Pharmacological characterization of mammalian D1 and D2 dopamine receptors expressed in Drosophila Schneider-2 cells

Mammalian D1 and D2 dopamine receptors were stably expressed in Drosophila Schneider-2 (S2) cells and screened for their pharmacological properties. Saturable, dose-dependent, high affinity binding of the D1-selective antagonist [3H]SCH-23390 was detected only in membranes from S2 cells induced to express rat dopamine D1 receptors, while saturable, dose-dependent, high affinity binding of the D2-selective antagonist [3H]methylspiperone was detected only in membranes from S2 cells induced to express rat dopamine D2 receptors. No specific binding of either radioligand could be detected in membranes isolated from uninduced or untransfected S2 cells. Both dopamine D1 and D2 receptor subtypes displayed the appropriate stereoselective binding of enantiomers of the nonselective antagonist butaclamol. Each receptor subtype also displayed the appropriate agonist stereoselectivities. The dopamine D1 receptor bound the (+)-enantiomer of the D1-selective agonist SKF38393 with higher affinity than the (-)-enantiomer, while the dopamine D2 receptor bound the (-)-enantiomer of the D2-selective agonist norpropylapomorphine with higher affinity than the (+)-enantiomer. At both receptor subtypes, dopamine binding was best characterized as occurring to a single low affinity site. In addition, the low affinity dopamine binding was also found to be insensitive to GTPgammaS and magnesium ions. Overall, the pharmacological profiles of mammalian dopamine D1 and D2 receptors expressed in Drosophila S2 cells is comparable to those observed for these same receptors when they are expressed in mammalian cell lines. A notable distinction is that there is no evidence for the coupling of insect G proteins to mammalian dopamine receptors. These results suggest that the S2 cell insect G system may provide a convenient source of pharmacologically active mammalian D1 and D2 dopamine receptors free of promiscuous G protein contaminants.     

Dopamine receptor subtype imbalance in schizophrenia

We have investigated the radioligand binding properties of D1 and D2 dopamine receptors in postmortem brains from schizophrenic patients. Consistent with previous reports, the schizophrenic population demonstrated a significant 56% increase in D2 dopamine receptor density. Importantly, the D1 dopamine receptor density was significantly reduced by 43%. These alterations in dopamine receptor densities resulted in a highly significant difference in the ratio of D2/D1 dopamine receptors between schizophrenic patients and controls. A correlation between D1 dopamine receptor density and age was apparent in the schizophrenic patients: D1 dopamine receptor density decreased markedly with age and the linear regressions of D1 dopamine receptor density versus age in both the controls and schizophrenic patients had similar slopes. These results may have clinical implications for the treatment of schizophrenia and tardive dyskinesia.     

Age-related reduction of extrastriatal dopamine D2 receptor measured by PET

Although the aging effect of dopamine D2 receptor in the striatum is well-documented, the effect of age on the extrastriatal dopamine D2 receptor has not been fully examined. Since the density of extrastriatal dopamine D2 receptor is very low, suitable ligands are limited. In this study, we used [11C]FLB 457 to quantify the extrastriatal dopamine D2 receptor in the living human brain. Twenty-seven healthy male subjects aged from 21 to 82 years participated in the positron emission tomography study. Extrastriatal [11C]FLB 457 binding was quantified with a reference tissue model using cerebellum as a reference region. Binding potentials corresponding to Bmax/Kd were used to evaluate age-related change. We found age-related decreases of D2 receptor binding in all measured extrastriatal regions. The decrease of D2 receptor binding was 13.8% per decade in frontal cortex, 12.0% in temporal cortex, 13.4% in parietal cortex, 12.4% in occipital cortex, 12.2% in hippocampus, and 4.8% in thalamus. These findings suggest that the amounts of D2 receptor declines in all brain regions as part of the normal aging process.     

Measurement of dopamine D2-like receptors in postmortem CNS and pituitary: differential regional changes in schizophrenia

In situ radioligand binding with autoradiography and anti-human dopamine D(2) receptor antibodies with Western blots have been used to measure the density of dopamine D(2)-like receptors in the caudate-putamen and pituitary from schizophrenic subjects who did or did not have residual antipsychotic drugs in their tissue at death. There was a significant decrease in the Ki for haloperidol displaceable [(125)I]iodosulpride binding in the pituitary (p < 0.01) and caudate-putamen (p < 0.05) from subjects with schizophrenia with residual drugs in their tissue. There was a significant decrease in the density of [(125)I]iodosulpride in the pituitary (p < 0.001) and a strong trend to a decrease in binding in the caudate-putamen (p = 0.055) from subjects with schizophrenia. By contrast, [(3)H]spiperone binding was decreased in the caudate-putamen (p < 0.05) with a trend to decreased binding in the pituitary (p = 0.07) from subjects with schizophrenia. There was no difference in the density of dopamine D(2) receptors in the caudate-putamen from subjects with schizophrenia (p = 0.31). All the findings on receptor densities were independent of drug status. [(125)I]iodosulpride binds to the dopamine D(2&3) receptors. We have shown that there is no change in the dopamine D(2) receptor in the caudate-putamen from subjects with schizophrenia and therefore, these data would be consistent with there being a decrease in the dopamine D(3) in the caudate-putamen from subjects with schizophrenia. Since dopamine D(3) receptors are absent or present at low concentrations in the pituitary, our data would suggest the dopamine D(2) receptor is decreased in that tissue from schizophrenic subjects.     

Solubilization of D2 Dopamine Receptor Coupled to Guanine Nucleotide Regulatory Protein from Bovine Striatum

D2 dopamine receptor from bovine striatum was solubilized in a form sensitive to guanine nucleotides, by means of a zwitterionic detergent, 3-[(3-cholamidopropyl)-dimethylammonio]-1-propane sulfonate (CHAPS). The presence of sodium ion markedly increased the solubilization yield. Treatment of the membranes with 10 mM CHAPS and 0.72 M NaCl solubilized 26% of the stereospecific [3H]spiperone binding sites in the original membrane preparations. The solubilized [3H]spiperone binding sites possessed characteristics of the D2 dopamine receptor: (a) localization of the site in the striatum but not in the cerebellum; (b) high affinity to nanomolar concentrations of [3H]spiperone; (c) displacement of [3H]spiperone binding by nanomolar concentrations of neuroleptics, but only by micromolar concentrations of dopamine and apomorphine; (d) equal activity of various dopamine agonists and antagonists in the soluble and membrane preparations. Guanine nucleotides decreased the affinity of the solubilized D2 dopamine receptor for dopamine agonists, but not for antagonists. The solubilized receptor complex was eluted in Sepharose CL-4B column chromatography as a large molecule, with a Stokes radius of approximately 90 A. These results indicate that the complex between the D2 dopamine receptor and GTP binding protein remains intact throughout the solubilization procedure.     

Evidence for the Importance of a Carboxyl Group in the Binding of Ligands to the D2 Dopamine Receptor

A series of group specific modifying reagents were tested for their effects on [3H]spiperone binding to brain D2 dopamine receptors to identify amino acid residues at the binding site of the D2 dopamine receptor that are critical for ligand binding. The dependence of ligand binding to the receptor on the pH of the incubation medium was also examined. N-Acetylimidazole, 5,5'-dithiobis(2-nitrobenzoic acid), 1,2-cyclohexanedione, and acetic anhydride had no specific effect on [3H]spiperone binding, indicating the lack of participation of tyrosine, free sulphydryl, arginine, or primary amino groups in ligand binding to the receptor. N,N'-Dicyclohexylcarbodiimide (DCCD) potently reduced the number of [3H]spiperone binding sites, indicating that a carboxyl group is involved in ligand binding to the receptor. The effects of DCCD could be prevented by prior incubation of the receptor with D2 dopamine receptor selective compounds. The pH-binding profile for [3H]spiperone binding indicated the importance of an ionising group of pKa 5.2 for ligand binding which may be the same carboxyl group. Diethyl pyrocarbonate, the histidine modifying reagent, also inhibited [3H]spiperone binding, reducing the affinity of the receptor for this ligand but the effects were not at the ligand binding site. From the effects of pH changes on ligand binding some evidence was obtained for a second ionising group (pKa 7.0) that specifically affects the binding of substituted benzamide drugs to the receptor. It is concluded that the D2 dopamine receptor binding site contains separate but over-lapping binding regions for antagonists such as spiperone and substituted benzamide drugs. The former region contains an important carboxyl group; the latter region contains another group that may be a second carboxyl group or a histidine.     

Adenosine and dopamine receptor antagonist binding in the rat ventral and dorsal striatum: lack of changes after a neonatal bilateral lesion of the ventral hippocampus.

There is experimental evidence from radioligand binding experiments for the existence of strong antagonistic interactions between different subtypes of adenosine and dopamine receptors in the striatum, mainly between adenosine A1 and dopamine D1 and between adenosine A2A and dopamine D2 receptors. These interactions seem to be more powerful in the ventral compared to the dorsal striatum, which might have some implications for the treatment of schizophrenia. The binding characteristics of different dopamine and adenosine receptor subtypes were analysed in the different striatal compartments (dorsolateral striatum and shell and core of the nucleus accumbens), by performing saturation experiments with the dopamine D1 receptor antagonist [125I]SCH-23982, the dopamine D2-3 receptor antagonist [3H]raclopride, the adenosine A1 receptor antagonist [3H]DPCPX and the adenosine A2A receptor antagonist [3H]SCH 58261. The experiments were also performed in rats with a neonatal bilateral lesion of the ventral hippocampus (VH), a possible animal model of schizophrenia. Both dopamine D2-3 and adenosine A2A receptors follow a similar pattern, with a lower density of receptors (40%) in the shell of the nucleus accumbens compared with the dorsolateral caudate-putamen. A lower density of adenosine A1 receptors (20%) was also found in the shell of the nucleus accumbens compared with the caudate-putamen. On the other hand, dopamine D1 receptors showed a similar density in the different striatal compartments. Therefore, differences in receptor densities cannot explain the stronger interactions between adenosine and dopamine receptors found in the ventral, compared to the dorsal striatum. No statistical differences in the binding characteristics of any of the different adenosine and dopamine receptor antagonists used were found between sham-operated and VH-lesioned rats.     

Dopamine D(3) receptors are down-regulated following heterologous endocytosis by a specific interaction with G protein-coupled receptor-associated sorting protein-1

The D(3) dopamine receptor is endocytosed through a heterologous mechanism mediated by phorbol esters. Here, we show that following this endocytosis the D(3) dopamine receptors fail to recycle and are instead targeted for degradation through an interaction with the G protein-coupled receptor (GPCR)-associated sorting protein-1 (GASP-1). Furthermore, we identified a specific binding motif in the C terminus common to the D(3) and D(2) that confers GASP-1 binding. shRNA knockdown of GASP-1 delayed post-endocytic degradation of both the D(2) and D(3) dopamine receptors. In addition, mutation of the D(2) and D(3) receptor C termini to resemble the D(4), which does not interact with GASP-1, not only inhibited GASP-1 binding but slowed degradation after endocytosis. Conversely, mutation of the C terminus of the D(4) to resemble that of the D(2) and D(3) facilitated GASP-1 binding and promoted post-endocytic degradation of the mutant D(4) receptor. Thus, we have identified a motif that is both necessary and sufficient to promote GASP-1 binding and receptor degradation. In addition, these data demonstrated that GASP-1 can mediate post-endocytic degradation of dopamine receptors that have been endocytosed not only as a consequence of dopamine activation but also as a consequence of activation by phorbol esters.     

Evidence that calmodulin binding to the dopamine D2 receptor enhances receptor signaling

The Ca2+ sensor calmodulin (CaM) regulates numerous proteins involved in G protein-coupled receptor (GPCR) signaling. CaM binds directly to some GPCRs, including the dopamine D2 receptor. We confirmed that the third intracellular loop of the D2 receptor is a direct contact point for CaM binding using coimmunoprecipitation and a polyHis pull-down assay, and we determined that the D2-like receptor agonist 7-OH-DPAT increased the colocalization of the D2 receptor and endogenous CaM in both 293 cells and in primary neostriatal cultures. The N-terminal three or four residues of D2-IC3 were required for the binding of CaM; mutation of three of these residues in the full-length receptor (I210C/K211C/I212C) decreased the coprecipitation of the D2 receptor and CaM and also significantly decreased D2 receptor signaling, without altering the coupling of the receptor to G proteins. Taken together, these findings suggest that binding of CaM to the dopamine D2 receptor enhances D2 receptor signaling.     

Altered Dopamine Receptor and Dopamine Transporter Binding and Tyrosine Hydroxylase mRNA Expression Following Perinatal NMDA Receptor Blockade

This study examined how perinatal phencyclidine (PCP) treatment would affect dopamine D2 receptor and dopamine transporter (DAT) binding at different stages after treatment cessation. Female rat pups received injections of PCP (10 mg/kg, s.c.) or saline on postnatal day (PN)7, 9 and 11. D2 receptor and transporter binding was examined at four time-points (PN12, 18, 32 and 96) following injections. PCP treatment altered D2 receptor binding throughout development, with a final end-point of 22-33% decreased binding at adulthood in the nucleus accumbens and caudate putamen (P < 0.01), accompanied by a small but significant increase in DAT binding in the caudate putamen. Tyrosine hydroxylase mRNA expression was also significantly increased by 25% (P < 0.05) in the ventral tegmental area of adult rats, suggesting that this model may produce a long-term increase in dopamine output. This study demonstrates that early insult to the brain from NMDA receptor hypofunction alters the dopaminergic system at different stages of development.     

Preparation of an affinity chromatography matrix for the selective purification of the dopamine D2 receptor from bovine striatal membranes

A ligand affinity matrix has been developed and utilized to purify the dopamine D2 receptor approx. 2100 fold from bovine striatal membranes. 3-[2-Aminoethyl]-8-[3-(4-fluorobenzoyl)propyl]-4-oxo-1-phenyl-1,3,8- triazaspiro[4.5]decan-4-one (AES) was synthesized and used to prepare the affinity matrix by coupling to epoxy-activated Sepharose 6B (AES-Sepharose). AES (Ki approximately 1.7 nM) is similar in potency to the parent compound, spiperone (Ki approximately 0.8 nM), in competing for [3H]spiperone-binding activity. AES has no significant potency in competing for the dopamine D1 receptor as assessed by competition for [3H]SCH23390 binding (Ki greater than 1 microM). Covalent photoaffinity labeling of the dopamine D2 receptor in bovine striatal membranes with N-(p-azido-m-[125I]iodophenethyl)spiperone [( 125I]N3-NAPS) was prevented by AES at nanomolar concentrations. The dopamine D2 receptor was solubilized from bovine striatal membranes using 0.25% cholate in the presence of high ionic strength, followed by precipitation and subsequent treatment with 0.5% digitonin. Nearly 100% of the [3H]spiperone-binding activity in the cholate-digitonin solubilized preparation was absorbed at a receptor-to-resin ratio of 2:1 (v/v). Dopamine D2 receptor was eluted from the affinity resin using a competing dopaminergic antagonist molecule, haloperidol. Recovery of dopamine D2 receptor activity from the affinity matrix was approx. 9% of the activity adsorbed to the resin. The [3H]spiperone-binding activity in AES-Sepharose affinity purified preparations is saturable and of high affinity (0.2 nM). Affinity-purified preparations maintain the ligand-binding characteristics of a dopamine D2 receptor as assessed by agonist and antagonist competition for [3H]spiperone binding.     

Purification and characterization of the recombinant human dopamine D2S receptor from Pichia pastoris

The human dopamine D2S receptor was expressed in the methylotrophic yeast Pichia pastoris, where the receptor with a molecular mass of approximately 40kDa exhibited specific and saturable binding properties. The dopamine antagonist [3H]spiperone showed an average dissociation constant K(d) of 0.6+/-0.17 nM for the dopamine D2S receptor. The receptor was solubilized using the non-ionic detergent dodecylmaltoside and purified by affinity chromatography using a Ni(2+) chelate (His-Trap) column or by batch extraction with an anti-FLAG M1 affinity resin. The receptor maintained its biological activity after solubilization and purification from the membrane protein fraction. A 244- or 185-fold enrichment, as judged by an increase in specific binding, was obtained after adsorption to the His-Trap or anti-FLAG materials, respectively.     

Characteristics of 3H-cis-flupenthixol binding in rat striatum

Characteristics of membrane receptor binding by 3H-cis-flupenthixol were examined in rat striatum. Using modifications of standard dopamine receptor binding techniques, it was possible to obtain 70% specific binding with 3H-cis-flupenthixol. Association and dissociation rates were very rapid, with equilibrium reached in 2 min and half-time of dissociation being 1 min. Analysis of saturation and competition studies using cis-flupenthixol and spiroperidol indicated that cis-flupenthixol binds to two striatal receptors with apparent KD's of 0.7 and 4.8 nM. It is suggested these represent D1 and D2 dopamine receptors respectively. The further characterization of the properties of cis-flupenthixol binding presented here should enable more detailed studies of multiple dopamine receptors to be designed.     

Distinct function of the cytoplasmic tail in human D1-like receptor ligand binding and coupling

To delineate the role of the cytoplasmic tail in the distinct binding and coupling properties of human dopamine D1-like receptors, chimeric receptors were generated in which the entire tail region of wild-type human D1A (or D1) and D1B (or D5) receptors was exchanged. The hD1A-D1BT, but not hD1B-D1AT, receptor expression was dramatically reduced compared with wild-type receptor expression. Swapping the cytoplasmic tail resulted in a full switch of dopamine binding affinity and constitutive activity, while dopamine potency decreased and agonist-mediated maximal activation of adenylyl cyclase increased for both chimeras. Hence, the cytoplasmic tail plays a crucial role in D1-like receptor expression, agonist binding affinity and constitutive activation but regulates in a distinct fashion the formation of D1A and D1B receptor active states upon dopamine binding.     

Design and synthesis of a piperazinylalkylisoxazole library for subtype selective dopamine receptor ligands

A piperazinylbutylisoxazole libary was designed, synthesized and screened for the binding affinities to dopamine D2, D3, and D4 receptors. Several ligands were identified to possess high binding affinity and selectivity for the D3 and D4 receptors over the D2 receptor. Compounds 6s and 6t showed K(i) values of 2.6 nM and 3.9 nM for the D3 receptor with 46- and 50-fold selectivity over the D2 receptor, respectively.     

Analogues of the dopamine D2 receptor antagonist L741,626: Binding, function, and SAR

A series of analogues of the dopamine D2 receptor antagonist L741,626 were synthesized and evaluated for binding and function at D2 family receptor subtypes. Several analogues showed comparable binding profiles to the parent ligand, however, in general, chemical modification served to reduce D2 binding affinity and selectivity.     

Novel Dopamine D2 Receptor Signaling through Proteins Interacting with the Third Cytoplasmic Loop

The diverse activities of dopamine D2-like receptors, including D2, D3, and D4 receptors, are mediated by proteins that interact with the third cytoplasmic loop and regulate receptor signaling, receptor trafficking, and apoptosis. Such interacting proteins include calmodulin, the N-methyl-d-aspartate receptor 2B subunit, calcium/calmodulin-dependent protein kinase II, prostate apoptosis response-4, and β-arrestins, which regulate receptor signaling and the pharmacological action through D2 receptor. The gene encoding the D2 receptor gives rise to two isoforms, termed the dopamine D2 receptor long isoform (D2L) and the dopamine D2 receptor short isoform; the latter lacks 29 amino acids of the D2L receptor within the third cytoplasmic loop. In this review, we first focus on novel functions of the hetero-oligomeric D1/D2 and D2/adenosine A_2A receptors. We next discuss novel signaling through proteins interacting with the D2 receptor third cytoplasmic loop and define the function of a novel binding protein, heart-type fatty acid binding protein, which interacts with the D2L third cytoplasmic loop.     

N-linked oligosaccharides are responsible for rat striatal dopamine D2 receptor heterogeneity

The glycoprotein nature of the binding subunit of the dopamine D2 receptor in rat striatum has been examined by photoaffinity labeling receptor preparations with N-(p-azido-m-[125I]iodophenethyl)spiperone followed by treatment of crude membrane receptor or receptor fractions isolated from sodium dodecyl sulfate (SDS) polyacrylamide gels with endo- and exoglycosidases. The major photoaffinity labeled protein migrates as a heterogeneous species on 10% SDS polyacrylamide gels and ranges from 130,000 to 75,000 relative molecular mass (Mr). This heterogeneity can be explained by glycosylation of the receptor by complex-type N-linked oligosaccharides. Three fractions of labeled receptor were isolated from SDS polyacrylamide gels over a range of 130,000 to 75,000 Mr; after digestion with peptide-N4-[N-acetyl-beta-glucosaminyl] asparagine amidase, all fractions yielded a single peptide approximately 40,000 Mr. Treatment of photoaffinity labeled membranes with alpha-mannosidase was without effect. The dopamine D2 receptor appears to contain substantial amounts of sialic acid as treatment of photoaffinity labeled membranes with neuraminidase increased the receptor mobility on SDS polyacrylamide gels to a species of 50,000-54,000 Mr. Treatment of the receptor with neuraminidase followed by endo-alpha-N-acetylgalactosaminidase did not change the electrophoretic migration pattern from that seen after neuraminidase treatment alone, suggesting that the binding peptide contains no serine- or threonine-linked oligosaccharides. A smaller binding peptide of approximately 31,000 Mr is also apparent in crude photoaffinity labeled membranes. This material also contains N-linked oligosaccharide. Complete removal of N-linked oligosaccharide from the dopamine D2 receptor did not change the rank order potency of agonist and antagonist compounds to compete for [3H]spiperone binding to crude membrane fractions. The dopamine D2 receptor represents a highly glycosylated neural receptor.     

Cloning and characterization of a Caenorhabditis elegans D2-like dopamine receptor

The neurotransmitter dopamine plays an important role in the regulation of behavior in both vertebrates and invertebrates. In mammals, dopamine binds and activates two classes of dopamine receptors, D1-like and D2-like receptors. However, D2-like dopamine receptors in Caenorhabditis elegans have not yet been characterized. We have cloned a cDNA encoding a putative C. elegans D2-like dopamine receptor. The deduced amino acid sequence of the cloned cDNA shows higher sequence similarities to vertebrate D2-like dopamine receptors than to D1-like receptors. Two splice variants that differ in the length of their predicted third intracellular loops were identified. The receptor heterologously expressed in cultured cells showed high affinity binding to [125I]iodo-lysergic acid diethylamide. Dopamine showed the highest affinity for this receptor among several amine neurotransmitters tested. Activation of the heterologously expressed receptor led to the inhibition of cyclic AMP production, confirming that this receptor has the functional property of a D2-like receptor. We have also analyzed the expression pattern of this receptor and found that the receptor is expressed in several neurons including all the dopaminergic neurons in C. elegans.     

Chemical modification reveals involvement of tyrosine in ligand binding to dopamine D1 and D2 receptors

The effect of tyrosine-alkylating agents on the ligand-binding properties of bovine striatal dopamine D1 and D2 receptors was investigated. The tyrosine-alkylating agents, p-nitrobenzenesulphonylfluoride (pNBSF) and tetranitromethane (TNM) caused a time-and dose-dependent loss of the binding of [3H]SCH-23390 and [3H]spiroperidol, ligands specific for dopamine D1 and D2 receptors, respectively. The two dopamine receptors, however, showed a differential sensitivity to inactivation by these agents. The mechanism of inhibition of the two receptors appears to be complex as treatment of membranes with pNBSF and TNM resulted in a decrease of both the Kd and the Bmax of ligand binding. Spiroperidol almost completely protected the TNM-induced inhibition of [3H]spiroperidol binding to dopamine D2 receptors whereas SCH-23390 afforded only partial protection of the [3H]SCH-23390 binding by TNM suggesting that more than one tyrosine groups may be involved in the D1 receptor binding activity.