High-yield covalent attachment of epidermal growth factor to its receptor by kinetically controlled, stepwise affinity cross-linking.
We report here the use of a stepwise affinity cross-linking technique in the specific covalent attachment of epidermal growth factor (EGF) to its receptor. A heterobifunctional cross-linking reagent, sulfo-N-succinimidyl 4-(fluorosulfonyl)benzoate (SSFSB), which contains a rapidly reacting sulfo-N-succinimidyl active ester and a much more slowly reacting aromatic fluorosulfonyl moiety, was synthesized and characterized. Murine EGF (mEGF) was modified by the cross-linker to yield as the major product a derivative of mEGF having the (fluorosulfonyl)benzoyl moiety attached covalently at the amino terminus. SSFSB-modified, 125I-labeled mEGF was separated from unreacted SSFSB by size-exclusion chromatography and applied to shed membrane vesicles from A431 human carcinoma cells. Binding of derivatized 125I-mEGF to vesicles led to high yields (greater than 60%) of covalent linkage of 125I-mEGF to the EGF receptor, as determined by measurement of the fraction of specifically bound radiolabel which comigrated with the EGF receptor in NaDodSO4-polyacrylamide gels. The specificity of affinity cross-linking was evident in the negligible degree of labeling of species other than the EGF receptor and in the retention of EGF-stimulated receptor kinase activity after cross-linking.
Woltjer
R L
RL
Department of Biochemistry, Vanderbilt University, Nashville, Tennessee 37235.
Weclas-Henderson
L
L
Papayannopoulos
I A
IA
Staros
J V
JV
D000818:Animals; D001345:Autoradiography; D003432:Cross-Linking Reagents / Q000494:pharmacology*; D004591:Electrophoresis, Polyacrylamide Gel; D004815:Epidermal Growth Factor / Q000302:isolation & purification / Q000378:metabolism*; D066246:ErbB Receptors / Q000302:isolation & purification / Q000378:metabolism*; D007457:Iodine Radioisotopes; D007700:Kinetics; D051379:Mice; D010766:Phosphorylation; D011505:Protein-Tyrosine Kinases / Q000378:metabolism*; D013388:Succinimides / Q000138:chemical synthesis / Q000494:pharmacology*
D003432:Cross-Linking Reagents; D007457:Iodine Radioisotopes; D013388:Succinimides; C076345:sulfo-N-succinimidyl-4-(fluorosulfonyl)benzoate; D004815:Epidermal Growth Factor; D066246:ErbB Receptors; D011505:Protein-Tyrosine Kinases