The Class-A GPCR Dopamine D2 Receptor Forms Transient Dimers Stabilized by Agonists: Detection by Single-Molecule Tracking

Abstract

Whether class-A G-protein coupled receptors (GPCRs) exist and work as monomers or dimers has drawn extensive attention. A class-A GPCR dopamine D2 receptor (D2R) is involved in many physiological and pathological processes and diseases, indicating its critical role in proper functioning of neuronal circuits. In particular, D2R homodimers might play key roles in schizophrenia development and amphetamine-induced psychosis. Here, using single-molecule imaging, we directly tracked single D2R molecules in the plasma membrane at a physiological temperature of 37 °C, and unequivocally determined that D2R forms transient dimers with a lifetime of 68 ms in its resting state. Agonist addition prolonged the dimer lifetime by a factor of ~1.5, suggesting the possibility that transient dimers might be involved in signaling.

Metastable GPCR dimers trigger the basal signal by recruiting G-proteins

“…To determine the lifetime of β2AR dimers using single color observations (with ATTO594-ACP-β2AR), the duration of each colocalisation-codiffusion event was measured 4,11,29 , and after the durations of many events were determined, histograms showing the distribution of colocalisation durations were obtained. All of the histograms obtained from the image sequences obtained at a time resolution of 33 ms could be fitted by single exponential functions, which provided decay time constants (lifetime; τ observed ).…”

“…All of the histograms obtained from the image sequences obtained at a time resolution of 33 ms could be fitted by single exponential functions, which provided decay time constants (lifetime; τ observed ). τ observed was then corrected for the photobleaching lifetime of the fluorescent probe ATTO594 ( τ bleach = 7.21 ± 0.81 s for 30 Hz [ n = 300] and 0.812 ± 0.041 s for 250 Hz [ n = 539]), to obtain the duration of apparent colocalisation ( τ apparent colocalisation ), using the equation [ τ observed -1 - 2 τ bleach -1 ] -1 4,11,29 . The effective dimer lifetime or the effective binding lifetime of proteins, τ , was evaluated by subtracting the duration of incidental colocalisation (without specific molecular interactions; τ incidental ) from τ apparent colocalisation .…”

“…The effective dimer lifetime or the effective binding lifetime of proteins, τ , was evaluated by subtracting the duration of incidental colocalisation (without specific molecular interactions; τ incidental ) from τ apparent colocalisation . For details, see our previous publications 4,29 . τ incidental was evaluated by using a monomer reference molecule, ACP linked to the transmembrane domain of the low-density lipoprotein receptor, called ACP-TM 29 , and was found to be 19 ± 0.46 ms (exponential lifetime; n = 241 examined dimers; 39 examined movies; No.…”

“…For details, see our previous publications 4,29 . τ incidental was evaluated by using a monomer reference molecule, ACP linked to the transmembrane domain of the low-density lipoprotein receptor, called ACP-TM 29 , and was found to be 19 ± 0.46 ms (exponential lifetime; n = 241 examined dimers; 39 examined movies; No. of Freedoms = 97).…”

In vivo actions of SCTR/AT1aR heteromer in controlling Vp expression and release via cFos/cAMP/CREB pathway in magnocellular neurons of PVN

“…Of note, the functional specificity of SCTR/AT1aR heteromer was fully demonstrated by intracerebroventricular injection of STM2 because it can only disrupt SCTR/AT1aR heterodimerization but not SCTR or AT1aR homodimerization. It is noteworthy also to mention that intracerebroventricular injection of TM peptides alone into mice could not affect basal Vp release, indicating that functional SCTR/AT1aR complex requires the presence of either ligands, which is similar to the ligand induction of oligomerization processes observed in β 2 ‐adrenoceptors, somatostatin receptor type 1 subtype b, chemokine receptor type 2/chemokine receptor type 5 receptors, as well as dopamine D 2 receptors (38–41),.…”