The EGF receptor has been the subject of many mutation studies which have focused on the questions of receptor activation and signal transduction. However, very little is known about the structure of the protein beyond the cDNA-based amino acid sequence. We have previously reported the variability of glycosylation as a function of cell culture time up to 710 days; now we report additional structural characterization of the receptor. In order to define the structural constraints needed for model building and to identify the source of the observed glycosylation heterogeneity, we have initiated a study to determine disulfide bond connectivity. To this end a tryptic peptide map was obtained using the following conditions: 2M GuHCl, 50mM Tris-HCl, pH6.5. Combining data from amino acid analysis and Bio-Ion 20 mass spectral analysis of the HPLC-purified tryptic fragments, we have identified the disulfide pairing of Cys271 and Cys283. Additionally, we have established that one of the twelve putative glycosylation sites Asn328 is not glycosylated. Identification of the other disulfide bond pairing residues and determination of the six glycosylation sites out of 12 possible are currently being carried out.
