Single Quantum Dot Tracking Unravels Agonist Effects on the Dopamine Receptor Dynamics

Abstract

D2 dopamine receptors (DRD2s) belong to a family of G protein-coupled receptors that modulate synaptic dopaminergic tone via regulation of dopamine synthesis, storage, and synaptic release. DRD2s are the primary target for traditional antipsychotic medications; dysfunctional DRD2 signaling has been linked to major depressive disorder, attention-deficit hyperactivity disorder, addiction, Parkinson’s, and schizophrenia. DRD2 lateral diffusion appears to be an important post-translational regulatory mechanism; however, the dynamic response of DRD2s to ligand-induced activation is poorly understood. Dynamic imaging of the long isoform of DRD2 (D2L) fused to an N-terminal antihemagglutinin (HA) epitope and transiently expressed in HEK-293 cells was achieved through a combination of a high-affinity biotinylated anti-HA antigen-binding fragment (Fab) and streptavidin-conjugated quantum dots (QD). Significant reduction (∼40%) in the rate of lateral diffusion of QD-tagged D2L proteins was observed under agonist (quinpirole; QN)-stimulated conditions compared to basal conditions. QN-induced diffusional slowing was accompanied by an increase in frequency, lifetime, and confinement of temporary arrest of lateral diffusion (TALL), an intrinsic property of single receptor lateral motion. The role of the actin cytoskeleton in QN-induced diffusional slowing of D2L was also explored. The observed dynamic changes appear to be a sensitive indicator of the receptor activity status and might also spatially and temporally shape the receptor-mediated downstream signaling. This dynamic information could potentially be useful in informing drug discovery efforts based on single-molecule pharmacology.

Membrane Nanoscopic Organization of D2L Dopamine Receptor Probed by Quantum Dot Tracking

“…The diffusion coefficient D 2–5 was calculated from the slope of the first 2–5 points of the MSD plot versus time with the equation: where σ x is the spot localization accuracy in one direction (i.e., is the ordinate at the origin of the linear fit). Trajectories of Qdots greater than the previously determined immobile particle threshold 5 × 10 −4 μm 2 /s [ 14 , 53 ] were used for statistical comparison via the nonparametric Mann–Whitney U test and Kolmogorov–Smirnov test. Merge and split events occurring in Qdot time-lapse image series were determined by using the fully functional linear assignment problem tracker in TrackMate [ 47 , 48 ] while allowing the detection of Qdot signal splitting and merging with a maximum displacement of 1 μm.…”

“…There has also been a growing interest in the lateral organization of GPCRs at the cell surface, as advances in single-molecule fluorescence microscopy and single-particle tracking (SPT) have enabled direct observation of the stochastic and dynamic behavior of individual GPCRs in real-time [ 7 , 8 , 9 , 10 , 11 , 12 ]. Current state-of-the-art knowledge derived from SPT studies is that (i) a vast majority of GPCRs maintain a diffusible surface pool [ 13 ], (ii) activation status controls mobility [ 9 , 10 , 14 ], (iii) agonist-dependent global decrease in the diffusion rate is not dependent on the GPCR subfamily or G protein coupling selectivity [ 10 ], and (iv) GPCRs can assemble into transient homo- and heterodimers, although the preponderance and lifetime of such oligomeric assemblies remain controversial [ 8 , 9 , 15 , 16 ]. Nevertheless, these results highlight the need for a continued inquiry into one of the most debated features of GPCR signaling—whether receptors are pre-coupled to signal transducers or engage them via random collisions facilitated by the plasma membrane specialized nanodomains [ 13 , 17 ].…”

“…Our recent research focus has been on characterizing lateral mobility and defining its signal transduction implications for the D2 subtype dopamine receptor (D2DR), a Class A GPCR [ 14 , 18 ]. D2 dopamine receptors are involved in the regulation of dopamine-dependent motor function, motivation, cognition, emotion, and neuroendocrine secretion [ 19 , 20 ].…”

“…D2 dopamine receptors exist as two alternative splice variants—long isoform (D2L) and short isoform (D2S), which differ in a 29-amino acid insert in the third intracellular loop [ 30 , 31 ]. A popular strategy to enable SPT of dopamine receptors and GPCRs, in general, involves the fusion of an epitope tag (e.g., hemagglutinin (HA; YPYDVPDYA), FLAG (DYKDDDDK), SNAP) to the extracellular N terminus, where it is well tolerated [ 10 , 14 , 32 , 33 , 34 , 35 ]. We adopted this strategy in our previous report by targeting the HA-D2L receptor construct with a high-affinity biotinylated anti-HA-antibody fragment (Fab) and streptavidin-conjugated quantum dots (SavQdots) [ 14 ].…”

“…A popular strategy to enable SPT of dopamine receptors and GPCRs, in general, involves the fusion of an epitope tag (e.g., hemagglutinin (HA; YPYDVPDYA), FLAG (DYKDDDDK), SNAP) to the extracellular N terminus, where it is well tolerated [ 10 , 14 , 32 , 33 , 34 , 35 ]. We adopted this strategy in our previous report by targeting the HA-D2L receptor construct with a high-affinity biotinylated anti-HA-antibody fragment (Fab) and streptavidin-conjugated quantum dots (SavQdots) [ 14 ]. The use of quantum dots, a colloquial term for nanometer-sized semiconductor crystals, allowed us to capitalize on their unique photophysical properties [ 36 , 37 ], including excellent photostability and high brightness, whereas relying on the anti-HA-Fab as the off-site targeting unit enabled monitoring dynamic changes in response to substrate binding.…”