posted on 2007-04-15, 00:00authored byEmily A. Smith, Thomas A. Bunch, Danny L. Brower
A method for measuring the microclustering of a class of
cell surface receptors called integrins is reported. Integrins are proteins involved in bidirectional signaling
across the cell membrane and are important in cell
adhesion, growth, and survival. Their activity is regulated
by changes in protein conformation and protein clustering. The developed in vivo clustering assay uses fluorescence resonance energy transfer (FRET) and has the
benefit of requiring a single cloning step to generate FRET
donors and acceptors that can be used to measure the
clustering of a series of integrin mutants. The FRET
reporters contain extracellular donor or acceptor fluorescent protein attached to native integrin cytoplasmic and
transmembrane domains, and these are expressed along
with wild-type or mutant integrins. Expression of the FRET
reporters has no affect on the ligand binding properties
of coexpressed integrins. FRET values are calculated for
cell lines spreading on ligand coated surfaces, and these
values are independent of fluorescent protein expression.
No FRET is observed in cell lines expressing the reporters
in the absence of integrins. Integrin-dependent FRET
values increase ∼2−3-fold when the integrins contain
mutations that result in increased ligand binding affinities.