK phosphorylation

G-protein coupled receptors (GPCRs) are a large family of receptors for a wide range of stimulants, including hormones, neurotransmitters, and taste and olfactory chemicals. Due to their broad involvement in cellular responses, GPCRs affect many important body functions both in health and disease. Compared to other receptor families, the GPCRs have been a rich source of extracellularly-acting pharmaceuticals, due largely to the fact that many GPCR ligands are small molecules when compared with ligands for other receptors, such as the tyrosine kinase receptor family. This has allowed the development of small molecule modulators of receptor function that act on specific GPCRs, such as those involved in cardiovascular regulation. However, at several levels, current screening technologies of drug development for GPCRs are lacking. Firstly, responses from many GPCRs, such as the Gi-coupled GPCRs, are not easily measured in large screening programs by current techniques. Secondly, there are few options for detecting agonists of orphan GPCRs. Thirdly, it is now clear that the signaling from GPCRs is more complex than once thought, and the measurement of Ca(2+) and cAMP can account for only a fraction of the biological information emanating from an activated GPCR. Studies of the discrete and sometimes separable activation of the Ras/Raf/Mek/ERK cascade by many GPCRs is likely to offer development of new agonists and antagonists, contribute to new pharmacologies from receptors, and raise the potential for novel drug candidates in this important area of biology. Downstream activation of the ERK pathway, with or without transactivation of growth factor receptors, has not been measurable by high throughput methodologies. This article presents recent advances and associated applications for screening of GPCRs and other receptor species through the rapid measurement of protein phosphorylation events, such as ERK phosphorylation, as new readouts for drug discovery.     

Direct linkage of EGF to its receptor: characterization and biological relevance

A small portion of the 125I-EGF that binds specifically to intact cells or isolated membrane from a variety of sources becomes directly and irreversibly linked to EGF receptors. This provides a simple technique for affinity labeling the EGF receptor. Membranes isolated from the human epidermoid carcinoma cell line A431, which possesses extraordinarily high numbers of EGF receptors, gave rise to three major direct linkage complexes of MW = 160,000, 145,000, and 115,000. The time course for information of each is similar, showing that 125I-EGF can form direct linkage complexes with several preexisting forms of the EGF receptor. The direct linkage of EGF to receptor is slow in comparison to 125I-EGF binding, but both processes have similar susceptibilities to competition by unlabeled EGF. EGF was modified chemically with the amino site-specific reagent, N-hydroxysuccinimidyl biotin. The biotinyl-EGF had a reduced capacity to engage in direct linkage complex formation with no concomitant reduction in its ability to bind to EGF receptors. Since native and biotinyl EGF have identical abilities to stimulate the uptake of 3H-thymidine into DNA when incubated with cultured murine 3T3 cells, the direct linkage of EGF to its receptor does not appear to play an important role in EGF-stimulated mitogenesis.     

Effect of short-term ethanol on the proliferative response of Swiss 3T3 cells to mitogenic growth factors

Both adaptive and deleterious responses of cells to ethanol are likely triggered by short-term interactions of the cells with ethanol. Many studies have demonstrated the direct effect of ethanol on growth factor-stimulated cell proliferation. Using Swiss 3T3 cells whose growth was inhibited by ethanol in a concentration-dependent manner, we further investigated the molecular mechanisms of acute ethanol treatment by examining its effect on EGF- and PDGF-mediated cellular signaling systems for the mitogenic function. Tyrosine autophosphorylation of the growth factor receptors was partially prevented by ethanol in intact cells. When ethanol was included before or after EGF stimulation, no effect on the receptor signaling was observed. Here we also report that ethanol inhibits activation of ERK induced by both EGF and PDGF. EGF-induced JNK activation was reduced but PDGF-induced rapid JNK activation was delayed by the addition of ethanol. The balance between its inhibitory and stimulatory effect on the signaling molecules might determine the rate of cell growth.     

The small-molecule iron transport inhibitor ferristatin/NSC306711 promotes degradation of the transferrin receptor

Iron delivery by transferrin (Tf) is accomplished through clathrin-mediated endocytosis of Tf receptors. The small molecule NSC306711 inhibits iron uptake from the Tf-TfR pathway. Here we show that the drug's mechanism of action is to induce internalization and degradation of unoccupied Tf receptors through an unexpected endocytic pathway. Unlike classical clathrin-mediated Tf receptor endocytosis, internalization promoted by NSC306711 is independent of clathrin and dynamin, and is sensitive to the cholesterol-depleting agents filipin and nystatin. The finding of this cholesterol-dependent Tf receptor internalization pathway through use of the small-molecule inhibitor sheds light on the pleiotropic nature of membrane trafficking dynamics and adds a complex dimension to our understanding of receptor regulation. Because of its unusual properties to inhibit iron uptake, we refer to NSC306711 as "ferristatin."     

Methods for Studying Endocytotic Pathways of Herpesvirus Encoded G Protein-Coupled Receptors

Endocytosis is a fundamental process involved in trafficking of various extracellular and transmembrane molecules from the cell surface to its interior. This enables cells to communicate and respond to external environments, maintain cellular homeostasis, and transduce signals. G protein-coupled receptors (GPCRs) constitute a family of receptors with seven transmembrane alpha-helical domains (7TM receptors) expressed at the cell surface, where they regulate physiological and pathological cellular processes. Several herpesviruses encode receptors (vGPCRs) which benefits the virus by avoiding host immune surveillance, supporting viral dissemination, and thereby establishing widespread and lifelong infection, processes where receptor signaling and/or endocytosis seem central. vGPCRs are rising as potential drug targets as exemplified by the cytomegalovirus-encoded receptor US28, where its constitutive internalization has been exploited for selective drug delivery in virus infected cells. Therefore, studying GPCR trafficking is of great importance. This review provides an overview of the current knowledge of endocytic and cell localization properties of vGPCRs and methodological approaches used for studying receptor internalization. Using such novel approaches, we show constitutive internalization of the BILF1 receptor from human and porcine γ-1 herpesviruses and present motifs from the eukaryotic linear motif (ELM) resources with importance for vGPCR endocytosis.     

A new human interferon-gamma mutant harboring EGF receptor-interfering sequence possesses high antiproliferative activity.

An interferon gamma (IFN-gamma) mutant gene harboring an additional epidermal growth factor (EGF) sequence at its 3' terminal was constructed and highly expressed in E. coli by using pBV220 as a vector. The new fusion protein-IFN-gamma-EGF possesses not only high antiviral activity, but also high antiproliferative effect as demonstrated by 3H-TdR incorporation method using CN II cells which are rich in EGF receptors.     

Real time differentiation of G-protein coupled receptor (GPCR) agonist and antagonist by two photon fluorescence laser microscopy

Receptor-based signaling mechanisms are the primary source of cellular regulation. The superfamily of G protein-coupled receptors (GPCR) is the largest and most ubiquitous of the receptor-mediated processes. Desensitization of G-protein-coupled receptors is a fundamental mechanism regulating the cellular response to agonists. We have recently studied the agonist and antagonist of the human melanocortin receptors (hMC1, hMC3, hMC4, and hMC5 receptors), the human delta opioid receptor, and the human gluacagon receptor with the help of synthetic fluorescent labeled ligands and fluorescent protein-labeled beta-arrestin-receptors that shed new insight on cellular signaling and rapid screening of drugs in real time. It was demonstrated that stimulation of these receptors by the cognate agonist triggers the rapid internalization of ligand-receptor complexes, while the interaction of the receptor with antagonists does not follow this pathway. Furthermore, receptor internalization is dependent upon beta-arrestin, which has been shown to be responsible for the rapid desensitization of cAMP-signaling processes.     

Chemical synthesis, folding, and structural insights into O-fucosylated epidermal growth factor-like repeat 12 of mouse Notch-1 receptor

Notch receptors are cell surface glycoproteins that play key roles in a number of developmental cascades in metazoa. The extracellular domains of Notch-1 receptors are composed of 36 tandem epidermal growth factor (EGF)-like repeats, many of which are modified at highly conserved consensus sites by an unusual form of O-glycan, with O-fucose. The O-fucose residues on certain EGF repeats may be elongated. In mammalian cells this can be a tetrasaccharide, Siaα2,3Galβ1,4GlcNAcβ1,3Fucα1→. This elongation process is initiated by the action of O-fucose-specific β1,3 N-acetylglucosaminyltransferases of the Fringe family. There is evidence that the addition of GlcNAc by Fringe serves as an essential modulator of the interaction of Notch with its ligands and the triggering of activation. Here we describe the efficient synthesis, folding, and structural characterization of EGF repeat 12 (EGF 12) of a mouse Notch-1 receptor bearing different O-fucose glycan chains. We demonstrate that the three disulfide bonds, Cys(456)-Cys(467) (C1-C3), Cys(461)-Cys(476) (C2-C4), and Cys(478)-Cys(487) (C5-C6) were correctly formed in the nonglycosylated as well as the O-fucosylated forms of EGF 12. Three-dimensional structural studies by NMR reveal that the methyl group of fucose is in close contact with ILe(475), Met(477), Pro(478) residues and this stabilizes the conformation of the antiparallel β-sheet of EGF 12. The addition of the GlcNAc residue on O-fucosylated EGF 12 induces a significant conformational change in the adjacent tripeptide sequence, Gln(462)Asn(463)Asp(464), which is a motif involved in the natural, enzymatic O-fucosylation at the conserved site (Cys(461)X(4)Ser/ThrCys(467)).     

Akt is involved in the inhibition of cell proliferation by EGF

Axin is a negative regulator of the Wnt/beta-catenin pathway and is involved in the regulation of axis formation and proliferation. Involvement of Axin in the regulation of other signaling pathways is poorly understood. In this study, we investigated the involvement of Akt in growth regulation by Axin in L929 fibroblasts stimulated by EGF. Akt activity was increased by EGF treatment and Ras activation, respectively. Both the EGF- and Ras-induced Akt activations were abolished by Axin induction, as revealed by both Western blot and immunocytochemical analyses. The proliferation and Akt activation induced by EGF were decreased by Axin induction, and the effects of EGF were abolished by treatment of an Akt-specific inhibitor. Therefore, Axin inhibits EGF-induced proliferation of L929 fibroblasts by blocking Akt activation.     

Epigallocatechin-3-gallate inhibits paracrine and autocrine hepatocyte growth factor/scatter factor-induced tumor cell migration and invasion

Aberrant activation of hepatocyte growth factor/scatter factor (HGF/SF) and its receptor, Met, is involved in the development and progression of many human cancers. In the cell-based screening assay, (-)epigallocatechin-3-gallate (EGCG) inhibited HGF/SF-Met signaling as indicated by its inhibitory activity on HGF/SF-induced cell scattering and uPA activation (IC50=15.8 microgram/ml). Further analysis revealed that EGCG at low doses specifically inhibited HGF/SF-induced tyrosine phosphorylation of Met but not epidermal growth factor (EGF)-induced phosphorylation of EGF receptor (EGFR). On the other hand, high-dose EGCG decreased both Met and EGFR proteins. We also found that EGCG did not act on the intracellular portion of Met receptor tyrosine kinase, i.e., it inhibited InlB-dependent activation of Met but not NGF-induced activation of Trk-Met hybrid receptor. This inhibition decreased HGF-induced migration and invasion by parental or HGF/SF-transfected B16F10 melanoma cells in vitro in either a paracrine or autocrine manner. Furthermore, EGCG inhibited the invasion/metastasis of HGF/SF-transfected B16F10 melanoma cells in mice. Our data suggest the possible use of EGCG in human cancers associated with dysregulated paracrine or autocrine HGF/SF-Met signaling.     

Arginine topology controls escape of minimally cationic proteins from early endosomes to the cytoplasm

Proteins represent an expanding class of therapeutics, but their actions are limited primarily to extracellular targets because most peptidic molecules fail to enter cells. Here we identified two small proteins, miniature protein 5.3 and zinc finger module ZF5.3, that enter cells to reach the cytosol through rapid internalization and escape from Rab5+ endosomes. The trafficking pathway mapped for these molecules differs from that of Tat and Arg(8), which require transport beyond Rab5+ endosomes to gain cytosolic access. Our results suggest that the ability of 5.3 and ZF5.3 to escape from early endosomes is a unique feature and imply the existence of distinct signals, encodable within short sequences, that favor early versus late endosomal release. Identifying these signals and understanding their mechanistic basis will illustrate how cells control the movement of endocytic cargo and may allow researchers to engineer molecules to follow a desired delivery pathway for rapid cytosolic access.     

Modulation of EGF binding and action by succinylated concanavalin A in fibroblast cell cultures

The role of the binding of succinylated concanavalin A to tissue culture cells in influencing epidermal growth factor (EGF)-mediated cell proliferation has been studied. Succinylated concanavalin A dramatically reduces the stimulation of 3T6 cells by EGF in Dulbecco's modified Eagle's medium (DME) containing insulin and vitamin B12 as additional growth factors, but no serum. Furthermore, binding studies using 125I-labeled EGF have shown that the binding of EGF to the cell surface is reduced upon addition of succinylated concanavalin A.     

Strategies for the purification of laminin-10 for studies on colon cancer metastasis

Signals from the epidermal growth factor (EGF) receptor family are thought to combine with integrin-dependent adhesion to laminins to contribute to disease progression and metastasis in cancer. To date, little is known about the mechanisms by which these signals interact. Recently, we have shown that the colon cancer cell line LIM1215 secretes and adheres to laminin-10 through multiple integrin receptors, and that EGF stimulates spreading and migration of these cells on the same substrate. Additionally laminin-10/11 has been shown by immunohistochemistry to be present at the invasive edge of moderately differentiated colon cancers. To enable detailed structure-function studies to be undertaken, it is important to be able to rapidly obtain highly purified native laminin-10 from bulk biological samples in reasonable yield. The development of a multidimensional micropurification scheme to achieve this is presented and compared with other reported methods for the purification of laminins.     

Multivalent Ligands for Inducing Receptor-Receptor Interactions

To obtain enchanced and/or unique cellular responses to peptide hormones by simultaneous activation of two different receptor systems, chimera peptide hormones in which enkephalin (EK) is connected through sarcosine oligomer (n) to the message segment of neurotensin [NT(8–13)] were synthesized. Interaction of these two receptor systems was studied by receptor-binding assay and determination of cAMP and cGMP level in cytosol of NG108-15 cells. It was found that EK-12-NT(8- 13) induced specific cell response in terms of the second messenger level. The specific cell response was explained in terms of the enhancement of receptor-receptor interaction in cytosol due to activation of nearby occurring receptors of different kinds. It was also found that EK-12- NT(8-13) has a high affinity toward neurotensin receptor but a low affinity toward enkephalin receptor. To strengthen and prolong the activity of peptide hormones by simultaneous binding with two or more receptors in the target cell, enkephalin/lipid conjugates were immobilized on the surface of polymerized liposomes. It was found that multivalent ligands with a high receptor affinity were synthesized, which was sensibly influenced by the presence of a spacer chain connecting the enkephalin unit to the lipid part. It was found that the introduction of anionic charges to the polymerized liposomes strongly affected the receptor affinity of immobilized ligands.     

Synergistic stimulation of early events and DNA synthesis by phorbol esters, polypeptide growth factors, and retinoids in cultured fibroblasts

12-O-Tetradecanoyl-phorbol-13-acetate (TPA), in the absence of serum, acts synergistically with a range of polypeptide growth factors to stimulate DNA synthesis in quiescent Swiss 3T3 cells. These growth factors include epidermal growth factor (EGF), insulin, and the peptide produced by BHK cells transformed by SV-40 virus (fibroblast-derived growth factor, FDGF). Retinoids also show mitogenic synergism with TPA or polypeptide growth factors. The spectrum of mitogenic synergisms displayed by TPA are similar to those of vasopressin, a pituitary peptide. However, TPA and vasopressin do not synergistically interact to stimulate DNA synthesis in quiescent 3T3 cells. This suggests that TPA and vasopressin act via an identical biochemical pathway. Several lines of evidence suggest rapid postreceptor convergence of the mitogenic mechanisms of action of the hormone and the tumor promotor. Thus, vasopressin and TPA both inhibit EGF binding to cellular receptors. Furthermore, TPA and vasopressin induce a similar array of early events in quiescent cells--most strikingly, identical stimulation of Rb+ influx. Stimulation of ion flux is suggested as the possible convergence point of the pathway by which TPA and vasopressin act as mitogens.     

Mitogenic effects of murine serum and fibroblast growth factor on EGF nonproliferative variants of 3T3.

The enhanced ability of murine serum to support growth of 3T3 cells, when compared with fetal calf serum, is also evident on variants of 3T3 cells lacking the ability to bind epidermal growth factor (EGF). Variant 3T3 cell lines unable to bind EGF also retain a mitogenic response to fibroblast growth factor.     

Disulfide‐Unit Conjugation Enables Ultrafast Cytosolic Internalization of Antisense DNA and siRNA

Development of intracellular delivery methods for antisense DNA and siRNA is important. Previously reported methods using liposomes or receptor‐ligands take several hours or more to deliver oligonucleotides to the cytoplasm due to their retention in endosomes. Oligonucleotides modified with low molecular weight disulfide units at a terminus reach the cytoplasm 10 minutes after administration to cultured cells. This rapid cytoplasmic internalization of disulfide‐modified oligonucleotides suggests the existence of an uptake pathway other than endocytosis. Mechanistic analysis revealed that the modified oligonucleotides are efficiently internalized into the cytoplasm through disulfide exchange reactions with the thiol groups on the cellular surface. This approach solves several critical problems with the currently available methods for enhancing cellular uptake of oligonucleotides and may be an effective approach in the medicinal application of antisense DNA and siRNA.     

Engineering of PDMS surfaces for use in microsystems for capture and isolation of complex and biomedically important proteins: epidermal growth factor receptor as a model system

Elastomers based on poly(dimethylsiloxane) (PDMS) are promising materials for fabrication of a wide range of microanalytical systems due to their mechanical and optical properties and ease of processing. To date, however, quantitative studies that demonstrate reliable and reproducible methods for attachment of binding groups that capture complex receptor proteins of relevance to biomedical applications of PDMS microsystems have not been reported. Herein we describe methods that lead to the reproducible capture of a transmembrane protein, the human epidermal growth factor (EGF) receptor, onto PDMS surfaces presenting covalently immobilized antibodies for EGF receptor, and subsequent isolation of the captured receptor by mechanical transfer of the receptor onto a chemically functionalized surface of a gold film for detection. This result is particularly significant because the physical properties of transmembrane proteins make this class of proteins a difficult one to analyze. We benchmark the performance of antibodies to the human EGF receptor covalently immobilized on PDMS against the performance of the same antibodies physisorbed to conventional surfaces utilized in ELISA assays through the use of EGF receptor that was (32)P-radiolabeled in its autophosphorylation domain. These results reveal that two pan-reactive antibodies for the EGF receptor (clones H11 and 111.6) and one phosphospecific EGF receptor antibody (clone pY1068) capture the receptor on both PDMS and ELISA plates. When using H11 antibody to capture EGF receptor and subsequent treatment with a stripping buffer (NaOH and sodium dodecylsulfate) to isolate the receptor, the signal-to-background obtained using the PDMS surface was 82 : 1, exceeding the signal-to-background measured on the ELISA plate (<48 : 1). We also characterized the isolation of captured EGF receptor by mechanical contact of the PDMS surface with a chemically functionalized gold film. The efficiency of mechanical transfer of the transmembrane protein from the PDMS surface was found to be 75-81%. However, the transfer of non-specifically bound protein was substantially less than 75%, thus leading to the important finding that mechanical transfer of the EGF receptor leads to an approximately four-fold increase in signal-to-background from 20 : 1 to 88 : 1. The signal-to-background obtained following mechanical transfer is also better than that obtained using ELISA plates and stripping buffer (<48 : 1). The EGF receptor is a clinically important protein and the target of numerous anticancer agents and thus these results, when combined, provide guidance for the design of PDMS-based microanalytical systems for the capture and isolation of complex and clinically important transmembrane proteins.