Supervisor: Prof. Ago Rinken, MD, PhD, Keemia Instituut
Opponent: Prof. Mika Scheinin, MD, PhD, University of Turku, Finland
"Assay systems for characterisation of subtype-selective binding and functional activity of ligands on dopamine receptors"
Dopamine has several functions in central nervous system including voluntary movement, feeding, affect, reward, sleep, attention, working memory, and learning. In the periphery, dopamine plays important physiological roles in the regulation of olfaction, retinal processes, hormonal regulation, cardiovascular functions, sympathetic regulation, immune system, and renal functions, among others. Dopaminergic ligands, substances acting on dopamine receptors, have remained an active area in the development of CNS drugs, agonists mainly in the treatment of Parkinson's disease and antagonist as antipsychotics in the treatment of schizophrenia.
Within current study the binding and function of dopamine receptors was studied in various model systems such as Sf9 insect cell line, striatal tissue of rat brain, CHO and fibroblast cell lines, and HEK293 cells.
Sf9 cells of insect origin was used a relatively "clean" cell line for studying the interaction between dopamine D1 and adenosine A1 receptors. In this cell line, A1-D1 receptor antagonistic interaction was revealed only when the cells expressed both Gi- and Gs- proteins necessary for the activity of either receptor, and on the level of cyclic AMP not on the level of agonist affinity, as previously reported. This suggests that the antagonistic interactions observed in various studies might be more complicated than previously thought.
High expression of dopamine D1 and D2 receptor expression was shown in rat striatum with proper ligand binding and G-protein coupling profiles. Nevertheless, in the level of second messenger, cAMP, the D1 receptor activation could be clearly detected, whereas the D2 receptor-mediated signalling remained hidden in [3H]cAMP assay. Therefore [3H]cAMP assay is suitable for selective discrimination of D1 receptor signalling events in rat striatal tissue.
Dopamine receptor binding of newly synthesised ligands was initially studied on D1 and D2 receptors expressed in fibroblast and CHO cell lines, respectively. The differences in cellular environment of the two expressed receptor subtypes, as well as a need to screen new ligands on all three major dopamine receptor subtypes - D1, D2L and D3 - however triggered the development of a more comparable assay system. The obtained HEK293 cell-based dopamine D1, D2L and D3 receptor stable cell lines were characterised for their binding and functional properties using subtype-selective and non-selective dopaminergic ligands. Functional properties of the receptors were studied on the level of G-protein activation (studing the effect of GTP_S binding on agonist affinity) and activation of adenylate cyclase (modulation of cyclic AMP level by agonists and antagonists). While GTP_S effectively modulated agonist binding to D1 and D2L receptor, the effect on D3 receptor binding was negligible, suggesting a weak interaction of D3 receptors and G-proteins. This was further supported by [3H]cAMP binding assay in which agonist response to D1 and D2L receptor activation was reproducibly reflected in modulation of the level of cAMP, whereas results of D3 receptor activation were contradictory. When receptor activation was measured in a dynamic cAMP mode using the BacMam Epac2-camps cAMP-biosensor we could reliably detect modulation of cAMP levels by all the three receptor subtypes, suggesting that the D3 receptor indeed was effectively coupled to its G-proteins despite the lack of GTP_S sensitivity and the lack of cAMP-response in [3H]cAMP assay.
Despite minor limitations of the developed dopamine receptor cell lines in functional assays, they were effectively used in screening of ligand binding and activity of series of new 1- and 3-substituted apomorphines and 3-azabicyclo[3.1.0]hexane derivatives in cooperation with other research groups involved in ligand synthesis.