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In-Depth Information
dimerize or not. If the M mutant that has to be tested retains a classic internalization
profile upon 5-HT exposure, it will be coexpressed with the D
100
A mutant. Upon 5-
HT exposure, if RASSL protomers appear to be internalized, one can conclude that
the Mmutant is capable to dimerize with the D
100
A protomers. This technique could
thus be used as a dimerization-screening test. 5-HT
4
mutants that have been de-
scribed to disrupt dimer formation (
Berthouze et al., 2007
) have to be assayed in
our procedure to validate this hypothesis. Moreover, due to the high conservation
of an aspartate in the third transmembrane domain of biogenic amine receptors
(
Strader et al., 1991
), corresponding to the D
100
or D
3.32
position, the point mutation
into alanine can easily be transposed in metabotropic serotonergic receptors
(
Kristiansen et al., 2000
) as well as other class A GPCR such as melanocortin-4 re-
ceptors (
Srinivasan, Santiago, Lubrano, Vaisse, & Conklin, 2007
) or histamine H1
receptors (
Bakker et al., 2004
), conferring similar RASSL properties to the mutated
receptors. Consequently, this dimerization-screening test could be extended to other
GPCR structurally related to the 5-HT
4
receptors.
We finally described a protocol to achieve relative quantification of 5-HT
4
R di-
mer formation using TR-FRET. Using this method, we can also assess heteromeriza-
tion with other GPCRs by competition assay. This technology is simple and reliable
and can be improved using covalent binding of the acceptor and donor fluorophores
to a small suicide enzyme inserted N-terminally in the GPCRs instead of the classical
tags (
Comps-Agrar et al., 2011
). Future developments will aim to use permeant sub-
strates of these enzymes to provide intracellular labeling of the dimers and follow
their trafficking.
SUMMARY
Dimerization process is essential for 5-HT
4
receptor function. We provide here
methods to analyze 5-HT
4
R dimer formation ranging from classical Western blotting
and coimmunoprecipitation protocols to cross internalization screening assay and
TR-FRET measurements. We intend to describe in details the experimental proce-
dures with the key points necessary to achieve precise and accurate studies regarding
5-HT
4
receptor dimerization.
Acknowledgments
This work was supported by grants from Centre National de la Recherche Scientifique
(CNRS), Institut National de la Sant´ et de la Recherche M´dicale (INSERM), Minist`re Fran-
¸ais de la Recherche (ANR Blanc-2006-0087-02—“GPCR dimers”), and Universit´ de
Montpellier.
cAMP quantification, FRET measurements, and ELISA were carried out using the
ARPEGE Pharmacology Screening Interactome facility at the Institute of Functional Geno-
mics (Montpellier, France).