Separation and reformation of cell surface dopamine receptor oligomers visualized in cells

We previously showed that dopamine receptors existed as homo- and heterooligomers, in cells and in brain tissue. We developed a method designed to study the formation and regulation of G protein coupled receptor (GPCR) oligomers in cells, using a GPCR into which a nuclear localization sequence (NLS) had been inserted. Unlike wildtype GPCRs, in the presence of agonist/antagonist ligands the GPCR–NLS is retained at the cell surface, and following ligand removal, the GPCR–NLS translocated from the cell surface. The D1 dopamine receptor expressed with either D2–NLS or D1–NLS receptors translocated to the nucleus, indicating hetero- or homo-oligomerization with the NLS-containing receptor. Using these tools, we now demonstrate that D1–D2 dopamine heterooligomers can be disrupted and the component receptors separated by dopamine and selective agonists that occupied one or both binding pockets. Subsequent agonist removal allowed the reformation of the heterooligomer. D1 receptor homooligomers could also be disrupted by agonist, but at higher concentrations than that required for the disruption of the D1–D2 heteromer. Dopamine D1 or D2 receptor antagonists had no effect on the integrity of the homo- or heterooligomer. We have also determined that the D1–D2 heterooligomer contains D1 homooligomers. These studies indicate that the populations of dopamine receptor oligomers at the cell surface are subject to conformational changes following agonist occupancy and are likely dynamically regulated following agonist activation.