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member of the Ras superfamily of proteins, a guanosine-nucleotide-binding
protein (small G protein also displaying a GTPase activity) closely related in
structure to the G
a
subunit of heterotrimeric G proteins (large GTPases).
These proteins have been shown to be strongly involved in cancer, with mu-
tated Ras stabilized into a constitutively active conformation, and they have
also been shown to be involved in some GPCR signaling cascades. Similar
to the assay previously described to measure binding of Eu-labeled GTP
analog to a large G protein, in this assay the authors have used a
terbium-labeled GTP (Tb-GTP).
25
However, no washing or filtration
steps are required, as the assay is based on the homogeneous QRET.
The Tb-GTP binding to Ras protects the fluorescence signal of terbium
from quenching by a quencher in solution, whereas the signal of the non-
bound fraction of Tb-GTP is quenched. Again, the time-resolved mea-
surement of the fluorescence signal increases the sensitivity of the assay
allowing HTS application.
3.3.2 Screening for G protein-independent pathways
After agonist stimulation, most GPCRs are phosphorylated by specific
GPCR kinases, and the recruitment of
b
-arrestins to the phosphorylated
GPCRs eventually terminates G protein signaling and leads to a coordi-
nated process of receptor desensitization, inactivation, internalization,
and, finally, either recycling to the membrane or lysosomal degradation.
b
-Arrestin proteins are also known to promote G protein-independent
signaling of GPCRs, including those involving mitogen-activated protein
kinases (MAPKs) such as ERK1/2, receptor and nonreceptor tyrosine
kinases, phosphatidylinositol 3-kinases, and probably many others. A sand-
wich format TR-FRET assay designed to detect and quantify phosphor-
ylation of ERK1/2 was recently marketed by Cisbio Bioassays.
17
It is based
on the use of two labeled antibodies, one directed against ERK bound to
the donor and the other one recognizing specifically the phosphorylated
form of the protein and bound to the acceptor. It should be emphasized
that MAPK signaling cascade events are temporally and spatially very well
controlled in the cells; thus some optimization should be done before using
this kit, as a single endpoint measurement of pERK could miss important
events occurring in the cells. Of interest, a proof-of-concept study to
measure recruitment of intrace lularproteinstoaGPCRhasbeen
performed.
33
This study relies on the insertion of small tags (HA, Flag,
or Myc) at the carboxy-terminal tail of the receptor and in the protein
of
interest. After mild solubilization of the membrane allowing the
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