Visualization of differential GPCR crosstalk in DRD1-DRD2 heterodimer upon different dopamine levels

Dopaminergic signaling is regulated by transient micromolar (phasic) and background nanomolar (tonic) dopamine releases in the brain. These dopamine signals can be differentially translated by dopamine receptor type 1 and type 2, DRD1 and DRD2, which are G protein-coupled receptors (GPCRs). In response to dopamine, DRD1 and DRD2 are known to mediate opposite functions on cAMP production via Gs and Gi protein signaling. Interestingly, they can form a heterodimer. However, receptor crosstalk between DRD1-DRD2 heterodimers has not been directly measured, but it was only inferred from measuring downstream signaling pathways. Here we develop fluorescent protein-based multicolor biosensors which can monitor individual activation states of DRD1 and DRD2, and apply them to directly monitor the functional crosstalk between DRD1-DRD2 heterodimers in live cells. Utilizing these powerful tools, we surprisingly discover differential crosstalk in the DRD1-DRD2 heterodimers upon different dopamine (DA) levels: DRD1 activation is selectively inhibited at micromolar DA levels, while DRD2 is inhibited only by nanomolar DA concentration, implying a novel function of the DRD1-DRD2 heterodimer upon different DA levels. Our results imply differential receptor crosstalk and novel functions of the DRD1-DRD2 heterodimer in response to physiological dopamine levels from nanomolar to micromolar dopamine concentrations.