Highly purified lysosomes, rough and smooth endoplasmic reticulum, and Golgi apparatus, as well as microvillus plasma membranes, bound 125I-labelled epidermal growth factor ([125I]EGF) with similar affinity. Scatchard plots for all the organelles were curvilinear. The apparent number of available binding sites per mg protein of intracellular organelles was 27–71% of that found in microvillus plasma membranes. The bound and free [125I]EGF were not degraded by any of the organelles.

Binding and dissociation of [125I]EGF in all organelles were dependent on the time and temperature of incubation. The specificity of [125I]EGF binding was similar in all organelles. The optimal pH for binding to lysosomes was 6·0, in contrast to 7·0 for all the other organelles. Exposure of different organelles to enzymes and protein-modifying reagents resulted in numerous binding differences between the intracellular organelles and microvillus plasma membranes. Covalent affinity labelling with [125I]EGF revealed two major proteins of 155 and 140(×103)Mr in all the organelles. The 155×103Mr protein was labelled predominantly in all organelles except rough endoplasmic reticulum, where both proteins were equally labelled. Addition of proteolytic inhibitors during isolation of organelles did not alter the pattern of [125I]EGF-labelled binding proteins found in the organelles.

EGF also stimulated phosphorylation of the 155 and 140(× 103)Mr proteins in all the organelles. The 155×103Mr protein was phosphorylated more than the 140×103Mr protein in microvillus plasma membranes and smooth endoplasmic reticulum, whereas the 140×103Mr protein was phosphorylated more than the 155×103Mr protein in lysosomes and both proteins were equally phosphorylated in rough endoplasmic reticulum. Several organelles also contained minor [125I]EGF-binding proteins that did not show phosphorylation response and proteins that showed phosphorylation response but did not bind [125I]EGF.

Thus, the present study demonstrates by a number of different criteria, that several intracellular organelles of term human placenta also contain EGF-binding and kinase activities.

Mouse epidermal growth factor (EGF) and urogastrone, the human form of EGF, are potent mitogens in a variety of cells of ectodermal and mesodermal origin (Carpenter & Cohen, 1979; Gospodarowicz, 1981). They also modulate a variety of differentiated cellular functions (Carpenter & Cohen, 1979; Gospodarowicz, 1981; Johnson et al. 1980a; Jones et al. 1982). The biological actions of EGF are presumed to be initiated by EGF binding to specific and high-affinity receptors present in outer cell membranes of responsive cells (Carpenter & Cohen, 1979; Gospodarowicz, 1981). Following binding to cell surface receptors, the EGF-receptor complexes are internalized (Carpenter & Cohen, 1979; Gospodarowicz, 1981). The internalization may represent simply a mechanism for degradation of EGF—receptor complexes, thus potentially limiting the cellular response to EGF (Carpenter & Cohen, 1976; Pastan & Willingham, 1982) and, or, it may also play a role by translocating domains of the EGF and, or, its receptors to parts of the cells where they can come in contact with intracellular organelles, including the nucleus. Supporting the latter possibility were the reports showing that: (1) the biological effects of EGF may require its internalization (Aharonov et al. 1978; Fox & Das, 1979; King et al. 1981); (2) the internalized EGF-receptor complexes are biologically active and they catalyse the phosphorylation of soluble proteins (Cohen & Fava, 1985); (3) regenerating rat liver cells accumulate intact EGF in their nuclei (Raper et al. 1985); (4) GH3 cells accumulate EGF in their nuclei in the presence of lysosomotropic agents (Johnson et al. 1980b; Savion et al. 1981); (5) EGF increases the phosphorylation of specific nuclear proteins in GH3 cells, and this effect was augmented under conditions that increase nuclear EGF accumulation (Johnson et al. 1982); (6) EGF induces perturbation of nuclear chromatin structure (Johnson et al. 1980a); (7) the Golgi uptake of [125I]EGF was enhanced under conditions that can fully support cell growth (Miskimins & Shimizu, 1982); (8) placental syncytio-trophoblasts accumulate [125I]EGF in their spherical and polymorphic endosomes, multivesicular bodies, nuclei, lysosomes, rough endoplasmic reticulum and Golgi apparatus (Lai et al. 1986; Chegini & Rao, 1986); and (9) highly purified EGF receptor can interact and nick supercoiled double-stranded DNA in an ATP-dependent manner (Mroczkowski et al. 1984). However, a more recent study demonstrated that the topoisomerase activity of the highly purified EGF receptor was due to contaminating protein copurifying with the receptor (Basu et al. 1985).

If the mechanism that mediates the biological activity of EGF involves the necessary internalization of the EGF and its receptors, and if the EGF receptors are synthesized and degraded as other membrane proteins, then it would not be incompatible with EGF receptors being present in intracellular organelles. In fact some of the recent studies have demonstrated the presence of intracellular EGF binding sites (Moriarity & Savage, 1980; Lev-Ran et al. 1984; Dunn & Hubbard, 1985). These studies, however, have neither attempted to isolate all the cellular organelles nor characterized the intracellular binding sites, in detail.

We have used term human placenta, which expresses a high level of EGF receptors in the microvillus plasma membranes (Rao et al. 1985), to investigate and characterize intracellular binding sites for EGF.

The sources of materials used in the present studies have been described (Rao et al. 1985). EGF was purified from mouse submaxillary gland and iodinated in our laboratory (Rao et al. 1984) using previously published procedures (Savage & Cohen, 1972; Davis, 1954; Matrisian et al.1982; Gospodarowicz, 1975). The specific activity of [125I]EGF ranged from 190 to 220 μCiμg−1. The EGF iodinated by the procedure of Comens et al. (1982) was used in covalent labelling of EGF receptors, whereas that obtained by the procedure of Carpenter & Cohen (1976) was used in the rest of the binding experiments.

Term human placentae from spontaneous normal vaginal deliveries were brought to the laboratory on ice. The reflected foetal membranes were cut and discarded. Placental tissue was scraped off from the underlying blood vessels and repeatedly washed with chilled physiological saline to remove blood.

Procedures to isolate intracellular organelles of placenta are based on those described for liver. However, we had to improvise a great deal in order to obtain pure fractions as differences exist between subcellular fractionation of term human placenta and liver. After about two and half years of trial-and-error experiments, the following isolation procedure was finally adopted (Fig. 1). Briefly, microvillus plasma membranes were first harvested by mechanical shearing followed by a series of centrifugations and exposure to 10mM-Mg2+ to aggregate intracellular organelles (Booth et al. 1980). Then the placental tissue was homogenized at 4°C in 10mM-Tris-HCl (pH7·0) containing 250 mM-sucrose and 1 mM-Caz+ (referred to as homogenizing buffer) with a polytron homogenizer at a setting of 6 using three 10-s bursts with short pauses in between. The homogenates were filtered through four layers of cheese-cloth and subjected to differential sedimentation and floatation discontinuous sucrose-gradient centrifugation steps to isolate various intracellular organelles (see Fig. 1).

Fig. 1.

Flow chart for the preparation of subcellular organelles from term human placenta.

Fig. 1.

Flow chart for the preparation of subcellular organelles from term human placenta.

The property of Mg2+ to aggregate intracellular organelles was used at two strategic stages in the fractionation procedure to reduce contamination by cell surface membranes. The first stage was resuspending the 15 000 g pellet in Mg2+-containing buffer and leaving it to settle for 3 h. At the end of this period, the supernatant containing surface membranes was removed and discarded. The settled fraction, containing primarily intracellular organelles, was washed free of Mg2+ and then subjected to sedimentation and floatation discontinuous sucrose-density gradient centrifugations to isolate lysosomes. The second place was stirring the 105000 g pellet for an hour in Mg2+-containing buffer and then centrifuging for 15 min at 15000 g. The pellet, containing primarily intracellular organelles, was washed free of Mg2+ and then centrifuged in discontinuous sucrose-density gradient to isolate rough and smooth endoplasmic reticulum and Golgi apparatus. Measurement of surface-membrane marker enzyme activity in fractions prepared with and without Mg2+ treatment revealed that the treatment was effective in reducing surface membrane contamination. Electron micrographs and thiamine pyrophosphatase activity presented in this paper also lend support to the above conclusion.

Immediately after isolation, the subcellular fractions were processed for electron microscopic examination as previously described by Chegini et al. (1984).

All the subcellular fractions were frozen in 1 ml samples at — 20°C. After digestion at 80°C in 100mM-NaOH containing 0·1% sodium dodecyl sulphate (SDS), the protein in the fractions was determined by the method of Lowry et al. (1951) using bovine serum albumin (BSA) as the standard.

Various marker enzymes were assayed using previously described procedures. Thiamine pyrophosphatase (EC 3.6.1.1) according to Bramley & Ryan (1978), glucose-6-phosphate dehydrogenase (EC 1.1.1.49) according to Kelly et al. (1955) and lactate dehydrogenase (EC 1.1.1.27) according to the procedure described in Worthington’s Enzyme Manual (1972). The inorganic phosphorus released in the thiamine pyrophosphatase assays was measured according to Martin & Doty (1949). Appropriate blanks were run in all the enzyme assays and corrections for these values were made.

Binding studies were performed at 22°C for 2h with 0·5nM-[125I]EGF in the presence and absence of 176-fold excess of unlabelled EGF in 0·2 ml of incubation medium, whose final composition was 5 mM-Tris. HC1 (pH 7·0), 125 mM-sucrose, 0’5 mM-Ca2+, 75 mM-NaCl and 0·5% bovine serum albumin (BSA). The free and bound [125I]EGF were separated by filtration across 0·22μm pore-size filters. The bound [12SI]EGF was counted in a Searle model 1197 autogamma counter with a counting efficiency of 82% for 125I. The data presented in this paper are specific binding, i.e. the difference between total and non-specific binding. Further details and variations from those described above are presented in figure and table legends.

Covalent labelling of EGF receptors and EGF-stimulated protein phosphorylation have been described (Rao et al. 1985). The molecular mass determination of proteins between 100000 and 200 000 are accurate to within 5000 in our systems.

Each experiment was conducted on subcellular organelles from the same batch, under identical conditions and at the same time. All the experiments were replicated two or three times on organelles from different batches. The values presented are the means and their standard errors of observations collected in all these experiments. Analysis of variance and Dunnet’s multiple range test (Steel & Torrie, 1960) were used in determining whether the observed differences between microvillus plasma membranes and intracellular organelles were significant. The significant differences are indicated by placing asterisks across the columns in various tables. Although the significant differences among the intracellular organelles was not indicated, they can be judged, at least in some cases, from the comparison of means and standard errors, and relating them to the significant differences found between microvillus plasma membranes and intracellular organelles.

Purity of subcellular organelles

Microvillus plasma membranes consisted largely of spherical and elongated vesicles, some of which contained electron-dense material (Fig. 2A). Very little or no contamination with other organelles was found in this fraction.

Fig. 2.

Electron micrographs of isolated microvillus plasma membranes (A), lysosomes (B), rough endoplasmic reticulum (C) and smooth endoplasmic reticulum (D). ×15 290. Bar, 1 μm.

Fig. 2.

Electron micrographs of isolated microvillus plasma membranes (A), lysosomes (B), rough endoplasmic reticulum (C) and smooth endoplasmic reticulum (D). ×15 290. Bar, 1 μm.

Lysosomal fraction consisted of vesicles of different sizes and shapes with single and sometimes multiple limiting membranes (Fig. 2B). Rough endoplasmic reticulum was occasionally seen in this fraction.

Rough endoplasmic reticulum fraction was characterized by vesicles of irregular profiles with bound ribosomes (Fig. 2C). A number of scattered mitochondria were seen among the vesicles.

Smooth endoplasmic reticulum consisted of empty smooth-surface vesicles, some of which contained amorphous electron-dense material (Fig. 2D). Round and elongated vesicles containing electron-dense material were seen in this fraction. These vesicles were smaller than but otherwise similar to those found in microvillus plasma membranes. There was no contamination of this fraction with either mitochondria or rough endoplasmic reticulum (Fig. 2D).

Vesicles of different sizes and shapes, and some flattened membranes were seen in the Golgi fraction (Fig. 3). Contamination with the other organelles was not evident in this fraction.

Fig. 3.

Electron micrograph of isolated Golgi apparatus. ×16700. Bar, 1 μm.

Fig. 3.

Electron micrograph of isolated Golgi apparatus. ×16700. Bar, 1 μm.

Marker enzyme activities were also measured to evaluate further the purity of the fractions. The S’ nucleotidase was distributed not only in plasma membranes but also in cytosol and other intracellular organelles of placenta (Rao et al. 1985; Madrid-Marina & Fox, 1986; Berry et al. 1986; and unpublished observations from our laboratory). Because of this, we surveyed 10 different marker enzyme activities in all the subcellular organelles of placenta. The results indicated that thiamine pyrophosphatase was the most reliable marker for plasma membranes. Since our primary concern was to determine the extent of plasma membrane contamination in the intracellular organelles, our findings on thiamine pyrophosphatase activity were fortuitous; its activity was enriched in microvillus plasma membranes from the original homogenate (Table 1). More importantly, very little or none of this enzyme activity was found in the intracellular organelles, suggesting that microvillus plasma membrane contamination in the intracellular organelles was minimal to non-existent. The same conclusion was reached after a careful inspection of subcellular organelles at the electron microscopic level (Figs 2AD, 3).

Table 1.

Marker enzyme activities in subcellular fractions of human placenta

Marker enzyme activities in subcellular fractions of human placenta
Marker enzyme activities in subcellular fractions of human placenta

We have mot found any marker enzymes intrinsic to intracellular organelles of placenta, and liver intracellular organelles markers were not useful. Therefore, we had to rely on the morphology of these organelles that showed little or no cross contamination. Cytosol contamination in the isolated membranous organelles, as judged by the measurement of glucose 6-phosphate and lactate dehydrogenases, was minimal (Table 1).

The procedures and improvisations used in isolation of pure fractions from term human placenta led to severe and unaccountable losses of fractions, which made the calculations of recoveries of fractions and bindings meaningless. In addition, because of the losses, all the properties of binding on all the fractions could not be studied.

Properties of [125I]EGF binding to different organelles

[125I]EGF binding to microvillus plasma membranes (Fig. 4A), lysosomes (Fig. 4B) and rough endoplasmic reticulum (Fig. 4C) was dependent on time and temperature of incubation. The binding reached a maximum by about 40 – 60 min at all three temperatures.

Fig. 4.

Time and temperature dependency of [125I]EGF binding to microvillus plasma membranes (A), lysosomes (B) and rough endoplasmic reticulum (C). Protein samples (5–10μg) of subcellular organelles were incubated at 4°C (▪), 22°C (▴) and 38°C (•) for varying lengths of time. The organelles incubated for 60 min at corresponding temperatures served as controls and the binding to them was taken as 100%.

Fig. 4.

Time and temperature dependency of [125I]EGF binding to microvillus plasma membranes (A), lysosomes (B) and rough endoplasmic reticulum (C). Protein samples (5–10μg) of subcellular organelles were incubated at 4°C (▪), 22°C (▴) and 38°C (•) for varying lengths of time. The organelles incubated for 60 min at corresponding temperatures served as controls and the binding to them was taken as 100%.

The organelles bound [125I]EGF partially dissociated as a function of time at 22°C in the presence and absence of excess unlabelled EGF in dilution media used for 50-fold dilution of the incubation mixture (Fig. 5A). In the presence of excess unlabelled EGF, however, the dissociation was considerably enhanced in all the organelles with significantly greater enhancement in microvillus plasma membranes as compared to the intracellular organelles (Fig. 5B).

Fig. 5.

Dissociation of microvillus plasma membrane-(•), lysosome-(▴) and rough endoplasmic reticulum-(▪) bound [125I]EGF in the absence (A) and presence (B) of excess unlabelled EGF in medium used for 50-fold dilution of incubation mixture. Subcellular organelles (100μgml−1 protein) were preincubated with [125I]EGF. Then the unbound [125IJEGF was removed by centrifugation (10000g, 20min). The pellets containing bound [125I]EGF were resuspended in homogenizing buffer. Samples were diluted 50-fold in homogenizing buffer containing no unlabelled EGF or 6·6 nM unlabelled EGF and then reincubated for indicated lengths of time at 22°C. The controls were filtered immediately after dilution and the binding in these tubes was considered 100%.

Fig. 5.

Dissociation of microvillus plasma membrane-(•), lysosome-(▴) and rough endoplasmic reticulum-(▪) bound [125I]EGF in the absence (A) and presence (B) of excess unlabelled EGF in medium used for 50-fold dilution of incubation mixture. Subcellular organelles (100μgml−1 protein) were preincubated with [125I]EGF. Then the unbound [125IJEGF was removed by centrifugation (10000g, 20min). The pellets containing bound [125I]EGF were resuspended in homogenizing buffer. Samples were diluted 50-fold in homogenizing buffer containing no unlabelled EGF or 6·6 nM unlabelled EGF and then reincubated for indicated lengths of time at 22°C. The controls were filtered immediately after dilution and the binding in these tubes was considered 100%.

The free and eluted bound [125I]EGF from microvillus plasma membranes and intracellular organelles was able to rebind to fresh microvillus plasma membranes as effectively as fresh [125I]EGF, suggesting that [125I]EGF was not degraded during the binding reaction (Table 2). The 20% decrease in rebindability of free [125I]EGF recovered from the media following incubation with microvillus plasma membranes was perhaps due to greater binding during the first incubation. In other words, [125I]EGF molecules that bind to the receptors well were taken up by microvillus plasma membranes receptors, leaving behind [125I]EGF molecules that bind less readily to the receptors. This is the best explanation we can offer at this time for this finding.

Table 2.

Lack of [125I]EGF degradation in subcellular organelles from term human placenta

Lack of [125I]EGF degradation in subcellular organelles from term human placenta
Lack of [125I]EGF degradation in subcellular organelles from term human placenta

The specific binding of [125I]EGF to microvillus plasma membranes (Fig. 6A), lysosomes (Fig. 6B), rough endoplasmic reticulum (Fig. 6C), smooth endoplasmic reticulum (Fig. 6D) and Golgi apparatus (Fig. 6E) increased with increasing concentrations of added [125I]EGF, reaching saturation at about 2·0 nM of added [125I]EGF. The Scatchard (1949) transformation and graphic display of the data (insets in Figs) show that the Scatchard plots are curvilinear. In some reports, this curvilinearity was suggested to be due to expression of non-interacting high- and low-affinity sites (King & Cuatrecases, 1982; Lai & Guyda, 1984). In others it was attributed to negative cooperativity (Carson et al. 1983). We favour the possibility of negative cooperativity because excess unlabelled EGF enhanced dissociation of microvillus plasma membrane, lysosomal and rough endoplasmic reticulum-bound [125I]EGF more than dilution alone (Fig. 5B). Realizing that the dissociation paradigm alone is not unequivocal proof of negative cooperativity, the apparent dissociation constants (Kd) were nevertheless calculated from the slopes derived from the first few points of the Scatchard plots. This is the best approximation of apparent Kd values when site-site interactions are involved and these values should be reasonably close to the Kd values of high-affinity sites when site-site interactions are not involved.

Fig. 6.

Dependence of [125I]EGF binding to microvillus plasma membranes (A), lysosomes (B), rough endoplasmic reticulum (C), smooth endoplasmic reticulum (D) and Golgi apparatus (E) on the [125I]EGF concentration in the incubation medium. Samples of 5 μg protein of subcellular organelles were incubated with increasing concentrations of [125I]EGF. Insets show the Scatchard (1949) plots of the binding data.

Fig. 6.

Dependence of [125I]EGF binding to microvillus plasma membranes (A), lysosomes (B), rough endoplasmic reticulum (C), smooth endoplasmic reticulum (D) and Golgi apparatus (E) on the [125I]EGF concentration in the incubation medium. Samples of 5 μg protein of subcellular organelles were incubated with increasing concentrations of [125I]EGF. Insets show the Scatchard (1949) plots of the binding data.

The apparent Kd values calculated from two to three Scatchard plots for each organelle, varied from 0·6×10−10 to 1·2×10−10M (Table 3). The apparent total number of available sites per mg protein calculated from the x-axis intercepts of low-slope lines was 52% in lysosomes, 38% in rough endoplasmic reticulum, 71% in smooth endoplasmic reticulum and 27% in Golgi apparatus, as compared to those in microvillus plasma membranes. Table 3 shows that the rate constants for association and dissociation (in the absence of excess unlabelled EGF) were similar for microvilli in microvillus plasma membranes, lysosomes and rough endoplasmic reticulum.

Table 3.

Equilibrium and kinetic constants for [125I]EGF binding to subcellular organelles from term human placenta

Equilibrium and kinetic constants for [125I]EGF binding to subcellular organelles from term human placenta
Equilibrium and kinetic constants for [125I]EGF binding to subcellular organelles from term human placenta

Increasing concentrations of unlabelled EGF inhibited [125I]EGF binding to all the subcellular organelles in a dose-dependent manner (Table 4). One hundred-fold higher concentrations of EGF, NGF and PDGF only moderately reduced [125I]-EGF binding. A variety of protein and peptide hormones, and prostaglandins (PGs), even at very high concentrations, had no effect on [125I]EGF binding.

Table 4.

Specificity of [125I]EGF binding to subcellular organelles from term human

Specificity of [125I]EGF binding to subcellular organelles from term human
Specificity of [125I]EGF binding to subcellular organelles from term human

Table 5 shows that an optimal pH for [125I]EGF binding to lysosomes was 6·0 and for all the other organelles it was 7·0. All the organelles exhibited binding losses below and above the optimal pH. However, the lysosomal binding losses at pH 9·0 were lower (P< 0·05) compared to microvillus plasma membranes.

Table 5.

Effect of incubation media pH on [125I]EGF binding to subcellular organelles from term human placenta

Effect of incubation media pH on [125I]EGF binding to subcellular organelles from term human placenta
Effect of incubation media pH on [125I]EGF binding to subcellular organelles from term human placenta

Preincubation of all the subcellular organelles at increasing temperature for 15 min resulted in similar and irreversible [125I]EGF-binding losses (Table 6). At 55°C, however, the lysosomal binding losses were significantly greater (P<0·05) compared to those of microvillus plasma membranes.

Table 6.

Thermal sensitivity of EGF binding sites in subcellular organelles from term human placenta

Thermal sensitivity of EGF binding sites in subcellular organelles from term human placenta
Thermal sensitivity of EGF binding sites in subcellular organelles from term human placenta

Pretreatment with trypsin virtually abolished [125I]EGF binding in all the intracellular organelles as well as in microvillus plasma membranes (Table 7). Simultaneous addition of soybean trypsin inhibitor completely reversed trypsin-induced binding losses in all organelles except lysosomes (P < 0·05). The incomplete reversal in lysosomes may be attributable to trypsin-like proteins in lysosomes, which may bind the soybean trypsin inhibitor making it less available to block exogenously added trypsin. DNase had little or no effect on binding losses from all organelles except lysosomes, where there was a 20% loss of binding (P<0·05). RNase, neuraminidase, lipase, phospholipases C and D had no effect on [125I]EGF binding in any of the organelles. Phospholipase A was able to reduce binding in all the organelles but with a lesser reduction in smooth endoplasmic reticulum (P < 0·05).

Table 7.

Effect of pretreatment of various subcellular organelles with different enzymes on subsequent [125I]EGF binding

Effect of pretreatment of various subcellular organelles with different enzymes on subsequent [125I]EGF binding
Effect of pretreatment of various subcellular organelles with different enzymes on subsequent [125I]EGF binding

Pretreatment with acetic anhydride reduced binding in all the organelles with a lesser reduction in smooth endoplasmic reticulum (P<0·05) (Table 8). Mercaptoethanol and TV-ethylmaleimide had no effect on binding in any organelle. p-Chloro-mercuribenzoate reduced binding in all the organelles except smooth endoplasmic reticulum (P<0 ·05). Dinitrofluorobenzene reduced binding only in rough endoplasmic reticulum (P<0 ·05). Tetranitromethane reduced binding in all the subcellular organelles to a similar extent.

Table 8.

Effect of pretreatment of subcellular organelles with various protein

Effect of pretreatment of subcellular organelles with various protein
Effect of pretreatment of subcellular organelles with various protein

Molecular mass of [125I]EGF-binding proteins

The property of irreversible, covalent and specific binding of some of the labelled EGF molecules in modified chloramine T iodinated preparations (Comens et al. 1982; Baker et al. 1979; Linsley et al. 1979) was used in determining the molecular mass of [125I]EGF-binding proteins.

Microvillus plasma membranes (lanes 1A –1C in Fig. 7), lysosomes (lanes 2A –2C), rough endoplasmic reticulum (lanes 3A –3C) and smooth endoplasmic reticulum (lanes 4A –4C), regardless of whether they were prepared in homogenizing buffers containing Ca2+ (lanes A of corresponding organelles), EGTA (B lanes) or a-2-macroglobulin (C lanes), contain two major (155 and 140(× 103)Mr) and several minor (210, 175 and 120(×103)Mr) [125I]EGF-binding proteins. The 210 and 175(× 103)Mr proteins were only found in microvillus plasma membranes, whereas the 120 ×103Mr protein was only found in lysosomes. The 155 ×103Mr protein was predominantly labelled in microvillus plasma membranes, lysosomes and smooth endoplasmic reticulum, whereas 155 and 140(×103)Mr proteins were labelled to an approximately equal extent in rough endoplasmic reticulum.

Fig. 7.

Autoradiography of covalently labelled EGF-binding proteins in microvillus plasma membranes (70μg protein, lanes 1A–1C), lysosomes (40μg protein, lanes 2A–2C), rough endoplasmic reticulum (80 μ g protein, lanes 3A–3C) and smooth endoplasmic reticulum (5 μg protein, lanes 4A–4C). The microvillus plasma membranes were prepared as previously described (Rao et al. 1985) and in the final step were resuspended in homogenizing buffer (HB) (lane 1A) or in the HB where Ca2+ was replaced with 1 mM-EGTA (lane IB) or 1/zgml−1 a’-2-macroglobulin (lane 1C). The intracellular organelles were prepared as indicated in the flow chart (Fig. 1) in HB (lane A) or in HB where Caz+ was replaced with EGTA (lane B) or a-2-macroglobulin (lane C) or the corresponding organelle. Approximately 38000, 18000, 41000 and 13000 disintsmin−1 were applied to each lane from microvillus plasma membranes, lysosomes, rough and smooth endoplasmic reticulum, respectively. About 4000 disintsmin−1 were applied to each lane from incubation containing unlabelled EGF.

Fig. 7.

Autoradiography of covalently labelled EGF-binding proteins in microvillus plasma membranes (70μg protein, lanes 1A–1C), lysosomes (40μg protein, lanes 2A–2C), rough endoplasmic reticulum (80 μ g protein, lanes 3A–3C) and smooth endoplasmic reticulum (5 μg protein, lanes 4A–4C). The microvillus plasma membranes were prepared as previously described (Rao et al. 1985) and in the final step were resuspended in homogenizing buffer (HB) (lane 1A) or in the HB where Ca2+ was replaced with 1 mM-EGTA (lane IB) or 1/zgml−1 a’-2-macroglobulin (lane 1C). The intracellular organelles were prepared as indicated in the flow chart (Fig. 1) in HB (lane A) or in HB where Caz+ was replaced with EGTA (lane B) or a-2-macroglobulin (lane C) or the corresponding organelle. Approximately 38000, 18000, 41000 and 13000 disintsmin−1 were applied to each lane from microvillus plasma membranes, lysosomes, rough and smooth endoplasmic reticulum, respectively. About 4000 disintsmin−1 were applied to each lane from incubation containing unlabelled EGF.

Stimulation of membrane protein phosphorylation by EGF

EGF stimulated phosphorylation of 140 and 155(× 103)Mr proteins in microvillus plasma membranes (lane 1 in Fig. 8), rough endoplasmic reticulum (lane 2), smooth endoplasmic reticulum (lane 3) and lysosomes (lane 4). It should be noted, however, that the 155 × 103Mr protein was phosphorylated more than the 140 × 103Mr protein in microvillus plasma membranes and smooth endoplasmic reticulum, whereas the 140 ×103Mr protein was phosphorylated more than 155 ×103Mr protein in lysosomes. Both the 140 and 155(×103)Mr proteins were phosphorylated equally (albeit less than in other organelles) in rough endoplasmic reticulum. EGF also stimulated phosphorylation of 45–60, 75 and 180(×103)Mr proteins in lysosomes and 76 × 103Mr protein in smooth endoplasmic reticulum.

Fig. 8.

The phosphorylation of microvillus plasma membrane (lanes 1), rough endo-plasmic reticulum (lanes 2), smooth endoplasmic reticulum (lanes 3) and lysosomes (lanes 4) proteins in the presence (+) and absence (—) of EGF. A 12 μg protein sample of organelles were applied to each lane.

Fig. 8.

The phosphorylation of microvillus plasma membrane (lanes 1), rough endo-plasmic reticulum (lanes 2), smooth endoplasmic reticulum (lanes 3) and lysosomes (lanes 4) proteins in the presence (+) and absence (—) of EGF. A 12 μg protein sample of organelles were applied to each lane.

Human term placentas by virtue of their abundant availability and tissue mass, and being rich in EGF receptors, represent an excellent tissue choice in which to investigate whether EGF binding sites are also present in the intracellular organelles. We have made tremendous efforts and used a diverse data base to ascertain whether plasma membrane contamination could explain intracellular organelle-EGF binding. The following findings: (1) thiamine pyrophosphatase distribution; (2) electron microscopy, which shows that all the fractions are quite different from each other; (3) differences in binding properties among various organelles; (4) the magnitude of binding, which in some organelles is 71% of microvillus plasma membranes; and (5) qualitative and quantitative differences among different organelles with respect to proteins covalently labelled with [125I]EGF and the pattern of proteins phosphorylated in response to EGF, rule out the possibility of contamination.

Various intracellular organelles and microvillus plasma membranes bound [125I]-EGF with a similar affinity. However, the apparent total number of available sites per mg protein of intracellular organelles was 27–71% of that found in microvillus plasma membranes. The bound and free [125I]EGF were not degraded in any of the organelles. The lack of [125I]EGF degradation, particularly in isolated lysosomes, may be due to alteration of lysosomes during the isolation procedures and, or, lack of extra lysosomal factors required for lysosomal degradation. It has previously been reported that isolated lysosomes from appropriate target tissues do not degrade human chorionic gonadotropin (Chegini et al. 1984; Rao et al. 1981) or prolactin (Ferland et al. 1984).

The organelles exhibited dependency of binding and dissociation on time and temperature of incubation. The Scatchard plots on all the organelles were curvilinear. The specificity of binding in all the organelles was similar. pH optima for lysosomal binding was 6 ·0, in contrast to 7 ·0 for all the other organelles. Lysosomal binding losses were lower at pH 9 ·0 and higher when preincubated for 15 min at 55°C.

Intracellular organelles exhibited numerous differences in binding among themselves and when compared to microvillus plasma membranes following pretreatment with various enzymes and protein reagents. These differences may not necessarily mean that the receptor molecules per se are different, as some of these differences could have come from differences in the membrane environment in which these receptors are located.

The use of covalent affinity labelling of EGF-binding proteins by chloramine T oxidized and iodinated EGF is a qualitative indication of the molecular species that are capable of binding EGF. All the intracellular organelles, and microvillus plasma membranes, have two major (140 and 155(× 103)Mr) [125I]EGF binding proteins. In addition, there were two minor (210 and 175(× 103)Mr) binding proteins in microvillus plasma membranes and one (120 ×103Mr) in rough endoplasmic reticulum. The 155 ×103Mr protein was predominantly labelled in microvillus plasma membranes, lysosomes and smooth endoplasmic reticulum, whereas both the proteins (140 and 155(× 103) Mr) were equally labelled in rough endoplasmic reticulum.

Replacing Ca2+ with EGTA or α-2-macroglobulin in buffers during the preparation of organelles did not change the pattern of multiple [125I]EGF binding proteins. This suggests that the multiple binding proteins found were not due to tissue proteases unless they have already acted before exposure of the tissue to various buffers, and that the multiple binding proteins are normal components of these organelles.

EGF stimulated phosphorylation of (155 and 140(× 103)Mr) proteins in all the organelles. But the amount of phosphorylation of one protein relative to the other was different in various organelles. For example, the 155 ×103Mr protein was phosphorylated more in microvillus plasma membranes and smooth endoplasmic reticulum; the 140 ×103Mr protein was phosphorylated more in lysosomes and both proteins were equally phosphorylated (albeit less than in other organelles) in rough endoplasmic reticulum. EGF also stimulated phosphorylation of 45–60, 75 and 180(×103)Mr proteins in lysosomes and 75 ×103Mr protein in smooth endoplasmic reticulum. Since the same amount of sample protein was applied to the gel lanes, the differences in the phosphorylation pattern may represent actual differences in the amounts of these phosphorylated proteins present in the samples.

Our data, i.e. similar molecular masses of the major [125I]EGF binding proteins and proteins phosphorylated in response to EGF in all the organelles, support the data of Cohen et al. (1982) that the EGF-binding domain and the kinase activity are functions of the same protein. The minor [125I] EGF-binding proteins that were not phosphorylated in response to EGF represent perhaps proteins that lack the kinase and, or, phosphorylation domains. The low molecular weight proteins that were phosphorylated in response to EGF but did not bind [125I]EGF suggest that these are protein substrates for EGF receptor kinase action. We cannot rule out the possibility that the level of detection of phosphorylation in the first case and [125I]EGF binding in the second case, are not sensitive enough to see these proteins.

There are at least three possibilities to explain the presence of EGF binding sites in the intracellular organelles. (1) Receptors in intracellular organelles represent those in a catabolic route following internalization. Binding with exogenously added [125I]EGF suggests that the endogenous bound ligand dissociated during the experimental procedures or during the internalization process. (2) Receptors in intracellular organelles represent newly synthesized receptors. (3) At least a portion of the intracellular organelle receptors are normal components. Internalized [125I]EGF might associate with these organelles leading to the generation of biological signals of EGF action. Our data presented in the accompanying paper (Chegini & Rao, 1986) and here show that indeed internalized [125I]EGF associates with the intracellular organelles and one of the earliest detectable biochemical signals generated in response to EGF binding, i.e. phosphorylation (Carpenter et al. 1978; King et al. 1980), is present in the intracellular organelles.

Among these three possibilities, the last one is perhaps the most provocative. Nevertheless, it should be given serious consideration although there is no definitive evidence to show whether internalized EGF and its receptors can or cannot act inside the cells.

We thank Mr Bryan Stadig and Dr S. Mitra for performing some of the experiments and Ms Yvonne Morris for typing the manuscript.

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