Functional effects of glycosylation at Asn-579 of the epidermal growth factor receptor.
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.
Whitson
Kristin B
KB
Department of Biological Sciences, Vanderbilt University, Nashville, Tennessee 37235, USA.
Whitson
Stefanie R
SR
Red-Brewer
Monica L
ML
McCoy
Austin J
AJ
Vitali
Angela A
AA
Walker
Francesca
F
Johns
Terrance G
TG
Beth
Albert H
AH
Staros
James V
JV
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
D000906:Antibodies; D008024:Ligands; D004815:Epidermal Growth Factor; D001216:Asparagine; D066246:ErbB Receptors