Energy metabolism of the inner cell mass and trophectoderm of the mouse blastocyst

Mammalian pre-implantation development culminates in the formation of the blastocyst consisting of two distinct cell lineages, approximately a third of the cells comprise the pluripotent inner cell mass (ICM) and the remainder the differentiated trophectoderm (TE). However, the contribution made by these two cell types to the overall energy metabolism of the intact blastocyst has received relatively little attention. In this study, the metabolism of the intact mouse blastocyst and isolated ICMs were determined in terms of total ATP formation (calculated from oxygen consumption and lactate formation), mitochondrial distribution and amino acid turnover to provide an indication of protein synthesis. The TE consumed significantly more oxygen, produced more ATP and contained a greater number of mitochondria than the ICM. Amino acid turnover was significantly greater (p<0.001) in the TE compared with the ICM. Specifically, there was a significant difference in the utilization of aspartate (p=0.020), glutamate (p=0.024), methionine (p=0.037), and serine (p=0.041) between the cells of the ICM and TE. These data suggest that the TE produces approximately 80% of the ATP generated and is responsible for 90% of amino acid turnover compared with the ICM. The major fate of the energy produced by the TE is likely to be the Na(+), K(+)ATPase (sodium pump enzyme) located on the TE basolateral membrane. In conclusion, the pluripotent cells of the ICM display a relatively quiescent metabolism in comparison with that of the TE.     

Rabbit blastocyst: Allocation of cells to the inner cell mass and trophectoderm

The proportion of total cells in the blastocyst allocated to the inner cell mass (ICM) and trophectoderm (TE) is important for future development and may be a sensitive indicator to evaluate culture conditions. The number of cells and their distribution within the two primary cell lineages were determined for the rabbit embryo developing in vivo after superovulation or nonsuperovulation or embryo transfer and compared with embryos developing in vitro. Comparisons were made with cultured embryos or embryos grown in vivo until 3.5, 4.0, and 4.5 days of age. Embryos from superovulated rabbits developed in vivo for 3.5, 4.0, and 4.5 days, respectively, had 361, 758, and 902 total cells (P<0.05), and in nonsuperovulated rabbits 130, 414, and 905 total cells (P<0.05), with increasing proportions of ICM cells over time (P<0.05). One-cell embryos recovered from superovulated females and transferred to nonsuperovulated recipients developed more slowly with 70, 299, and 550 total cells after 3.5, 4.0, and 4.5 days of culture (P<0.05), respectively. The proportion of ICM cells increased with age of the embryo. Corresponding values for one-cell embryos cultured in vitro resulted in 70, 299, and 550 total cells (P<0.05). However, in vitro culture of morula-stage embryos in the presence of fetal bovine serum for 24 hr did not delay growth. In addition, the proportions of ICM/total cells were 0.17, 0.25, and 0.29 for embryos developing in vitro at 3.5, 4.0, and 4.5 days, respectively, similar to those for embryos developing in vivo at each of the three recovery times. These data establish for the first time the number and proportion of cells allocated to the ICM of the rabbit embryo developing in vivo or under defined conditions in vitro. © 1995 Wiley-Liss, Inc.     

Epidermal growth factor and transforming growth factor-alpha induce differential processing of the epidermal growth factor receptor

The capacity of epidermal growth factor (EGF) or transforming growth factor-alpha (TGF-alpha) to induce internalization and degradation of the EGF receptor was compared in NIH-3T3 cells expressing the human EGF receptor. This study was initiated following the observation that TGF-alpha was much less efficient relative to EGF in generating a Mr = 125,000 amino-terminally truncated degradation product from the mature EGF receptor (EGF-dependent generation of this degradation product is described in S.J. Decker, J. Biol. Chem., 264:17641-17644). Pulse-chase experiments revealed that EGF generally stimulated EGF receptor degradation to a greater extent than TGF-alpha. Both ligands induced EGF receptor internalization to similar degrees. However, recovery of [125I]-EGF binding following incubation with EGF or TGF-alpha was much faster for TGF-alpha treated cells. Recovery of [125I]-EGF binding after TGF-alpha treatment did not appear to require protein synthesis. Tyrosine phosphorylation of EGF receptor from cells treated with TGF-alpha decreased more rapidly following removal of TGF-alpha compared to cells treated similarly with EGF. These data suggest that EGF routes the EGF receptor directly to a degradative pathway, whereas TGF-alpha allows receptor recycling prior to degradation, and that tyrosine phosphorylation could play a role in this differential receptor processing.     

Outgrowth promoting factor for the inner cell mass of the mouse blastocyst

When cultured individually, isolated inner cell masses (ICMs) of the mouse blastocyst form an outer layer of endoderm and remain in suspension as spherical structures for several days. Conditioned media from certain teratocarcinoma- and blastocyst-derived cell lines contain one or more outgrowth promoting factors (OPF) which facilitate attachment and outgrowth of ICMs, in some cases prior to endoderm formation. OPF adheres to culture dishes and presumably promotes ICM cell outgrowth by alteration of the substratum. Analyses indicate that the active material is a nondialyzable protein which is stable to freezing and thawing, a wide range of pH, and extended incubation at 37°C. However, inactivation takes place at 60°C for 20 min. Although these properties of OPF are in some ways similar to those of various proteins which have been implicated in cell adhesion and spreading, differences between OPF and most of these other proteins are evident. The detection of OPF activity in conditioned medium from primary cultures of embryonic and some extraembryonic cell types suggests that the factor is biologically significant, perhaps, for example, in the spreading of parietal endoderm cells along the surface of the trophoblast layer.     

UBPY-mediated epidermal growth factor receptor (EGFR) de-ubiquitination promotes EGFR degradation

Whereas poly-ubiquitination targets protein substrates for proteasomal degradation, mono-ubiquitination is known to regulate protein trafficking in the endosomal system and to target cargo proteins for lysosomal degradation. The role of the de-ubiquitinating enzymes AMSH and UBPY in endosomal trafficking of cargo proteins such as the epidermal growth factor receptor (EGFR) has only very recently been the subject of study and is already a matter of debate. Although one report (Mizuno, E., Iura, T., Mukai, A., Yoshimori, T., Kitamura, N., and Komada, M. (2005) Mol. Biol. Cell 16, 5163-5174) concludes that UBPY negatively regulates EGFR degradation by de-ubiquitinating the EGFR on endosomes, another report (Row, P. E., Prior, I. A., McCullough, J., Clague, M. J., and Urbe, S. (2006) J. Biol. Chem. 281, 12618-12624) concludes that UBPY-mediated EGFR de-ubiquitination is essential for EGFR degradation. Here, we demonstrate that Usp8/UBPY, the mammalian ortholog of budding yeast Ubp4/Doa4, constitutively co-precipitates in a bivalent manner with the EGFR. Moreover, UBPY is a substrate for Src-family tyrosine kinases that are activated after ligand-induced EGFR activation. Using overexpression of three different recombinant dominant negative UBPY mutants (UBPY C748A mutant, UBPY 1-505, and UBPY 640-1080) in NIH3T3 and HEK293 cells, we demonstrate that UBPY affects both constitutive and ligand-induced (i) EGFR ubiquitination, (ii) EGFR expression levels, and (iii) the appearance of intermediate EGFR degradation products as well as (iv) downstream mitogen-activated protein kinase signal transduction. Our findings provide further evidence in favor of the model that UBPY-mediated EGFR de-ubiquitination promotes EGFR degradation.     

Role of transforming growth factor-alpha and the epidermal growth factor receptor in embryonic rat testis development

Embryonic testis development requires the morphogenesis of cords and growth of all cell populations to allow organ formation. It is anticipated that coordination of the growth and differentiation of various cell types involves locally produced growth factors. The current study was an investigation of the hypothesis that transforming growth factor-alpha (TGF-alpha) is involved in regulating embryonic testis growth. TGF-alpha has previously been shown to function in the postnatal testis. TGF-alpha and other members of the epidermal growth factor (EGF) family act through the epidermal growth factor receptor (EGFR) to stimulate cell proliferation and tissue morphogenesis. To understand the potential actions of TGF-alpha in the embryonic testis, general cell proliferation was investigated. Characterization of cell proliferation in the rat testis throughout embryonic and postnatal development indicated that each cell type has a distinct pattern of proliferation. Germ cell growth was transiently suppressed around birth. Interstitial cell growth was high embryonically and decreased to low levels around birth. A low level of Sertoli cell proliferation was observed at the onset of testis cord formation. Sertoli cell proliferation in early embryonic development was low; the levels were high later in embryonic development and remained high until the onset of puberty. Both TGF-alpha and the EGFR were shown to be expressed in the embryonic and postnatal rat and mouse testis. Perturbation of TGF-alpha function using neutralizing antibodies to TGF-alpha on testis organ cultures dramatically inhibited the growth of both embryonic and neonatal testis. TGF-alpha antibodies had no effect on cord formation. The TGF-alpha antibody was found to be specific for TGF-alpha in Western blots when compared to EGF and heregulin. Testis growth was also inhibited by perturbation of EGFR signaling using an EGFR kinase inhibitor. Therefore, TGF-alpha appears to influence embryonic testis growth but not morphogenesis (i.e., cord formation). Treatment of embryonic testis organ cultures with exogenous TGF-alpha also perturbed development, leading to an increased proliferation of unorganized cells. Testis from EGFR and TGF-alpha knockout mice were analyzed for testis morphology. TGF-alpha knockout mice had no alterations in testis phenotype, while EGFR knockout mice had a transient decrease in the relative amount of interstitial cells before birth. Observations suggest that there may be alternate or compensatory factors that allow testis growth to occur in the apparent absence of TGF-alpha actions in the mutant mice. In summary, the results obtained suggest that TGF-alpha is an important factor in the regulation of embryonic testis growth, but other factors will also be involved in the process.     

Immunohistochemical localization of epidermal growth factor receptor (EGF-r) and transforming growth factor alpha (TGF-alpha) in developing human ovarian follicles

In this study, we aimed to detect the distribution of epidermal growth factor receptor (EGF-r) and transforming growth factor alpha in ovarian follicles at different stages. Indirect immunohistochemical methods and EGF-r polyclonal and TGF-alpha monoclonal antibodies were used; tissues were examined with light microscope. While dense collection of both growth factors were observed in primordial follicles, there was a strong reaction especially for EGF-r in follicles. Strong reactivity for EGF-r and moderate reactivity for TGF-alpha were observed in the nearby connective tissue. In examinations of primary follicles for EGF-r presence only, dye uptake was moderate in oocytes and dense in apical and basal cytoplasm of follicle cells. Reactivity was moderate in the nearby connective tissue. In the corpus luteum, there was weak reaction for both growth factors. But in stromal cells, reaction was strong. In degenerated follicle cells and in stroma of atretic follicles, reaction was positive for both growth factors; but EGF-r reactivity was more obvious. While strong staining was observed for both factors especially in granulosa cells surrounding the oocyte in Graafian follicle, moderate TGF-alpha reactivity was determined in oocyte cytoplasm. In conclusion, it is possible that EGF-r and TGF-alpha have ortocrine and paracrine effects on development and regression of human ovarian follicles.     

Hypoxia inducible factor activates the transforming growth factor-alpha/epidermal growth factor receptor growth stimulatory pathway in VHL(-/-) renal cell carcinoma cells

Bi-allelic-inactivating mutations of the VHL tumor suppressor gene are found in the majority of clear cell renal cell carcinomas (VHL(-/-) RCC). VHL(-/-) RCC cells overproduce hypoxia-inducible genes as a consequence of constitutive, oxygen-independent activation of hypoxia inducible factor (HIF). While HIF activation explains the highly vascularized nature of VHL loss lesions, the relative role of HIF in oncogenesis and loss of growth control remains unknown. Here, we report that HIF plays a central role in promoting unregulated growth of VHL(-/-) RCC cells by activating the transforming growth factor-alpha (TGF-alpha)/epidermal growth factor receptor (EGF-R) pathway. Dominant-negative HIF and enzymatic inhibition of EGF-R were equally efficient at abolishing EGF-R activation and serum-independent growth of VHL(-/-) RCC cells. TGF-alpha is the only known EGF-R ligand that has a VHL-dependent expression profile and its overexpression by VHL(-/-) RCC cells is a direct consequence of HIF activation. In contrast to TGF-alpha, other HIF targets, including vascular endothelial growth factor (VEGF), were unable to stimulate serum-independent growth of VHL(-/-) RCC cells. VHL(-/-) RCC cells expressing reintroduced type 2C mutants of VHL, and which retain the ability to degrade HIF, fail to overproduce TGF-alpha and proliferate in serum-free media. These data link HIF with the overproduction of a bona fide renal cell mitogen leading to activation of a pathway involved in growth of renal cancer cells. Moreover, our results suggest that HIF might be involved in oncogenesis to a much higher extent than previously appreciated.     

Immunohistochemical localization of epidermal growth factor, transforming growth factor-alpha and growth factor-beta s in the caprine peri-implantation period

Control over the action of steroid hormones in the uterus and conceptus during the initial period of gestation appears to be regulated locally by growth factors. This study involved immunohistochemical detection of epidermal growth factor (EGF), transforming growth factor-alpha (TGF-alpha) and transforming growth factor-beta s (TGF-beta s), to determine their role in the caprine peri-implantation period. Epidermal growth factor was expressed in the luminal and glandular endometrial epithelium of goats on all days studied (Days 22 to 30 post coitum), but it was not detected in trophoblastic cells or in other embryonic structures. Between Days 22 and 30 post coitum, TGF-alpha was detected in the epithelial cells and superficial stroma of the uterus and in the trophoendodermic cells of the embryo. Transforming growth factor-beta s expression, observed in the endometrium, embryo and extraembryonic membranes on Day 22 post coitum, decreased by Day 24 post coitum and disappeared in the embryo by Day 30 post coitum, while remaining in the other structures. The presence of these growth factors during the peri-implantation period in the goat suggests their participation in proliferation and differentiation phenomena which occur during implantation and embryonic development.     

Immunocytochemical localization of the epidermal growth factor receptor in mouse testis

The distribution of the epidermal growth factor receptor (EGFR) in mouse testis was ascertained by immunocytochemical methodology using a polyclonal antibody (RK2) shown previously to recognize the cytoplasmic domain of the human (A431 cells), murine (Swiss 3T3 cells), and chicken (CK 109 cells) EGFR. Initial studies performed to determine the usefulness of this antibody as a probe of the murine EGFR in testis employed two murine cell lines, TM4 and MA10, of Sertoli cell and Leydig cell origin, respectively, in which a physiological response of EGF and specific binding of iodinated EGF has been demonstrated. Western blotting in membrane preparations of TM4 and MA10 revealed only one prominent band at 170 kDa. Immunocytochemical localization in TM4 and MA10 cells illustrated a plasma membrane distribution of the receptor. Western blotting of membrane fractions prepared from testis also revealed a specific band at 170 kDa. In the intact testis, the EGFR was immunolocalized specifically in Leydig cells and Sertoli cells only. These results suggest that the involvement of EGF action in spermatogenesis may occur at the level of the somatic components of the testes, principally in the Leydig and Sertoli cells.     

Pharmacogenomics of epidermal growth factor receptor (EGFR) tyrosine kinase inhibitors

The EGFR is a validated anticancer target whose successful exploitation has added novel agents to our current treatment protocols. Subsets of patients have shown to benefit the most from these therapies, and though these differential responses have yet to be completely defined, they are mostly of genetic nature. Egfr amplifications have shown to increase sensitivity to both small molecule inhibitors and specific monoclonal antibodies targeting the EGFR. A somatic/germline egfr intron 1 CA repeat sequence polymorphism has shown to have an important role in the control of EGFR protein expression, and has been linked to an increased risk of familial breast cancer, a worse outcome in patients with colorectal cancer, and anti-EGFR treatment efficacy in preclinical models. Egfr activating mutations have been recently described in lung cancer linking a cluster of genotypes with sensitivity to EGFR tyrosine kinase pharmacological inhibition. Despite the initial excitement that this discovery elicited, follow-up reports have not unequivocally confirmed this finding, and these drugs have been solidly efficacious both in individual patients and in diseases generally lacking egfr mutations such as pancreas cancer. We are witnessing exciting developments in the field of the pharmacogenomics of cancer, and this has particularly evolved in the area pertaining EGFR tyrosine kinase inhibitors. This review will discuss the background and currently available preclinical and clinical data.     

CD95 death receptor and epidermal growth factor receptor (EGFR) in liver cell apoptosis and regeneration

Recent evidence suggests that signaling pathways towards cell proliferation and cell death are much more interconnected than previously thought. Whereas not only death receptors such as CD95 (Fas, APO-1) can couple to both, cell death and proliferation, also growth factor receptors such as the epidermal growth factor receptor (EGFR) are involved in these opposing kinds of cell fate. EGFR is briefly discussed as a growth factor receptor involved in liver cell proliferation during liver regeneration. Then the role of EGFR in activating CD95 death receptor in liver parenchymal cells (PC) and hepatic stellate cells (HSC), which represent a liver stem/progenitor cell compartment, is described summarizing different ways of CD95- and EGFR-dependent signaling in the liver. Here, depending on the hepatic cell type (PC vs. HSC) and the respective signaling context (sustained vs. transient JNK activation) CD95-/EGFR-mediated signaling ends up in either liver cell apoptosis or cell proliferation.     

Epidermal growth factor and transforming growth factor alpha bind differently to the epidermal growth factor receptor

Epidermal growth factor (EGF) and transforming growth factor alpha (TGF alpha) compete with each other for binding to the EGF receptor. These two growth factors have similar actions, but there are distinguishable differences in their biological activities. It has never been clear how this one receptor can mediate different responses. A monoclonal antibody to the EGF receptor (13A9) has been identified which has only small effects on the binding of EGF to the EGF receptor, but which has very large effects on the binding of TGF alpha to the EGF receptor; 5 micrograms/mL antibody has been shown to totally block 0.87 microM TGF alpha from binding to purified EGF receptor and to lower both the high- and low-affinity binding constants of TGF alpha binding to EGF receptor on A431 cells by about 10-fold. The 13A9 antibody causes a 2.5-fold stimulation of the tyrosine kinase activity of partially purified EGF receptor, compared to a 4.0-fold stimulation of the tyrosine kinase activity by EGF under the same conditions. The data suggest either that the antibody stabilizes a conformation of the EGF receptor which is not favorable for TGF alpha binding or that it blocks a part of the surface of the receptor which is necessary for TGF alpha binding but not EGF binding.     

Endosomal accumulation of the activated epidermal growth factor receptor (EGFR) induces apoptosis

Endocytosis positively and negatively regulates cell surface receptor signaling by temporally and spatially controlling interactions with downstream effectors. This process controls receptor-effector communication. However, the relationship between receptor endocytic trafficking and cell physiology is unclear. In MDA-MB-468 cells, cell surface EGF receptors (EGFRs) promote cell growth, whereas intracellular EGFRs induce apoptosis, making these cells an excellent model for studying the endocytic regulation of EGFR signaling. In addition, MDA-MB-468 cells have limited EGFR degradation following stimulation. Here, we report that in MDA-MB-468 cells the phosphorylated EGFR accumulates on the limiting membrane of the endosome with its carboxyl terminus oriented to the cytoplasm. To determine whether perturbation of EGFR trafficking is sufficient to cause apoptosis, we used pharmacological and biochemical strategies to disrupt EGFR endocytic trafficking in HeLa cells, which do not undergo EGF-dependent apoptosis. Manipulation of HeLa cells so that active EGF·EGFRs accumulate on the limiting membrane of endosomes reveals that receptor phosphorylation is sustained and leads to apoptosis. When EGF·EGFR complexes accumulated in the intraluminal vesicles of the late endosome, phosphorylation of the receptor was not sustained, nor did the cells undergo apoptosis. These data demonstrate that EGFR-mediated apoptosis is initiated by the activated EGFR from the limiting membrane of the endosome.     

Multidomain binding of transforming growth factor alpha to the epidermal growth factor receptor.

Solubilized epidermal growth factor receptor (EGF-R) has been used in an extension of the Geysen epitope mapping protocol in order to provide additional insight into the amino acid residues in human transforming growth factor alpha (hTGF alpha) which are critical to recognition and binding. Overlapping heptapeptides which encompassed the 50 amino acid primary sequence of hTGF alpha were synthesized on a polyethylene solid phase, and the amount of detergent-solubilized EGF-R bound to each peptide was measured using ELISA. EGF-R appeared to bind reproducibly to four heptapeptides cognate to sequences in both the N- and C-domains of hTGF alpha (residues 22-28, 28-34, 36-42, and 44-50). Visualization of these four regions on three-dimensional solution phase structures of hTGF alpha, derived from 1H NMR measurements [Kline, T.-P., Brown, F.K., Brown, S.C., Jeffs, P.W., Kopple, K.D., & Mueller, L. (1990) Biochemistry 29, 7805-7813], indicated that the peptide segments are located on a single face of the protein and suggested the presence of a potential receptor binding cavity. If peptide segments within both the N- and C-domains of hTGF alpha are involved in binding to EGF-R, then this has direct consequences for possible molecular mechanisms by which receptor activation might take place. For example, the observed conformational flexibility in the six NMR-derived hTGF alpha structures due to variations in the main-chain torsion angles of Val-33, in combination with the involvement of residues from both domains in the proposed binding cavity, may imply that receptor activation results from interdomain reorientation in the protein ligand.(ABSTRACT TRUNCATED AT 250 WORDS)     

Binding of epidermal growth factor and transforming growth factor-alpha in mammalian preimplantation embryos

Preimplantation embryos of the pig (Days 11 to 15), cow (Days 14 to 16), sheep (Day 14) and pony (Day 16) bind epidermal growth factor (EGF) specifically. Binding was not detected in embryos of the rabbit at Day 5 or 6 or the hamster at Day 3. Transforming growth factor-alpha displaced [(125)I] EGF in pig, cow and pony embryos almost as much as unlabeled EGF. The binding affinities of EGF ranged from 12 to 233 pM in pig and cow embryos. The range of species and binding features indicate that the EGF family may play a significant role in mammalian preimplantation development.     

Ligand-induced lysosomal epidermal growth factor receptor (EGFR) degradation is preceded by proteasome-dependent EGFR de-ubiquitination

Studies on the differential routing of internalized epidermal growth factor receptors (EGFRs) induced by EGF, TGF alpha, and the superagonist EGF-TGF alpha chimera E4T suggested a correlation between receptor recycling and their mitogenic potency. EGFR sorting to lysosomes depends on its kinase domain and its ubiquitination by Cbl proteins. Proteasomes have also been proposed to regulate EGFR degradation, but the underlying mechanism remains obscure. Here we evaluated EGFR activation, Cbl recruitment, EGFR ubiquitination and degradation in response to EGF, TGF alpha, and E4T. We also determined the fate of activated EGFRs and Cbl proteins by using v-ATPase (bafilomycin A1) and proteasome (lactacystin) inhibitors. Our results demonstrate that E4T and TGF alpha provoke decreased Cbl recruitment, EGFR ubiquitination and EGFR degradation compared with EGF. Furthermore, bafilomycin treatment blocks EGFR but not c-Cbl degradation. In contrast, lactacystin treatment blocks EGF-induced c-Cbl degradation but does not block EGFR degradation, even though lactacystin causes a minor delay in EGFR degradation. Surprisingly, even though bafilomycin completely blocks EGFR degradation, it does not prevent EGFR de-ubiquitination upon prolonged EGF stimulation. Strikingly, when combined with bafilomycin, lactacystin treatment stabilizes the ubiquitinated EGFR and prevents its de-ubiquitination. We conclude that the enhanced EGFR recycling that has been observed in HER-14 cells following TGF alpha or E4T stimulation correlates with decreased EGFR ubiquitination and EGFR degradation, and that proteasomal activity is required for de-ubiquitination of the EGFR prior to its lysosomal degradation.     

Transforming growth factor-alpha short-circuits downregulation of the epidermal growth factor receptor

Transforming growth factor-alpha (TGFalpha) is an epidermal growth factor receptor (EGFR) ligand which is distinguished from EGF by its acid-labile structure and potent transforming function. We recently reported that TGFalpha induces less efficient EGFR heterodimerization and downregulation than does EGF (Gulliford et al., 1997, Oncogene, 15:2219-2223). Here we use isoform-specific EGFR and ErbB2 antibodies to show that the duration of EGFR signalling induced by a single TGFalpha exposure is less than that induced by equimolar EGF. The protein trafficking inhibitor brefeldin A (BFA) reduces the duration of EGF signalling to an extent similar to that seen with TGFalpha alone; the effects of TGFalpha and BFA on EGFR degradation are opposite, however, with TGFalpha sparing EGFR from downregulation but BFA accelerating EGF-dependent receptor loss. This suggests that BFA blocks EGFR recycling and thus shortens EGF-dependent receptor signalling, whereas TGFalpha shortens receptor signalling and thus blocks EGFR downregulation. Consistent with this, repeated application of TGFalpha is accompanied by prolonged EGFR expression and signalling, whereas similar application of EGF causes receptor downregulation and signal termination. These findings indicate that constitutive secretion of pH-labile TGFalpha may perpetuate EGFR signalling by permitting early oligomer dissociation and dephosphorylation within acidic endosomes, thereby extinguishing a phosphotyrosine-based downregulation signal and creating an irreversible autocrine growth loop.