Membrane receptor, for example, G-protein-coupled receptors (GPCRs), 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, 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.
Study of GPCR Homo- and Heteroreceptor Complexes in Specific Neuronal Cell Populations Using the In Situ Proximity Ligation Assay
Patrizia Ambrogini;
2021
Abstract
Membrane receptor, for example, G-protein-coupled receptors (GPCRs), 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, 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.I documenti in IRIS sono protetti da copyright e tutti i diritti sono riservati, salvo diversa indicazione.
