Biology Reference
In-Depth Information
HA- and FLAG-5-HT 4 receptors for dimer formation, indicating the possible exis-
tence of heterodimers between 5-HT 4 and
b 2 -adrenergic receptors.
7.3 DISCUSSION
In this chapter, we first described protocols that are classically used in biochemistry
of GPCRs such as Western blotting and coimmunoprecipitation. This part is impor-
tant for us, as detecting 5-HT 4 receptors on gel from transient expression in cell lines
is a tricky point. Indeed, when this receptor is overexpressed it appears in numerous
bands, corresponding to different glycosylation states and maturation. The receptor
populations that have reached the plasma membrane are more homogenous in term
of sugar maturation, thus starting from a membrane preparation improve the results.
One alternative could be to use kits to purify membrane proteins such as Qproteome
Plasma Membrane Protein Kit (Qiagen, #37601), which gives good results in our
hands, is less time-consuming but more expensive. Adding a deglycosylation step
was also a plus to clarify the migration profile of 5-HT 4 receptors. The challenge
was to find a replacement to the N-Glycosidase F from Roche that was discontinued.
This enzyme was efficient in numerous buffers and the actual replacement enzymes
(Peptide-N-Glycosidase F, PNGase F) from different suppliers are not as effective.
Migration on long gels is another trick to separate more easily complexes of similar
size that helps us to show the possibility of heteromerization between 5-HT 4 R splice
variants.
We then described a method to functionally assess the formation of dimers by
cross desensitization of the RASSL-5-HT 4 mutant that is unable to bind 5-HT
( Claeysen et al., 2003 ). In the presence of WT 5-HT 4 receptor and upon 5-HT expo-
sure, the D 100 A mutant is cointernalized with the WT receptor, thus providing a nice
way to show D 100 A/WT dimers. A key step in this procedure is the cells to incubate
with the primary antibodies prior to fixation. This procedure avoids the small per-
meabilization that could be induced by paraformaldehyde. Antibodies in contact
with intact cells will label only plasma membrane receptors and not receptors that
are in the synthesis pathway. Consequently, the dimers that are immunolabeled
and endocyted originate without doubt from the plasma membrane. Interestingly,
our cross desensitization assay using the D 100 A mutant and the WT 5-HT 4 receptor
could also be used to assess whether a particular 5-HT 4 mutant named “M” is able to
FIGURE 7.4—Cont'd determined by ELISA. (D) Competition FRET experiments. A constant
amount of WT HA-5-HT 4 R and FLAG-5-HT 4 R was expressed and the FRET corresponding to
their association was determined in the presence of increasing amounts of Myc-tagged
GPCRs belonging to different classes. The FRET signal was plotted as a function of cell
surface expression of the Myc-tagged receptors determined by ELISA. Challenger GPCRs, 5-
HT 7 R and 5-HT 4 R, serotonin receptor subtypes 7 and 4, respectively;
b 2 -adrenergic
receptor; GABA B2 R, subunit of the GABA B receptor that reaches the cell surface alone (GB 2 ).
b 2 -AR,
Search WWH ::




Custom Search