Computer-assisted image analysis of caveolin-1 involvement in the internalization process of adenosine A(2A)-dopamine D-2 receptor heterodimers

A functional aspect of the Horizontal Molecular Networks has been experimentally investigated, namely the heteromerisation between adenosine A2A and dopamine D2 receptors and the possible role of caveolin-1 in the co-trafficking of these molecular complexes. This study has been carried out by means of computer assisted image analysis procedure of laser images of membrane immunoreactivity of caveolin-1, A2A, D1 and D2 receptors obtained in two clones of CHO cells, one transfected with A2A and dopamine D1 receptors and the other one with A2A and D2 receptors. Cells were treated for three hours with the D1 receptor agonist SKF 38393 (10 µM), the D2-D3 receptor agonist quinpirole (50 µM) and the A2A receptor agonist CGS 21680 (200 nM). In A2A-D1 co-transfected cells caveolin-1 was found to co-localize with both A2A and D1 receptors and treatment with SKF 38393 induced internalization of caveolin-1 and D1 receptors, with a preferential internalization of D1 receptors co-localized with caveolin-1. In the A2A-D2 co-transfected cells caveolin-1 was found to co-localize with both A2A and D2 receptors and either CGS 21680 or quinpirole treatment induced internalization of caveolin-1, A2A and D2 receptors, with a preferential internalization of A2A and D2 receptors co-localized with caveolin-1. The results suggest that A2A and D2 receptors and caveolin-1 likely interact forming a macro-complex that internalizes upon agonist treatment. These observations are discussed in the frame of receptor oligomerization and of the possible functional role of caveolin-1 in the process of co-internalization and hence in controlling the permanence of receptors at plasma membrane level (pre-requisite for Receptor Mosaic organisation and plastic adjustments) and in the control of receptor desensitisation.