High-yield covalent attachment of epidermal growth factor to its receptor by kinetically controlled, stepwise affinity cross-linking

Abstract

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.

Identification of Residues of the Epidermal Growth Factor Receptor Proximal to Residue 45 of Bound Epidermal Growth Factor

“…The absence of an identifiable signal in cycle two, which corresponds to Lys 465 of the receptor sequence, combined with the observation that this site is not cleaved by LysC, argues strongly that Lys 465 is the point of attachment of the receptor to Lys 45 of bound N1pE/H22Y/R45K-mEGF. 2 In this, as in our previous affinity cross-linking studies (13,14), we have observed specifically bound, but not covalently cross-linked 125 I-EGF present through the WGL-agarose purification step, which under the best of circumstances takes many hours and subjects the ligandreceptor complex to enormous dilution. The integrity of the ligandreceptor complex under such conditions can, in part, be explained by the very high rates of association observed for the hormone with the receptor (19).…”

“…Affinity Cross-linking of 125 I-N1pE/H22Y/R45K-mEGF to EGF Receptor in A431 Membrane Vesicles-Analytical scale affinity cross-linking was carried out as described by Woltjer et al (13), with modifications noted in the legend for Fig. 1.…”

“…We exploited this aspect of the chemistry of the hormone in a study in which the N terminus of wild-type mEGF was modified with SSFSB, a heterobifunctional cross-linking reagent, and affinity crosslinked to the receptor (13). The site of cross-linking was identified by Edman degradation of a purified fragment of the receptor containing the cross-link to EGF, revealing Tyr 101 as a receptor residue adjacent to the N terminus of bound EGF (14).…”

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, stepwise affinity cross-linking technique to achieve high yields of the EGF receptor covalently linked to the N terminus of murine EGF (mEGF) (30). Here we report purification of cross-linked species and conditions for denaturation and tryptic digestion of 125I-mEGF-linked receptor.…”

“…mEGF was prepared as described (31); 1251-mEGF was prepared as described (32), using 1 mg of unlabeled mEGF per ml as a carrier for radiolabeled ligand. The cross-linking reagent sulfo-N-succinimidyl-4-(fluorosulfonyl)benzoate (SSFSB) was synthesized as described (30). A431 cells were grown to confluence in Dulbecco's modified Eagle's medium (GIBCO) supplemented with 10%o calf serum (GIBCO).…”