Binding, internalization, and degradation of epidermal growth factor by balb 3T3 and BP3T3 cells: relationship to cell density and the stimulation of cell proliferation.

Epidermal growth factor (EGF) stimulates the growth of both benzo[a]pyrene-transformed Balb 3T3 cells (BP3T3) and untransformed Balb 3T3 cells. We describe here the binding, internalization, and degradation of [125I]-EGF by BP3T3 cells and 3T3 cells. Binding of [125I]-EGF reaches a maximum after 45 to 90 minutes incubation at 37 degrees C. In both BP3T3 and 3T3 cells the extent of EGF binding required to stimulate DNA synthesis is density dependent; sparse cultures require a 15-30% occupancy to elicit a maximal response whereas dense cultures require a 70-85% occupancy. At physiological concentrations the total binding of [125I]-EGF to 3T3 cells is higher than to BP3T3 cells, and this difference increases at higher cell densities. The rate of degradation of [125I]-EGF is directly proportional to the total [125I]-EGF binding in each cell type. This supports the hypothesis that one cause of the diminished serum requirement of BP3T3 cells is a reduced rate of utilization of serum growth factors.     

Transforming growth factor-alpha binds to the epidermal growth factor receptor in gastric mucosa.

The ability of transforming growth factor-alpha (TGF-alpha) to interact with the gastric mucosal epidermal growth factor (EGF) receptor was investigated using a mucosal membrane preparation. TGF-alpha inhibited specific binding of [125I]EGF to its receptor, but the IC50 for TGF-alpha was at least 100 fold greater than that observed for unlabeled EGF. Cross-linking studies revealed no attachment of [125I]TGF-alpha to EGF-receptor size components, and the unlabeled TGF-alpha was only weakly effective in inhibiting cross-linking of [125I]EGF to the 170 kDa receptor. However, when the cytosolic fraction was reconstituted with the membrane preparation, an enhancement in binding of [125I]TGF-alpha to the EGF receptor occurred in a manner dependent on the concentration of cytosolic protein. Hence the binding characteristics of TGF-alpha to the EGF receptor in gastric mucosa are different from those for EGF.     

Inhibition of epidermal growth factor-induced phosphorylation by trifluoperazine

We studied the effects of a series of drugs on A431, a cell line with well-characterized growth factor requirements and epidermal growth factor (EGF) receptors. The major [32PO4]-labeled protein immunoprecipitated with anti-phosphotyrosine antibodies from EGF-stimulated A431 cells was the EGF receptor. Both the quantity of [32PO4]-labeled EGF receptor immunoprecipitated and the phosphotyrosine content of total [32PO4]-labeled proteins were reduced by the addition during EGF stimulation of trifluoperazine (TFP). TFP had little effect on the binding, internalization, and processing of [125I]-EGF. In addition to the effects on phosphorylation, TFP inhibited cell growth both in the presence and absence of EGF. Morphologically, TFP blocked EGF-induced ruffling. TFP did not alter the EGF-stimulated phosphatidylinositol turnover. In an in vitro experiment using A431 cell membranes, TFP did not inhibit phosphorylation of the EGF receptor.     

Competition of a growth stimulating-/cholecystokinin (CCK) releasing-peptide (monitor peptide) with epidermal growth factor for binding to 3T3 fibroblasts

The growth stimulating-/cholecystokinin (CCK) releasing-peptide (monitor peptide) is a peptide purified from rat bile-pancreatic juice on the basis of its stimulatory activity toward pancreatic enzyme secretion. Its multiple functions and peptide sequence suggested that it is distinct from epidermal growth factor (EGF). However, we found that the peptide competes with [125I]-EGF in the binding to Swiss 3T3 fibroblast cells to almost the same extent as unlabeled EGF does. [125I]-EGF binding was inhibited by 50% by the peptide at 82.8 ng/ml and by unlabeled EGF at 71.4 ng/ml. This suggests that the growth stimulating effect of the peptide on 3T3 fibroblasts is mediated via the EGF receptor, and also suggests that the partial homologous sequence between monitor peptide and EGF is required for the receptor binding, or that the EGF receptor has a broad ligand specificity.     

Developments in the mechanism of growth factor action: activation of protein kinase by epidermal growth factor

The interaction between epidermal growth factor (EGF) and its target cells has been used as a model for studying the regulation of cell proliferation. Many of the details of binding and subsequent internalization and degradation of this growth factor have been elucidated by following the fate of [125I]EGF in the presence of responsive cells. To investigate the membrane-localized biochemical consequences of EGF-receptor complex formation, a subcellular membrane system has been developed. In this system, EGF enhances phosphorylation of its receptor as well as other endogenous proteins. This EGF-stimulable protein kinase activity is not separated from the EGF receptor activity either by detergent solubilization or by affinity purification of the solubilized membranes. The data suggest that the EGF-binding activity and EGF-sensitive protein kinase activity reside in a single membrane protein.     

Epidermal growth factor enhancement of insulin-like growth factor-I-induced down-regulation of insulin-like growth factor I receptor in cultured placental trophoblastic cells.

Epidermal growth factor (EGF) and insulin-like growth factor I (IGF-I) synergistically stimulate placental lactogen (hPL) secretion by placental cells. To understand the mechanism of actions we have investigated a possible heterologous regulatory effect of EGF and IGF-I on each other's receptors. Pretreatment of the cells with IGF-I had no effect on [125I]-EGF binding or the down-regulation of EGF receptor. Pretreatment of the cells with EGF, concomitantly with IGF-I, had no effect on [125I]-IGF-I binding but it augmented the IGF-I down-regulation of IGF-I receptor. The time required to initiate the IGF-I-induced down-regulation of IGF-I receptor was reduced by 4 h in the presence of EGF. IGF-I-down-regulated decreased (P less than 0.05) receptor numbers were further decreased (p less than 0.05) in the presence of EGF. These results suggested that the synergistic effect of EGF and IGF-I seen in hPL secretion by placental cells is not due to direct heterologous hormone-receptor interactive effects. However, the effects seen may be due to a differentiating effect of EGF sensitizing the cells for responsiveness to IGF-I.     

Inhibition of epidermal growth factor/transforming growth factor-alpha-stimulated cell growth by a synthetic peptide

Estrogen-stimulated growth of the human mammary adenocarcinoma cell line MCF-7 is significantly inhibited by monoclonal antibodies to the epidermal growth factor (EGF) receptor that act as antagonists of EGF's mitogenic events by competing for high-affinity EGF receptor binding sites. These antibodies likewise inhibit the EGF or transforming growth factor-alpha (TGF-alpha)-stimulated growth of these MCF-7 cells. An analogous pattern of specific EGF or TGF-alpha growth inhibitory activity was obtained using a synthetic peptide analog encompassing the third disulfide loop region of TGF-alpha, but containing additional modifications designed for increased membrane affinity [( Ac-D-hArg(Et)2(31),Gly32,33]HuTGF-alpha(31-43)NH2). The growth factor antagonism by this synthetic peptide was specific in that it inhibited EGF, TGF-alpha, or estrogen-stimulated growth of MCF-7 cells but did not inhibit insulin-like growth factor-1 (IGF-1)-stimulated cell growth. Altogether, these results suggest that a significant portion of the estrogen-stimulated growth of these MCF-7 cells is mediated in an autocrine/paracrine manner by release of EGF or TGF-alpha-like growth factors. The TGF-alpha peptide likewise inhibited EGF- but not fibroblast growth factor (FGF)- or platelet-derived growth factor (PDGF)-stimulated growth of NIH-3T3 cells in completely defined media; but had no effect on growth or DNA synthesis of G0-arrested cells, nor did it effect growth of NR-6 cells, which are nonresponsive to EGF. Although this synthetic peptide did not directly compete with EGF for cell surface receptor binding, it exhibited binding to a cell surface component (followed by internalization), which likewise was not competed by EGF. The peptide did not directly inhibit EGF-stimulated phosphorylation of the EGF receptor, nor did it inhibit phosphorylation of an exogenous substrate, angiotensin II, by activated EGF receptor. The TGF-alpha peptide did, however, affect the structure of laminin as manifested by laminin self-aggregation; this affect on laminin may, in turn, have a modulatory effect on EGF-mediated cell growth.     

Epidermal growth factor (EGF)-stimulated tyrosine phosphorylation and EGF receptor degradation in cells expressing EGF receptors truncated at residue 973.

Epidermal growth factor (EGF)-stimulated tyrosine phosphorylation of proteins was examined in cells expressing wild-type (WT-EGFR) EGF receptors or EGF receptors truncated at residue 973 (973-EGFR). A much broader spectrum of tyrosine phosphorylated proteins was found following EGF treatment of 973-EGFR expressing cells compared with cells expressing wild-type receptors. Several additional ras GTPase activating protein-associated tyrosine phosphorylated proteins were found in EGF-treated 973-EGFR cells relative to WT-EGFR cells. Additional tyrosine-phosphorylated proteins were also found to co-immunoprecipitate with phospholipase C gamma 1 (PLC gamma 1) following EGF treatment of cells expressing 973-EGFR relative to cells expressing WT-EGFR. EGF-stimulated tyrosine phosphorylation of PLC gamma 1 was found in cells expressing WT-EGFR, but not in cells expressing 973-EGFR. WT-EGF receptor from EGF-treated cells bound well to bacterially expressed src homology (SH) regions of PLC gamma 1 and to a lesser extent to bacterially expressed GTPase activating protein SH regions. No binding of 973-EGF receptor to SH regions of either protein could be detected. EGF treatment greatly reduced the half-life of WT-EGFR, but had relatively little effect on the half-life of 973-EGFR. EGF induced internalization of 973-EGFR at a slower rate than WT-EGFR and caused the appearance of discrete receptor degradation products for both cell types. The data indicate that truncation of the EGF receptor at residue 973 alters receptor substrate specificity, decreases the rate of receptor internalization, and has an inhibitory effect on receptor degradation.     

Monoclonal antibody that immunoreacts with a subclass of human receptors for epidermal growth factor

Spleen cells from BALBc mice immunized with human epidermoid carcinoma A431 cells were fused with mouse myeloma P3NP cells. One of the isolated hybridoma lines, B4G7, secreted a monoclonal antibody of the IgG class which inhibited the binding of [125I]-EGF to A431 cells and human fibroblasts, but not to mouse 3T3 cells. This inhibition was partial (65-70%) and Scatchard analysis of the EGF binding data suggested that the B4G7 antibody interacts preferentially with a low-affinity class of EGF receptors. This monoclonal antibody specifically precipitated EGF receptors (Mr = 170,000 and 155,000) of A431 cells which were directly crosslinked with [125I]-EGF. It also precipitated EGF receptors from cells whose surface proteins were labeled with 125I, from cells grown in the presence of [35S]-methionine or [32P]-orthophosphate, and from membrane fractions phosphorylated in vitro with [32P]-gamma-ATP. Receptors subjected to EGF-induced phosphorylation, both in vivo and in vitro, were also precipitated. The B4G7 antibody blocked approximately 70% of the EGF receptors in human fibroblasts, but did not stimulate DNA synthesis in these cells. However, in the presence of this antibody, cells showed the full mitogenic response to EGF, presumably through the unblocked receptors that are likely to be of the high-affinity type.     

Effect of glucocorticoid on epidermal growth factor receptor in human salivary gland adenocarcinoma cell line HSG

Human salivary gland adenocarcinoma (HSG) cells treated with 10(-6) M triamcinolone acetonide for 48 h exhibited a 1.7- to 2.0-fold increase in [125I]human epidermal growth factor (hEGF) binding capacity as compared with untreated HSG cells. Scatchard analysis of [125I]EGF binding data revealed that the number of binding sites was 83,700 (+/- 29,200) receptors/cell in untreated cells and 160,500 (+/- 35,500) receptors/cell in treated cells. No substantial change in receptor affinity was detected. The dissociation constant of the EGF receptor was 0.78 (+/- 0.26).10(-9) M for untreated cells, whereas it was 0.93 (+/- 0.31).10(-9)M for treated cells. The triamcinolone acetonide-induced increase in [125I]EGF binding capacity was dose-dependent between 10(-9) and 10(-6)M, and maximal binding was observed at 10(-6)M. EGF receptors on HSG cells were affinity-labeled with [125I]EGF by use of the cross-linking reagent disuccinimidyl suberate (DSS). The cross-linked [125I]EGF was 3-4% of the total [125I]EGF bound to HSG cells. The affinity-labeled EGF receptor was detected as a specific 170 kDa band in the autoradiograph after SDS-polyacrylamide gel electrophoresis (SDS-PAGE). Densitometric analysis revealed that triamcinolone acetonide amplified the intensity of this band 2.0-fold over that of the band of untreated cells. EGF receptor synthesis was also measured by immunoprecipitation of [3H]leucine-labeled EGF receptor protein with anti-hEGF receptor monoclonal antibody. Receptor synthesis was increased 1.7- to 1.8-fold when HSG cells were treated with 10(-8)-10(-6)M triamcinolone acetonide for 48 h. When the immunoprecipitated, [35S]methionine-pulse-labeled EGF receptor was analyzed by SDS-PAGE and fluorography, the newly synthesized EGF receptor was detected at the position of 170 kDa; and treatment of HSG cells with triamcinolone acetonide resulted in a 2.0-fold amplification of this 170 kDa band. There was no significant difference in turnover rate of EGF receptor between treated and untreated HSG cells. These results demonstrate that the triamcinolone acetonide-induced increase in [125I]EGF binding capacity is due to the increased synthesis of EGF receptor protein in HSG cells.     

Binding of epidermal growth factor by human colon carcinoma cell (Caco-2) monolayers

The objective of this study was to investigate whether Caco-2 cells bind and internalize epidermal growth factor (EGF). [125I]EGF was presented to the apical (AP) or basolateral (BL) side of Caco-2 monolayers, grown on microporous membranes, at different times in culture. At day 10, [125I]EGF binding (at 37 degrees C) to the BL membrane was 2-3 times greater than binding to the AP membrane. Of that [125I]EGF bound to the AP membrane 76% was internalized within 3 h while internalization from the BL membrane was 90%. At lower temperatures membrane-bound [125I]EGF increased while internalization decreased. At day 16, AP and BL binding decreased and then remained constant through day 25. [125I]EGF was bound to the BL membrane of 10 days old monolayers with a Kd of 0.67 nM. There was a single binding site whose numbers in the BL membrane was about 5500/cell.     

Production of growth factors by type 5 adenovirus transformed rat embryo cells

Transformation of Sprague-Dawley rat embryo (RE) cells and a cloned Fischer rat embryo cell line (CREF) with wild-type (Ad5) or a temperature-sensitive DNA-minus mutant (H5ts125) of type 5 adenovirus results in a reduction in binding of epidermal growth factor (EGF) to cell surface receptors. A reduction in EGF binding is also seen in a Syrian hamster embryo cell line transformed by a hexon mutant of Ad5. In contrast, a human embryonic kidney cell line (293) transformed by sheared Ad5 DNA or transfected clones of KB cells expressing the E1 transforming region of Ad5 do not show a decrease in receptor binding. When cocultivated, the adenovirus transformed rat cells were able to induce the growth in agar of normal CREF cells. Medium from Ad5 transformed RE cells stimulated the growth in agar of CREF cells and also inhibited [125I]-EGF binding in CREF cells. When fractionated by gel filtration, two peaks of [125I]-EGF inhibiting activities were obtained with apparent molecular weights of 35,000 and 16,000. These results provide the first evidence that cells transformed by an adenovirus can produce a growth factor(s) that inhibits EGF-receptor binding and induces anchorage-independent growth of normal cells.     

Transforming growth factor-beta modulates the high-affinity receptors for epidermal growth factor and transforming growth factor-alpha

The epidermal growth factor (EGF) receptor mediates the induction of a transformed phenotype in normal rat kidney (NRK) cells by transforming growth factors (TGFs). The ability of EGF and its analogue TGF-alpha to induce the transformed phenotype in NRK cells is greatly potentiated by TGF-beta, a polypeptide that does not interact directly with binding sites for EGF or TGF-alpha. Our evidence indicates that TGF-beta purified from retrovirally transformed rat embryo cells and human platelets induces a rapid (t 1/2 = 0.3 h) decrease in the binding of EGF and TGF-alpha to high-affinity cell surface receptors in NRK cells. No change due to TGF-beta was observed in the binding of EGF or TGF-alpha to lower affinity sites also present in NRK cells. The effect of TGF-beta on EGF/TGF-alpha receptors was observed at concentrations (0.5-20 pM) similar to those at which TGF-beta is active in promoting proliferation of NRK cells in monolayer culture and semisolid medium. Affinity labeling of NRK cells and membranes by cross-linking with receptor-bound 125I-TGF-alpha and 125I-EGF indicated that both factors interact with a common 170-kD receptor structure. Treatment of cells with TGF-beta decreased the intensity of affinity-labeling of this receptor structure. These data suggest that the 170 kD high-affinity receptors for EGF and TGF-alpha in NRK cells are a target for rapid modulation by TGF-beta.     

The E5 oncoprotein of human papillomavirus type 16 transforms fibroblasts and effects the downregulation of the epidermal growth factor receptor in keratinocytes.

To determine the function of the E5 open reading frame (ORF) of the human papillomaviruses (HPVs), rodent fibroblast cell lines were transfected with the E5 ORF of HPV type 6 (HPV-6) and HPV-16 expressed from an exogenous promoter. Transfected fibroblasts were transformed to colony formation in soft agar, and the transformation frequency was increased by epidermal growth factor (EGF) but not by platelet-derived growth factor. In a transitory assay, the E5 ORFs from both HPV-6 and HPV-16 were mitogenic in primary human foreskin epithelial cells (keratinocytes) and acted synergistically with EGF. Investigation of keratinocytes expressing HPV-16 E5 showed that the number of endogenous EGF receptors (EGFRs) per cell was increased two- to fivefold. Immunofluorescence microscopy of HPV-16 E5-expressing keratinocytes indicated that there was an apparent delay in the internalization and degradation of EGFRs compared with controls. Kinetic studies with [125I]EGF showed that the ligand underwent normal internalization and degradation in both HPV-16 E5-expressing and control keratinocytes, but in E5-expressing cells, a greater number of receptors recycled back to the cell surface within 1 to 6 h of ligand binding. Finally, ligand-stimulated phosphorylation of the EGFR on tyrosine, an indication of receptor kinase activity, was of greater magnitude in the HPV-16 E5-expressing keratinocytes than in control cells, although the basal level of receptor phosphorylation was similar.     

Localization and quantification of epidermal growth factor receptors on single cells by confocal laser scanning microscopy.

We have established a method for quantifying binding of fluorescence-labeled growth factors to their receptors on single cells in situ with the confocal laser scanning microscope (CLSM). Biotinylated epidermal growth factor (EGF) coupled to phycoerythrin-labeled anti-biotin was used to compare the levels of fluorescence on three different cell types for which the number of EGF factors was known from Scatchard analysis of [125I]-EGF binding. The results showed that as few as 10,000 receptors/cell were detectable above back-ground. This method will provide a rapid and quantifiable alternative to autoradiography for ligand binding to single cells in situ.     

Calcium effects on epidermal growth factor receptor-mediated endocytosis in normal and SV40-transformed human fibroblasts

Lowering of extracellular Ca²⁺ levels will reversibly arrest the growth of human fibroblasts (WI38). Simian virus₄₀(SV₄₀)-transformed WI38 cells do not exhibit this Ca²⁺-dependent arrest. One possibility for this difference in Ca²⁺ requirement is that extracellular or surface membrane-bound Ca²⁺ may be required for growth factor receptor-mediated endocytosis and this Ca²⁺ requirement may differ in normal versus transformed cells. In this study we have evaluated the role of Ca²⁺ in the binding, internalization, and degradation of epidermal growth factor (EGF) in the WI38 and SV₄₀ WI38 cell. The binding of [¹²⁵I]EGF to the cell surface is not significantly altered by lowering of Ca²⁺ to 10^?5-M levels in either the normal or transformed cell. At this Ca²⁺ level, growth of the normal cell is inhibited. The subsequent internalization of EGF is reduced nearly threefold in the normal cell but not in the transformed cell following Ca²⁺ deprivation. Degradation of the EGF-receptor complex is also sensitive to Ca²⁺. A twofold reduction in the rate of release of acid-soluble ¹²⁵I occurs in the normal but not the transformed cell under conditions of lowered medium Ca²⁺. In contrast, 2-chloro-10-3-aminopropyl phenothiazine (CP), an inhibitor of the Ca²⁺-dependent regulator protein calmodulin, causes an inhibition of [¹²⁵I]EGF internalization and degradation in both the normal and transformed WI38 cell, and a marked inhibition of [¹²⁵I]EGF binding to the cell surface receptor of the transformed cell but not the normal cell.     

Ligand-induced activation of A431 cell epidermal growth factor receptors occurs primarily by an autocrine pathway that acts upon receptors on the surface rather than intracellularly

A431 cells express high numbers of epidermal growth factor (EGF) receptors and produce a ligand for these receptors, transforming growth factor-alpha (TGF-alpha). We have obtained evidence that the EGF receptors on these cells may be activated through an "autocrine" pathway by ligand and have investigated whether activation of phosphorylation of the receptor by the endogenously produced TGF-alpha occurs intracellularly or at the cell surface. When A431 cells were cultured under serum-free conditions, in the absence of exogenous ligand, EGF receptors were found to have a basal level of phosphorylation. When cells were labeled by culturing with 32Pi in the continuous presence of monoclonal antibodies that block binding of TGF-alpha to the EGF receptor, phosphorylation decreased to 30 +/- 10% of the basal level. This reduction could not be accounted for by the decrease in receptor content attributable to down-regulation and catabolism of EGF receptors that resulted from the binding of anti-receptor monoclonal antibodies. The reduction in receptor phosphorylation mediated by antibody was accompanied by the accumulation of increased levels of secreted TGF-alpha species in the culture medium. We also pulse-labeled A431 cells for 15 min with [35S]cysteine and immunoprecipitated the cell lysate with anti-phosphotyrosine antibody after various chase periods. Tyrosine-phosphorylated EGF receptor became detectable after 40 min of chase and reached a maximum after 4-6 h; these times are in agreement with the intervals required for EGF receptors to reach the cell surface after synthesis and then to achieve maximal expression. In addition, only the 170-kDa, mature EGF receptor species, and not the 160-kDa intracellular precursor, was immunoprecipitated with the anti-phosphotyrosine antibody. The results of these pulse-chase experiments and the finding that anti-receptor monoclonal antibody can block receptor phosphorylation suggest that activation of EGF receptors can result from the binding of an endogenous ligand (presumably TGF-alpha), which occurs at the cell surface and not during receptor biosynthesis and intracellular processing.     

Epidermal growth factor (EGF) stimulates EGF receptor synthesis

Epidermal growth factor (EGF) binds to the extracellular domain of a specific 170,000-dalton transmembrane glycoprotein; this results in rapid removal of both ligand and receptor from the cell surface. In WB cells, a rat hepatic epithelial cell line, ligand-directed receptor internalization leads to receptor degradation. We tested whether the EGF receptor was replenished at a constitutive or enhanced rate following EGF binding by immunoprecipitating biosynthetically labeled EGF receptor from cells cultured with [35S]methionine. EGF stimulated receptor synthesis within 2 h in a dose-dependent manner; this was particularly evident when examining the nascent form of the receptor. To determine the site of EGF action, total WB cell RNA was transferred to nitrocellulose paper after electrophoresis and was hybridized to cDNA probes from both the external and cytoplasmic coding regions of the human EGF receptor. EGF increased receptor mRNA by 3-5-fold. Therefore, at least in some cells, the surface action of EGF that leads to EGF receptor degradation is counterbalanced by a positive effect on receptor synthesis.     

Characterization of structural domains of the human epidermal growth factor receptor obtained by partial proteolysis

Partial cleavage with trypsin has been used to study the structure of the epidermal growth factor (EGF) receptor purified from human carcinoma cells. Following affinity labeling of the receptor with 125I-EGF or the ATP analogue 5'-p-fluorosulfonyl benzoyl[14C]adenosine, metabolic labeling with [35S]methionine, [3H]glucosamine, or [32P]orthophosphate, or in vitro autophosphorylation with [gamma-32P]ATP, tryptic cleavage defines the following three regions of the 180-kDa receptor protein: 1) a 125-kDa trypsin-resistant domain which contains sites of glycosylation, EGF binding, and an EGF-specific threonine phosphorylation site; 2) an adjacent 40-kDa fragment which contains serine and threonine phosphorylation sites and is further cleaved to a 30-kDa trypsin-resistant domain; and 3) a terminal 15-kDa portion of the receptor that contains the sites of tyrosine phosphorylation and is degraded to small fragments in the presence of trypsin. Both the 125- and 40-kDa regions of the EGF receptor appear to be required for receptor-associated protein kinase activity since separation of these regions by tryptic cleavage abolishes this activity, and both regions are specifically labeled with an ATP affinity analogue, suggesting that both are involved in ATP binding. Additional 63- and 48-kDa phosphorylated fragments are generated upon trypsin treatment of EGF receptor from EGF-treated cells. The potential usefulness of partial tryptic cleavage in studying the EGF receptor and the possible biological function of the 30-kDa trypsin-resistant fragment of the receptor are discussed.     

The effect of epidermal growth factor (EGF) on progestin secretion and cyclic AMP synthesis in cultured luteal cells from pregnant rats

It is reported that steroid synthesis in ovarian cells is affected by epidermal growth factor (EGF). We cultured luteal cells from pregnant rats for 2 days with or without EGF, followed by incubation of the cells with or without stimulants (hCG, forskolin and dibutyryl cyclic AMP) for 5 hours. The levels of progesterone, 20 alpha-hydroxy-pregn-4-en-3-one (20 alpha-dihydroprogesterone) and cyclic AMP (cAMP) in the media were assayed. EGF had no effect on the basal levels of progesterone, 20 alpha-dihydroprogesterone and cAMP, but it suppressed these levels which were increased by the stimulants. We investigated binding capacity of [125I]-EGF to ovarian tissue of pregnant rats. Ovarian tissue had specific binding sites for EGF. The maximum number of binding sites was 2.38 fmol/mg tissue and the Kd value was 0.547 nM. It was indicated that EGF modified the reactivity of luteal cells to stimulants; counteracting the tropic effect of gonadotropins. It was shown that this effect of EGF might be exerted through its receptor in luteal cells.