Temperature-dependent lateral and transverse distribution of the epidermal growth factor receptor in A431 plasma membranes

Summary To elucidate further the structure and molecular dynamics of the epidermal growth factor receptor, temperature-dependent aggregation and extracellular protrusion of the epidermal growth factor receptor in isolated plasma membranes from A431 cells were examined by fluorescence energy-transfer techniques. Epidermal growth factor was labeled at the amino terminus with either fluorescein isothiocyanate or tetramethylrhodamine isothiocyanate. A radionuclide receptor displacement assay demonstrated the bioactivity of these derivatives. Aggregation of the epidermal growth factor receptor was measured by determining the increase in fluorescence energy transfer between receptorbound fluorescein and tetramethylrhodamine-labeled epidermal growth factor. Energy transfer between receptor-bound fluorescent derivatives was reversibly greater at 37 than 4°C, indicating temperature-dependent aggregation of the receptor. The extracellular protrusion of the epidermal growth factor receptor was calculated from the magnitude of energy transfer between receptorbound fluorescein labeled epidermal growth factor and 5-(N-dodecanoylamino)-eosin partitioned into the lipid membrane at 4 and 37°C. No significant change in the distance of closest approach between the N-terminus of epidermal growth factor and the plasma membrane was observed at 4°C (69±2 Å) and 37°C (67±2 Å). Thus, the extracellular protrusion of the occupied epidermal growth factor receptor did not change detectably upon receptor aggregation.     

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

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

The role of microfilaments in the capping of epidermal growth factor receptor in A431 cells

Capping of the EGF receptor (EGF-R) on the surface of suspended and adherent epidermoid carcinoma cells, A431, is studied. It was induced at 20 degrees C after treating cells with monoclonal antibody to the EGF receptor followed by the second antibody conjugated with FITC. Accumulation of cortical actin under the caps was detected by rhodamine-phalloidin. Destruction of the actin stress-fiber-like bundles was observed during incubation of cells with the ligands at 0 degrees C. Two processes appear to take place at 20 degrees C: redistribution of the EGF-R with cortical actin into the caps within 15-30 min and reconstruction of cytoplasmic actin bundles over 45-60 min. Dihydrocytochalasin B prevented cap formation in adherent cells, but small patches of EGF-R colocalized with actin aggregates under plasma membrane were observed. The function of different actin-containing cytoskeleton structures in the process of capping is discussed.     

Biosynthesis of the epidermal growth factor receptor in A431 cells.

A monoclonal antibody R1 against the human epidermal growth factor receptor has been used to study biosynthesis in the carcinoma cell line A431. Two glycoproteins of apparent mol. wts. 95 000 and 160 000 were immunoprecipitated from cells labelled for short times with [35S]methionine or [3H]mannose. Pulse-chase studies show the 160 000 mol. wt. glycoprotein to be a precursor of the 175 000 mol. wt. receptor, but do not establish a precursor role for the 95 000 mol. wt. glycoprotein. Limited proteolysis, peptide mapping, endoglycosidase digestion and the use of monensin and tunicamycin show that the 95 000 mol. wt. glycoprotein is structurally related to the 160 000 mol. wt. glycoprotein and that both glycoproteins have approximately 22 000 - 28 000 mol. wt. of oligosaccharide side chains. Monensin blocks conversion of the 160 000 to the 175 000 mol. wt. mature receptor, a process which involves complexing several of its N-linked oligosaccharide chains. Pulse-chase studies showed that an immunoprecipitable polypeptide of 115 000 mol. wt., or 95 000 mol. wt., in the presence of monensin, was secreted into the medium at late chase times. The possible mechanisms for the origins of all the receptor-related polypeptides are discussed.     

Transient epidermal growth factor (EGF)-dependent suppression of EGF receptor autophosphorylation during internalization.

To study the activity of the epidermal growth factor (EGF) receptor during EGF-directed internalization, liver epithelial cells were exposed to EGF at 37 degrees C for various periods of time, washed, and homogenized at 0 degrees C. EGF receptor autophosphorylation was assessed in homogenates using [gamma-32P]ATP. Autophosphorylation was stimulated 3- to 6-fold in homogenates of cells incubated with EGF (100 ng/ml) for 15 min but was at or below basal levels in homogenates of cells treated with EGF for 2.5-5 min. This was surprising because immunoblotting revealed that EGF receptor phosphotyrosine (P-Tyr) content in intact cells was near maximal from 30 s to 5 min after EGF treatment. Excess EGF (1 microgram/ml), added after homogenization but prior to the assay, increased autophosphorylation in homogenates of cells that had not been treated with EGF, but failed to increase activity in homogenates of cells treated with EGF in culture for 2.5-5 min. Suppression of tyrosine phosphorylation of an exogenous kinase substrate was also observed at times paralleling the suppression of EGF receptor autophosphorylation. The transient suppression of receptor autophosphorylation in the cell-free assay was not explained by persistent occupation of autophosphorylation sites by phosphate added in the intact cells. The sites were greater than 80% dephosphorylated during the homogenization. Additionally phosphatase inhibition that prevented the normal loss of EGF receptor P-Tyr in intact cells at 15 min did not affect the pattern of early (2.5-5 min) suppression and later (15 min) stimulation of autophosphorylation measured in the cell-free assay. The suppression was not explained by activation of protein kinase C in that depletion of greater than 95% of cellular protein kinase C activity by an 18-h incubation of cells with 10 microM 12-O-tetradecanoylphorbol 13-acetate (TPA) did not affect the early suppression of autophosphorylation in EGF-treated cells. Moreover, under the conditions tested, activation of protein kinase C by short-term treatment (0.5-10 min) with TPA or angiotensin II did not appreciably alter subsequent autophosphorylation in the cell-free assay. In contrast, a 30 degrees C preincubation of homogenates from cells with suppressed EGF receptor autophosphorylation led to the recovery of the ability of EGF to stimulate EGF receptor autophosphorylation. These results suggest that a rapid reversible protein kinase C-independent process prevents detection of EGF receptor kinase activity during an early phase of EGF-dependent receptor internalization.     

Proteolytic cleavage of epidermal growth factor receptor. A Ca2+-dependent, sulfhydryl-sensitive proteolytic system in A431 cells

The Mr = 160,000 epidermal growth factor (EGF) receptor in A431 cells is partially cleaved during membrane isolation to a Mr = 145,000 polypeptide containing both EGF binding and phosphate acceptor sites. We show that the proteolytic degradation of the EGF receptor depends upon the presence of Ca2+ in the medium used to scrape the cells from the substratum. Only the high molecular weight form of the receptor is detected in membranes prepared in the absence of Ca2+. Ca2+-dependent proteolysis occurs rapidly (t1/2 approximately 5 min) following cell scraping. Proteolysis results in a decrease in EGF-dependent phosphorylation of the receptor while retaining EGF binding capacity. In addition, membranes containing the uncleaved form of the receptor reveal a substantial increase in EGF-dependent phosphorylation of proteins with Mr approximately 80, 89, and 185 X 10(3). In the presence of Ca2+, addition of iodoacetic acid to the scraping medium strongly inhibits receptor fragmentation, whereas other inhibitors (phenylmethylsulfonyl fluoride, leupeptin, and pepstatin) have no effect. The results implicate a role for a Ca2+-dependent, SH-sensitive protease in EGF receptor degradation. Prevention of proteolysis yields membrane preparations with highly active EGF-dependent kinase system.     

Recycling of epidermal growth factor in A431 cells

The fate of epidermal growth factor (EGF) after internalization by A431 cells was studied. First, cells containing 125I-EGF-receptor complexes in endosomes were obtained. Subsequent incubation of the cells at 37 degrees C resulted in the recycling of 125I-EGF from endosomes to the cell surface in the receptor-bound state and the gradual release of recycled ligand into the medium. The excess of unlabeled EGF blocked both rebinding and re-internalization of recycled 125I-EGF to produce enhanced accumulation of ligand in the medium. The rate of recycling was shown to be much higher than that of EGF degradation.     

Ligand-mediated autophosphorylation activity of the epidermal growth factor receptor during internalization

The association of EGF with its receptor in endosomes isolated from rat liver homogenates was assessed biochemically by polyethylene glycol precipitation and morphologically by electron microscope radioautography. The proportion of receptor-bound ligand in endosomes at 15 min after the injection of doses of 0.1 and 1 microgram EGF/100 g body weight was 57%. This value increased to 77% for the dose of 10 micrograms EGF injected. Quantitative electron microscope radioautography carried out on endosomes isolated at 15 min after the injection of 10 micrograms 125I-EGF demonstrated that most radiolabel was over the endosomal periphery thereby indicating that ligand- receptor complexes were in the bounding membrane but not in intraluminal vesicles of the content. EGF receptor autophosphorylation activity during internalization was evaluated in plasmalemma and endosome fractions. This activity was markedly but transiently reduced on the cell surface shortly after the administration of saturating doses of EGF. The same activity, however, was augmented and prolonged in endosomes for up to 30 min after EGF injection. The transient desensitization of cell surface activity was not due to prior in vivo phosphorylation since receptor dephosphorylation in vitro failed to restore autophosphorylation activity. Transient desensitization of cell surface autophosphorylation activity coincided with a diminished capacity for endocytosis of 125I-EGF with endocytosis returning to normal after the restoration of cell surface autophosphorylation activity. The inhibition of cell surface autophosphorylation activity and the activation of endosomal autophosphorylation activity coincident with downregulation suggest that EGF receptor traffic is governed by ligand-regulated phosphorylation activity.     

Partition of epidermal growth factor receptors on freeze-fractured plasma membranes of A431 cells is affected by the ligand

The fracture immunolabel technique, which permits assessment of the partition of transmembrane proteins with the inner or outer leaflets of the freeze-fractured membrane, was used to analyze the behavior on fracture of epidermal growth factor (EGF) receptors over the plasma membranes of A431 cells. The receptors partition mainly with the outer leaflet of the freeze-fractured plasma membranes, whereas they become associated with the inner leaflet when they are occupied by the ligand. This modified partition is even more evident after receptor clustering induced by incubation with EGF at 37 degrees C. Treatment with 12-O-tetradecanoylphorbol-13-acetate (TPA) decreases the number of receptors over both inner and outer leaflets. An effect similar to that induced by the ligand is obtained when receptor aggregation is achieved using anti-receptor monoclonal antibodies (MAb). The modified partition therefore indicates receptor activation and appears to be a consequence of receptor cross-linking rather than to reflect a conformational change of the receptor molecule. Parallel immunolabeling with anti-phosphotyrosine antibodies of freeze-fractured EGF-treated A431 cells reveals that the receptors, when activated, are associated only with the inner leaflet of the plasma membrane.     

Biosynthesis of the epidermal growth factor receptor in human epidermoid carcinoma-derived A431 cells

Using human-specific antibody reagents, we have examined the biosynthesis of the epidermal growth factor receptor in human epidermoid carcinoma-derived A431 cells. Four Mr species (Mr = 70,000, 95,000, 135,000, and 145,000) are detected when cells are subjected to a brief pulse of L-[35S]methionine; an Mr = 165,000 species is detected after 45-60 min of exposure of cells to radiolabel. In pulse-chase experiments, the four lower Mr species appear to bear a precursor relation to the Mr = 165,000 protein. The molecule acquires N-linked oligosaccharide cotranslationally, and two of the species (Mr = 95,000 and 145,000) are susceptible to digestion with endo-beta-N-acetylglucosaminidase H. The Mr = 145,000 and Mr = 165,000 proteins, which become labeled with 125I-epidermal growth factor after treatment of intact cells with a bifunctional cross-linking reagent, are phosphorylated at serine and threonine on identical tryptic peptides.     

Effects of gangliosides GM3 and De-N-acetyl GM3 on epidermal growth factor receptor kinase activity and cell growth.

Previously it was reported (Bremer, E.G., Schlessinger, J., and Hakomori, S.-I. (1986) J. Biol. Chem. 261, 2434-2440) that ganglioside GM3 inhibited epidermal growth factor (EGF)-stimulated phosphorylation of the EGF receptor in Triton X-100-treated preparations of human epidermoid carcinoma (A431) cell membranes. In addition, these authors reported that GM3 inhibited the growth of A431 cells. In contrast, a modified ganglioside, de-N-acetyl GM3, enhanced the EGF-dependent tyrosine kinase activity of the EGF receptor. In this work and in subsequent studies (Hanai, N., Dohi, T., Nores, G. A., and Hakomori, S.-I. (1988) J. Biol. Chem. 263, 6296-6301), the tyrosine kinase activity of the receptor from A431 cell membranes was assayed in the presence of Triton X-100. In this report, we confirm that GM3 inhibited and de-N-acetyl GM3 stimulated EGF receptor autophosphorylation in the presence of Triton X-100. However, in the absence of detergents, ganglioside GM3 inhibited EGF-stimulated receptor autophosphorylation, whereas de-N-acetyl GM3 had no effect on EGF-stimulated receptor autophosphorylation. The effects of these gangliosides on receptor autophosphorylation were measured in both A431 cell plasma membranes and in 3T3 cell membranes permeabilized to [32P]ATP by a freeze-thaw procedure, in intact A431 cells permeabilized with alamethicin, and in intact A431 cells grown in the presence of [32P]orthophosphate. Thus, the inhibitory effect of GM3 on receptor autophosphorylation was demonstrated in the presence and in the absence of detergent; the stimulatory effect of de-N-acetyl GM3 was observed only in the presence of detergent. We also demonstrate that ganglioside GM3 inhibited EGF-stimulated growth of transfected murine fibroblasts (3T3) that express the gene for human EGF receptor (Velu, T. J., Beguinot, L., Vass, W. C., Zhang, K., Pastan, I., and Lowy, D. R. (1989) J. Cell. Biochem. 39, 153-166). De-N-acetyl ganglioside GM3 had no effect on the growth of these cells. Growth of control fibroblasts, which lack endogenous EGF receptors (Pruss, R. M., and Herschman, H. R. (1977) Proc. Natl. Acad. Sci. U.S.A. 74, 3918-3921), was not affected by the presence of either ganglioside. Similarly, ganglioside GM3, but not de-N-acetyl ganglioside GM3, inhibited the EGF-dependent incorporation of [3H]thymidine into DNA by transfected fibroblasts. Incorporation of labeled thymidine into DNA of control fibroblasts was not affected by the presence of either ganglioside. These studies indicate that ganglioside GM3, but not its deacetylated analogue, can affect EGF receptor kinase activity in intact membranes.(ABSTRACT TRUNCATED AT 400 WORDS)     

Relation of epidermal growth factor receptor concentration to growth of human epidermoid carcinoma A431 cells

The relation between the concentration of epidermal growth factor (EGF) receptor/kinase and effects of EGF on cell proliferation has been studied using variant A431 cells and antagonist anti-EGF receptor monoclonal antibodies. Clonal A431 cell variants selected for escape from the EGF-mediated growth inhibition of parental A431 cells all have reduced concentrations of EGF receptor/kinase; Harvey sarcoma virus-transformed A431 cells, which have escaped from EGF-mediated growth inhibition, also have reduced EGF receptors. Three clonal variants which have reacquired EGF-mediated growth inhibition have 2- to 4-fold more EGF receptor than their respective parent variant. A biphasic response with stimulation at low and inhibition at high concentrations of EGF was especially evident in revertants of clone 29. Three separate antagonist monoclonal anti-EGF receptor antibodies block the growth inhibitory effects of EGF and uncover EGF-mediated growth stimulation. These studies indicate that in A431 cell variants a continuum of ligand-activated EGF receptors determines proliferative responses from low concentrations of active receptors under basal conditions to intermediate concentrations causing growth stimulation to high concentrations, causing inhibition of cell proliferation.     

Ligand-induced association of epidermal growth factor receptor to the cytoskeleton of A431 cells

Recently, we have obtained evidence in favor of a structural interaction between the epidermal growth factor (EGF) receptor and the Triton X-100-insoluble cytoskeleton of epidermoid carcinoma A431 cells. Here we present a further analysis of the properties of EGF receptors attached to the cytoskeleton. Steady-state EGF binding studies, analyzed according to the Scatchard method, showed that A431 cells contain two classes of EGF-binding sites: a high-affinity site with an apparent dissociation constant (KD) of 0.7 nM (7.5 x 10(4) sites per cell) and a low-affinity site with a KD of 8.5 nM (1.9 x 10(6) sites per cell). Non-equilibrium binding studies revealed the existence of two kinetically distinguishable sites: a fast-dissociating site, with a dissociation rate constant (k-1) of 1.1 x 10(-3) s-1 (1.0-1.3 x 10(6) sites per cell) and a slow-dissociating site, with a k-1 of 3.5 x 10(-5) s-1 (0.6-0.7 x 10(6) sites per cell). The cytoskeleton of A431 cells was isolated by Triton X-100 extraction. Scatchard analysis revealed that approximately 5% of the original number of receptors were associated with the cytoskeleton predominantly via high-affinity sites (KD = 1.5 nM). This class of receptors is further characterized by the presence of a fast-dissociating component (k-1 = 2.0 x 10(-3) s-1) and a slow-dissociating component (k-1 = 9.1 x 10(-5) s-1). The distribution between fast and slow sites of the cytoskeleton was similar to that of intact cells (65% fast and 35% slow sites). Incubation of A431 cells for 2 h at 4 degrees C in the presence of EGF resulted in a dramatic increase in the number of EGF receptors associated to the cytoskeleton. These newly cytoskeleton-associated receptors appeared to represent low-affinity binding sites (KD = 7 nM). Dissociation kinetics also revealed an increase of fast-dissociating sites. These results indicate that at 4 degrees C EGF induces the binding of low-affinity, fast-dissociating sites to the cytoskeleton of A431 cells.     

Transforming growth factor beta modulates phosphorylation of the epidermal growth factor receptor and proliferation of A431 cells.

Transforming growth factor beta (TGF-beta) increased the phosphorylation of the epidermal growth factor (EGF) receptor and inhibited the growth of A431 cells. Incubation with TGF-beta induced maximal EGF receptor phosphorylation to levels 1.5-fold higher than controls. Phosphorylation increased more prominently (4-5-fold) on tyrosine residues as determined by phosphoamino acid analysis and antiphosphotyrosine antibody immunoblotting. The kinase activity of EGF receptor was also elevated 2.5-fold when cells were cultured in the presence of TGF-beta. The antiproliferative effect of TGF-beta on A431 cells was accompanied by prolongation of G0-G1 phase and by morphological changes. TGF-beta augmented the growth inhibition of A431 cells which could be induced by EGF. In parallel, the specific EGF-induced increase in total phosphorylation of the EGF receptor was also augmented in the presence of TGF-beta. In cells cultured with TGF-beta, the phosphorylation of EGF receptor tyrosines induced by 20-min exposure to EGF was further increased 2-3-fold, suggesting additive effects upon receptor phosphorylation. EGF receptor activation by TGF-beta is characterized by kinetics quite distinct from that induced by EGF and therefore appears to take place through an independent mechanism. The TGF-beta-induced elevation in the phosphorylation of the EGF receptor may have a role in the augmented growth inhibition of A431 cells observed in the presence of EGF and TGF-beta.     

Different forms of the epidermal growth factor receptor kinase have different autophosphorylation sites

Limited proteolysis converts the native (Mr 170 000) epidermal growth factor (EGF) receptor to the Mr 150 000 form of the receptor. Calcium-activated, neutral protease (purified to homogeneity from beef lung), chymotrypsin, and elastase were all similarly effective in generating the 150-kilodalton (150-kDa) form of the receptor in detergent-solubilized, membrane vesicles shed from A-431 cells. The rate of autophosphorylation with [gamma-32P]ATP of the 150-kDa form was only 10% of the rate with the native receptor. This decreased rate was not due to loss of kinase activity, since the phosphorylation of angiotensin was virtually unchanged after limited proteolysis of the native receptor kinase. However, maps of elastase-produced peptides from 170-kDa forms and elastase-generated 150-kDa forms of the EGF receptor showed that the major autophosphorylation sites in these two forms were totally different. Confirming this difference in autophosphorylation sites was the finding that the 32P label in the autophosphorylated native receptor could not be recovered in the 150-kDa form following proteolysis. This label was quantitatively recovered in 30-15-kDa peptide fragments generated simultaneously with the 150-kDa form of the receptor. Therefore, the decreased autophosphorylation of the 150-kDa form results from the loss of preferred autophosphorylation sites on the native receptor. Only 1-3% of the phosphate incorporated in the native receptor during autophosphorylation could be found on the 150-kDa autophosphorylation sites. Hence, autophosphorylation of the tyrosine sites in the 150-kDa form of the EGF receptor is markedly enhanced by removing the major sites autophosphorylated on the native form of the receptor.     

Effect of 3T3 plasma membranes on cells exposed to epidermal growth factor

Epidermal growth factor (EGF) induced DNA synthesis in non-confluent, G₀-arrested Swiss 3T3 fibroblasts is partially blocked by plasma membranes isolated from the EGF receptor deficient NR-6 Swiss 3T3 cell line. This inhibition could be due to either a steric block of the receptor by the membranes, a membrane induced down regulation of the EGF receptor, or a signal generated by membrane binding which is antagonistic towards the mitogenic signal generated by EGF. Binding measurements utilizing ¹²⁵I-labeled EGF demonstrated that membranes do not block either the EGF induced down regulation of the receptor or alter the number of receptors on the surface. These results suggest that the membranes exert their inhibitory effect via generation of a signal which is antagonistic to the EGF induced mitogenic signal, with the result expressed as a reduced mitogenic response.     

Characterization of the interaction of 5'-p-fluorosulfonylbenzoyl adenosine with the epidermal growth factor receptor/protein kinase in A431 cell membranes

Treatment of membrane vesicles from A431 cells, a human epidermoid carcinoma line, with the affinity label 5'-p-fluorosulfonylbenzoyl [8-14C]adenosine (5'-p-FSO2Bz[14C]Ado) results in an inhibition of the epidermal growth factor (EGF)-stimulable protein kinase and in the modification of proteins having the same molecular weight (Mr = 170,000 and 150,000) as the receptor for EGF (Buhrow, S. A., Cohen, S., and Staros, J. V. (1982) J. Biol. Chem. 257, 4019-4022). Modification of the vesicles with 5'-p-FSO2BzAdo inhibits not only the EGF-stimulated phosphorylation of endogenous membrane proteins but also the EGF-stimulated phosphorylation of an exogenous synthetic tyrosine-containing peptide substrate. This indicates that the EGF-stimulable protein kinase is modified by 5'-p-FSO2BzAdo at a site affecting catalytic activity. Membrane vesicles were treated with 5'-p-FSO2Bz-[14C]Ado to affinity label the kinase, then the EGF receptor was purified by affinity chromatography on immobilized EGF. The EGF receptor thus purified contains the 5'-p-SO2Bz[14C]Ado moiety. These data strongly support our hypothesis that the EGF receptor and EGF-stimulable kinase are two parts of the same polypeptide chain.     

Compartmentalization dynamics of the epidermal growth factor in A431 cells

Dynamics of compartmentalization of epidermal growth factor (EGF) in human carcinoma A431 cells during the first hour after initiation of endocytosis was examined by methods of the organelle fractionation on a 20% Percoll gradient and of the microfluorimetric visualization of endocytosis of rhodamine-labeled EGF (EGF-R). EGF was revealed in small vesicles localized in the peripheral region of cytoplasm in a few minutes after endocytosis initiation. During centrifugation in Percoll these vesicles (endosomes), with an average density of 1.038 g/ml, were seen co-sedimented with Golgi membranes. By one hour after initiation of endocytosis, EGF-R was accumulated in perinuclear zone, in a trans-Golgi region, as numerous big luminous centres that were apparently MB-endosomes and had the same density in Percoll as did small peripheral endosomes. Such centres appeared in several cells already within 5-10 minutes. In A431 cells EGF did not reach lysosomes within 60 minutes, because no accumulation of 125I-EGF was shown in lysosome corresponding regions of Percoll gradient (average density 1.070 g/ml).     

Visualization of epidermal growth factor receptor in cryosections of cultured A431 cells by immuno-gold labeling

Cryo-ultramicrotomy in combination with immuno-gold labeling has been demonstrated to present a powerful tool in the visualization of extra- and intracellular located antigens. We have applied this method to localize epidermal growth factor (EGF) receptor in cultured A431 human epidermoid carcinoma cells. However, both the labeling efficiency, maintenance of antigenicity, and the recognizability of the ultrastructure in cryosections are highly dependent upon the fixation procedures. Using 125I-EGF or a consecutive labeling with a monoclonal anti EGF-receptor antibody, rabbit-anti-mouse antibody and 125I-protein A, it was shown that maintenance of antigenicity was optimal using 2% paraformaldehyde as a fixative, whereas under these conditions also the recognizability of ultrastructure was sufficient. After appropriate fixation and labeling, gold particles were observed associated with various regions of the plasma membrane, including coated pits, and with various types of vesicles, including coated vesicles, intracellular vesicular membranes, multi-vesicular bodies and lysosomes. The results indicate that this method allows a visualization of EGF-receptors and resolution of the EGF-receptor processing pathway at the electron microscopic level, independent of the internalization process of labeled ligands.     

Blood group-active carbohydrate chains on the receptor for epidermal growth factor of A431 cells

The antigens expressed on the carbohydrate chains of the receptor for epidermal growth factor of A431 cells were studied by immunoblotting with monoclonal antibodies. Blood group A and the Type 1 based blood group ALeb and Lea antigens were detected as well as antigens associated with unsubstituted, monofucosylated and difucosylated Type 2 blood group chains. The Lea and the difucosylated Type 2 antigen activities were abolished by treating the blotted receptor with endo-beta-galactosidase, indicating that they are expressed on backbone structures of poly-lacto/neolacto type. (The term 'poly-lacto/neolacto' is used here to describe oligosaccharide backbone structures consisting of repeating Type 1, Gal beta 1-3GlcNAc (lacto) or Type 2, Gal beta 1-4GlcNAc (neolacto) sequences.) The glycosidic linkage of oligosaccharides to protein was investigated using Pronase digests of the receptor biosynthetically labelled with [3H]glucosamine or [3H]fucose. The oligosaccharides were alkali-resistant, consistent with N- rather than O-glycosidically linked chains. A proportion of [3H]fucose-labelled glycopeptides was susceptible to endo-beta-galactosidase, confirming the immunoblotting experiment using antibodies against the Lea and the difucosylated Type 2 antigenic determinants. Oligosaccharides were released from the [3H]fucose- and [3H]-glucosamine-labelled glycopeptides by hydrazinolysis. Chromatography of the oligosaccharides on Bio-Gel P6 and Concanavalin A columns indicated a spectrum of oligosaccharides which include those of high mannose type labelled with [3H]glucosamine, and a mixture of oligosaccharides labelled with [3H]fucose and [3H]glucosamine of bi- and multiantennary complex types of which a subpopulation is susceptible to digestion with endo-beta-galactosidase.