Human DRD1 Stable Cell Line - HEK293

As a hormone and neurotransmitter unable to permeate the cell membrane, dopamine has traditionally been thought to transmit signals solely by binding to and activating dopamine receptors (members of the G protein-coupled receptor/GPCR family) located on the plasma membrane. Here, using novel nanobody-based biosensors, researchers demonstrated for the first time that the dopamine D1 receptor (D1DR, also known as DRD1)-a key mediator of dopamine signaling in the brain and kidney-functions not only at the plasma membrane but can also be activated at the Golgi apparatus in the presence of the ligand. Evidence provided by the researchers indicates that the activation of the Golgi-localized D1DR pool depends on organic cation transporter 2 (OCT2, a dopamine transporter); this finding explains how membrane-impermeable dopamine gains access to the subcellular D1DR pool. It was further confirmed that dopamine can activate Golgi-localized D1DRs on medium spiny neurons in the mouse striatum, a process dependent on OCT2 function. Additionally, the researchers introduced a new method to selectively investigate compartmentalized D1DR signaling by employing a nanobody-based chemical recruitment system to inhibit Gαs coupling. Using this strategy, they discovered that Golgi-localized D1DRs can regulate cAMP production and mediate local protein kinase A (PKA) activation.

Through directed evolution of Nb80, researchers obtained a high-affinity nanobody (Nb6B9) capable of stably binding the active conformation of the β2-adrenergic receptor (β2AR). Given that the binding site for Nb6B9 on β2AR is highly conserved among other aminergic receptors such as β1AR and D1DR, the researchers hypothesized that this nanobody could also serve as a conformation-sensitive biosensor for the real-time detection of activated D1DR in living cells (Figure 1a). In HeLa cells expressing SNAP-tagged D1DR, GFP-fused Nb6B9 (Nb6B9-GFP) was diffusely distributed in the cytoplasm (Figure 1b, 0 min). Upon stimulation with 10 μM dopamine (DA), Nb6B9-GFP was rapidly recruited, first accumulating at the plasma membrane and subsequently translocating to the Golgi apparatus (Figure 1b, 2 min). The recruitment of Nb6B9-GFP to the plasma membrane and Golgi apparatus was dose-dependent, with onset concentrations of 10 nM and 100 nM DA, respectively (Figure 1c). Collectively, these data indicate that the addition of extracellular DA activates the pool of D1DRs located at the Golgi apparatus. In contrast to the observations in HeLa cells, treatment of D1DR-expressing HEK293 cells with 10 μM DA resulted in Nb6B9-GFP recruitment exclusively to the plasma membrane (Figure 1b, bottom panel; Figure 1d, 2 min).

Figure 1. Conformational biosensor detects activated D1DR at the plasma membrane and the Golgi upon dopamine (DA) stimulation. (Puri N M, et al., 2022)