Basal signaling activity of human dopamine D2L receptor demonstrated with an ecdysone-inducible mammalian expression...

Basal signaling activity of the dopamine D2L (long isoform) receptor was investigated using an ecdysone-inducible mammalian expression system. Whereas basal signaling activity had been demonstrated for the D1, D2S (short isoform), D3, and D5 receptor subtypes, this issue has yet to be clearly resolved for the D2L receptors. An ecdysone-inducible mammalian expression system was used to express the dopamine D2L receptor in human embryonic kidney cells (HEK293). The two ecdysone analogs, muristerone A and ponasterone A, induced D2L receptor expression dose-dependently from 120 (non-induced) to 2000 fmol/mg protein which is similar to physiological D2 receptor density in dopaminergic brain regions. With this approach, we demonstrate significant basal D2L receptor activity. Basal and agonist-stimulated signaling activity, determined with assays of cAMP levels and [35S]GTPgammaS binding to assess G protein coupling, correlated with the level of receptor expression. Furthermore, among several antagonists tested, only fluphenazine and trifluoperazine exhibited clearly detectable inverse agonist activity. This differs from the D2S receptors, where most antagonists were reported to display full inverse agonism. Differences between inverse agonist effects of D2L and D2S antagonists may be relevant to the treatment of diseases involving dopamine D2 receptors.