Biology Reference
In-Depth Information
11.
At specified times, stop the binding reaction by adding 2 ml of cold 50 mM
Tris-HCl, pH 7.4 to each binding reaction and immediately filter it on the glass
fiber circles placed on the manifold.
12.
Rinse the filters twice with 2 ml of cold 50 mM Tris-HCl, pH 7.4.
13.
Add the glass fiber circle to SARSTEDT tubes or scintillation tubes (then add
5 ml of scintillation liquid) to prepare for reading.
14.
Assess receptor-bound radioactivity with the appropriate counter.
9.2.3
Analysis and interpretation of results
Analysis of dissociation kinetic data can easily be performed using commercial soft-
ware, such as GraphPad 5 Prism (or higher versions). Prior to importing data into
GraphPad, specific binding needs to be calculated by subtracting nonspecific binding
from total binding. Moreover, reporting the data as fold over the binding at dissociation
kinetic time “0” (i.e., at binding equilibrium, without no cold ligand), or basal, will
permit normalization between experiments. Once the binding data is analyzed, it
can be plotted as
y
-axis against dissociation time (in this example, 0, 2, 5, 15, and
30 min). Data points can be further analyzed by nonlinear regression, using a
“dissociation-one-phase exponential decay” fit by constraining both the initial
binding (Y0) to 100 and maximum dissociation at (e.g., 30 min time) or nonspecific
binding (NS) at infinite time to an average common values. The time at which half of
the ligand has dissociated, or half-life (
t
1/2
), is calculated from ln(2)/
K
,where
K
rep-
resents the dissociation rate constant (
K
off
) and is an indicator of the affinity between
the ligand and its receptor. Once the nonlinear regression analysis is performed,
statistical comparisons (extra sum-of-square F test) between the best-fit values can
be evaluated assuming the null hypothesis that
K
is the same for all data sets. Rejection
of the null hypothesis (i.e.,
K
differs for each data sets) is thus interpreted as allosteric
modulation of the radioligand binding on receptors as compared to control conditions.
Representative examples of the effects GTP
g
S and PDC113.824 (an allosteric
modulator of PGF2
a
receptor FP) on [
3
H]-PGF2
a
binding to FP are shown in
Figs. 9.2 and 9.3
. Dissociation rates of [
3
H]-PGF2
a
binding from FP were increased
by almost threefold (
K
differed for each data sets,
P
0.001) in the presence of
GTP
g
S, suggesting a negative allosteric modulation of FP binding to its ligand by
the functional uncoupling of the G protein to the receptor. Similarly, dissociation
of [
3
H]-PGF2
a
binding to FP is increased by the allosteric modulator PDC (
t
1/2
of
2.55 min vs. 1.55,
P
<
0.05). On the other hand, positive allosteric modulation of
ligand binding to its receptor can be detected in the context of a receptor heterodimer,
as shown in
Fig. 9.4
, where dissociation rate of [
3
H]-PGF2
a
binding to FP increases
(
t
1/2
of 3.40 vs. 5.13 min,
P
<
0.001) in the presence of the angiotensin II (Ang II)
type I receptor (AT1R). Finally, this technique can also be used in an endogenous
context, where overexpression of receptors is not required. When the affinity of a
receptor for its ligand is increased by the presence of the receptor partner,
t
1/2
will
increase compared to the receptor of interest expressed alone. The opposite is also
true. In both cases,
<
if allosteric communication occurs between receptors,
the
