Biology Reference
In-Depth Information
4 (5-HT
4
) receptors and required to obtain full receptor activity. Several techniques
have been developed to analyze dimer formation and properties. Due to our involve-
ment in deciphering 5-HT
4
R transduction mechanisms, we improved and set up new
procedures to study 5-HT
4
R dimers, by classical methods or modern tools. This
chapter presents detailed protocols to detect 5-HT
4
R dimers by Western blotting
and coimmunoprecipitation, including the optimizations that we routinely carry
out. We developed an innovative method to achieve functional visualization
of 5-HT
4
R dimers by immunofluorescence, taking advantage of the 5-HT
4
-RASSL
(receptor activated solely by synthetic ligand) mutant that was engineered in the
laboratory. Finally, we adapted the powerful time-resolved FRET technology to
assess a relative quantification of dimer formation and affinity.
INTRODUCTION
Serotonin type 4 receptors (5-HT
4
Rs) belong to the extended family of serotonin re-
ceptors, which counts 15 different types of receptors involved in a wide range of
physiological processes (
Berger, Gray, & Roth, 2009
). All, except 5-HT
3
receptor
that is an ionic channel, are G protein-coupled receptors (GPCRs) activating G
s
-,
G
i
-, or G
q
-dependent pathways and G protein-independent signaling cascades
(
Barnes & Sharp, 1999; Bockaert, Claeysen, Becamel, Dumuis, & Marin, 2006;
Millan, Marin, Bockaert, & Mannoury la Cour, 2008
). Homodimerization of seroto-
nin receptors has been described for the 5-HT
1A
(
Gorinski et al., 2012
), 5-HT
1B/D
(
Lee et al., 2000
), 5-HT
2A
(
Lukasiewicz, Faron-Gorecka, Kedracka-Krok, &
Dziedzicka-Wasylewska, 2011
), 5-HT
2C
(
Herrick-Davis, Grinde, &
Mazurkiewicz, 2004
), 5-HT
4
(
Berthouze et al., 2005
), and 5-HT
7
(
Renner et al.,
2012
), suggesting that all metabotropic serotonergic receptors form constitutive
homodimers. This dimerization process is essential for receptor function. Indeed,
the full activity of 5-HT
2C
and 5-HT
4
receptors has been obtained with the binding
of two molecules of ligand and one G protein per dimer (
Herrick-Davis, Grinde,
Harrigan, & Mazurkiewicz, 2005; Pellissier et al., 2011
).
Our team has been involved in the first pharmacological description of 5-HT
4
receptors (
Dumuis, Bouhelal, Sebben, Cory, & Bockaert, 1988
), in the cloning of
some splice variants (
Claeysen, Sebben, Becamel, Bockaert, & Dumuis, 1999;
Claeysen, Sebben, Journot, Bockaert, & Dumuis, 1996
) and in the characterization
of several original signaling pathways following the activation of these receptors
(
Bockaert, Claeysen, Compan, & Dumuis, 2011
). For years, we have developed
methods and tools to detect 5-HT
4
receptor dimers that we present in the succeeding
text. Ranging from classical to more sophisticated methods, we intend to provide
here the hints and tips that facilitate 5-HT
4
R dimerization study.
We will describe the following procedures:
1.
Cell transfection
2.
Detection of 5-HT
4
R dimers by Western blot