Structural insights into the human D1 and D2 dopamine receptor signaling complexes.

The D1- and D2-dopamine receptors (D1R and D2R), which signal through Gs and Gi, respectively, represent the principal stimulatory and inhibitory dopamine receptors in the central nervous system. D1R and D2R also represent the main therapeutic targets for Parkinson's disease, schizophrenia, and many other neuropsychiatric disorders, and insight into their signaling is essential for understanding both therapeutic and side effects of dopaminergic drugs. Here, we report four cryoelectron microscopy (cryo-EM) structures of D1R-Gs and D2R-Gi signaling complexes with selective and non-selective dopamine agonists, including two currently used anti-Parkinson's disease drugs, apomorphine and bromocriptine. These structures, together with mutagenesis studies, reveal the conserved binding mode of dopamine agonists, the unique pocket topology underlying ligand selectivity, the conformational changes in receptor activation, and potential structural determinants for G protein-coupling selectivity. These results provide both a molecular understanding of dopamine signaling and multiple structural templates for drug design targeting the dopaminergic system.

Ligand recognition and allosteric regulation of DRD1-Gs signaling complexes.

Dopamine receptors, including D1- and D2-like receptors, are important therapeutic targets in a variety of neurological syndromes, as well as cardiovascular and kidney diseases. Here, we present five cryoelectron microscopy (cryo-EM) structures of the dopamine D1 receptor (DRD1) coupled to Gs heterotrimer in complex with three catechol-based agonists, a non-catechol agonist, and a positive allosteric modulator for endogenous dopamine. These structures revealed that a polar interaction network is essential for catecholamine-like agonist recognition, whereas specific motifs in the extended binding pocket were responsible for discriminating D1- from D2-like receptors. Moreover, allosteric binding at a distinct inner surface pocket improved the activity of DRD1 by stabilizing endogenous dopamine interaction at the orthosteric site. DRD1-Gs interface revealed key features that serve as determinants for G protein coupling. Together, our study provides a structural understanding of the ligand recognition, allosteric regulation, and G protein coupling mechanisms of DRD1.

Pharmacologic neuroimaging of the ontogeny of dopamine receptor function.

Characterization of the ontogeny of the cerebral dopaminergic system is crucial for gaining a greater understanding of normal brain development and its alterations in response to drugs of abuse or conditions such as attention-deficit hyperactivity disorder. Pharmacological MRI (phMRI) was used to determine the response to dopamine transporter (DAT) blockers cocaine and methylphenidate (MPH), the dopamine releaser D-amphetamine (AMPH), the selective D1 agonist dihydrexidine, and the D2/D3 agonist quinpirole in young (<30 days old) and adult (>60 days old) rats. In adult rats, cocaine (0.5 mg/kg i.v.) or MPH (2 mg/kg) induced primarily positive cerebral blood volume (rCBV) changes in the dopaminergic circuitry, but negative rCBV changes in the young animals. Microdialysis measurements in the striatum showed that young rats have a smaller increase in extracellular dopamine in response to cocaine than adults. The young rats showed little rCBV response to the selective D1 agonist dihydrexidine in contrast to robust rCBV increases observed in the adults, whereas there was a similar negative rCBV response in the young and adult rats to the D2 agonist quinpirole. We also performed a meta-analysis of literature data on the development of D1 and D2 receptors and the DAT. These data suggest a predominance of D2-like over D1-like function between 20 and 30 days of age. These combined results suggested that the dopamine D1 receptor is functionally inhibited at young age.

Ultrastructural localization and function of dopamine D1-like receptors in the substantia nigra pars reticulata and the internal segment of the globus pallidus of parkinsonian monkeys.

The motor symptoms of Parkinson's disease (PD) are commonly attributed to striatal dopamine loss, but reduced dopamine innervation of basal ganglia output nuclei, the internal globus pallidus (GPi) and the substantia nigra pars reticulata (SNr) may also contribute to symptoms and signs of PD. Both structures express dopamine D1 and D5 receptors under normal conditions, and we have recently demonstrated that their local activation reduces neuronal discharge rates and enhances bursts and oscillatory activity in both nuclei of normal monkeys [M.A. Kliem et al. (2007)J. Neurophysiol., 89, 1489-1500]. Here, we determined the ultrastructural localization and function of D1-like receptors in 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)-treated parkinsonian monkeys. In both normal and MPTP-treated monkeys, most of the D1 and D5 receptor immunoreactivity was associated with unmyelinated axons, but we also found significant postsynaptic D5 receptor immunostaining in dendrites of GPi and SNr neurons. A significant proportion of axonal D1 immunostaining was bound to the plasma membrane in both normal and MPTP-treated monkeys. Local microinjections of the D1/D5 receptor agonist SKF82958 significantly reduced discharge rates in GPi and SNr neurons, while they increased burst firing and oscillatory activity in the 3-15-Hz band in SNr, but not in GPi, of parkinsonian monkeys. Together with our recent findings from normal monkeys, these data provide evidence that functional D1/D5 receptors are expressed in GPi and SNr in both normal and parkinsonian states, and that their activation by endogenous dopamine (under normal conditions) or dopamine receptor agonists (in parkinsonism) may regulate basal ganglia outflow.

Time-dependent exposure of nicotine and smoke modulate ultrasubcellular organelle localization of dopamine D1 and D2 receptors in the rat caudate-putamen.

The correlation of the subcellular localization of dopamine D(1) and D(2) receptors (DA D(1) R, DA D(2) R) with nicotine addiction has not been studied. We demonstrated the ultrasubcellular organelle localization of DA D(1) and D(2) Rs in the caudate-putamen (CPu) area of rat brain in vivo exposed to nicotine (3 mg/day; oral) and passive cigarette smoking (500 ml each; 3 times/day) for 1, 4, and 12 weeks, respectively. Our results revealed DA D(1) R localization in the presynaptic and postsynaptic dendrites, endocytic vesicles, and secretory granules, and DA D(2) R localization in the presynaptic dendrites and vesicles. DA D(1) R immunogold particles were highly decreased in the secretory granules of CPu, and increased in the postsynaptic area and vesicles after prolonged nicotine and smoking exposures, suggesting the strong influence of long time smoking and nicotine exposures on DA D(1) R subcellular organelle localization. DA D(2) R immunoreactivity was comparatively less changed than that of the DA D(1) R. Western blot analysis also showed the differential expression of DA D(1) and D(2) R proteins upon nicotine and smoking exposures as compared to the untreated controls. Taken together, the results for the first time suggests the execution of addictive behavior of nicotine through modulation of mesolimbic dopaminergic system targeting subcellular organelle of DA D(1) and D(2) Rs in the CPu of adult rat brain that may lead to novel therapeutic approaches related to nicotine's neuropsychological disorders including drug addiction.

Pharmacological analysis demonstrates dramatic alteration of D1 dopamine receptor neuronal distribution in the rat analog of L-DOPA-induced dyskinesia.

We have associated behavioral, pharmacological, and quantitative immunohistochemical study in a rat analog of l-DOPA-induced dyskinesia to understand whether alterations in dopamine receptor fate in striatal neurons may be involved in mechanisms leading to movement abnormalities. Detailed analysis at the ultrastructural level demonstrates specific alterations of dopamine D(1) receptor (D(1)R) subcellular localization in striatal medium spiny neurons in l-DOPA-treated 6-hydroxydopamine-lesioned rats with abnormal involuntary movements (AIMs). This includes exaggerated D(1)R expression at the plasma membrane. However, D(1)R retains ability of internalization, as a challenge with the potent D(1)R agonist SKF-82958 induces a strong decrease of labeling at membrane in animals with AIMs. Since a functional cross talk between D(1)R and D(3)R has been suggested, we hypothesized that their coactivation by dopamine derived from l-DOPA might anchor D(1)R at the membrane. Accordingly, cotreatment with l-DOPA and the D(3)R antagonist ST 198 restores normal level of membrane-bound D(1)R. Together, these results demonstrate that AIMs are related to abnormal D(1)R localization at the membrane and intraneuronal trafficking dysregulation, and suggest that strategies aiming at disrupting the D(1)R-D(3)R cross talk might reduce l-DOPA-induced dyskinesia by reducing D(1)R availability at the membrane.

Differences in ultrastructural localization of dopaminergic D1 receptors between dorsal striatum and nucleus accumbens in the rat.

Dopaminergic receptors of the D1 type are highly expressed in the dorsal striatum and nucleus accumbens. In the dorsal striatum, they are rarely observed on presynaptic terminals. However, their subcellular localization in the nucleus accumbens core and shell had not been compared to that of dorsal striatum. Here we investigated the subcellular localization of D1 receptors in these three brain regions using immunogold labeling and electron microscopy. We showed that, among all presynaptic terminals forming asymmetric contact with dendritic processes, the percentage of D1R immunoreactive terminals was low in the dorsal striatum (8.2%), but reached in the nucleus accumbens core and shell 25.5 and 29%, respectively. These observations are consistent with electrophysiological studies, which showed that D1 stimulation inhibits the response of target neurons to glutamatergic input via presynaptic mechanisms in the nucleus accumbens but not in the dorsal striatum.

Mammal-like striatal functions in Anolis. II. Distribution of dopamine D(1) and D(2) receptors, and a laminar pattern of basal ganglia sub-systems.

We used in situ autoradiographic ligand binding methods to determine the occurrence and distribution of dopamine D(1) and D(2) receptor sub-types in the anole lizard, Anolis carolinensis. Both were present and exhibited pharmacological specificity characteristics similar to those described for mammals. However, unlike in mammals where in the neostriatum [outside the nucleus accumbens/olfactory tubercle complex (NA/OT)] these receptors exhibit only slight dorsolateral (D(2) high, D(1) low) to ventromedial (D(1 )high, D(2) low) gradients that co mingle extensively, in the anole striatum outside the NA/OT there was a striking laminar pattern, with little if any overlap between D(2) (high in a dorsal band) and D(1) (high ventral to the D(2) band) distributions. As D(1) receptors are related to the direct and D(2) to the indirect basal ganglia (BG) subsystems in mammals, we also determined anole striatal distributions of pre-proenkephalin mRNA, a marker for striatal efferents to the indirect BG subsystem in mammals. Here, too, there was a striking laminar pattern, with pre-proenkephalin mRNA in a band similar to that seen for D(2) receptors. The crisp neuroanatomical separation between these classic BG subsystem markers in Anolis striatum make this species attractive for the study of such systems' functions during behavior.

Microanatomy of the dopaminergic system in the rainbow trout retina.

We have investigated the morphology of dopaminergic interplexiform cells as well as the distribution of two classes of dopamine receptors in the retina of the rainbow trout. Interplexiform cells were visualized using an antiserum against tyrosine hydroxylase and PAP immunocytochemistry. In whole amounts, these cells have a density of between 91 and 182 cells per mm2 with highest values in the lower temporal quadrant. Their cell bodies lie at the inner margin of the inner nuclear layer with only 12-17 cells per retina displaced to the ganglion cell layer. There are three levels of stratification in the inner plexiform layer, one at the distal and proximal borders respectively, and one in the middle. They arise mostly from a radially oriented, stout primary dendrite. Tangential processes are about 1 micron in diameter and show a number of varicosities. The density of processes is greatest in sublayer 5, but no major difference in the general organization is apparent between the three sublayers. In the outer retina, there are two levels of dense ramification confined to the layer of horizontal cells. Light and electron microscopic analysis shows synaptic input to horizontal cells, but not to photoreceptors. The distribution of D1 receptors was assessed by studying the binding pattern of a specific, fluorescent-labelled antagonist, SCH 23390, in unfixed frozen sections. We found displaceable binding in the inner and outer plexiform layers and in the region of horizontal cell perikarya. We used an anti-peptide antibody directed to an extracellular domain of the rat D2 receptor and a fluorescent secondary antiserum to study the localization of D2 receptors. In addition to marked label in both plexiform layers, the outer, and especially the inner segments of rods and cones show specific immunoreactivity. In addition, there is distinct label at the level of the horizontal cell bodies; in the inner retina, specific fluorescence is found in somata of some amacrine cells. The significance of the connectivity pattern and the distribution of the two receptor types is discussed with respect to the role of dopamine in controlling adaptational processes in the outer retina, such as retinomotor movements and changes in horizontal cell morphology and physiology.

Effect of adding the D1 agonist CY 208-243 to chronic bromocriptine treatment. I: Evaluation of motor parameters in relation to striatal catecholamine content and dopamine receptors.

A group of four cynomolgus monkeys previously rendered parkinsonian by the toxin 1-methyl-4-phenyl,1,2,3,6-tetrahydropyridine (MPTP) were observed in locomotion cages equipped with photocells during four periods of 7 days during which they received saline or two doses of the D1 agonist CY 208-243. The larger dose of 0.5 mg/kg produced a significant increase in locomotion in three of four animals. A second group of eight monkeys also previously rendered parkinsonian by MPTP and having received no other treatment were given a daily treatment of bromocriptine 1.66 mg/kg orally daily during 4 weeks. In four of the animals, after a week on bromocriptine alone, the D1 agonist CY 208-243 was added in increasing doses of 0.05, 0.1, and 0.5 mg/kg. The motor response as measured by locomotion, hand dexterity, and a disability score improved progressively at least in some of the animals on bromocriptine alone. The addition of CY 208-243 produced a more striking improvement of all three parameters, which appeared to be dose dependent. Biochemical analysis of the brain of these animals plus one control and one MPTP untreated monkey showed a > 90% loss of dopamine in the striatum in six of the eight treated monkeys. Both D2 and D1 dopamine receptors were increased in density by denervation, but both treatments abolished this increase for the D2 receptors while increasing the affinity of the D1 receptors.