Computer-assisted image analysis of caveolin-1 involvement in the internalization process of adenosine A2A-dopamine D2 receptor heterodimers

A functional aspect of horizontal molecular networks has been investigated experimentally, namely the heteromerization between adenosine A(2A) and doparnine D-2 receptors and the possible role of caveolin-1 in the cotrafficking 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, A(2A), D-1, and D-2 receptors obtained in two clones of Chinese hamster ovary cells-one transfected with A(2A) and dopamine D, receptors and the other one with A(2A) and D-2 receptors. Cells were treated for 3 h with 10 mu M D-1 receptor agonist SKF 38393,50 mu M D-2-D-3 receptor agonist quinpirole, and 200 nM A(2A) receptor agonist CGS 21680. In A(2A)-D-1-cotransfected cells, caveolin-1 was found to colocalize with both A2A and D, receptors and treatment with SKF 38393 induced internalization of caveolin-1 and D-1 receptors, with a preferential internalization of D, receptors colocalized. with caveolin-1. In A(2A)-D-2-cotransfected cells, caveolin-1 was found to colocalize with both A2A and D2 receptors and either CGS 21680 or quinpirole treatment induced internalization of caveolin-1 and A2A and D2 receptors, with a preferential internalization of A2A and D2 receptors colocalized with caveolin-1. The results suggest that A2A and D2 receptors and caveolin-1 likely interact forming a macrocomplex 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 the plasma membrane level (prerequisite for receptor mosaic organization and plastic adjustments) and in the control of receptor desensitization.