ABSTRACT
Title
Intercellular transfer of components of plasma membrane associated integrative mechanisms: focus on A2A-D2 heteromers and tetraspanin CD9.
Authors
L.F. Agnati1, M. Guescini2, G. Leo3, V. Stocchi2, M. Mantuano2, P. Tibollo2, C. Carone3, F. Ciruela4, D. Guidolin5, S. Genedani3, K. Fuxe6
1IRCCS Lido Venezia, Italy;
2Department of Biomolecular Sciences, University of Urbino Italy;
3Department of Biomedical Sciences, University of Modena Italy;
4Department of Biochemistry, University of Barcelona Spain;
5Department of Anatomy, University of Padova Italy;
6Department of Neurosciences KI, Stockholm Sweden.
Abstract
Receptor-receptor interactions between GPCRs especially in Receptor Mosaics (RMs; higher-order receptor oligomers) represent an important integrative mechanism in the plasma membrane. In several instances, RMs can operate as input units in Horizontal Molecular Networks (HMNs). Recently, it has been demonstrated the special role played by tetraspanins (a class of membrane proteins regarded to have scaffolding functions) in the HMNs. As a matter of fact, not only tetraspanins can associate with one another at membrane level forming a ‘tetraspanin web’ but they can also transiently interact with several other proteins, e.g., GPCRs.
Against this background the demonstration of the intercellular transfer of components of HMNs is of high interest. We will here show the exosome-mediated transfer of A2A and D2 receptors and of tetraspanin CD9 in cell cultures.
HEK-293T cells were transiently transfected with the plasmid cDNA encoding the D2L receptor-CFP (D2LR-CFP) or A2A receptor-YFP (A2AR-YFP). Twenty-four hours after transfection the two differentially transfected HEK-293T cells were put into co-culture and fixed after another 24 hours. Western blotting analysis of the micro-vesicles released by transfected cells revealed the presence of A2AR-YFP, D2R-CFP and of tetraspanin CD9. Furthermore, these micro-vesicles were characterized as exosomes by means of the specific exosome-markers Tsg101 and Alix.
Confocal laser scanning microscopy was performed using a Leica MultibandTCS SP2 with an AOBS microscope (Leica Microsystems, Mannheim, Germany). CFP was excited with the 458 nm laser while YFP was excited with the 514 nm laser. After 24-hour of co-culture of HEK-293T cells transfected with D2LR-CFP (cyan) or A2AR-YFP (yellow), confocal microscopy analysis showed the presence of cells expressing the two types of receptor in spite of being singly transfected. This result demonstrates that receptors can be inter-cellularly transferred via exosomes.
Finally,using acceptor photo-bleaching FRET technique, the direct interaction was demonstrated between A2AR-YFP and D2R-CFP in cells showing the two receptor types.
The inter-cellular transfer of GPCRs and tetraspanin CD9 deserves attention as it increases the plasticity of the plasma membrane associated integrative mechanisms by opening up the possibility that a reciprocal inter-cellular tuning of the receptor-mediated signal transduction systems takes place. In this way the integrative actions of entire cell networks can become enriched. This phenomenon mediated via exosomes has interesting implications especially for neuropathology as well as for new strategies in drug development.
Against this background the demonstration of the intercellular transfer of components of HMNs is of high interest. We will here show the exosome-mediated transfer of A2A and D2 receptors and of tetraspanin CD9 in cell cultures.
HEK-293T cells were transiently transfected with the plasmid cDNA encoding the D2L receptor-CFP (D2LR-CFP) or A2A receptor-YFP (A2AR-YFP). Twenty-four hours after transfection the two differentially transfected HEK-293T cells were put into co-culture and fixed after another 24 hours. Western blotting analysis of the micro-vesicles released by transfected cells revealed the presence of A2AR-YFP, D2R-CFP and of tetraspanin CD9. Furthermore, these micro-vesicles were characterized as exosomes by means of the specific exosome-markers Tsg101 and Alix.
Confocal laser scanning microscopy was performed using a Leica MultibandTCS SP2 with an AOBS microscope (Leica Microsystems, Mannheim, Germany). CFP was excited with the 458 nm laser while YFP was excited with the 514 nm laser. After 24-hour of co-culture of HEK-293T cells transfected with D2LR-CFP (cyan) or A2AR-YFP (yellow), confocal microscopy analysis showed the presence of cells expressing the two types of receptor in spite of being singly transfected. This result demonstrates that receptors can be inter-cellularly transferred via exosomes.
Finally,using acceptor photo-bleaching FRET technique, the direct interaction was demonstrated between A2AR-YFP and D2R-CFP in cells showing the two receptor types.
The inter-cellular transfer of GPCRs and tetraspanin CD9 deserves attention as it increases the plasticity of the plasma membrane associated integrative mechanisms by opening up the possibility that a reciprocal inter-cellular tuning of the receptor-mediated signal transduction systems takes place. In this way the integrative actions of entire cell networks can become enriched. This phenomenon mediated via exosomes has interesting implications especially for neuropathology as well as for new strategies in drug development.