Bipartite inhibition of Drosophila epidermal growth factor receptor by the extracellular and transmembrane domains of Kekkon1

In Drosophila, signaling by the epidermal growth factor receptor (EGFR) is required for a diverse array of developmental decisions. Essential to these decisions is the precise regulation of the receptor's activity by both stimulatory and inhibitory molecules. To better understand the regulation of EGFR activity we investigated inhibition of EGFR by the transmembrane protein Kekkon1 (Kek1). Kek1 encodes a molecule containing leucine-rich repeats (LRR) and an immunoglobulin (Ig) domain and is the founding member of the Drosophila Kekkon family. Here we demonstrate with a series of Kek1-Kek2 chimeras that while the LRRs suffice for EGFR binding, inhibition in vivo requires the Kek1 juxta/transmembrane region. We demonstrate directly, and using a series of Kek1-EGFR chimeras, that Kek1 is not a phosphorylation substrate for the receptor in vivo. In addition, we show that EGFR inhibition is unique to Kek1 among Kek family members and that this function is not ligand or tissue specific. Finally, we have identified a unique class of EGFR alleles that specifically disrupt Kek1 binding and inhibition, but preserve receptor activation. Interestingly, these alleles map to domain V of the Drosophila EGFR, a region absent from the vertebrate receptors. Together, our results support a model in which the LRRs of Kek1 in conjunction with its juxta/transmembrane region direct association and inhibition of the Drosophila EGFR through interactions with receptor domain V.     

Receptor heterodimerization: essential mechanism for platelet-derived growth factor-induced epidermal growth factor receptor transactivation

Previous studies showed that the epidermal growth factor receptor (EGFR) can be transactivated by platelet-derived growth factor (PDGF) stimulation and that EGFR transactivation is required for PDGF-stimulated cell migration. To investigate the mechanism for cross talk between the PDGF beta receptor (PDGFbetaR) and the EGFR, we stimulated rat aortic vascular smooth muscle cells (VSMC) with 20 ng of PDGF/ml. Transactivation of the EGFR, defined by receptor tyrosine phosphorylation, occurred with the same time course as PDGFbetaR activation. Basal formation of PDGFbetaR-EGFR heterodimers was shown by coimmunoprecipitation studies, and interestingly, disruption of this receptor heterodimer abolished EGFR transactivation. Breakdown of the heterodimer was observed when VSMC were pretreated with antioxidants or with a Src family kinase inhibitor. Disruption of heterodimers decreased ERK1 and ERK2 activation by PDGF. Although PDGF-induced PDGFbetaR activation was abolished after pretreatment with 1 microM AG1295 (a specific PDGF receptor kinase inhibitor), EGFR transactivation was still observed, indicating that PDGFbetaR kinase activity is not required. In conclusion, our data demonstrate that the PDGFbetaR and the EGFR form PDGFbetaR-EGFR heterodimers basally, and we suggest that heterodimers represent a novel signaling complex which plays an important role in PDGF signal transduction.     

MICAL-like1 mediates epidermal growth factor receptor endocytosis

Small GTPase Rabs are required for membrane protein sorting/delivery to precise membrane domains. Rab13 regulates epithelial tight junction assembly and polarized membrane transport. Here we report that Molecule Interacting with CasL (MICAL)-like1 (MICAL-L1) interacts with GTP-Rab13 and shares a similar domain organization with MICAL. MICAL-L1 has a calponin homology (CH), LIM, proline rich and coiled-coil domains. It is associated with late endosomes. Time-lapse video microscopy shows that green fluorescent protein-Rab7 and mcherry-MICAL-L1 are present within vesicles that move rapidly in the cytoplasm. Depletion of MICAL-L1 by short hairpin RNA does not alter the distribution of a late endosome/lysosome-associated protein but affects the trafficking of epidermal growth factor receptor (EGFR). Overexpression of MICAL-L1 leads to the accumulation of EGFR in the late endosomal compartment. In contrast, knocking down MICAL-L1 results in the distribution of internalized EGFR in vesicles spread throughout the cytoplasm and promotes its degradation. Our data suggest that the N-terminal CH domain associates with the C-terminal Rab13 binding domain (RBD) of MICAL-L1. The binding of Rab13 to RBD disrupts the CH/RBD interaction, and may induce a conformational change in MICAL-L1, promoting its activation. Our results provide novel insights into the MICAL-L1/Rab protein complex that can regulate EGFR trafficking at late endocytic pathways.     

Signaling by the Drosophila epidermal growth factor receptor pathway during development

In 1997 we wrote a review entitled "A thousand and one roles for the Drosophila epidermal growth factor (EGF) receptor (DER/EGFR)." We are not there yet in terms of the number of developmental roles assigned to this receptor in Drosophila. Nevertheless, DER has certainly emerged as one of the key players in development, since it is used repeatedly to direct cell fate choices, cell division, cell survival, and migration. A battery of activating ligands and an inhibitory ligand achieves this versatility. For the ligands that are produced as membrane-bound precursors, trafficking and processing are the key regulatory steps, determining the eventual temporal and spatial pattern of receptor activation. In most cases DER is activated at a short range, in the cells adjacent to the ones producing the active ligand. This activation dictates a binary choice. In some instances DER is also activated over a longer range, and multiple cell fate choices may be induced, according to its level of activation. A battery of negative feedback loops assures the limited range of DER induction. The distinct responses to DER activation in the different tissues depend upon combinatorial interactions with other signaling pathways and tissue-specific factors, at the level of target-gene regulation.     

Activation of BAD by therapeutic inhibition of epidermal growth factor receptor and transactivation by insulin-like growth factor receptor

Novel cancer chemotherapeutics are required to induce apoptosis by activating pro-apoptotic proteins. Both epidermal growth factor (EGF) and insulin-like growth factor (IGF) provide potent survival stimuli in many epithelia, and activation of their receptors is commonly observed in solid human tumors. Here we demonstrate that blockade of the EGF receptor by a new drug in phase III clinical trails for cancer, ZD1839, potently induces apoptosis in mammary epithelial cell lines and primary cultures, as well as in a primary pleural effusion from a breast cancer patient. We identified the mechanism of apoptosis induction by ZD1839. We showed that it prevents cell survival by activating the pro-apoptotic protein BAD. Moreover, we demonstrate that IGF transactivates the EGF receptor and that ZD1839 blocks IGF-mediated phosphorylation of MAPK and BAD. Many cancer therapies kill tumor cells by inducing apoptosis as a consequence of targeting DNA; however, the threshold at which apoptosis can be triggered through DNA damage is often different from that in normal cells. Our results indicate that by targeting a growth factor-mediated survival signaling pathway, BAD phosphorylation can be manipulated therapeutically to induce apoptosis.     

In vivo inhibition of epidermal growth factor receptor autophosphorylation prevents receptor internalization

The question whether epidermal growth factor (EGF)-induced receptor endocytosis requires the prior autophosphorylation via the EGF receptor (EGFR) kinase domain has been a matter of long-standing debate. In the airway epithelial cell line NCI-H292, the EGFR kinase domain inhibitor BIBW 2948 BS was found to inhibit both autophosphorylation and subsequent internalization of the endogenous EGFR with similar IC₅₀ values. Applying an ex vivo EGFR internalization assay in a clinical study, the in vivo effect of inhalatively administered BIBW 2948 BS was determined directly at the targeted receptor in airway tissues from COPD patients. In these experiments, the in vivo inhibition of the EGFR kinase domain prevented the EGF-induced internalization of EGFR.     

A radioimmunoassay for human epidermal growth factor receptor

The development of a radioimmunoassay (RIA) for the human epidermal growth factor receptor solubilized with nonionic detergents which employs iodinated epidermal growth factor (125I-EGF) as the specific ligand is described. A monoclonal antibody (R1) that binds specifically to human EGF receptors [Waterfield, M. D., et al. (1982) J. Cell Biochem. 20, 149-161] was used to separate solubilized receptors saturated with 125I-EGF from free ligand by absorption to protein A-Sepharose, and the bound radioactivity was determined. The RIA was linear when increasing amounts of solubilized membrane protein were added and, when compared to the standard polyethylene glycol assay, was more reproducible. In addition, the background nonspecific binding obtained in the presence of a hundred-fold excess of unlabeled EGF was less in the RIA. Substitution of normal mouse serum for the monoclonal antibody gave very low nonspecific background ligand binding and avoided the use of large amounts of unlabeled EGF in the assay. Two major classes of binding sites for EGF were observed in membrane preparations from the cervical carcinoma cell line A431 or from normal human placental tissue. These were present in approximately equal amounts, with apparent dissociation constants of 4 X 10(-10) and 4 X 10(-9) M. Upon solubilization with the nonionic detergent Triton X-100, only one class of EGF binding sites was detected in both cases, with a dissociation constant of 3 X 10(-8) M. The RIA can be used to monitor receptor purification and for quantitation of receptor number and affinity in various cell types.     

Solubilization of membrane receptor for epidermal growth factor.

The membrane receptor for epidermal growth factor (EGF) has been solubilized from A-431 tumor cells using Triton X-100. Operational criteria used to define solubilization include failure of the binding activity to be pelleted after centrifugation at 90,000 x g for 1.5 hrs and the requirement for polyethylene glycol precipitation to detect ¹²⁵I-EGF: receptor complexes on membrane filters. Properties of the solubilized EGF are characterized and compared to the properties of the particulate receptor. The specific binding capacity of the solubilized EGF receptor was 8.0 picomoles ¹²⁵I-EGF bound per mg protein--approximately 60% of the binding capacity of particulate receptor preparations. Also, solubilization of the EGF receptor resulted in a 10-fold decrease in the affinity of the receptor for ¹²⁵I-EGF.     

Transactivation of epidermal growth factor receptor by insulin-like growth factor 1 requires basal hydrogen peroxide

Insulin-like growth factor (IGF-1) plays an important role in prostate cancer development. Recent studies suggest that IGF-1 has mitogenic action through epidermal growth factor receptor (EGFR). However, the mechanism remains largely unknown. Here, we demonstrated in prostate cancer DU145 cells that IGF-1 induced EGFR transactivation, leading to ERK activation. Matrix metalloproteinase-mediated shedding of heparin-binding EGF is involved in this process. Antioxidants and catalase inhibited IGF-1-stimulated EGFR phosphorylation, indicating that H(2)O(2) is required for EGFR activation. However, exogenous H(2)O(2) did not activate EGFR or IGF-1R in DU145 cells. IGF-1 did not induced production of H(2)O(2) in DU145 cells. Our results suggest that transactivation of EGFR by IGF-1 requires basal intracellular H(2)O(2) in DU145 cells.     

Mechanism of inhibition of the Drosophila and mammalian EGF receptors by the transmembrane protein Kekkon 1

The transmembrane protein Kekkon 1 (Kek1) has previously been shown to act in a negative feedback loop to downregulate the Drosophila Epidermal Growth Factor Receptor (DER) during oogenesis. We show that this protein plays a similar role in other DER-mediated developmental processes. Structure-function analysis reveals that the extracellular Leucine-Rich Repeat (LRR) domains of Kek1 are critical for its function through direct association with DER, whereas its cytoplasmic domain is required for apical subcellular localization. In addition, the use of chimeric proteins between Kek1 extracellular and transmembrane domains fused to DER intracellular domain indicates that Kek1 forms an heterodimer with DER in vivo. To characterize more precisely the mechanism underlying the Kek1/DER interaction, we used mammalian ErbB/EGFR cell-based assays. We show that Kek1 is capable of physically interacting with each of the known members of the mammalian ErbB receptor family and that the Kek1/EGFR interaction inhibits growth factor binding, receptor autophosphorylation and Erk1/2 activation in response to EGF. Finally, in vivo experiments show that Kek1 expression potently suppresses the growth of mouse mammary tumor cells derived from aberrant ErbB receptors activation, but does not interfere with the growth of tumor cells derived from activated Ras. Our results underscore the possibility that Kek1 may be used experimentally to inhibit ErbB receptors and point to the possibility that, as yet uncharacterized, mammalian transmembrane LRR proteins might act as modulators of growth factor signalling.     

Enzyme-linked immunosorbent assay for the epidermal growth factor receptor

An enzyme-linked immunosorbent assay (ELISA) for the epidermal growth factor (EGF) receptor was developed using three different antibody preparations, one of which is commercially available. Using one of the antisera (986), the assay could detect as few as 200 × 10⁶ receptors. This is equal to 0.332 fmol. This sensitivity means that a minimum of 100 A-431 cells (human carcinoma) or 5,000 normal cells are needed to quantitate the number of EGF receptors. Two of the antisera (986 and 451) recognized EGF receptors from placental tissue. EGF receptors from as little as 667 ng of placental membrane protein were detectable. The assay is highly species specific, with the sensitivity for the EGF receptor from different species dependent on the antiserum used. The commercial antibody, 29.1, had especially strong reactivity against pig and dog EGF receptors. An ELISA using this antibody had the capacity to detect the number of EGF receptors in 10 μg of liver membrane protein. The assay is sensitive to receptor conformation. The binding of antisera 986 and 451 to 1% sodium dodecyl sulfate (SDS)-denatured receptor was reduced. The binding of antibody 29.1 was impaired by the presence of 1% Triton X-100 but not the same levels of Tween-20 or SDS. In addition to being a sensitive technique for the quantitation of the EGF receptor, this assay is very rapid, taking a total of 4 h. The microtiter dish format also allows hundreds of samples to be assayed at once. By using the appropriate antiserum and standards, the EGF receptor can be quantitated in tissues from humans, dogs, pigs, and mice.     

Novel mechanism for regulation of epidermal growth factor receptor endocytosis revealed by protein kinase A inhibition

Current models put forward that the epidermal growth factor receptor (EGFR) is efficiently internalized via clathrin-coated pits only in response to ligand-induced activation of its intrinsic tyrosine kinase and is subsequently directed into a lysosomal-proteasomal degradation pathway by mechanisms that include receptor tyrosine phosphorylation and ubiquitylation. Herein, we report a novel mechanism of EGFR internalization that does not require ligand binding, receptor kinase activity, or ubiquitylation and does not direct the receptor into a degradative pathway. Inhibition of basal protein kinase A (PKA) activity by H89 and the cell-permeable substrate peptide Myr-PKI induced internalization of 40-60% unoccupied, inactive EGFR, and its accumulation into early endosomes without affecting endocytosis of transferrin and mu-opioid receptors. This effect was abrogated by interfering with clathrin function. Thus, the predominant distribution of inactive EGFR at the plasma membrane is not simply by default but involves a PKA-dependent restrictive condition resulting in receptor avoidance of endocytosis until it is stimulated by ligand. Furthermore, PKA inhibition may contribute to ligand-induced EGFR endocytosis because epidermal growth factor inhibited 26% of PKA basal activity. On the other hand, H89 did not alter ligand-induced internalization of EGFR but doubled its half-time of down-regulation by retarding its segregation into degradative compartments, seemingly due to a delay in the receptor tyrosine phosphorylation and ubiquitylation. Our results reveal that PKA basal activity controls EGFR function at two levels: 1) residence time of inactive EGFR at the cell surface by a process of "endocytic evasion," modulating the accessibility of receptors to stimuli; and 2) sorting events leading to the down-regulation pathway of ligand-activated EGFR, determining the length of its intracellular signaling. They add a new dimension to the fine-tuning of EGFR function in response to cellular demands and cross talk with other signaling receptors.     

Metalloprotease inhibitor blocks angiotensin II-induced migration through inhibition of epidermal growth factor receptor transactivation

In vascular smooth muscle cells (VSMCs), angiotensin II (AngII) induces transactivation of the EGF receptor (EGFR) which involves a metalloprotease that stimulates processing of heparin-binding EGF from its precursor. However, the identity and pharmacological sensitivity of the metalloprotease remain unclear. Here, we screened the effects of several metalloprotease inhibitors on AngII-induced EGFR transactivation in VSMCs. We found that an N-phenylsulfonyl-hydroxamic acid derivative [2R-[(4-biphenylsulfonyl)amino]-N-hydroxy-3-phenylpropinamide] (BiPS), previously known as matrix metalloprotease (MMP)-2/9 inhibitor, markedly inhibited AngII-induced EGFR transactivation, whereas the MMP-2 or -9 inhibition by other MMP inhibitors failed to block the transactivation. BiPS markedly inhibited AngII-induced ERK activation and protein synthesis without affecting AngII-induced intracellular Ca2+ elevation. VSMC migration induced by AngII was also inhibited not only by an EGFR inhibitor but also by BiPS. Thus, BiPS is a specific candidate to block AngII-induced EGFR transactivation and subsequent growth and migration of VSMCs, suggesting its potency to prevent vascular remodeling.     

Aldosterone stimulates epidermal growth factor receptor expression

The steroid hormone aldosterone plays an important role during pathological tissue modifications, similar to cardiovascular or renal fibrosis. The underlying mechanisms for the pathological actions are not understood. Interaction of aldosterone with the epidermal growth factor (EGF) receptor is an attractive hypothesis to explain pathological tissue remodeling elicited by aldosterone, because (i) mineralocorticoids can sensitize cells for EGF, (ii) mineralocorticoid receptor (MR)-antagonists reduce EGFR-mRNA expression, (iii) EGFR itself supports the development of cardiovascular or renal fibrosis, and (iv) signaling elements involved in the pathological action of aldosterone (similar to ERK1/2 or NFkB) are typical downstream modules during EGF signaling. In addition, an interaction of aldosterone and EGF with respect to ERK1/2 activation has been described. Here we show that aldosterone stimulates EGFR expression in renal tissue of adrenalectomized rats and in human renal primary cell cultures. Furthermore, Chinese hamster ovary (CHO) cells normally devoid of EGFR or MR express EGFR after transfection with human MR (CHO-MR cells) but not after transfection with human glucocorticoid receptor (CHO-GR cells). In CHO-MR cells, EGFR-expression is up-regulated by aldosterone and inhibited by spironolactone. CHO-MR cells but not CHO-GR cells respond with ERK1/2 phosphorylation to EGF exposure. The responsiveness to other peptide hormones was virtually not affected. These data suggest that EGFR is an aldosterone-induced protein and is involved in the manifold (patho)biological actions of aldosterone.     

Phosphatidylinositol-4-phosphate 5-kinase-1beta is essential for epidermal growth factor receptor-mediated endocytosis

Phosphatidylinositol-4,5-bisphosphate (PIP(2)) is known to play an important role in signal transduction and membrane trafficking. We show that one enzyme responsible for PIP(2) production, phosphatidylinositol-4-phosphate 5-kinase type 1beta (PIPKbeta), is essential for epidermal growth factor receptor (EGFR)-mediated endocytosis. Expression of murine PIPKbeta in NR6 cells expressing EGFR strikingly increased receptor internalization. Moreover, the kinase was shown to form an immunoprecipitable complex with EGFR. Expression of either a truncated kinase or a kinase dead mutant inhibited EGFR endocytosis and also blocked the membrane recruitment of PIPKbeta and both clathrin light chain and dynamin. Our results delineate a novel mechanism by which PIPKbeta regulates receptor-mediated endocytosis and receptor tyrosine kinase membrane traffic.