Abstract
We have exploited the differences in binding affinities of the chicken epidermal growth factor (EGF) receptor for EGF and transforming growth factor alpha (TGF alpha) to study the role of the B-loop beta-sheet of these ligands in receptor recognition and activation. Although EGF and TGF alpha share similar secondary and tertiary structures imposed by three highly conserved intramolecular disulfide bonds, they have only 30-40% overall sequence identity. The B-loop beta-sheet is the major structural element in EGF and TGF alpha, but sequence similarity in this region is low. To investigate its role in receptor binding, we constructed two chimeric growth factors (mEGF/hTGF alpha 21-30 and mEGF/hTGF alpha 21-32) composed of the murine EGF (mEGF) amino acid sequence with residues 21-30 of the B-loop beta-sheet replaced by the equivalent residues of human TGF alpha (hTGF alpha); in chimera mEGF/hTGF alpha 21-32, asparagine 32, which lies at the boundary of the amino and carboxyl domains of mEGF, was also replaced by its hTGF alpha counterpart (valine). In initial studies using unpurified medium, it was found that the recombinant growth factors exhibited differing mitogenic potencies (mEGF/hTGF alpha 21-32 > mEGF/hTGF alpha 21-30 > mEGF) when assayed on chicken fibroblasts, even though they were equivalent in mitogenesis assays using cells expressing the human EGF receptor. After purification, mEGF/hTGF alpha 21-32 was found to be 50 times more potent than mEGF in the chick fibroblast mitogenesis assay and exhibited a 10-fold increase in relative affinity for the chicken EGF receptor; both growth factors still exhibited equivalent mitogenic and receptor binding activity when tested on cells expressing human EGF receptors. We conclude that the B-loop beta-sheet of hTGF alpha is an important determinant of EGF receptor binding affinity and biological activity.
Highlights
From the Cancer Research Campaign Medical Oncology Unit, Southampton General Hospital, Tremona Road, Southampton S016 6YD, United Kingdom
To investigate its role in receptor binding, we constructed two chimeric growth factors composed of the murine epidermal growth factor (EGF) amino acid sequence with residues 21-30 of the B-Ioop p-sheet replaced by the equivalent residues of human transforming growth factor a (TGFa); in chimera mEGF/ hTGFa21-32' asparagine 32, which lies at the boundary of the amino and carboxyl domains of mEGF, was replaced by its hTGFa counterpart
In initial studies using unpurified medium, it was found that the recombinant growth factors exhibited differing mitogenic potencies when assayed on chicken fibroblasts, even though they were equivalent in mitogenesis assays using cells expressing the human EGF receptor
Summary
Materials-Standard mEGF was purified from murine submaxillary glands according to the method of Savage and Cohen [30] and was a gift from Dr P Moores (Commonwealth Scientific and Industrial Research Organization, Australia). This was used as immunogen for production of a polyclonal sheep anti-mEGF antiserum. 250 jJ.l/well of 125I_labeled mEGF (20 ng/ml, 3.7 mBq/ILg)in PBS, 1% (w/v) BSA, 0.02% (w/v) sodium azide was added to all wells, and the cells incubated at 22 "C for 4 h After this time, buffer containing unbound material was removed from the trays by pipette, and the cells were washed with PBS, 0.1% BSA. Nonspecific binding, determined in the presence of an excess of unlabeled mEGF, was less than 5 (NR6/HER) and 20% (CEF) of total bound ligand
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