Computer-assisted image analysis of caveolin-1 involvement in the internalization process of adenosine A_2A-dopamine D₂ receptor heterodimers

This paper is dedicated to Luciano Martini, Professor of Endocrinology in Milano and to Faustino Savoldi, Professor of Neurolgy in Pavia. A functional aspect of horizontal molecular networks has been investigated experimentally, namely the heteromerization between adenosine A_2A and dopamine 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, D₁, and D₂ receptors obtained in two clones of Chinese hamster ovary cells - one transfected with A_2A and dopamine D₁ receptors and the other one with A_2A and D₂ receptors. Cells were treated for 3 h with 10 μM D₁ receptor agonist SKF 38393,50 μM D ₂-D₃ 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 A_2A and D₁ receptors and treatment with SKF 38393 induced internalization of caveolin-1 and D₁ receptors, with a preferential internalization of D₁ receptors colocalized with caveolin-1. In A_2A-D₂-cotransfected cells, caveolin-1 was found to colocalize with both A_2A and D₂ receptors and either CGS 21680 or quinpirole treatment induced internalization of caveolin-1 and A _2A and D₂ receptors, with a preferential internalization of A_2A and D₂ receptors colocalized with caveolin-1. The results suggest that A_2A 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.