Abstract:
Fibronectin (FN) matrix is crucial for cell and tissue functions
during embryonic development, wound healing, and oncogenesis.
Assembly of FN matrix fibrils requires FN domains that
mediate interactions with integrin receptors and with other FN
molecules. In addition, regulation of FN matrix assembly
depends on the first two FN type III modules, III1 and III2, which
harbor FN-binding sites. We propose that interactions between
these two modules sequester FN-binding sites in soluble FN and
that these sites become exposed by FN conformational changes
during assembly. To test the idea that III1–2 has a compact conformation,
we constructed CIIIY, a conformational sensor of
III1–2 based on fluorescent resonance energy transfer between
cyan and yellow fluorescent proteins conjugated at its N and C
termini. We demonstrate energy transfer in CIIIY and show that
fluorescent resonance energy transfer was eliminated by proteolysis
and by treatment with mild denaturants that disrupted
intramolecular interactions between the two modules. We also
show that mutations of key charged residues resulted in conformational
changes that exposed binding sites for the N-terminal
70-kDa FN fragment. Collectively, these results support a conformation-dependent
mechanism for the regulation of FN
matrix assembly by III1–2.
