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.

Calcium signaling cascade links dopamine D1-D2 receptor heteromer to striatal BDNF production and neuronal growth.

Although the perturbation of either the dopaminergic system or brain-derived neurotrophic factor (BDNF) levels has been linked to important neurological and neuropsychiatric disorders, there is no known signaling pathway linking these two major players. We found that the exclusive stimulation of the dopamine D1-D2 receptor heteromer, which we identified in striatal neurons and adult rat brain by using confocal FRET, led to the activation of a signaling cascade that links dopamine signaling to BDNF production and neuronal growth through a cascade of four steps: (i) mobilization of intracellular calcium through Gq, phospholipase C, and inositol trisphosphate, (ii) rapid activation of cytosolic and nuclear calcium/calmodulin-dependent kinase IIalpha, (iii) increased BDNF expression, and (iv) accelerated morphological maturation and differentiation of striatal neurons, marked by increased microtubule-associated protein 2 production. These effects, although robust in striatal neurons from D5(-/-) mice, were absent in neurons from D1(-/-) mice.

Calcium signaling by dopamine D5 receptor and D5-D2 receptor hetero-oligomers occurs by a mechanism distinct from that for dopamine D1-D2 receptor hetero-oligomers.

In this report, we investigated whether the D5 dopamine receptor, given its structural and sequence homology with the D1 receptor, could interact with the D2 receptor to mediate a calcium signal similar to the G(q/11) protein-linked phospholipase C-mediated calcium signal resulting from the coactivation of D1 and D2 dopamine receptors within D1-D2 receptor heterooligomers. Fluorescent resonance energy transfer experiments demonstrated close colocalization of cell surface D5 and D2 receptors (<100 A), indicating hetero-oligomerization of D5 and D2 receptors in cells coexpressing both receptors. Coactivation of D5 and D2 receptors within the D5-D2 hetero-oligomers activated a calcium signal.

Trafficking of preassembled opioid mu-delta heterooligomer-Gz signaling complexes to the plasma membrane: coregulation by agonists.

The cellular site of formation, Galpha-coupling preference, and agonist regulation of mu-delta opioid receptor (OR) heterooligomers were studied. Bioluminescence resonance energy transfer (BRET) showed that mu-deltaOR heterooligomers, composed of preformed mu and delta homooligomers, interacted constitutively in the endoplasmic reticulum (ER) with Galpha-proteins forming heteromeric signaling complexes before being targeted to the plasma membrane. Compared to muOR homooligomers, the mu-delta heterooligomers showed higher affinity and efficiency of interaction for Gz over Gi, indicating a switch in G-protein preference.

Using ligand-induced conformational change to screen for compounds targeting G-protein-coupled receptors.

The authors describe a novel drug strategy designed as a primary screen to discover either antagonist or agonist compounds targeting G-protein-coupled receptors (GPCRs). The incorporation of a nuclear localization sequence (NLS, a 5 amino acid substitution), in a location in helix 8 of the GPCR structure, resulted in ligand-independent receptor translocation from the cell surface to the nucleus. Blockade of the GPCR-NLS translocation from the cell surface was achieved by either antagonist or agonist treatments, each achieving their result in a sensitive concentration-dependent manner.

Dopamine receptor oligomerization visualized in living cells.

G protein-coupled receptors occur as dimers within arrays of oligomers. We visualized ensembles of dopamine receptor oligomers in living cells and evaluated the contributions of receptor conformation to the dynamics of oligomer association and dissociation, using a strategy of trafficking a receptor to another cellular compartment. We incorporated a nuclear localization sequence into the D1 dopamine receptor, which translocated from the cell surface to the nucleus.