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

Abstract

The role of lateral mobility and nanodomain organization of G protein-coupled receptors in modulating subcellular signaling has been under increasing scrutiny. Investigation of D2 dopamine receptor diffusion dynamics is of particular interest, as these receptors have been linked to altered neurotransmission in affective disorders and represent the primary target for commonly prescribed antipsychotics. Here, we applied our single quantum dot tracking approach to decipher intrinsic diffusion patterns of the wild-type long isoform of the D2 dopamine receptor and its genetic variants previously identified in several cohorts of schizophrenia patients. We identified a subtle decrease in the diffusion rate of the Val96Ala mutant that parallels its previously reported reduced affinity for potent neuroleptics clozapine and chlorpromazine. Slower Val96Ala variant diffusion was not accompanied by a change in receptor-receptor transient interactions as defined by the diffraction-limited quantum dot colocalization events. In addition, we implemented a Voronoї tessellation-based algorithm to compare nanoclustering of the D2 dopamine receptor to the dominant anionic phospholipid phosphatidylinositol 4,5-bisphosphate in the plasma membrane of live cells.

Ligand-Free Signaling of G-Protein-Coupled Receptors: Physiology, Pharmacology, and Genetics

“…These processes determine the signaling response [50] accounting for distinct effects of biased agonists and antagonists [3,4,6,29]. Powerful methods probing conformational states reveal dynamics and cellular location of signaling receptor aggregates [10,[51][52][53][54]. Spatial confinement in nanodomains at the cell membrane or intracellular compartments confers additional signaling specificity [44,51,54].…”

“…Also, morphine causes rapid Ca ++ influx in µ opioid receptor (MOR) transfected HEK293 cells over the first 10 sec, followed by sustained intracellular Ca ++ release [61]. GPCR activation commonly leads to clustering into nano-domains, a process that modulates signaling, as shown with the DRD2 receptor [54]. Pre-coupling with a noncanonical G protein followed by agonist activation resulted in enhanced agonist stimulated coupling to the canonical G protein [48], suggesting a priming effect that could occur during the ago-R* or the ligand-free R* states.…”

Nanoscale imaging of quantum dot dimers using time-resolved super-resolution microscopy combined with scanning electron microscopy

“…including light emitting diodes [1,2], television displays [3], photovoltaics [4], lasers [5], biological labels [6,7], and other quantum technologies [8]. In many applications, the QDs are formed into close-packed assemblies, such as thin films or matrices, in which electronic interactions can occur among neighboring QDs.…”

Interaction of Proteins with Biomembranes

“…By using a single quantum dot tracking approach, Kovtun et al [3] studied the lateral diffusion, nanodomain formation, and their implications in signal transduction of the D2 subtype dopamine receptor (D2DR), a class A G-protein-coupled receptor (GPCR), which has naturally occurring genetic variants in schizophrenia. The authors found a significant decrease in the diffusion dynamics of the Val96Ala D2L schizophrenia variant.…”