ARAP1 association with CIN85 affects epidermal growth factor receptor endocytic trafficking

Background information. ARAP1 is an Arf (ADP‐ribosylation factor)‐directed GAP (GTPase‐activating protein) that inhibits the trafficking of EGFR (epidermal growth factor receptor) to the early endosome. To further understand the function of ARAP1, we sought to identify proteins that interact with ARAP1. Results. Here we report that ARAP1 associates with the CIN85 (Cbl‐interacting protein of 85 kDa). Arg⁸⁶ and Arg⁹⁰ of ARAP1 and the SH3 (Src homology 3) domains of CIN85 are necessary for the interaction. We found that a mutant of ARAP1 with reduced affinity for CIN85 does not efficiently rescue the effect of reduced ARAP1 expression on EGFR trafficking to the early endosome. Reduced expression of CIN85 has a similar effect as reduced expression of ARAP1 on traffic of the EGFR. Cbl proteins regulate the endocytic trafficking of the EGFR by mediating ubiquitination of the EGFR. Overexpression of ARAP1 reduced ubiquitination of the EGFR by Cbl and slowed Cbl‐dependent degradation of the EGFR. Reduced expression of ARAP1 accelerated degradation of EGFR but did not affect the level of ubiquitination of the receptor that was detected. Conclusion. ARAP1 interaction with CIN85 regulates endocytic trafficking of the EGFR and affects ubiquitination of EGFR. We propose a model in which the ARAP1‐CIN85 complex drives exit of EGF—EGFR–Cbl complex from a pre‐early endosome into a pathway distinct from the early endosome/lysosome pathway.     

CIN85 regulates dopamine receptor endocytosis and governs behaviour in mice

Despite extensive investigations of Cbl‐interacting protein of 85 kDa (CIN85) in receptor trafficking and cytoskeletal dynamics, little is known about its functions in vivo. Here, we report the study of a mouse deficient of the two CIN85 isoforms expressed in the central nervous system, exposing a function of CIN85 in dopamine receptor endocytosis. Mice lacking CIN85 exon 2 (CIN85^Δex2) show hyperactivity phenotypes, characterized by increased physical activity and exploratory behaviour. Interestingly, CIN85^Δex2 animals display abnormally high levels of dopamine and D2 dopamine receptors (D2DRs) in the striatum, an important centre for the coordination of animal behaviour. Importantly, CIN85 localizes to the post‐synaptic compartment of striatal neurons in which it co‐clusters with D2DRs. Moreover, it interacts with endocytic regulators such as dynamin and endophilins in the striatum. Absence of striatal CIN85 causes insufficient complex formation of endophilins with D2DRs in the striatum and ultimately decreased D2DR endocytosis in striatal neurons in response to dopamine stimulation. These findings indicate an important function of CIN85 in the regulation of dopamine receptor functions and provide a molecular explanation for the hyperactive behaviour of CIN85^Δex2 mice.     

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.     

Sphingolipids are involved in N-methyl-N'-nitro-N-nitrosoguanidine-induced epidermal growth factor receptor clustering

Previously we have found that N-methyl-N'-nitro-N-nitrosoguanidine (MNNG), an alkylating agent, can induce the clustering of cellular surface receptors including tumor necrosis factor receptor (TNFR) and epidermal growth factor receptor (EGFR). Since sphingolipids, especially ceramide, have been suggested as major players in ligand-induced receptor clustering, their involvement in this ligand-independent, chemical-induced receptor clustering was evaluated. It was shown that MNNG-induced EGFR clustering occurred primarily at lipid rafts, as nystatin, which can disrupt lipid raft structure, significantly decreasing MNNG-induced EGFR clustering. Lipidomic studies revealed that MNNG treatment induced profound changes in sphingolipids metabolism, which were not the same as those induced by EGF treatment. Acid sphingomyelinase (ASM) is responsible for hydrolyzing sphingomyelin to generate ceramide, and it was demonstrated that MNNG treatment caused ASM distribution changing from diffused state to concentrated area of cells, which colocalized with lipid rafts. Nystatin treatment also abolished the redistribution of ASM. In addition, blockage of ceramide production by ASM inhibitor imipramine interrupted MNNG-induced receptor clustering. Taken together, these data suggested that sphingolipids are involved in MNNG-induced receptor clustering; however, the specific species involved may be different from those involved in EGF-mediated receptor clustering.     

Ligand-induced clathrin-mediated endocytosis of the keratinocyte growth factor receptor occurs independently of either phosphorylation or recruitment of eps15

Keratinocyte growth factor receptor (KGFR) is a receptor tyrosine kinase expressed on epithelial cells. Following ligand binding, KGFR is rapidly activated and internalized by clathrin-mediated endocytosis. Among the possible receptor substrates which could be involved in the regulation of KGFR endocytosis and down-modulation, we analyzed here the eps15 protein in view of the proposed general role of eps15 in regulating clathrin-mediated endocytosis as well as that of eps15 tyrosine phosphorylation in the control of regulated endocytosis. Immunoprecipitation and Western blot analysis showed that activated KGFR was not able to phosphorylate eps15, suggesting that eps15 is not a receptor substrate. Double immunofluorescence and confocal microscopy revealed that activated KGFR, differently from epidermal growth factor receptor (EGFR), did not induce recruitment of eps15 to the cell plasma membrane. Microinjection of a monoclonal antibody directed against the C-terminal DPF domain which contains the AP2 binding region of eps15 led to inhibition of both pathways of receptor-mediated endocytosis, the EGFR ligand-induced endocytosis and the transferrin constitutive endocytosis, but did not appear to block the KGFR ligand-induced internalization. Taken together our results indicate that the clathrin-mediated uptake of KGFR is not mediated by eps15.     

Characterization of a monoclonal antibody directed against the epidermal growth factor receptor binding site

Summary In this work a new monoclonal antibody (mAb), designated MGR1, which recognizes the epidermal growth factor receptor (EGF-R) binding site, is described. The main characteristic of this mAb is its ability to discriminate between cells that express normal levels of EGF-R from cells with overexpression, the detectability threshold by immunocytochemical tests being 5 × 10⁴ receptors/cell of 10 µm diameter. MGR1 was found to inhibit EGF binding on the relevant target cells, and vice versa its binding was inhibited by EGF, which indicated that MGR1 recognizes the EGF receptor binding site. MGR1 exerted an inhibitory effect on both the in vitro and in vivo growth of cells with EGF-R overexpression, but had no effect on cells with a normal expression of the receptor. Tumour growth inhibition in athymic mice was also obtained on already implanted tumours. MGR1 therefore seems to be an adequate reagent for the development of immunotherapeutical approaches suitable for the treatment of tumours with EGF-R overexpression.     

Novel monoclonal antibodies recognizing the active conformation of epidermal growth factor receptor

The precise regulation of epidermal growth factor receptor (EGFR) is crucial for its function in cellular growth control. Although many antibodies against EGFR have been developed and used to analyze its regulation and function, it is not yet easy to analyze activated EGFR specifically. Activated EGFR has been mainly detected by its phosphorylation state using anti-phospho-EGFR and anti-phosphotyrosine antibodies. In the present study, we have established novel monoclonal antibodies which recognize the activated EGFR independently of its phosphorylation. Our antibodies detected active state of EGFR in immunoprecipitation and immunofluorescence, by recognizing the epitopes which are exposed through the conformational change induced by ligand-binding. Furthermore, we found that our antibodies preferentially detected the conformation of constitutively active EGFR mutants found in lung cancer cell lines. These results indicate that our antibodies may become novel research and diagnostic tools for detecting and analyzing the conformation of active EGFR in various cells and tissues.     

The Cbl RING finger C-terminal flank controls epidermal growth factor receptor fate downstream of receptor ubiquitination

Evolutionarily conserved sequences of the E3/protein-ubiquitin ligase Cbl regulate epidermal growth factor receptor (EGF-R) signaling and degradation. These sequences encompass Cbl's tyrosine kinase-binding domain, linker region, RING finger (RF), and an uncharacterized flank C-terminal to the RF (residues 420-436). The latter domain, designated the RF tail, extends beyond Cbl's ubiquitin-conjugating enzyme (Ubc)-binding domain and has no known function. We report structure-function studies evaluating the impact of Cbl RF tail truncations on EGF-R fate in HEK 293 cells. All of the truncation mutants exhibit greatly reduced binding to activated EGF-R and lack proline-rich sequences that mediate direct Cbl association with SH3 proteins such as Grb2, yet a subset of mutants collectively enhances EGF-R ubiquitination, downregulation, and degradation. Significantly, EGF-R degradation correlates better with RF tail-dependent degradation of the Cbl substrate Sprouty2 than with EGF-R ubiquitination: expression of the RF tail truncation mutant Cbl 1-433 enhanced EGF-R ubiquitination while impeding Sprouty2 degradation, and Cbl 1-433 failed to enhance EGF-R downregulation or degradation. Our results suggest that EGF-R fate is controlled by a checkpoint downstream of receptor ubiquitination whose regulation by the Cbl RF tail may require Sprouty2 degradation.     

Two different stages of epidermal growth factor (EGF) receptor endocytosis are sensitive to free ubiquitin depletion produced by proteasome inhibitor MG132

Numerous studies implicate proteasomes in the regulation of EGF receptor (EGFR) endocytosis on the basis of the ability of inhibitors to decrease EGFR degradation, but the exact mechanisms remain obscure. We demonstrated that EGFR itself is not a direct target for proteasome, since it is delivered to lysosomes intact. Evidence is presented that the inhibitory effect of MG132 on EGF degradation is due mostly to free ubiquitin depletion resultant from the suppression of proteasomal functioning by MG132. By subcellular fractionation, we show two MG132-sensitive steps in the EGFR degradation pathway: sorting from early (EE) to late (LE) endosomes, and late stage of LE maturation. MG132 treatment resulted in stabilization of EGFR tyrosine phosphorylation and its association with c-Cbl. Nevertheless, ubiquitination of EGFR at late stages of endocytosis was significantly lower than that in control cells. Highly ubiquitinated forms of EGFR demonstrated more sensitivity to MG132 treatment.     

Dynamic distribution of epidermal growth factor during mouse embryo peri-implantation

Embryo implantation depends on the synchronized development of the blastocyst and the endometrium. This process is highly controlled by the coordinated action of the steroid hormones: estrogen and progesterone. By autocrine, paracrine or juxtacrine routes, some growth factors or cytokines are involved in this steroidal regulation pathway. Here we report the effects of epidermal growth factor (EGF) on embryo implantation in the mouse, the expression and distribution patterns of EGF protein in the mouse blastocyst, ectoplacental cone (EPC) and peri-implantation uterus on days 1-8 of gestation.By RT-PCR and dot blot, we found that EGF and its receptor (EGFR) are co-expressed in the blastocyst and peri-implantational uteri of pregnant days 2-8 (D2-D8) mice. Injection of EGF antibody into a uterine horn on the third day of pregnancy (D3) significantly reduced the number of mouse embryos that implanted on D8, indicating EGF have a function in the mouse embryo implantation.Further investigation by using indirect immunofluorescence and confocal microscope was made to trace EGF and EGFR protein localization during the mouse embryo implantation. EGF and EGFR are co-localized in the blastocyst, and in the secondary trophoblastic giant cells (SGC) of the EPC. At the pre-implantation stage, the distribution of EGF protein in the mouse uterus changes from epithelium to stroma. On D1 of pregnancy, EGF is mainly distributed in uterine stroma and myometrium. On D2, it is present in the uterine epithelium. On D3, it changes again from the uterine epithelium to the stroma. By D4, EGF is predominantly in the stroma. This dynamic distribution correlates with the proliferation activity of uterine cells at each period. On D6-D8 of embryo implantation, EGF 3 protein accumulates at the uterine mesometrial pole, a region that contributes to the trophoblastic invasiveness and placentation.This temporal and spatial localization of EGF protein in the mouse uterus implicates the cytokine in the regulation of trophoblastic invasiveness and uterine receptiveness.     

Signaling pathways activated by epidermal growth factor receptor or fibroblast growth factor receptor differentially regulate branching morphogenesis in fetal mouse submandibular glands

Although growth factor signaling is required for embryonic development of organs, individual signaling mechanisms regulating these organotypic processes are just beginning to be defined. We compared signaling activated in fetal mouse submandibular glands (SMGs) by three growth factors, epidermal growth factor (EGF), fibroblast growth factor (FGF) 7, or FGF10, and correlated it with specific events of branching morphogenesis. Immunoblotting showed that EGF strongly stimulated phosphorylation of extracellular signal-regulated kinase-1/2 (ERK-1/2) and weakly stimulated phosphorylation of phospholipase C γ 1 (PLC γ 1) and phosphatidylinositol-3 kinase (PI3K) in cultured E14 SMG. However, FGF7 and FGF10 stimulated phosphorylation of both PLC γ 1 and PI3K, but elicited only minimal phosphorylation of ERK-1/2. Morphological study of mesenchyme-free SMG epithelium cultured in Matrigel revealed that EGF induced cleft formation of endpieces, that FGF7 stimulated both cleft formation and stalk elongation, but that FGF10 induced only stalk elongation. In mesenchyme-free SMG epithelium cultured with EGF, FGF7 and FGF10, U0126 (MEK inhibitor) completely blocked cleft formation, whereas U73122 (PLC γ 1 inhibitor) suppressed stalk elongation. These finding suggest that EGF stimulates cleft formation and drives branch formation via ERK-1/2, and that FGF7 stimulates both cleft formation and stalk elongation via PLC γ 1 and partly via ERK-1/2, but that FGF10 stimulates stalk elongation mainly via PLC γ 1.     

Localization of epidermal growth factor (EGF) and its receptor (EGFR) during postnatal testis development in the alpaca (Lama pacos)

The objective of the present studies was to determine the localization of epidermal growth factor (EGF) and the epidermal growth factor receptor (EGFR) in testicular tissue collected from male alpacas at 12 and 24 months of age. In the testes of 12-month-old alpacas, positive staining for EGF was not detected. EGFR was localized to Leydig cells within the 12-month-old alpaca testis, but staining was absent within seminiferous tubules. At 24 months of age, EGF was localized to Leydig cells, peritubular myoid cells, Sertoli cells and germ cells of the alpaca testis, with a preferential adluminal compartment staining within the seminiferous tubules. EGFR was also localized to the Leydig cells, peritubular myoid cells, Sertoli cells and germ cells within the 24-month-old alpaca testis, but staining within the tubules was primarily within the basal compartment. Results indicate distinct temporal and spatial regulation of EGF and EGFR in the alpaca testis and support a potential role for EGF and its related ligands in alpaca testis development and spermatogenesis.     

CIN85 associates with TNF receptor 1 via Src and modulates TNF-alpha-induced apoptosis

CIN85 is a multidomain protein that associates with receptors carrying tyrosine kinase domains. Here we report that it is also a component of the signaling complex associated with tumor necrosis factor receptor 1 (TNFR1), which lacks a tyrosine kinase domain. This was established by showing that CIN85 was co-precipitated with TNFR1, TRADD, cIAP-1 and TARF1/2, but not with FADD, RIP, caspase-8 or TRAF6. However, CIN85 did not bind directly to the cytoplasmic domain of TNFR1 (TNFR1-CYT) but to Src family kinases, Cbl and the p85alpha subunit of phosphatidylinositol 3-kinase (PI3-K p85alpha). Src bound directly to TNFR1-CYT, but Cbl and PI3-K p85alpha did not. A human cell line ectopically expressing CIN85 was 10 times more susceptible to TNF-alpha-induced apoptosis than control cells, which expressed identical levels of TNFR1 on their surface. However, the susceptibility of these two cell lines to CD95-induced apoptosis was the same. The three SH3 domains of CIN85 were essential for this increased susceptibility to apoptosis and its proline-rich regions were also required for maximal effect. TNF-alpha treatment recruited CIN85 to the TNFR1 signaling complex. Taken together, these results indicate that CIN85 associates with TNFR1 via Src and modulates TNF-alpha-induced apoptosis.     

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.     

Cross-talk between the calcium-sensing receptor and the epidermal growth factor receptor in Rat-1 fibroblasts

The calcium-sensing receptor (CaR) is a G-protein-coupled receptor that is activated by extracellular calcium (Cao2+). Rat-1 fibroblasts have been shown to proliferate and increase ERK activity in response to elevation of [Ca2+]o, and these responses are dependent on functional CaR expression. In this report, we examined the role of cross-talk between the CaR and the epidermal growth factor receptor (EGFR) in mediating these responses in Rat-1 cells. This report shows that AG1478, a specific inhibitor of the EGFR kinase, significantly inhibits the increase in proliferation induced by elevated Cao2+. Furthermore, we show that AG1478 acts downstream or separately from G protein subunit activation of phospholipase C. AG1478 significantly inhibits Cao2+-stimulated ERK phosphorylation and in vitro kinase activity. A similar inhibition of ERK phosphorylation was observed in response to the inhibitor AG494. In addition, treatment with inhibitors of metalloproteases involved in shedding of membrane anchored EGF family ligands substantially inhibited the increase in ERK activation in response to elevated Cao2+. This is consistent with the known expression of TGFalpha by Rat-1 cells. These results indicate that EGFR transactivation is an important component of the CaR-mediated response to increased Cao2+ in Rat-1 fibroblasts and most likely involves CaR-mediated induction of regulated proteolysis and ligand shedding.     

The 18 kDa cytosolic acid phosphatase from bovine liver has phosphotyrosine phosphatase activity on the autophosphorylated epidermal growth factor receptor

In this paper we demonstrate that the cytosofic low-M_r acid phosphatase purified from bovine liver has phosphotyrosine protein phosphatase acitivity on ³²P-autophosphorylated epidermal growth factor (EGF) receptor. This activity was significantly inhibited by orthovanadate and p-hydroxymercuribenzoate; the latter result indicates that free sulfhydryl groups are required for phosphotyrosine phosphatase activity. The enzyme was active in a broad pH range, with maximum activity between pH 5.5 and 7.5. The apparent K_m for ³²P-EGF receptor dephosphorylation was 4 nM. The enzyme appeared to be specific for phosphotyrosine in that it dephosphorylated the autophosphorylated EGF receptor and L-phosphotyrosine, but not ³²P-Ser-casein, L-phosphoserine or L-phosphothreonine. These data suggest that the cytosolic low-M_r acid phosphatase might play a regulatory role in EGF receptor-dependent transmembrane signalling.