Mutations within subdomain II of the extracellular region of epidermal growth factor receptor selectively alter TGF alpha binding.
The interactions of epidermal growth factor (EGF) and transforming growth factor alpha (TGF alpha) with the epidermal growth factor receptor (EGFR) were examined by insertion mutagenesis of the receptor. Seventeen insertions were made throughout a construct containing only the extracellular domain. This truncated receptor (sEGFR) was secreted and had a dissociation constant similar to that of the full-length solubilized receptor. Receptors with insertions within subdomain III were not secreted. Two receptors with insertions at positions 291 and 474, which border subdomain III, have significantly decreased binding to both EGF and TGF alpha relative to wild type. This confirms previous work demonstrating that subdomain III forms the primary binding site for EGF and TGF alpha. Four of the mutants within subdomain II had a decreased binding to TGF alpha relative to wild type, but had wild type binding to EGF. These results suggest that a region within subdomain II may selectively regulate the binding of TGF alpha. Two receptors which contained insertions within subdomains II and IV, approximately equidistant from the center of subdomain III, bound twofold more ligand molecules than wild type receptor, with an affinity similar to that of wild type receptor. These findings suggest that insertion at these positions allows the access of more than one ligand molecule to the binding site.
Harte
M T
MT
Department of Biochemistry and Molecular Biology, Medical College of Ohio, Toledo 43699-0008, USA.
Gentry
L E
LE
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D002478:Cells, Cultured; D004815:Epidermal Growth Factor / Q000378:metabolism; D066246:ErbB Receptors / Q000235:genetics / Q000378:metabolism*; D015151:Immunoblotting; D008969:Molecular Sequence Data; D016254:Mutagenesis, Insertional; D011233:Precipitin Tests; D011485:Protein Binding; D011487:Protein Conformation; D011994:Recombinant Proteins / Q000378:metabolism; D017384:Sequence Deletion; D013329:Structure-Activity Relationship; D016211:Transforming Growth Factor alpha / Q000378:metabolism*
D011994:Recombinant Proteins; D016211:Transforming Growth Factor alpha; D004815:Epidermal Growth Factor; D066246:ErbB Receptors