Biosynthesis and glycosylation of the epidermal growth factor receptor in human tumor-derived cell lines A431 and Hep 3B.

Abstract

Biosynthesis of the receptor for epidermal growth factor was investigated in two human tumor-derived cell lines, Hep 3B and A431. When grown in the presence of tunicamycin, both cells expressed a receptor-related species p135, the presumptive aglycosylated form of the biosynthetic precursor, gp145, of the mature form of the receptor, gp165, expressed at the cell surface. Two additional receptor-related species, p15 and p70, were detected when A431, but not Hep 3B, cells were treated with tunicamycin. Furthermore, digestion of the A431 receptor-related proteins with endoglycosidase F resulted in the detection of these three aglycosylated species. P70 appears to be the aglycosylated form of gp95, the presumptive intracellular precursor of the receptorrelated species gpl20 that is secreted by A431 but not Hep 3B cells; gpl20 has a complex pattern of N-linked glycosylation, with consequent molecular weight and charge heterogeneity. P115 may be the aglycosylated form of a third biosynthetic intermediate, possibly a gp135 species detected in the early time points of pulse-chase labeling. Alternatively, p115 and gp135 may be derived co-or post-translationally by Ca2+-mediated proteolysis from p135 and gp145, respectively. The implications of the complexity of the biosynthesis of this molecule with regard to the multiple opportunities it affords the cell to modulate cell proliferation are discussed.Epidermal growth factor (EGF) enhances epidermal cell growth and keratinization in vivo (9) and proliferation of meso-and ectodermally derived cells in vitro (8, 23). The mitogenic response of cells to EGF is triggered by its binding to specific cellular receptors, which are subsequently internalized (8) and at least partially degraded in lysosomes (12). Whether cleavage of the C terminus of the EGF receptor by an apparently ubiquitous Ca2+-dependent protease has some function, for example that of generating a second messenger, in relaying the mitogenic signal remains an intriguing if unresolved question (19,58). The EGF receptor is also a tyrosine-specific protein kinase (53), as are at least three other hormone receptors (15,26,45) and several of the transforming proteins encoded by RNA tumor viruses of the Rous sarcoma gene family (2,4,10,24,32,41,44,54,57). This suggests that phosphorylation of tyrosine may underlie a more general mechanism of growth control. The link between proteins that regulate normal growth and cellular transformation is further strengthened by recent findings of sequence homology between virally encoded proteins and growth factor-related molecules. The v-erb B protein of avian erythroblastosis virus, for example, shares extensive homology with a portion of the EGF receptor that includes the ATP-binding site of the presumptive kinase domain (3,14).It is now clear that growth factors and growth factorrelated molecules also play a role during early embryonic development. Several groups have detected growth factors or growth factor receptors in mammalian fetal tissue (38, 42), and we have recently demon...

Expression and biosynthetic variation of the epidermal growth factor receptor in human hepatocellular carcinoma-derived cell lines.

“…Although all species detected during pulse-chase labeling served as substrates for endo H, the MW of the gp145 biosynthetic intermediate was reduced to a greater extent than that of gp150 (data not shown), suggesting that only a portion of the N-linked oligosaccharides of the mature gp150 receptor molecule were converted from the high-mannose to the complex carbohydrate configuration. Processing of N-linked oligosaccharides may occur to a lesser or greater degree, giving rise to subtle differences in MWs in other cells as well culture supernatants (7,36), the p135, p115, and p70 species (7), were clearly detectable.…”

A novel dileucine lysosomal-sorting-signal mediates intracellular EGF-receptor retention independently of protein ubiquitylation

“…EGFR-protein stability was measured in infected cells after they had been pulse-labeled for 15 minutes with [ 35 S]-amino-acids. Thirty minutes after cells were switched to non-radioactive chase medium, wild-type and 679-AA receptors both exhibited a mobility shift, which was expected because of the N-linked oligosaccharide content of the EGFR (Carlin and Knowles, 1984). Pulse-labeled wild-type receptors disappeared within three hours in cells that had been infected with the E3-13.7-positive virus (Fig.…”

A Tyrosine-Based Signal Plays a Critical Role in the Targeting and Function of Adenovirus RIDα Protein

“…7A). The EGFR acquires seven to nine N-linked high-mannose oligosaccharides cotranslationally that are processed to complex carbohydrates during Golgi body maturation, resulting in retarded migration on SDS-polyacrylamide gels (5). A molecular weight species corresponding to the high-mannose oligosaccharide EGFR precursor was detected in cells cotransfected with an empty vector control or plasmids encoding wild-type or mutant viral protein in cells that were harvested immediately after a 30-min pulse-label (Fig.…”