Direct identification of residues of the epidermal growth factor receptor in close proximity to the amino terminus of bound epidermal growth factor.
We have recently developed a kinetically controlled, step-wise affinity cross-linking technique for specific, high-yield, covalent linkage of murine epidermal growth factor (mEGF) via its N terminus to the EGF receptor. EGF receptor from A431 cells was cross-linked to radiolabeled mEGF (125I-mEGF) by this technique and the 125I-mEGF-receptor complex was purified and denatured. Tryptic digestion of this preparation gave rise to a unique radiolabeled peptide that did not comigrate with trypsin-treated 125I-mEGF in SDS/Tricine gels but that could be immunoprecipitated with antibodies to mEGF. The immunoprecipitated peptide was isolated by electrophoresis in SDS/Tricine gels, eluted, and sequenced. The sequence was found to correspond to that of a tryptic peptide of the EGF receptor beginning with Gly-85, which is in domain I, a region N terminal to the first cysteine-rich region of the receptor. Selective loss of signal in the 17th sequencing cycle suggests that the point of attachment of N-terminally modified 125I-mEGF to the receptor is Tyr-101. The data presented here provide identification by direct protein microsequencing of a site of interaction of EGF and the EGF receptor.
Woltjer
R L
RL
Department of Biochemistry, Vanderbilt University, Nashville, TN 37235.
Lukas
T J
TJ
Staros
J V
JV
D000595:Amino Acid Sequence; D000818:Animals; D001665:Binding Sites; D002460:Cell Line; D002846:Chromatography, Affinity; D003432:Cross-Linking Reagents; D003545:Cysteine / Q000032:analysis; D004815:Epidermal Growth Factor / Q000302:isolation & purification / Q000378:metabolism*; D066246:ErbB Receptors / Q000302:isolation & purification / Q000378:metabolism*; D006801:Humans; D007457:Iodine Radioisotopes; D051379:Mice; D008969:Molecular Sequence Data; D019000:Phosphotyrosine; D011487:Protein Conformation; D014443:Tyrosine / Q000031:analogs & derivatives
D003432:Cross-Linking Reagents; D007457:Iodine Radioisotopes; D019000:Phosphotyrosine; D014443:Tyrosine; D004815:Epidermal Growth Factor; D066246:ErbB Receptors; D003545:Cysteine