Detection, Analysis, and Quantification of GPCR Homo- and Heteroreceptor Complexes in Specific Neuronal Cell Populations Using the In Situ Proximity Ligation Assay
Por:
Borroto-Escuela D.O., Narvaez M., Valladolid-Acebes I., Shumilov K., Di Palma M., Wydra K., Schaefer T., Reyes-Resina I., Navarro G., Mudó G., Filip M., Sartini S., Friedland K., Schellekens H., Beggiato S., Ferraro L., Tanganelli S., Franco R., Belluardo N., Ambrogini P., Pérez de la Mora M., Fuxe K.
Publicada:
1 ene 2018
Resumen:
GPCR's receptosome operates via coordinated changes between the receptor
expression, their modifications and interactions between each other.
Perturbation in specific heteroreceptor complexes and/or their
balance/equilibrium with other heteroreceptor complexes and
corresponding homoreceptor complexes is considered to have a role in
pathogenic mechanisms. Such mechanisms lead to mental and neurological
diseases, including drug addiction, depression, Parkinson's disease, and
schizophrenia. To understand the associations of GPCRs and to unravel
the global picture of their receptor-receptor interactions in the brain,
different experimental detection techniques for receptor-receptor
interactions have been established (e.g., co-immunoprecipitation based
approach). However, they have been criticized for not reflecting the
cellular situation or the dynamic nature of receptor-receptor
interactions. Therefore, the detection and visualization of GPCR
homo-and heteroreceptor complexes in the brain remained largely unknown
until recent years, when a well-characterized in situ proximity ligation
assay (in situ PLA) was adapted to validate the receptor complexes in
their native environment. The in situ PLA protocol presented here can be
used to visualize GPCR receptor-receptor interactions in cells and
tissues in a highly sensitive and specific manner. We have developed a
combined method using immunohistochemistry and PLA, particularly aimed
to monitor interactions between GPCRs in specific neuronal cell
populations. This allows the analysis of homo-and heteroreceptor
complexes at a cellular and subcellular level. The method has the
advantage that it can be used in clinical specimens, providing
localized, quantifiable homo-and heteroreceptor complexes detected in
single cells. We compare the advantages and limitations of the methods,
underlining recent progress and the growing importance of these
techniques in basic research. We discuss also their potential as tools
for drug development and diagnostics.
Filiaciones:
Borroto-Escuela D.O.:
Department of Neuroscience, Karolinska Institutet, Stockholm, Sweden
Observatorio Cubano de Neurociencias, Grupo Bohío-Estudio, Yaguajay, Cuba
Department of Biomolecular Science, Section of Physiology, University of Urbino, Urbino, Italy
Narvaez M.:
Facultad de Medicina, Instituto de Investigación Biomédica de Málaga, Universidad de Málaga, Málaga, Spain
Valladolid-Acebes I.:
The Rolf Luft Research Center for Diabetes and Endocrinology, Karolinska Institutet, Karolinska University Hospital L1, Stockholm, Sweden
Shumilov K.:
Department of Neuroscience, Karolinska Institutet, Stockholm, Sweden
Di Palma M.:
Department of Biomolecular Science, Section of Physiology, University of Urbino, Urbino, Italy
Wydra K.:
Laboratory of Drug Addiction Pharmacology, Institute of Pharmacology, Polish Academy of Sciences, Kraków, Poland
Schaefer T.:
Department of Neuroscience, Karolinska Institutet, Stockholm, Sweden
Molecular & Clinical Pharmacy, Friedrich-Alexander-Universität Erlangen, Erlangen, Germany
Reyes-Resina I.:
CIBERNED, Centro de Investigación Biomédica en Red sobre Enfermedades Neurodegenerativas, Instituto de Salud Carlos III, Madrid, Spain
Institute of Biomedicine of the University of Barcelona (IBUB), Barcelona, Spain
Department of Biochemistry and Molecular Biomedicine, Faculty of Biology, University of Barcelona, Barcelona, Spain
Navarro G.:
CIBERNED, Centro de Investigación Biomédica en Red sobre Enfermedades Neurodegenerativas, Instituto de Salud Carlos III, Madrid, Spain
Institute of Biomedicine of the University of Barcelona (IBUB), Barcelona, Spain
Department of Biochemistry and Physiology, Faculty of Pharmacy, University of Barcelona, Barcelona, Spain
Mudó G.:
Department of Experimental Biomedicine and Clinical Neurosciences, University of Palermo, Palermo, Italy
Filip M.:
Laboratory of Drug Addiction Pharmacology, Institute of Pharmacology, Polish Academy of Sciences, Kraków, Poland
Sartini S.:
Department of Biomolecular Science, Section of Physiology, University of Urbino, Urbino, Italy
Friedland K.:
Molecular & Clinical Pharmacy, Friedrich-Alexander-Universität Erlangen, Erlangen, Germany
Schellekens H.:
APC Microbiome Institute, University College Cork, Cork, Ireland
Department of Anatomy and Neuroscience, University College Cork, Cork, Ireland
Beggiato S.:
Department of Medical Sciences, University of Ferrara, Ferrara, Italy
Ferraro L.:
Department of Medical Sciences, University of Ferrara, Ferrara, Italy
Tanganelli S.:
Department of Life Sciences and Biotechnology (SVEB), University of Ferrara, Ferrara, Italy
Franco R.:
CIBERNED, Centro de Investigación Biomédica en Red sobre Enfermedades Neurodegenerativas, Instituto de Salud Carlos III, Madrid, Spain
Institute of Biomedicine of the University of Barcelona (IBUB), Barcelona, Spain
Department of Biochemistry and Molecular Biomedicine, Faculty of Biology, University of Barcelona, Barcelona, Spain
Belluardo N.:
Department of Experimental Biomedicine and Clinical Neurosciences, University of Palermo, Palermo, Italy
Ambrogini P.:
Department of Biomolecular Science, Section of Physiology, University of Urbino, Urbino, Italy
Pérez de la Mora M.:
Instituto de Fisiología Celular, Universidad Nacional Autónoma de México, Mexico City, Mexico
Fuxe K.:
Department of Neuroscience, Karolinska Institutet, Stockholm, Sweden
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