Enhancement of epidermal growth factor signaling and activation of SRC kinase by gangliosides

In a recent study, inhibition of cellular ganglioside synthesis blocked growth factor-induced fibroblast proliferation. Conversely, enrichment of cell membrane gangliosides by ganglioside preincubation enhanced growth factor-elicited cell proliferation. In the absence of serum and growth factors, NeuNAcalpha2-3Galbeta1-3GalNAcbeta1-4(NeuNAcalpha2-3)Galbeta1-4Glcbeta1-1Cer (G(D1a)) acted like a growth factor when cells were pretreated with the ganglioside, stimulating proliferation of normal human dermal fibroblasts and Swiss 3T3 fibroblasts. In contrast, growth inhibition was observed when high concentrations of gangliosides were continuously present in the culture medium during incubation of fibroblasts with growth factors (Li, R., Manela, J., Kong, Y., and Ladisch, S. (2000) J. Biol. Chem. 275, 34213-34223). Here, we investigated the mechanisms whereby gangliosides elicit proliferation-coupled signaling in normal human dermal fibroblasts. Incubation of the fibroblasts with G(D1a) enhanced epidermal growth factor (EGF) receptor autophosphorylation and Ras and MAPK activation in a dose-dependent manner. Exposure of the cells to G(D1a) also enhanced the phosphorylation of Elk-1 by the activated MAPK. Brief pretreatment of the cells with PD98059 blocked the enhancing effect of gangliosides on EGF-induced MAPK activation. In the absence of serum and growth factors, G(D1a) incubation induced phosphorylation of Src kinase, Ras activation, and phosphorylation of MAPK and Elk-1 in a dose-dependent manner. The activation of Src kinase was confirmed by enhanced Src kinase activity. Brief treatment of the cells with PP1 blocked the activation of Src kinase and MAPK. Again, PD98059 treatment inhibited ganglioside-elicited MAPK phosphorylation. Among the gangliosides tested, G(D1a), was the most active molecule, whereas lactosylceramide was the least active one, indicating relative structural specificity of the ganglioside action. In conclusion, gangliosides promote fibroblast proliferation through enhancement of growth factor signaling and activation of Src kinase.     

Ligand-induced EGF receptor oligomerization is kinase-dependent and enhances internalization

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.     

Potentiation of Epidermal Growth Factor-Mediated Oncogenic Transformation by Sialidase NEU3 Leading to Src Activation

We previously demonstrated that sialidase NEU3, a key glycosidase for ganglioside degradation, is up-regulated in various human cancers, leading to increased cell invasion, motility and survival of cancer cells possibly through activation of EGF signaling. Its up-regulation is also important for promotion of the stage of colorectal carcinogenesis in vivo in human NEU3 transgenic mice treated with azoxymethane for the induction of aberrant crypt foci in the colon mucosa, accompanied by enhanced phosphorylation of EGF receptor (EGFR). To address whether the activation of EGF signaling by the sialidase is associated with oncogenic transformation, we here analyzed the effects of overexpression of NEU3 and EGFR in NIH-3T3 cells. When NEU3 was stably transfected with or without EGFR, it was associated with significant increases in clonogenic growth, clonogenicity on soft agar and in vivo tumor growth in nude mice either with or without the receptor overexpression in the presence of EGF, compared with the levels in their vector controls. Despite the fact that the endogenous level of EGFR is known to be extremely low in these cells, NEU3 significantly enhanced the phosphorylation of Akt and ERK, as well as that of the receptor. The NEU3-mediated activation was largely abrogated by the EGFR inhibitor AG1478 or PD153035, but significant clonogenic growth still remained. NEU3 was then found to activate Src kinase, and the clonogenicity was completely suppressed by an Src inhibitor, PP2. The activity-null mutants failed to activate Src and EGFR, indicating that ganglioside modulation by NEU3 may be necessary for the activation. NEU3 and Src were co-immunoprecipitated with EGFR in NEU3- and EGFR- transfected cells. These findings identify NEU3 as an essential participant in tumorigenesis through the EGFR/Src signaling pathway and a potential target for inhibiting EGFR-mediated tumor progression.     

Ganglioside depletion and EGF responses of human GM3 synthase-deficient fibroblasts

Recognition of important roles of gangliosides in normal and abnormal cell function has motivated pharmacological modification of cellular ganglioside content. However, constitutive depletion of gangliosides in untransformed human cells has not been reported. In this context, the recent identification of a kindred carrying a point mutation in the GM3 synthase [ST3Gal5, Siat9] gene (Simpson MA, Cross H, Proukakis C, Priestman DA, Neville DC, Reinkensmeier G, Wang H, Wiznitzer M, Gurtz K, Verganelaki A, Pryde A, Patton MA, Dwek RA, Butters TD, Platt FM, Crosby AH. 2004. Infantile-onset symptomatic epilepsy syndrome caused by a homozygous loss-of-function mutation of GM3 synthase. Nat Genet. 36:1225-1229) provided an opportunity to explore this possibility. We established primary cultures of skin fibroblasts of three patients homozygous for this autosomal recessive defect. They exhibited a 93% reduction in ganglioside content (0.8 +/- 0.2 nmol lipid-bound sialic acid per 10(7) cells versus 12.7 +/- 1.3 nmol per 10(7) normal fibroblasts). Importantly, this marked reduction was not compensated by the activation of an alternate pathway of ganglioside synthesis, as occurs in murine GM3 synthase knockout fibroblasts. Cell morphology appeared unaffected, but under stringent conditions EGF-induced proliferation and migration of the mutant fibroblasts were reduced by 80% and 60%, respectively. Probing potential explanations, we found that EGF binding (effective membrane EGF receptor (EGFR) number) was reduced by 52% (to 6.2 +/- 1.9 from 12.8 +/- 2.0 pmol/10(8) normal fibroblasts, P < 0.01), despite normal total EGFR protein. EGFR activation was likewise reduced as was EGF-induced Rho/Rac1 phosphorylation, which is associated with cell migration. We conclude that this GM3 synthase point mutation almost completely depletes human fibroblast cellular gangliosides, dampens membrane EGFR activation, and modulates related critical cell functions such as proliferation and migration. These cells offer a valuable model for the study of ganglioside modulation of cell function.     

The hominoid-specific oncogene TBC1D3 activates Ras and modulates epidermal growth factor receptor signaling and trafficking

Hominoid- and human-specific genes may have evolved to modulate signaling pathways of a higher order of complexity. TBC1D3 is a hominoid-specific oncogene encoded by a cluster of eight paralogs on chromosome 17. Initial work indicates that TBC1D3 is widely expressed in human tissues ( Hodzic, D., Kong, C., Wainszelbaum, M. J., Charron, A. J., Su, X., and Stahl, P. D. (2006) Genomics 88, 731-736 ). In this study, we show that TBC1D3 expression has a powerful effect on cell proliferation that is further enhanced by epidermal growth factor (EGF) in both human and mouse cell lines. EGF activation of the Erk and protein kinase B/Akt pathways is enhanced, both in amplitude and duration, by TBC1D3 expression, whereas RNA interference silencing of TBC1D3 suppresses the activation. Light microscopy and Western blot experiments demonstrate that increased signaling in response to EGF is coupled with a significant delay in EGF receptor (EGFR) trafficking and degradation, which significantly extends the life span of EGFR. Moreover, TBC1D3 suppresses polyubiquitination of the EGFR and the recruitment of c-Cbl. Using the Ras binding domain of Raf1 to monitor GTP-Ras we show that TBC1D3 expression enhances Ras activation in quiescent cells, which is further increased by EGF treatment. We speculate that TBC1D3 may alter Ras GTP loading. We conclude that the expression of TBC1D3 generates a delay in EGFR degradation, a decrease in ubiquitination, and a failure to recruit adapter proteins that ultimately dysregulate EGFR signal transduction and enhance cell proliferation. Altered growth factor receptor trafficking and GTP-Ras turnover may be sites where recently evolved genes such as TBC1D3 selectively modulate signaling in hominoids and humans.     

Effects of gangliosides GM3 and De-N-acetyl GM3 on epidermal growth factor receptor kinase activity and cell growth.

Previously it was reported (Bremer, E.G., Schlessinger, J., and Hakomori, S.-I. (1986) J. Biol. Chem. 261, 2434-2440) that ganglioside GM3 inhibited epidermal growth factor (EGF)-stimulated phosphorylation of the EGF receptor in Triton X-100-treated preparations of human epidermoid carcinoma (A431) cell membranes. In addition, these authors reported that GM3 inhibited the growth of A431 cells. In contrast, a modified ganglioside, de-N-acetyl GM3, enhanced the EGF-dependent tyrosine kinase activity of the EGF receptor. In this work and in subsequent studies (Hanai, N., Dohi, T., Nores, G. A., and Hakomori, S.-I. (1988) J. Biol. Chem. 263, 6296-6301), the tyrosine kinase activity of the receptor from A431 cell membranes was assayed in the presence of Triton X-100. In this report, we confirm that GM3 inhibited and de-N-acetyl GM3 stimulated EGF receptor autophosphorylation in the presence of Triton X-100. However, in the absence of detergents, ganglioside GM3 inhibited EGF-stimulated receptor autophosphorylation, whereas de-N-acetyl GM3 had no effect on EGF-stimulated receptor autophosphorylation. The effects of these gangliosides on receptor autophosphorylation were measured in both A431 cell plasma membranes and in 3T3 cell membranes permeabilized to [32P]ATP by a freeze-thaw procedure, in intact A431 cells permeabilized with alamethicin, and in intact A431 cells grown in the presence of [32P]orthophosphate. Thus, the inhibitory effect of GM3 on receptor autophosphorylation was demonstrated in the presence and in the absence of detergent; the stimulatory effect of de-N-acetyl GM3 was observed only in the presence of detergent. We also demonstrate that ganglioside GM3 inhibited EGF-stimulated growth of transfected murine fibroblasts (3T3) that express the gene for human EGF receptor (Velu, T. J., Beguinot, L., Vass, W. C., Zhang, K., Pastan, I., and Lowy, D. R. (1989) J. Cell. Biochem. 39, 153-166). De-N-acetyl ganglioside GM3 had no effect on the growth of these cells. Growth of control fibroblasts, which lack endogenous EGF receptors (Pruss, R. M., and Herschman, H. R. (1977) Proc. Natl. Acad. Sci. U.S.A. 74, 3918-3921), was not affected by the presence of either ganglioside. Similarly, ganglioside GM3, but not de-N-acetyl ganglioside GM3, inhibited the EGF-dependent incorporation of [3H]thymidine into DNA by transfected fibroblasts. Incorporation of labeled thymidine into DNA of control fibroblasts was not affected by the presence of either ganglioside. These studies indicate that ganglioside GM3, but not its deacetylated analogue, can affect EGF receptor kinase activity in intact membranes.(ABSTRACT TRUNCATED AT 400 WORDS)     

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.     

A novel positive feedback loop mediated by the docking protein Gab1 and phosphatidylinositol 3-kinase in epidermal growth factor receptor signaling

The Gab1 protein is tyrosine phosphorylated in response to various growth factors and serves as a docking protein that recruits a number of downstream signaling proteins, including phosphatidylinositol 3-kinase (PI-3 kinase). To determine the role of Gab1 in signaling via the epidermal growth factor (EGF) receptor (EGFR) we tested the ability of Gab1 to associate with and modulate signaling by this receptor. We show that Gab1 associates with the EGFR in vivo and in vitro via pTyr sites 1068 and 1086 in the carboxy-terminal tail of the receptor and that overexpression of Gab1 potentiates EGF-induced activation of the mitogen-activated protein kinase and Jun kinase signaling pathways. A mutant of Gab1 unable to bind the p85 subunit of PI-3 kinase is defective in potentiating EGFR signaling, confirming a role for PI-3 kinase as a downstream effector of Gab1. Inhibition of PI-3 kinase by a dominant-interfering mutant of p85 or by Wortmannin treatment similarly impairs Gab1-induced enhancement of signaling via the EGFR. The PH domain of Gab1 was shown to bind specifically to phosphatidylinositol 3,4,5-triphosphate [PtdIns(3,4,5)P3], a product of PI-3 kinase, and is required for activation of Gab1-mediated enhancement of EGFR signaling. Moreover, the PH domain mediates Gab1 translocation to the plasma membrane in response to EGF and is required for efficient tyrosine phosphorylation of Gab1 upon EGF stimulation. In addition, overexpression of Gab1 PH domain blocks Gab1 potentiation of EGFR signaling. Finally, expression of the gene for the lipid phosphatase PTEN, which dephosphorylates PtdIns(3,4, 5)P3, inhibits EGF signaling and translocation of Gab1 to the plasma membrane. These results reveal a novel positive feedback loop, modulated by PTEN, in which PI-3 kinase functions as both an upstream regulator and a downstream effector of Gab1 in signaling via the EGFR.     

Asp-960/Glu-961 Controls the Movement of the C-terminal Tail of the Epidermal Growth Factor Receptor to Regulate Asymmetric Dimer Formation

The epidermal growth factor (EGF) receptor is a tyrosine kinase that dimerizes in response to ligand binding. Ligand-induced dimerization of the extracellular domain of the receptor promotes formation of an asymmetric dimer of the intracellular kinase domains, leading to stimulation of the tyrosine kinase activity of the receptor. We recently monitored ligand-promoted conformational changes within the EGF receptor in real time using luciferase fragment complementation imaging and showed that there was significant movement of the C-terminal tail of the EGF receptor following EGF binding (Yang, K. S., Ilagan, M. X. G., Piwnica-Worms, D., and Pike, L. J. (2009) J. Biol. Chem. 284, 7474–7482). To investigate the structural basis for this conformational change, we analyzed the effect of several mutations on the kinase activity and luciferase fragment complementation activity of the EGF receptor. Mutation of Asp-960 and Glu-961, two residues at the beginning of the C-terminal tail, to alanine resulted in a marked enhancement of EGF-stimulated kinase activity as well as enhanced downstream signaling. The side chain of Asp-960 interacts with that of Ser-787. Mutation of Ser-787 to Phe, which precludes this interaction, also leads to enhanced receptor kinase activity. Our data are consistent with the hypothesis that Asp-960/Glu-961 represents a hinge or fulcrum for the movement of the C-terminal tail of the EGF receptor. Mutation of these residues destabilizes this hinge, facilitating the formation of the activating asymmetric dimer and leading to enhanced receptor signaling.     

Flotillin-1/reggie-2 protein plays dual role in activation of receptor-tyrosine kinase/mitogen-activated protein kinase signaling

Our previous work has shown that the membrane microdomain-associated flotillin proteins are potentially involved in epidermal growth factor (EGF) receptor signaling. Here we show that knockdown of flotillin-1/reggie-2 results in reduced EGF-induced phosphorylation of specific tyrosines in the EGF receptor (EGFR) and in inefficient activation of the downstream mitogen-activated protein (MAP) kinase and Akt signaling. Although flotillin-1 has been implicated in endocytosis, its depletion affects neither the endocytosis nor the ubiquitination of the EGFR. However, EGF-induced clustering of EGFR at the cell surface is altered in cells lacking flotillin-1. Furthermore, we show that flotillins form molecular complexes with EGFR in an EGF/EGFR kinase-independent manner. However, knockdown of flotillin-1 appears to affect the activation of the downstream MAP kinase signaling more directly. We here show that flotillin-1 forms a complex with CRAF, MEK1, ERK, and KSR1 (kinase suppressor of RAS) and that flotillin-1 knockdown leads to a direct inactivation of ERK1/2. Thus, flotillin-1 plays a direct role during both the early phase (activation of the receptor) and late (activation of MAP kinases) phase of growth factor signaling. Our results here unveil a novel role for flotillin-1 as a scaffolding factor in the regulation of classical MAP kinase signaling. Furthermore, our results imply that other receptor-tyrosine kinases may also rely on flotillin-1 upon activation, thus suggesting a general role for flotillin-1 as a novel factor in receptor-tyrosine kinase/MAP kinase signaling.     

Regulation of EGF-induced ERK/MAPK activation and EGFR internalization by G protein-coupled receptor kinase 2

G protein-coupled receptor kinases （GRKs） mediate agonist-induced phosphorylation and desensitization of various G protein-coupled receptors （GPCRs）. We investigate the role of GRK2 on epidermal growth factor （EGF） receptor signaling, including EGF-induced extracellular signal-regulated kinase and mitogen-activated protein kinase （ERK/MAPK） activation and EGFR internalization. Immunoprecipitation and immunofluorescence experiments show that EGF stimulates GRK2 binding to EGFR complex and GRK2 translocating from cytoplasm to the plasma membrane in human embryonic kidney 293 cells. Western blotting assay shows that EGF-induced ERK/MAPK phosphorylation increases 1.9-fold, 1.1-fold and 1.5fold （P〈0.05） at time point 30, 60 and 120 min, respectively when the cells were transfected with GRK2,suggesting the regulatory role of GRK2 on EGF-induced ERK/MAPK activation. Flow cytometry experiments show that GRK2 overexpression has no effect on EGF-induced EGFR internalization, however, it increases agonist-induced G protein-coupled δ5 opioid receptor internalization by approximately 40% （P〈0.01）. Overall,these data suggest that GRK2 has a regulatory role in EGF-induced ERK/MAPK activation, and that the mechanisms underlying the modulatory role of GRK2 in EGFR and GPCR signaling pathways are somewhat different at least in receptor internalization.     

EGF transregulates opioid receptors through EGFR-mediated GRK2 phosphorylation and activation

G protein-coupled receptor (GPCR) kinases (GRKs) are key regulators of GPCR function. Here we demonstrate that activation of epidermal growth factor receptor (EGFR), a member of receptor tyrosine kinase family, stimulates GRK2 activity and transregulates the function of G protein-coupled opioid receptors. Our data showed that EGF treatment promoted DOR internalization induced by DOR agonist and this required the intactness of GRK2-phosphorylation sites in DOR. EGF stimulation induced the association of GRK2 with the activated EGFR and the translocation of GRK2 to the plasma membrane. After EGF treatment, GRK2 was phosphorylated at tyrosyl residues. Mutational analysis indicated that EGFR-mediated phosphorylation occurred at GRK2 N-terminal tyrosyl residues previously shown as c-Src phosphorylation sites. However, c-Src activity was not required for EGFR-mediated phosphorylation of GRK2. In vitro assays indicated that GRK2 was a direct interactor and a substrate of EGFR. EGF treatment remarkably elevated DOR phosphorylation in cells expressing the wild-type GRK2 in an EGFR tyrosine kinase activity-dependent manner, whereas EGF-stimulated DOR phosphorylation was greatly decreased in cells expressing mutant GRK2 lacking EGFR tyrosine kinase sites. We further showed that EGF also stimulated internalization of mu-opioid receptor, and this effect was inhibited by GRK2 siRNA. These data indicate that EGF transregulates opioid receptors through EGFR-mediated tyrosyl phosphorylation and activation of GRK2 and propose GRK2 as a mediator of cross-talk from RTK to GPCR signaling pathway.     

Plakophilin-2 Promotes Tumor Development by Enhancing Ligand-Dependent and -Independent Epidermal Growth Factor Receptor Dimerization and Activation

Epidermal growth factor (EGF) receptor (EGFR) has been implicated in tumor development and invasion. Dimerization and autophosphorylation of EGFR are the critical events for EGFR activation. However, the regulation of EGF-dependent and EGF-independent dimerization and phosphorylation of EGFR has not been fully understood. Here, we report that cytoplasmic protein plakophilin-2 (PKP2) is a novel positive regulator of EGFR signaling. PKP2 specifically interacts with EGFR via its N-terminal head domain. Increased PKP2 expression enhances EGF-dependent and EGF-independent EGFR dimerization and phosphorylation. Moreover, PKP2 knockdown reduces EGFR phosphorylation and attenuates EGFR-mediated signal activation, resulting in a significant decrease in proliferation and migration of cancer cells and tumor development. Our results indicate that PKP2 is a novel activator of the EGFR signaling pathway and a potential new drug target for inhibiting tumor growth.     

Ganglioside induces caveolin-1 redistribution and interaction with the epidermal growth factor receptor

Although caveolin-1 is thought to facilitate the interaction of receptors and signaling components, its role in epidermal growth factor receptor (EGFR) signaling remains poorly understood. Ganglioside GM3 inhibits EGFR autophosphorylation and may thus affect the interaction of caveolin-1 and the EGFR. We report here that endogenous overexpression of GM3 leads to the clustering of GM3 on the cell membrane of the keratinocyte-derived SCC12 cell line and promotes co-immunoprecipitation of caveolin-1 and GM3 with the EGFR. Overexpression of GM3 does not affect EGFR distribution but shifts caveolin-1 to the detergent-soluble, EGFR-containing region; consistently, caveolin-1 is retained in the detergent-insoluble membrane when ganglioside is depleted. GM3 overexpression inhibits EGFR tyrosine phosphorylation and receptor dimerization and concurrently increases both the content and tyrosine phosphorylation of EGFR-associated caveolin-1, providing evidence that tyrosine phosphorylation of caveolin-1 inhibits EGFR signaling. Consistently, depletion of ganglioside both increases EGFR phosphorylation and prevents the EGF-induced tyrosine phosphorylation of caveolin-1. GM3 also induces delayed serine phosphorylation of EGFR-unassociated caveolin-1, suggesting a role for serine phosphorylation of caveolin-1 in regulating EGFR signaling. These studies suggest that GM3 modulates the caveolin-1/EGFR association and is critical for the EGF-induced tyrosine phosphorylation of caveolin-1 that is associated with its inhibition of EGFR activation.     

Role of somatostatin receptor 1 and 5 on epidermal growth factor receptor mediated signaling

Epidermal growth factor (EGF) regulates normal and tumor cell proliferation via epidermal growth factor receptor (EGFR) phosphorylation, homo- or heterodimerization and activation of mitogen-activated protein kinases (MAPKs) and PI3K/AKT cell survival pathways. In contrast, SST via activation of five different receptor subtypes inhibits cell proliferation and has been potential target in tumor treatment. To gain further insight for the effect of SSTRs on EGFR activated signaling, we determine the role of SSTR1 and SSTR1/5 in human embryonic kidney (HEK) 293 cells. We here demonstrate that cells transfected with SSTR1 or SSTR1/5 negatively regulates EGF mediated effects attributed to the inhibition of EGFR phosphorylation, MAPKs as well as the cell survival signaling. Furthermore, SSTR effects were significantly enhanced in cells when EGFR was knock down using siRNA or treated with selective antagonist (AG1478). Most importantly, the presence of SSTR in addition to modulating signaling pathways leads to the dissociation of the constitutive and EGF induced heteromeric complex of EGFR/ErbB2. Furthermore, cells cotransfected with SSTR1/5 display pronounced effect of SST on the signaling and dissociation of the EGFR/ErbB2 heteromeric complex than the cells expressing SSTR1 alone. Taken together this study provides the first evidence that the presence of SSTR controls EGF mediated cell survival pathway via dissociation of ErbB heteromeric complex. We propose that the activation of SSTR and blockade of EGFR might serve novel therapeutic approach in inhibition of tumor proliferation.     

Utilization of a receptor reserve for effective amplification of mitogenic signaling by an epidermal growth factor mutant deficient in receptor activation

The idea of a receptor reserve in mediating cellular function is well known but direct biochemical evidence has not been easy to obtain. This study stems from our results showing that L15 of epidermal growth factor (EGF) is important in both EGF receptor (EGFR) binding and activation, and the L15A analog of human EGF (hEGF) partially uncouples EGFR binding from EGFR activation (Nandagopal et al., [1996] Protein Engng 9:781-788). We address the cellular mechanism of mitogenic signal amplification by EGFR tyrosine kinase in response to L15A hEGF. L15A is partially impaired in receptor dimerization, shown by chemical cross-linking and allosteric activation of EGFR in a substrate phosphorylation assay. Immunoprecipitation experiments reveal, however, that L15A can induce EGFR autophosphorylation in intact murine keratinocytes by utilizing spare receptors, the ratio of total phosphotyrosine content per receptor being significantly lower than that elicited by wild-type. This direct biochemical evidence, based on function, of utilization of a receptor reserve for kinase stimulation suggests that an EGF variant can activate varying receptor numbers to generate the same effective response. L15A-activated receptors can stimulate mitogen-activated protein kinase (MAPK) that is important for mitogenesis. The lack of linear correlation between levels of receptor dimerization, autophosphorylation, and MAPK activation suggests that signal amplification is mediated by cooperative effects. Flow cytometric analyses show that the percentages of cells which proliferate in response to 1 nM L15A and their rate of entry into S-phase are both decreased relative to 1 nM wild-type, indicating that MAPK activation alone is insufficient for maximal stimulation of mitogenesis. Higher concentrations of L15A reverse this effect, indicating that L15A and wild-type differ in the number of receptors each activates to induce the threshold response, which may be attained by cooperative activation of receptor dimers/oligomers by van der Waal's weak forces of attraction. The maintenance of a receptor reserve underscores an effective strategy in cell survival.     

EGF receptor function is required in late G(1) for cell cycle progression induced by bombesin and bradykinin

We examined therole of epidermal growth factor (EGF) receptor (EGFR) tyrosine kinaseactivation in G protein-coupled receptor (GPCR) agonist-inducedmitogenesis in Swiss 3T3 and Rat-1 cells. Addition of EGFR tyrosinekinase inhibitors (e.g., tyrphostin AG-1478) abrogated bombesin-inducedextracellular signal-regulated kinase (ERK) activation in Rat-1 cellsbut not in Swiss 3T3 cells, indicating the importance of cell contextin determining the role of EGFR in ERK activation. In strikingcontrast, treatment with tyrphostin AG-1478 markedly (~70%)inhibited DNA synthesis induced by bombesin in both Swiss 3T3 and Rat-1cells. Similar inhibition of bombesin-induced DNA synthesis in Swiss3T3 cells was obtained using four structurally different inhibitors ofEGFR tyrosine kinase. Furthermore, kinetic analysis indicates that EGFRfunction is necessary for bombesin-induced mitogenesis in mid-lateG₁ in both Swiss 3T3 and Rat-1 cells. Our results indicatethat EGFR kinase activity is necessary in mid-late G₁ forpromoting the accumulation of cyclins D1 and E and implicate EGFRfunction in the coupling of GPCR signaling to the activation of thecell cycle.

     

Signal characteristics of G protein-transactivated EGF receptor.

The epidermal growth factor receptor (EGFR) tyrosine kinase recently was identified as providing a link to mitogen-activated protein kinase (MAPK) in response to G protein-coupled receptor (GPCR) agonists in Rat-1 fibroblasts. This cross-talk pathway is also established in other cell types such as HaCaT keratinocytes, primary mouse astrocytes and COS-7 cells. Transient expression of either Gq- or Gi-coupled receptors in COS-7 cells allowed GPCR agonist-induced EGFR transactivation, and lysophosphatidic acid (LPA)-generated signals involved the docking protein Gab1. The increase in SHC tyrosine phosphorylation and MAPK stimulation through both Gq- and Gi-coupled receptors was reduced strongly upon selective inhibition of EGFR function. Inhibition of phosphoinositide 3-kinase did not affect GPCR-induced stimulation of EGFR tyrosine phosphorylation, but inhibited MAPK stimulation, upon treatment with both GPCR agonists and low doses of EGF. Furthermore, the Src tyrosine kinase inhibitor PP1 strongly interfered with LPA- and EGF-induced tyrosine phosphorylation and MAPK activation downstream of EGFR. Our results demonstrate an essential role for EGFR function in signaling through both Gq- and Gi-coupled receptors and provide novel insights into signal transmission downstream of EGFR for efficient activation of the Ras/MAPK pathway.     

Human mammary epithelial cells rapidly exchange empty EGFR between surface and intracellular pools

Binding of ligand to the epidermal growth factor receptor (EGFR) initiates a series of processes including activation of the intrinsic EGFR tyrosine kinase, receptor autophosphorylation, and the assembly of active signaling complexes at the plasma membrane. Concomitantly, receptor trafficking is initiated, and the receptor is ultimately delivered to the lysosome, where it is degraded. Virtually all studies on EGFR trafficking have used fibroblasts and transformed cells. Because EGFR exerts a potent effect on the physiology of epithelial cells, we examined the regulation of EGFR activity and trafficking in nontransformed human mammary epithelial cells (HMEC). We found that HMEC that displayed a luminal phenotype were largely unresponsive to EGF and maintained a majority of their EGFR at the cell surface. In contrast, HMEC with a basal phenotype were highly responsive to EGF and, at steady state in the absence of exogenous ligand, distributed empty EGFR into intracellular pools. Maintenance of the intracellular pools was a direct consequence of specific and rapid endocytosis of the empty EGFR. The trafficking pattern was EGFR specific, used coated pits, and did not require receptor tyrosine kinase activity. Such an mechanism redistributes EGFR signaling potential among different membrane domains and into vesicles with unique biochemical microenviroments. In addition, our data show that EGFR endocytosis can be regulated in the absence of ligand binding and receptor activation in a cell-type-specific manner. J. Cell. Physiol. 180:448–460, 1999. © 1999 Wiley-Liss, Inc.