Interactions among adenosine deaminase, adenosine A 1 receptors and dopamine D 1 receptors in stably cotransfected fibroblast cells and neurons

Abstract

The role of adenosine deaminase in the interactions between adenosine A 1 and dopamine D 1 receptors was studied in a mouse fibroblast cell line stably cotransfected with human D 1 receptor and A 1 receptor cDNAs (A 1D 1 cells). Confocal laser microscopy analysis showed a high degree of adenosine deaminase immunoreactivity on the membrane of the A 1D 1 cells but not of the D 1 cells (only cotransfected with human D 1 receptor cDNAs). In double immunolabelling experiments in A 1D 1 cells and cortical neurons a marked overlap in the distribution of the A 1 receptor and adenosine deaminase immunoreactivities and of the D 1 receptor and adenosine deaminase immunoreactivities was found. Quantitative analysis of A 1D 1 cells showed that adenosine deaminase immunoreactivity to a large extent colocalizes with A 1 and D 1 receptor immunoreactivity, respectively. The A 1 receptor agonist caused in A 1D 1 cells and in cortical neurons coaggregation of A 1 receptors and adenosine deaminase, and of D 1 receptors and adenosine deaminase. The A 1 receptor agonist-induced aggregation was blocked by R-deoxycoformycin, an irreversible adenosine deaminase inhibitor. The competitive binding experiments with the D 1 receptor antagonist [ 3H]SCH-23390 showed that the D 1 receptors had a better fit for two binding sites for dopamine, and treatment with the A 1 receptor agonist produced a disappearance of the high-affinity site for dopamine at the D 1 receptor. R-Deoxycoformycin treatment, which has previously been shown to block the interaction between adenosine deaminase and A 1 receptors, and which is crucial for the high-affinity state of the A 1 receptor, also blocked the A 1 receptor agonist-induced loss of high-affinity D 1 receptor binding. The conclusion of the present studies is that the high-affinity state of the A 1 receptor is essential for the A 1 receptor-mediated antagonistic modulation of D 1 receptors and for the A 1 receptor-induced coaggregates of A 1 and adenosine deaminase, and of D 1 and adenosine deaminase. Thus, the confocal experiments indicate that both A 1 and D 1 receptors form agonist-regulated clusters with adenosine deaminase, where the presence of a structurally intact adenosine deaminase bound to A 1 receptors is important for the A 1–D 1 receptor–receptor interaction at the level of the D 1 receptor recognition.