Evidence for noncanonical neurotransmitter activation: Norepinephrine as a dopamine D₂-like receptor agonist

The Gαi/o-coupled dopamine D₂-like receptor family comprises three subtypes: the D₂ receptor (D2R), with short and long isoform variants (D2SR and D2LR), D₃ receptor (D3R), and D₄ receptor (D4R), with several polymorphic variants. The common overlap of norepinephrine innervation and D₂-like receptor expression patterns prompts the question of a possible noncanonical action by norepinephrine. In fact, previous studies have suggested that norepinephrine can functionally interact with D4R. To our knowledge, significant interactions between norepinephrine and D2R or D3R receptors have not been demonstrated. By using radioligand binding and bioluminescent resonance energy transfer (BRET) assays in transfected cells, the present study attempted a careful comparison between dopamine and norepinephrine in their possible activation of all D₂-like receptors, including the two D2R isoforms and the most common D4R polymorphic variants. Functional BRET assays included activation of G proteins with all Gαi/o subunits, adenylyl cyclase inhibition, and β arrestin recruitment. Norepinephrine acted as a potent agonist for all D₂-like receptor subtypes, with the general rank order of potency of D3R > D4R ≥ D2SR ≥ D2L. However, for both dopamine and norepinephrine, differences depended on the Gαi/o protein subunit involved. The most striking differences were observed with Gαi2, where the rank order of potencies for both dopamine and norepinephrine were D4R > D2SR = D2LR ≫ D3R. Furthermore the results do not support the existence of differences in the ability of dopamine and norepinephrine to activate different human D4R variants. The potency of norepinephrine for adrenergic α_2A receptor was only about 20-fold higher compared with D3R and D4R across the three functional assays.