Dopamine D1-D2 receptor Heteromer-mediated calcium release is desensitized by D1 receptor occupancy with or without signal activation: dual functional regulation by G protein-coupled receptor kinase 2.

We identified that activation of the G(q)-linked dopamine D1-D2 receptor hetero-oligomer generates a PLC-dependent intracellular calcium signal. Confocal FRET between endogenous dopamine D1 and D2 receptors in striatal neurons confirmed a physical interaction between them. Pretreatment with SKF 83959, which selectively activates the D1-D2 receptor heteromer, or SKF 83822, which only activates the D1 receptor homo-oligomer, led to rapid desensitization of the D1-D2 receptor heteromer-mediated calcium signal in both heterologous cells and striatal neurons. This desensitization response was mediated through selective occupancy of the D1 receptor binding pocket. Although SKF 83822 was unable to activate the D1-D2 receptor heteromer, it still permitted desensitization of the calcium signal. This suggested that occupancy of the D1 receptor binding pocket by SKF 83822 resulted in conformational changes sufficient for desensitization without heteromer activation. Bioluminescence resonance energy transfer and co-immunoprecipitation studies indicated an agonist-induced physical association between the D1-D2 receptor heteromeric complex and GRK2. Increased expression of GRK2 led to a decrease in the calcium signal with or without prior exposure to either SKF 83959 or SKF 83822. GRK2 knockdown by siRNA led to an increase in the signal after pretreatment with either agonist. Expression of the catalytically inactive and RGS (regulator of G protein signaling)-mutated GRK2 constructs each led to a partial recovery of the GRK2-attenuated calcium signal. These results indicated that desensitization of the dopamine D1-D2 receptor heteromer-mediated signal can occur by agonist occupancy even without activation and is dually regulated by both the catalytic and RGS domains of GRK2.

The role of palmitoylation in directing dopamine D1 receptor internalization through selective endocytic routes.

We previously determined that D1 receptors can endocytose through caveolae, a subset of lipid rafts, in addition to internalization via a clathrin-dependent pathway. In this report, we investigated the potential role that palmitoylation might have on directing D1 receptor internalization through either a clathrin or caveolar-dependent route. Through whole cell binding analysis and sucrose gradient fractionation studies, we demonstrated that although palmitoylation of the D1 receptor was not required for agonist-independent localization to caveolae, agonist induced internalization kinetics of a de-palmitoylated D1 receptor were accelerated ∼8-fold in comparison to wild-type D1 receptor and were very similar to that observed for clathrin-dependent D1 receptor internalization. Additionally, inhibition of the clathrin mediated pathway led to significant attenuation in the extent of agonist induced internalization of the de-palmitoylated D1 receptor, suggesting the de-palmitoylated D1 receptor was directed to a clathrin-dependent internalization pathway. Taken together, these data suggest that palmitoylation may be involved in directing agonist-dependent D1 receptor internalization through selective endocytic routes.

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.

Neuronal Gq/11-coupled dopamine receptors: an uncharted role for dopamine.

There is strong evidence for the existence of Gq/11-coupled dopamine receptors in the brain but the mechanism by which dopamine signaling activates Gq/11, or its roles in neuronal function, are only just beginning to be understood. The importance of such a pathway is underlined by putative links between dopamine-regulated phosphoinositide signaling and several central nervous system disorders that include schizophrenia, addiction and Parkinson's disease.

MIF-1 and its peptidomimetic analogs attenuate haloperidol-induced vacuous chewing movements and modulate apomorphine-induced rotational behavior in 6-hydroxydopamine-lesioned rats.

Two melanocyte-stimulating hormone release inhibiting factor-1 (MIF-1) also known as L-prolyl-L-leucyl-glycinamide (PLG) peptidomimetic analogs, 3(R)-[[[2(S)-pyrrolidinyl]carbonyl]-amino]-3-(butyl)-2-oxo-1-pyrrolidineacetamide trifluoroacetate (A) and 3(R)-[[[2(S)-pyrrolidinyl]carbonyl]amino]-3-(benzyl)-2-oxo-1-pyrrolidineacetamide trifluoroacetate (B), were evaluated for their ability to modulate dopaminergic activity by measuring apomorphine-induced rotations in 6-hydroxydopamine (6-OHDA)-lesioned rats, and haloperidol (HP)-induced vacuous chewing movements (VCMs) in rats; animal models of Parkinson's disease (PD) and human tardive dyskinesia (TD), respectively. In the 6-OHDA model, both analogs were found to potentiate the contralateral rotational behavior induced by apomorphine dose-dependently and with approximately the same potency. Furthermore, each analog was able to significantly attenuate HP-induced VCMs with almost equal efficacy. The potency and efficacy of these analogs were significantly greater than their parent compound, PLG. These results suggest that both analogs can modulate dopaminergic activity in vivo, likely by the same mechanisms recruited by PLG previously reported.

Design and synthesis of photoaffinity-labeling ligands of the L-prolyl-L-leucylglycinamide binding site involved in the allosteric modulation of the dopamine receptor.

Pro-Leu-Gly-NH(2) (PLG), in addition to its endocrine effects, possesses the ability to modulate dopamine D(2) receptors within the central nervous system. However, the precise binding site of PLG is unknown. Potential photoaffinity-labeling ligands of the PLG binding site were designed as tools to be used in the identification of the macromolecule that possesses this binding site. Six different photoaffinity-labeling ligands were designed and synthesized on the basis of the gamma-lactam PLG peptidomimetic 1. The 4-azidobenzoyl and 4-azido-2-hydroxybenzoyl photoaffinity-labeling moieties were placed at opposite ends of PLG peptidomimetic 1 to generate a series of ligands that potentially could be used to map the PLG binding site. All of the compounds that were synthesized possessed activity comparable to or better than PLG in enhancing [(3)H]-N-propylnorapomorphine agonist binding to dopamine receptors. Photoaffinity ligands that were cross-linked to the receptor preparation produced a modulatory effect that was either comparable to or greater than the increase in agonist binding produced by the respective ligands that were not cross-linked to the dopamine receptor. The results indicate that these photoaffinity-labeling agents are binding at the same allosteric site as PLG and PLG peptidomimetic 1.

Specific binding of photoaffinity-labeling peptidomimetics of Pro-Leu-Gly-NH2 to the dopamine D2L receptor: evidence for the allosteric modulation of the dopamine receptor.

The present study was undertaken to investigate the mechanistic role of l-prolyl-l-leucyl-glycinamide (PLG) in modulating agonist binding to the dopamine D(2L) receptor. Competition and displacement assays indicate that the photoaffinity-labeling peptidomimetics of PLG, 3(R)-[(4(S)-(4-azido-2-hydroxy-benzoyl) amino-2(S)-pyrrolidinylcarbonyl)amino]-2-oxo-1-pyrrolidineacetamide hydrochloride (1a) and 3(R)-[(4(S)-(4-azido-2-hydroxy-5-iodo-benzoyl)amino-2(S)-pyrrolidinylcarbonyl)amino]-2-oxo-1-pyrrolidineacetamide hydrochloride (1b) bind at the same site as PLG. Autoradiography was used to establish the covalent binding of [(125)I]-1b to an approximately 51kDa protein in bovine striatal membranes. Western blot analysis with a dopamine D(2L)-specific antibody, in combination with autoradiography, following a two-dimensional gel separation, suggested this approximately 51kDa protein to be the dopamine D(2L) receptor. Further evidence for binding of 1b to dopamine D(2L) was provided by samples immunoprecipitated with the D(2L) antibody. These samples were analyzed by western blotting in parallel with autoradiography of [(125)I]-1b labeled protein. Both methods revealed bands at approximately 51kDa. Furthermore, PLG is shown to compete with 1b for binding to the dopamine D(2L) receptor as determined by autoradiography, as well as competition experiments with PLG and 1a. Collectively, these findings suggest the successful development of a photoaffinity-labeling agent, compound 1b, that has been used to elucidate the interaction of PLG specifically with the dopamine D(2L) receptor.

Desensitization of the dopamine D1 and D2 receptor hetero-oligomer mediated calcium signal by agonist occupancy of either receptor.

When dopamine D1 and D2 receptors were coactivated in D1-D2 receptor hetero-oligomeric complexes, a novel phospholipase C-mediated calcium signal was generated. In this report, desensitization of this Gq/11-mediated calcium signal was demonstrated by pretreatment with dopamine or with the D1-selective agonist (+/-)-6-chloro-7,8-dihydroxy-1-phenyl-2,3,4,5-tetrahydro-1H-3-benzazepine hydrobromide (SKF-81297) or the D2-selective agonist quinpirole. Desensitization of the calcium signal mediated by D1-D2 receptor hetero-oligomers was initiated by agonist occupancy of either receptor subtype even though the signal was generated only by occupancy of both receptors. The efficacy, potency, and rate of calcium signal desensitization by agonist occupancy of the D1 receptor (t1/2, approximately 1 min) was far greater than by the D2 receptor (t1/2, approximately 10 min). Desensitization of the calcium signal was not mediated by depletion of calcium stores or internalization of the hetero-oligomer and was not decreased by inhibiting second messenger-activated kinases. The involvement of G protein-coupled receptor kinases 2 or 3, but not 5 or 6, in the desensitization of the calcium signal was shown, occurring through a phosphorylation independent mechanism. Inhibition of Gi protein function associated with D2 receptors increased D1 receptor-mediated desensitization of the calcium signal, suggesting that cross-talk between the signals mediated by the activation of different G proteins controlled the efficacy of calcium signal desensitization. Together, these results demonstrate the desensitization of a signal mediated only by hetero-oligomerization of two G protein-coupled receptors that was initiated by agonist occupancy of either receptor within the hetero-oligomer, albeit with differences in desensitization profiles observed.

Modulation of agonist binding to human dopamine receptor subtypes by L-prolyl-L-leucyl-glycinamide and a peptidomimetic analog.

The present study was undertaken to investigate the role of the hypothalamic tripeptide L-prolyl-L-leucyl-glycinamide (PLG) and its conformationally constrained analog 3(R)-[(2(S)-pyrrolidinylcarbonyl)amino]-2-oxo-1-pyrrolidineacetamide (PAOPA) in modulating agonist binding to human dopamine (DA) receptor subtypes using human neuroblastoma SH-SY5Y cells stably transfected with respective cDNAs. Both PLG and PAOPA enhanced agonist [3H]N-propylnorapomorphine (NPA) and [3H]quinpirole binding in a dose-dependent manner to the DA D2L,D2S, and D4 receptors. However, agonist binding to the D1 and D3 receptors and antagonist binding to the D2L receptors by PLG were not significantly affected. Scatchard analysis of [3H]NPA binding to membranes in the presence of PLG revealed a significant increase in affinity of the agonist binding sites for the D2L, D2S, and D4 receptors. Analysis of agonist/antagonist competition curves revealed that PLG and PAOPA increased the population and affinity of the high-affinity form of the D2L receptor and attenuated guanosine 5'-(beta,gamma-imido)-triphosphate-induced inhibition of high-affinity agonist binding sites for the DA D2L receptor. Furthermore, direct NPA binding with D2L cell membranes pretreated with suramin, a compound that can uncouple receptor/G protein complexes, and incubated with and without DA showed that both PLG and PAOPA had only increased agonist binding in membranes pretreated with both suramin and DA, suggesting that PLG requires the D2L receptor/G protein complex to increase agonist binding. These results suggest that PLG possibly modulates DA D2S, D2L, and D4 receptors in an allosteric manner and that the coupling of D2 receptors to the G protein is essential for this modulation to occur.