title: Functional effects of glycosylation at Asn-579 of the epidermal growth factor receptor.
abstract: We have investigated functional effects of glycosylation at N(579) of the epidermal growth factor receptor (EGFR). Our previous study showed that the population of cell-surface expressed EGFRs in A431 cells, a human epidermoid carcinoma cell line, is composed of two subpopulations that differ by glycosylation at N(579) [Zhen et al. (2003) Biochemistry 42, 5478-5492]. To characterize the subpopulation of receptors not glycosylated at N(579), we established a 32D cell line expressing a point mutant of the EGFR (N579Q), which cannot be glycosylated at this position. Analysis of epitope accessibility suggests that the lack of glycosylation at N(579) weakens auto-inhibitory tether interactions, and cross-linking experiments suggest a somewhat elevated level of preformed N579Q-EGFR dimers in the absence of ligand relative to wild-type EGFR (WT-EGFR). However, ligand drives the majority of N579Q-EGFR dimerization, suggesting that untethering, while necessary, is not sufficient to drive dimerization. Ligand-binding experiments reveal a much greater fraction of N579Q-EGFRs in a high-affinity state compared to the fraction of WT-EGFRs in a high-affinity state. However, differences in the kinetic association and dissociation rates indicate that the high-affinity states of the WT and the N579Q receptors are distinct. EGF-stimulated phosphorylation in cells expressing N579Q-EGFRs results in notable differences in the pattern of tyrosine phosphorylated proteins compared with that obtained in cells expressing WT-EGFRs. Moreover, although WT-EGFRs confer cell survival in 32D cells in the absence of interleukin-3 and EGF, we found that receptors lacking glycosylation at N(579) do not. This is the first study of which we are aware to show that selective glycosylation of a specific N-glycosylation site can produce two functionally distinct receptors.
authors.affiliation: Department of Biological Sciences, Vanderbilt University, Nashville, Tennessee 37235, USA.
authors.forename: Kristin B
authors.initials: KB
authors.lastname: Whitson
authors.forename: Stefanie R
authors.initials: SR
authors.lastname: Whitson
authors.forename: Monica L
authors.initials: ML
authors.lastname: Red-Brewer
authors.forename: Austin J
authors.initials: AJ
authors.lastname: McCoy
authors.forename: Angela A
authors.initials: AA
authors.lastname: Vitali
authors.forename: Francesca
authors.initials: F
authors.lastname: Walker
authors.forename: Terrance G
authors.initials: TG
authors.lastname: Johns
authors.forename: Albert H
authors.initials: AH
authors.lastname: Beth
authors.forename: James V
authors.initials: JV
authors.lastname: Staros
mesh_terms: D000906:Antibodies; D001216:Asparagine / Q000235:genetics / Q000378:metabolism*; D045744:Cell Line, Tumor; D049109:Cell Proliferation; D002470:Cell Survival; D004815:Epidermal Growth Factor / Q000378:metabolism; D066246:ErbB Receptors / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D006031:Glycosylation; D006801:Humans; D007700:Kinetics; D008024:Ligands; D017354:Point Mutation; D011487:Protein Conformation
chemical_list: D000906:Antibodies; D008024:Ligands; D004815:Epidermal Growth Factor; D001216:Asparagine; D066246:ErbB Receptors