A. Richter et al., CONTRIBUTION OF THE TRANSFORMING GROWTH-FACTOR-ALPHA B-LOOP BETA-SHEET TO BINDING AND ACTIVATION OF THE EPIDERMAL GROWTH-FACTOR RECEPTOR, The Journal of biological chemistry, 270(4), 1995, pp. 1612-1616
We have exploited the differences in binding affinities of the chicken
epidermal growth factor (EGF) receptor for EGF and transforming growt
h 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 on
ly 30-40% overall sequence identity. The B-loop beta-sheet is the majo
r structural element in EGF and TGF alpha, but sequence similarity in
this region is low. To investigate its role in receptor binding, we co
nstructed two chimeric growth factors (mEGF/hTGF alpha(21-40) and mEGF
/hTGF alpha(21-32)) composed of the murine EGF (mEGF) amino acid seque
nce with residues 21-30 of the B-loop beta-sheet replaced by the equiv
alent residues of human TGF alpha (hTGF alpha); in chimera mEGF/hTGF a
lpha(21-32), asparagine 32, which lies at the boundary of the amino an
d carboxyl domains of mEGF, was also replaced by its hTGF alpha counte
rpart (valine). In initial studies using unpurified medium, it was fou
nd that the recombinant growth factors exhibited differing mitogenic p
otencies (mEGF/hTGF alpha(21-32) > mEGF/hTGF alpha(21-30) > mEGF) when
assayed on chicken fibroblasts, even though they were equivalent in m
itogenesis assays us ing cells expressing the human EGF receptor, Afte
r purification, mEGF/hTGF alpha(21-32) was found to be 50 times more p
otent than mEGF in the chick fibroblast mitogenesis assay and exhibite
d a 10-fold increase in relative affinity for the chicken EGF receptor
; both growth factors still exhibited equivalent mitogenic and recepto
r 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.