Biology Reference
In-Depth Information
comparison of
t
1/2
values can validate these interactions. However, it is possible that no
differences in
t
1/2
values would be detected. Differences in the inhibition constant
K
i
might also be observed.
9.3
DISCUSSION
Here, we provide a simple method that can be added to the armamentarium to char-
acterize allosteric interactions in GPCRs, in both heterologous or homologous ex-
pression systems as measured with many different types of potential allosteric
modulators, including ligands, G protein, or dimer partners. The technique is not lim-
ited to GPCRs and should have general applicability for other receptors as well. The
advantages and difficulties of the approach are summarized in
Box 9.1
.
Box 9.1
Advantages and Pitfalls of This Method
Advantages
- Our method provides a simple way to assess the presence of an allosteric receptor dimer or
other allosteric effects with either known or new ligands or interacting proteins. Especially
when performed using isolated membranes, a change in dissociation kinetics of the labeled
orthosteric ligand can only be explained by an allosteric interaction between receptors.
- This technique is relatively easy to perform and could be used to simply detect the formation
of a dimer, as immunoprecipitation of GPCRs can be a tedious process due to the instability
of dimers in cell lysates or the poor specificity of antibodies directed against them.
- The technique can be used with endogenous receptors and native cells or primary tissues. No
special equipment is required for cellular imaging. Validation of such allosteric interactions
between receptors would, however, require depletion of one or both of the endogenous
partners (e.g., siRNA or shRNA).
- Alternatively to adding an excess of unlabeled competitor (preferably the same as the
orthosteric labeled one), in the reaction to promote the dissociation, one can also dilute the
reaction medium (more than 10 times) to promote dissociation at different time point.
Pitfalls
- Our technique requires radioligands, which are often expensive, and certain risks are
inherent in their use. However, high-affinity fluorescent ligands might work equally well.
- It is preferable to use the same orthosteric unlabeled ligand to displace the labeled one, in
order to avoid the possibility that allosteric modulation influences binding of these two
ligands differently. If not possible, the dilution method for assessing the kinetics of
dissociation of the labeled ligand should be used.
- Allosterism or asymmetry in binding affinities may not necessarily transmitted to the cell in
the same way; that is, a change in dissociation kinetics does not necessarily mean a similar
change in signaling downstream of the receptor.
- False negatives might be observed; no change in half-time of ligand dissociation does not
necessarily mean that there is no allosteric modulation; it may simply suggest another
mechanism of action. It is advisable to examine receptor binding and signaling allostery from
multiple vantage points.
