Receptor-Receptor Interactions in the Central Nervous System

353 Pages·2018·10.461 MB·English

by  KJELL FUXE, Dasiel O. Borroto-Escuela

Neuromethods 140
Kjell Fuxe
Dasiel O. Borroto-Escuela Editors
Receptor-Receptor
Interactions
in the Central
Nervous System
N
euromethods
Series Editor:
Wolfgang Walz
University of Saskatchewan
Saskatoon, Canada
For further volumes:
http://www.springer.com/series/7657
Receptor-Receptor Interactions
in the Central Nervous System
Edited by
Kjell Fuxe
Department of Neuroscience, Karolinska Institutet, Stockholm, Sweden
Dasiel O. Borroto-Escuela
Department of Neuroscience, Karolinska Institutet, Stockholm, Sweden
Editors
Kjell Fuxe Dasiel O. Borroto-Escuela
Department of Neuroscience Department of Neuroscience
Karolinska Institutet Karolinska Institutet
Stockholm, Sweden Stockholm, Sweden
ISSN 0893-2336 ISSN 1940-6045 (electronic)
Neuromethods
ISBN 978-1-4939-8575-3 ISBN 978-1-4939-8576-0 (eBook)
https://doi.org/10.1007/978-1-4939-8576-0
Library of Congress Control Number: 2018947383
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v
Preface
G protein-coupled receptor (GPCR) can form homo- and heteroreceptor complexes with
allosteric receptor–receptor interactions representing a novel molecular integrative mecha-
nism in the central nervous system (CNS) [1, 2]. This takes place through direct physical
interactions between co-expressed GPCRs either by physical association between the same
receptor (homoreceptor complex) or different GPCR types (heteroreceptor complex) and
with or without the participation of adapter proteins [3]. There are many impressive exam-
ples demonstrating GPCR–GPCR heteromerization, which through allosteric receptor–
receptor interactions alters the GPCR protomer recognition, signaling, and trafficking.
These changes in the receptor protomers produce marked increases in diversity and bias of
GPCR function.
One emerging concept in neuropsychopharmacology is that a dysfunction of the allo-
steric receptor–receptor interactions contributes to disease progression in mental and neu-
rological disorders [4–10]. So far their stoichiometry and topology are unknown within the
heteromer formed as well as the number of adapter proteins participating, including their
architecture. The heteromers in the CNS should therefore be described as heteroreceptor
complexes [2].
The overall architecture of the global GPCR heterodimer network shows that inter alia
D2R are hub components forming more than ten heterodimer pairs [11]. It should be
underlined that GPCR can also form complexes with ion channel receptors modulating, for
instance, the synaptic glutamate and GABA receptor signaling [12–14]. GPCR-tyrosine
receptor kinase (RTK) heteroreceptor complexes also exist [7, 15] in which the GPCR
protomer can modulate the trophic function of the receptor tyrosine kinase (RTK) through
allosteric modulation over the GPCR–RTK interface. Thus, GPCRs may modulate tro-
phism and structural plasticity via allosteric modulation of RTK function in GPCR–RTK
heteroreceptor complexes.
The contributors to this book will cover the large number of methodologies used to
unravel the receptor–receptor interactions in heteroreceptor complexes in the CNS from
the molecular to the network level.
The first three chapters deal with biochemical binding techniques, receptor autoradiog-
raphy, and (35S)GTP gammaS binding in autoradiography which were used already in the
early work. The biochemical binding experiments in CNS membrane preparations in the
early 1980s led to the concept of receptor–receptor interactions in the plasma membrane
(Chap. 1). The autoradiography makes possible an improved regional analysis and the use
of more intact brain membranes present in the brain sections. This may help maintain the
intramembrane receptor–receptor interaction (Chaps. 2 and 3). Chapter 4 deals with the
superfused synaptosome technique to understand the function of the receptor–receptor
interactions at the presynaptic level. Chapter 5 gives the protocols for dissociating and
maintaining primary neurons from the hippocampus in order to study the transactivation
of the FGFR1 by muscarinic cholinergic receptors. Methods to determine neurite out-
growth and protein phosphorylation are also described. They are needed to determine the
function of this RTK–GPCR interaction (Chap. 5). Protocols for electrophysiological
vii
viii Preface
approaches to determine GPCR–RTK interactions are found in Chap. 6. Patch-clamp
protocols are provided to analyze the GPCR–RTK interaction.
At the network level, the protocols for the microdialysis technique are provided used to
study the role of the receptor–receptor interactions in the brain circuits of the awake freely
moving rat (Chap. 7). In Chap. 8, behavioral methods are given to determine the role of
receptor–receptor interactions in fear and anxiety at the network level. The methods to use
small interference RNA knockdown in rats to determine the function of receptor–receptor
interactions in anxiety- and depression-like behaviors are provided in Chap. 9. Protocols for
the use of double fluorescent knock-in mice are provided to investigate subcellular distribu-
tion of endogenous mu-delta opioid heteromers in primary hippocampal cultures from this
mouse model (Chap. 10). Their coexistence in various brain regions can also be elegantly
demonstrated.
Moving into biochemical methods Chap. 11 gives the protocols for coimmunoprecipita-
tion (Co-IP) and protein affinity purification assays to determine D2R-associated protein
complexes. Their D2R–protein interactions like D2R–Disc1 interactions play a significant
role in regulating the D2R signaling. In Chap. 12, the protocols for BRET are introduced to
determine receptor heterodimers in cellular models. Protocols are also given for assays to
establish the existence of heterotrimers. These assays build on the use of a combination of
BRET and bimolecular fluorescence complementation (BiFc) or of a sequential BRET-F RET
(SRET) procedure. The restraints of the energy transfer assays are discussed and how they can
be complemented by in situ proximity ligation assay (PLA). A more detailed analysis of a
dimerization-induced BiFc is given in Chap. 13 introducing a high-resolution approach
using confocal laser microscopy vs a low-resolution approach using automated cell imaging
with a multi-mode plate reader. In Chap. 14, the protocols for flow cytometry- based FRET
(fcFRET) are given. This technique will allow the detection and quantification of dynamic
receptor–receptor interactions based on the analysis of the fc-FRET.
In Chap. 15, a novel strategy is given to study GPCR dimerization in living cells. It is
a protein complementation assay involving a reconstitution of a luminescent protein, a
so- called nanoluciferase. Complementary nanoluciferase fragments are fused to a receptor
pair, which can combine to form a reporter if dimerization takes place. Another new method
is provided in Chap. 16 to study GPCR dimerization based on proximity biotinylation.
Its advantages and disadvantages versus other methods are discussed. In Chap. 17, a new
method is presented for the conformational profiling of GPCRs using the fluorescein arsen-
ical hairpin (FIAsH) binders as energy acceptors with the tetracysteine binding tag in dif-
ferent positions within the intracellular domains of the 5-HT2A receptor. This method uses
a FIAsH-BRET approach with the Renilla luciferase fused to the C-terminal tail of the
5-HT2A receptor.
In Chap. 18, a detailed description of the GPCR-hetnet is given. The G protein-c oupled
receptor heterocomplex network database (GPCR-hetnet) is a database designed to store
information on GPCR heteroreceptor complexes and their allosteric receptor–receptor inter-
actions. It is an expert-authored and peer-reviewed database of well-documented GPCR–
GPCR, GPCR–receptor tyrosine kinase, and GPCR–ionotropic receptor/ligand-g ated ion
channel interactions. This chapter describes in a basic protocol how to use, navigate, and
browse through the GPCR-hetnet database to identify the clusters in which a receptor
protomer of interest is involved, while further applicability is also described and introduced.
In Chap. 19, detailed protocols are given for the use of the in situ proximity ligation
assay in the analysis of homo- and heteroreceptor complexes in the CNS. It involves the use
of confocal laser microscopy and quantitation of the density of PLA-positive clusters per
Preface ix
cell in the sampled fields of various brain regions. Protocols for demonstrating the brain
functions of receptor oligomers are presented in Chap. 20 using receptor interface interacting
peptides. The D1R-NMDAR heteromer is given as an example.
The book ends with a description of the methodology to study GPCR dimers and
higher order oligomers at the single-molecule level through super-resolution level (Chap. 21).
Photoactivated localization microscopy using photoactivatable dyes was employed to visualize
the spatial organization of the homo-heteroreceptor complexes and perform quantitation
of their composition in terms of monomers, dimers, trimers, etc.
Stockholm, Sweden Kjell Fuxe
Dasiel O. Borroto-Escuela
References
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Contents
Series Preface . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . v
Preface . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . vii
Contributors . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . xv
1 Analysis and Quantification of GPCR Allosteric Receptor–Receptor
Interactions Using Radioligand Binding Assays: The A2AR-D2R
Heteroreceptor Complex Example . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1
Dasiel O. Borroto-Escuela, Miguel Pérez de la Mora, Michele Zoli,
Fabio Benfenati, Manuel Narvaez, Alicia Rivera, Zaida Díaz-Cabiale,
Sarah Beggiato, Luca Ferraro, Sergio Tanganelli, Patrizia Ambrogini,
Malgorzata Filip, Fang Liu, Rafael Franco, Luigi F. Agnati,
and Kjell Fuxe
2 Analysis and Quantification of GPCR Heteroreceptor Complexes
and Their Allosteric Receptor–Receptor Interactions Using Radioligand
Binding Autoradiography . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 15
Manuel Narvaez, Fidel Corrales, Ismel Brito, Ismael Valladolid-Acebes,
Kjell Fuxe, and Dasiel O. Borroto-Escuela
3 On the Study of D R-MOR Receptor–Receptor Interaction in the Rat
4
Caudate Putamen: Relevance on Morphine Addiction . . . . . . . . . . . . . . . . . . . . 25
Alicia Rivera, Alejandra Valderrama-Carvajal, Diana Suárez-Boomgaard,
Kirill Shumilov, M. Ángeles Real, Kjell Fuxe, and Belén Gago
4 Use of Superfused Synaptosomes to Understand the Role
of Receptor–Receptor Interactions as Integrative Mechanisms in Nerve
Terminals from Selected Brain Region . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 41
Sarah Beggiato, Sergio Tanganelli, Tiziana Antonelli,
Maria Cristina Tomasini, Kjell Fuxe, Dasiel O. Borroto-Escuela,
and Luca Ferraro
5 Detection of Fibroblast Growth Factor Receptor 1 (FGFR1) Transactivation
by Muscarinic Acetylcholine Receptors (mAChRs) in Primary Neuronal
Hippocampal Cultures Through Use of Biochemical and Morphological
Approaches . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 57
Valentina Di Liberto, Giuseppa Mudó, Dasiel O. Borroto-Escuela,
Kjell Fuxe, and Natale Belluardo
6 Electrophysiological Approach to GPCR–RTK Interaction Study
in Hippocampus of Adult Rats . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 71
Davide Lattanzi, David Savelli, Michael Di Palma, Stefano Sartini,
Silvia Eusebi, Dasiel O. Borroto-Escuela, Riccardo Cuppini, Kjell Fuxe,
and Patrizia Ambrogini
xi