Identification of residues of the epidermal growth factor receptor proximal to residue 45 of bound epidermal growth factor.
A triple mutant of murine epidermal growth factor (mEGF), N1Q/H22Y/R45K-mEGF, was constructed by site-directed mutagenesis, expressed, purified, and characterized for use in an affinity cross-linking study to identify aminoacyl residues of the EGF receptor adjacent to a residue in the carboxyl-terminal domain of bound EGF thought to be important in distinguishing between EGF and transforming growth factor-alpha in their recognition by the receptor. Cyclization of Gln1 to form pyroglutamate (pE) limited the site of cross-linking in the mutant to Lys45, permitting identification of receptor residues that are proximal to this residue of bound EGF. The resulting N1pE/H22Y/R45K-mEGF was shown to be comparable to wild-type mEGF in receptor binding and stimulation of receptor autophosphorylation. 125I-Labeled N1pE/H22Y/R45K-mEGF was reacted with the heterobifunctional cross-linking reagent sulfo-N-succinimidyl-4-(fluorosulfonyl)benzoate, and the resulting modified EGF was incubated with A431 membrane vesicles bearing EGF receptors. Incubation resulted in specific cross-linking of the labeled N1pE/H22Y/R45K-mEGF to EGF receptors. The resulting cross-linked complex was then partially purified, denatured, reduced, and carboxyamidomethylated. Digestion with endoprotease LysC resulted in a unique radiolabeled peptide that could be immunoprecipitated using antibodies to mEGF. This immunoprecipitated fragment was purified by gel electrophoresis and subjected to microsequencing. The resulting sequence was matched to that of a LysC fragment of the receptor, which begins with Thr464 and is near the interface of receptor subdomains III and IV. Loss of signal at cycle 2 suggests that the point of attachment of cross-linked N1pE/H22Y/R45K is Lys465 of the receptor.
Summerfield
A E
AE
Department of Molecular Biology, Vanderbilt University, Nashville, Tennessee 37235, USA.
Hudnall
A K
AK
Lukas
T J
TJ
Guyer
C A
CA
Staros
J V
JV
D000595:Amino Acid Sequence; D000818:Animals; D001665:Binding Sites; D003432:Cross-Linking Reagents; D004815:Epidermal Growth Factor / Q000737:chemistry*; D066246:ErbB Receptors / Q000737:chemistry*; D051379:Mice; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D011485:Protein Binding
D003432:Cross-Linking Reagents; D004815:Epidermal Growth Factor; D066246:ErbB Receptors