Direct identification of residues of the epidermal growth factor receptor in close proximity to the amino terminus of bound epidermal growth factor

Randall L. Woltjer, Thomas J. Lukas, James V. Staros

Research output: Contribution to journalArticle

34 Scopus citations

Abstract

We have recently developed a kinetically controlled, step-wise affinity cross-linking technique for specific, high-yield, covalent linkage of marine 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.

Original languageEnglish (US)
Pages (from-to)7801-7805
Number of pages5
JournalProceedings of the National Academy of Sciences of the United States of America
Volume89
Issue number16
DOIs
StatePublished - Jan 1 1992

Keywords

  • Binding site
  • Cross-linking
  • Protein sequencing

ASJC Scopus subject areas

  • General

Fingerprint Dive into the research topics of 'Direct identification of residues of the epidermal growth factor receptor in close proximity to the amino terminus of bound epidermal growth factor'. Together they form a unique fingerprint.

  • Cite this