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. We also demonstrated that this signaling cascade was activated in adult rat brain, although with regional specificity, being largely limited to the nucleus accumbens. This dopaminergic pathway regulating neuronal growth and maturation through BDNF may have considerable significance in disorders such as drug addiction, schizophrenia, and depression.

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. However, unlike what is observed for D1 receptors, which activate extensive calcium mobilization only within a complex with the D2 receptors, a robust calcium signal was triggered by D5 receptors expressed alone. Hetero-oligomerization with the D2 receptor attenuated the ability of the D5 receptor to trigger a calcium signal. The D5 and D5-D2-associated calcium signals were G(q/11) protein-linked and phospholipase C-mediated but were also critically dependent on the influx of extracellular calcium through store-operated calcium channels, unlike the calcium release triggered by D1-D2 heterooligomers. Collectively, these results demonstrate that calcium signaling through D5-D2 receptor hetero-oligomers occurred through a distinct mechanism to achieve an increase in intracellular calcium levels.

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. GPCR-NLS translocation and blockade occurred regardless of the identity of the G-protein-coupling, and thus this assay is also ideally suited for identification of compounds targeting orphan GPCRs. The GPCR-NLS trafficking was visualized by fusion to fluorescent detectable proteins. Quantification of this effect was measured by determining the density of cell surface receptors, using enzyme fragment complementation in a manner suitable for high-throughput screening. Thus, the authors have developed a cellular assay for GPCRs suitable for compound screening without requiring prior identification of an agonist or knowledge of G-protein-coupling.

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 D(1) dopamine receptor expressed with either D(2)-NLS or D(1-)NLS receptors translocated to the nucleus, indicating hetero- or homo-oligomerization with the NLS-containing receptor. Using these tools, we now demonstrate that D(1)-D(2) 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. D(1) receptor homooligomers could also be disrupted by agonist, but at higher concentrations than that required for the disruption of the D(1)-D(2) heteromer. Dopamine D(1) or D(2) receptor antagonists had no effect on the integrity of the homo- or heterooligomer. We have also determined that the D(1)-D(2) heterooligomer contains D(1) 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.

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. Treatment with DAMGO or deltorphin II led to coregulated internalization of both receptors, whereas DPDPE and DSLET had no effect on mu-delta internalization. Staggered expression resulted in non-interacting mu and delta receptors, even though both receptors were colocalized at the cell surface. Agonists failed to induce BRET between staggered receptors, and resulted in internalization solely of the receptor targeted by agonist. Thus, mu-deltaOR heterooligomers form and preferentially associate with Gz to generate a signaling complex in the ER, and have a distinct agonist-internalization profile compared to either mu or delta homooligomers.

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. Receptor inverse agonists blocked this translocation, retaining the modified receptor, D1-nuclear localization signal (NLS), at the cell surface. D1 co-translocated with D1-NLS to the nucleus, indicating formation of homooligomers. (+)-Butaclamol retained both receptors at the cell surface, and removal of the drug allowed translocation of both receptors to the nucleus. Agonist-nonbinding D1(S198A/S199A)-NLS, containing two substituted serine residues in transmembrane 5 also oligomerized with D1, and both were retained on the cell surface by (+)-butaclamol. Drug removal disrupted these oligomerized receptors so that D1 remained at the cell surface while D1(S198A/S199A)-NLS trafficked to the nucleus. Thus, receptor conformational differences permitted oligomer disruption and showed that ligand-binding pocket occupancy by the inverse agonist induced a conformational change. We demonstrated robust heterooligomerization between the D2 dopamine receptor and the D1 receptor. The heterooligomers could not be disrupted by inverse agonists targeting either one of the receptor constituents. However, D2 did not heterooligomerize with the structurally modified D1(S198A/S199A), indicating an impaired interface for their interaction. Thus, we describe a novel method showing that a homogeneous receptor conformation maintains the structural integrity of oligomers, whereas conformational heterogeneity disrupts it.