Dasiel O Borroto-Escuela, Michael Di Palma, Ismel Brito and Kjell Fuxe
Posters-Accepted Abstracts: Biol Syst Open Access
G Protein Coupled Receptors (GPCRs) and Receptor Tyrosine Kinases (RTK) play critical roles in cellular processes and signaling and have been shown to form homo and heteroreceptor complexes with diverge biochemical and/or pharmacological activities. However, despite of extensive experimental results supporting the formation of GPCR and/or RTK homo and heteroreceptor complexes in heterologous systems, the existence of such homo and heteroreceptor complexes in their native environment remains largely unknown, mostly because of the lack of appropriate methodology. For instance, until recent years the methods that have been developed to study receptor-receptor interactions require that genetic constructs be expressed in the cells to enable detection of the receptor interactions, thus excluding the use of tissue samples. In order to demonstrate in native tissue the existence of GPCR and/or RTK homo and heteroreceptor complexes, especially in a manner that can be generally applicable to different receptor pairs, a well-characterized in situ proximity ligation assay (in situ PLA) has been adapted to confirm the existence of GPCR and/or RTK homo and heteroreceptor complexes in brain slices ex vivo. We also describe the in situ PLA procedure as a high selectivity and sensitivity assay to image GPCR and/or RTK homo and heteroreceptor complexes in brain sections by confocal microscopy and how the assay is performed. We point out as well the method advantages and disadvantages and compare it to other available techniques.