Functional Effects of Glycosylation at Asn-579 of the Epidermal Growth Factor Receptor

Authors:
  • SWStefanie R. Whitson
    Stefanie R. Whitson
  • MRMonica L. Red-Brewer
    Monica L. Red-Brewer
  • AMAustin J. McCoy
    Austin J. McCoy
  • AVAngela A. Vitali
    Angela A. Vitali
  • FWFrancesca Walker
    Francesca Walker
  • ABAlbert H. Beth
    Albert H. Beth
  • JSJames V. Staros
    James V. Staros
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Abstract
We have investigated functional effects of glycosylation at N579 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 N579 [Zhen et al. (2003) Biochemistry 42, 5478−5492]. To characterize the subpopulation of receptors not glycosylated at N579, 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 N579 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 N579 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.

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