Abstract. We have followed the internalization pathway of both epidermal growth factor (EGF) and its receptor in human epidermoid carcinoma (A431) cells. Using EGF conjugated with horseradish peroxidase and anti-receptor monoclonal antibodies (TL5 and EGFR 1) coupled either directly or indirectly to colloidal gold we have identified an extensive elaboration of endosomal compartments, consisting of a peripheral branching network of tubular cisternae connected to vacuolar elements that contain small vesicles and a pericentriolar compartment consisting of a tubular cisternal network connected to multivesicular bodies. Immunocytochemistry on frozen thin sections using receptor-specific antibody-gold revealed that at 4"C in the presence of EGF, receptors were mainly on the plasma membrane and, to a lesser extent, within some elements of both the peripheral and pericentriolar endosomal compartments. Upon warming to 37"C there was an EGF-dependent redistribution of most binding sites, first to the peripheral endosome compartment and then to the pericentriolar compartment and lysosomes. Upon warming only to 20"C the ligand-receptor complex accumulated in the pericentriolar compartment.Acid phosphatase cytochemistry identifies hydrolytic activity only within secondary lysosomes and trans cisternae of the Golgi stacks. Together these observations suggest that the prelysosomal endosome compartment extends to the pericentriolar complex and that the transfer of EGF receptor complexes to the acid phosphatase-positive lysosome involves a discontinuous, temperature-dependent step. EPIDERMAL growth factor (EGF) 1 is a potent mitogenic polypeptide, which, upon binding to specific receptors on the plasma membrane of its target cell, initiates a chain ofintracellular events that have been studied extensively (1, 4-6, 12, 26, 28, 35, 38). By the use of labeled derivatives of EGF it has been shown both biochemically and morphologically that ceil-bound EGF is rapidly internalized by its target cells into an endosomal compartment and is subsequently degraded in lysosomes (5,16,17,30, 34). In most cell types continuous incubation at 37"C in the presence of EGF results not only in the degradation of EGF but also in the down-regulation of the EGF receptor. This led to the idea, for which Krupp et at. (29) morphological evidence that down-regulation of the EGF receptor in KB cells is due to receptor internalization into an endosomal compartment followed by degradation in lysosomes.Much of the work on internalization and processing of the EGF receptor and its ligand has been carried out in epidermoid carcinoma (A431) cells, mainly because of the large number of receptors for EGF that these cells express ( 1-3 × 106/cell). However, although EGF receptors have recently been identified by immunocytochemistry in these cells (3), the morphological pathway of internalization of the EGF receptor has so far not been documented. Two monoclonal antibodies, TL5 (35) and EGFR1 (39), have been described that recognize, respectively, the carbohydrate do...

The distribution of cell surface receptors for transferrin-iron and epidermal growth factor (EGF) on the surface of cultured epitheloid (A431) cells has been identified by immunocytochemical electron microscopy. The patterns of movement displayed by these two receptor populations as they transfer to their sites of internalization on the cell surface are different. The movement of recycling transferrin receptors over the surface is ligand-independent whereas EGF receptors are more stable residents and remain monodisperse until they bind ligand.Prior to uptake transferrin receptors cluster, predominantly within existing clathrin-coated pits while the aggregates formed by EGF ligand-receptor complexes induce new membrane invag inations. These results are discussed in relation to receptor populations concerned with constitutive, high capacity uptake processes and receptors involved in signal transduction.

A radioimmunoassay was used to measure the concentration of epidermal growth factor (EGF) in mouse milk throughout lactation. The EGF content of mouse milk increased steadily from birth to a concentration of 427 micrograms/l at day 8 of lactation. These high levels were maintained until the approach of weaning, when values decreased from day 17 to 130 micrograms/l at day 22. Milk samples chromatographed on a Biogel P10 column gave a major peak of immunoreactivity at the point at which pure standard EGF was eluted. The origin of milk EGF is unknown, but the high concentrations of this peptide identified in mouse milk suggest that it must play a role in the neonate.

No abstract

SummaryThe concentration of urogastrone (URO) and lactose was measured in breast secretions from four women. URO levels were high in precolostrum and fell markedly around the time of parturition to relatively constant low levels in mature milk. When milk samples were fractionated on a Biogel PI0 column, the major peak of immunoreactivity occurred at the point at which pure URO eluted.There was good correspondence between the fall in URO levels at birth and the increase in lactose levels, suggesting that the decrease in URO was a dilution effect as the osmotic influence of lactose increased. Abbreviations EGF, epidermal growth factor RIA, radioimmunoassay URO, urogastroneHuman milk is a food source specifically tailored to the needs of the newborn infant and fulfis all the child's nutritional requirements for the first 4-6 months of life and up to three-quarters of his needs from 6-12 months of age (33). For a great many years cow's milk has substituted for human milk in feeding the newborn infant. With this use came an interest in comparing the composition of the two milks in an attempt to justify the continued use of this substitution (for reviews see 15 and 32); however, it is now commonly accepted that human milk is of greater nutritional value in feeding the newborn infant than cow's milk (1, 21). Furthermore, milk does not just f u l f i a nutritional role, but contains antibody molecules and other antimicrobial factors that are involved in the transfer of passive immunity (27, 28), and hormones and growth factors of unknown function (3,23,31). The concentration of hormones and growth factors in milk varies between species, presumably reflecting the varying needs of the neonate. Although we do not understand the role these molecules play in the neonate, it becomes important to compare their levels throughout lactation.In the study to be reported here we monitored the concentration of URO (the human equivalent of mouse EGF) in precolostrum, colostrum, and mature milk, from human donors. MATERIALS AND METHODSMilk samples. Milk was obtained from five individuals at various stages of pregnancy and lactation. Samples from four of the volunteers (CK, AR, JL, and KD) were supplied during antenatal and postnatal periods and were assayed after up to 12 months in storage at -40°C. The 5th donor (CL) only provided mature milk samples, and these were assayed within 4-8 wk of collection.Milk was manually expressed into the sterile universal containers provided, and stored in a domestic freezer compartment until they were transferred to storage at -40°C in the laboratory.Samples were centrifuged at 9000 x g in a Microfuge (Beckman RIIC Ltd.) and the aqueous phase withdrawn from below the floating lipid layer and assayed for URO activity.For the purposes of this study, we considered all milk produced before delivery as being precolostrum, milk produced during the first 48 h after delivery as colostrum, and milk produced after the first 48 h postpartum as mature milk. Donors CK, AR, and JL who were hypersecretory produced ...