Abstract
TGF-ß1, -ß2, and -ß3 are small, secreted
signaling proteins. They share 71-80% sequence identity
and signal through the same receptors, yet the isoform-specific
null mice have distinctive phenotypes and are inviable. The
replacement of the coding sequence of TGF-ß1 with TGF-ß3
and TGF-ß3 with TGF-ß1 led to only partial rescue of the
mutant phenotypes, suggesting that intrinsic differences
between them contribute to the requirement of each in vivo.
Here, we investigated whether the previously reported
differences in the flexibility of the interfacial helix and
arrangement of monomers was responsible for the differences in activity by generating two chimeric proteins in which residues
54-75 in the homodimer interface were swapped. Structural analysis of these using NMR and functional analysis using a dermal
fibroblast migration assay showed that swapping the interfacial region swapped both the conformational preferences and activity.
Conformational and activity differences were also observed between TGF-ß3 and a variant with four helix-stabilizing residues
from TGF-ß1, suggesting that the observed changes were due to increased helical stability and the altered conformation, as
proposed. Surface plasmon resonance analysis showed that TGF-ß1, TGF-ß3, and variants bound the type II signaling receptor,
TßRII, nearly identically, but had small differences in the dissociation rate constant for recruitment of the type I signaling
receptor, TßRI. However, the latter did not correlate with conformational preference or activity. Hence, the difference in activity
arises from differences in their conformations, not their manner of receptor binding, suggesting that a matrix protein that
differentially binds them might determine their distinct activities.
Tr
signaling proteins. They share 71-80% sequence identity
and signal through the same receptors, yet the isoform-specific
null mice have distinctive phenotypes and are inviable. The
replacement of the coding sequence of TGF-ß1 with TGF-ß3
and TGF-ß3 with TGF-ß1 led to only partial rescue of the
mutant phenotypes, suggesting that intrinsic differences
between them contribute to the requirement of each in vivo.
Here, we investigated whether the previously reported
differences in the flexibility of the interfacial helix and
arrangement of monomers was responsible for the differences in activity by generating two chimeric proteins in which residues
54-75 in the homodimer interface were swapped. Structural analysis of these using NMR and functional analysis using a dermal
fibroblast migration assay showed that swapping the interfacial region swapped both the conformational preferences and activity.
Conformational and activity differences were also observed between TGF-ß3 and a variant with four helix-stabilizing residues
from TGF-ß1, suggesting that the observed changes were due to increased helical stability and the altered conformation, as
proposed. Surface plasmon resonance analysis showed that TGF-ß1, TGF-ß3, and variants bound the type II signaling receptor,
TßRII, nearly identically, but had small differences in the dissociation rate constant for recruitment of the type I signaling
receptor, TßRI. However, the latter did not correlate with conformational preference or activity. Hence, the difference in activity
arises from differences in their conformations, not their manner of receptor binding, suggesting that a matrix protein that
differentially binds them might determine their distinct activities.
Tr
Original language | English |
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Pages (from-to) | 5737-5749 |
Number of pages | 13 |
Journal | Biochemistry |
Volume | 53 |
Issue number | 36 |
Early online date | 25 Aug 2014 |
DOIs | |
Publication status | Published - 16 Sept 2014 |