Evidence for Noncanonical Neurotransmitter Activation: Norepinephrine as a Dopamine D₂-Like Receptor Agonist

Abstract

The Gai/o-coupled dopamine D 2 -like receptor family comprises three subtypes: the D 2 receptor (D2R), with short and long isoform variants (D2SR and D2LR), D 3 receptor (D3R), and D 4 receptor (D4R), with several polymorphic variants. The common overlap of norepinephrine innervation and D 2 -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 D 2 -like receptors, including the two D2R isoforms and the most common D4R polymorphic variants. Functional BRET assays included activation of G proteins with all Gai/o subunits, adenylyl cyclase inhibition, and b arrestin recruitment. Norepinephrine acted as a potent agonist for all D 2 -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 Gai/o protein subunit involved. The most striking differences were observed with Gai2, where the rank order of potencies for both dopamine and norepinephrine were D4R . D2SR 5 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 a 2A receptor was only about 20-fold higher compared with D3R and D4R across the three functional assays.

Luciferase Complementation Based-Detection of G-Protein-Coupled Receptor Activity

“…All of the characteristics of D1R ligands are in agreement with previous reports [23,24]. D2R pharmacology was also in agreement with a previous study [25], in that norepinephrine showed less potency (pEC 50 5.220.14) relative to DA (pEC 50 6.610.10) and a selective D2R antagonist eticlopride showed complete blockade of 10M DA pre-activation and high potency (pIC 50 9.470.46). Albeit narrower luminescence drug-induced ranges, furimazine showed similar potencies and efficacies (Supplementary Figure 2C & D) Supplementary data.…”

“…The EC 50 values were also in close agreement with reports using different methods for measuring G-protein activation and receptor engagement [2628]. Overall, for the D1R and D2R, the potencies and efficacies of tested ligands were in agreement with previous reports [23,25,29,30], indicating the utility of the assay configuration for testing pharmacology of other receptors. It also needs to be pointed out that in this study coelenterazine H, as opposed to furimazine, was mainly used to characterize the complementation assays since coelenterazine H is much more cost-effective and affords a very good dynamic range.…”

Key role of the dopamine D 4 receptor in the modulation of corticostriatal glutamatergic neurotransmission

“…However, those experiments were performed with synthetic DA receptor agonists. We recently reported that DA is significantly more potent at activating any D 4 receptor (D 4.2 , D 4.4 , and D 4.7 ) than D 2 receptors (D 2S or D 2L ) ( 25 ), and we are obtaining results that indicate that DA is also more potent at activating D 4 receptors (including D 4.7 ) than D 2 -D 4 receptor heteromers (in preparation). Therefore, the inability of D 4.7 receptors to heteromerize should lead to a gain of function.…”

Differential regulation of phasic dopamine release in the forebrain by the VTA noradrenergic receptor signaling

“…D 2 ‐like receptors—which potently inhibit DAergic neurons—have similar affinity ranges for both DA and NA, with any catecholamine binding resulting in their activation and downstream signal transduction, especially in the presence of higher (i.e., micromolar) concentrations of DA and NA (Sánchez‐Soto et al . ). Indeed, existing evidence from FSCV studies strongly suggested the possibility of cross‐activation of D 2 R as a potential mechanism mediating the inhibitory effects of high concentrations of NA in the VTA (Park et al .…”