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A dopamine D2 receptor mutation was recently identified in a family with a novel hyperkinetic movement disorder. That allelic variant D2-I212F is a constitutively active and G protein–biased receptor. We now describe mice engineered using CRISPR-Cas9–mediated gene editing technology to carry the D2-I212F variant. Drd2I212Fmice exhibited gait abnormalities resembling those in other mouse models of chorea and/or dystonia and had striatal D2 receptor expression that was decreased approximately 30% per Drd2I212Fallele. Electrically evoked inhibitory postsynaptic conductances in midbrain dopamine neurons and striatum from Drd2I212Fmice, caused by G protein activation of potassium channels, exhibited slow kinetics (e.g., approximately four- to sixfold slower decay) compared with Drd2+/+mice. Current decay initiated by photolytic release of the D2 antagonist sulpiride from CyHQ-sulpiride was also ∼fourfold slower in midbrain slices from Drd2I212Fmice than Drd2+/+mice. Furthermore, in contrast to Drd2+/+mice, in which dopamine is several-fold more potent at neurons in the nucleus accumbens than in the dorsal striatum, reflecting activation of Gαoversus Gαi, dopamine had similar potencies in those two brain regions of Drd2I212Fmice. Repeated cocaine treatment, which decreases dopamine potency in the nucleus accumbens of Drd2+/+mice, had no effect on dopamine potency in Drd2I212Fmice. The results demonstrate the pathogenicity of the D2-I212F mutation and the utility of this mouse model for investigating the role of pathogenic DRD2variants in early-onset hyperkinetic movement disorders.SIGNIFICANCE STATEMENTThe first dopamine receptor mutation to cause a movement disorder, D2-I212F, was recently identified. The mutation makes receptor activation of G protein–mediated signaling more efficient. To confirm the pathogenesis of D2-I212F, this study reports that mice carrying this mutation have gait abnormalities consistent with the clinical phenotype. The mutation also profoundly alters D2 receptor expression and function in vivo. This mouse model will be useful for further characterization of the mutant receptor and for evaluation of potential therapeutic drugs.

We previously proposed that the dopamine D2 receptor–interacting protein S100B binds to a putative S100B-binding motif at residues R233–L240 toward the N terminus of the third intracellular loop. We used in vitro pull-down assays with FLAG-tagged fragments of the rat dopamine D2 receptor third intracellular loop (D2-IC3) and in vitro-synthesized S100B to evaluate this hypothesis. Our results indicate that the putative S100B-binding motif is neither necessary nor sufficient for strong binding of S100B to D2-IC3. Instead, two residues at the junction of the fifth membrane-spanning domain and the cytoplasmic extension of that α-helical domain, K211-I212, are required for robust, calcium-sensitive binding of S100B. This is also the approximate location of previously identified determinants for the binding of arrestin and calmodulin. A D2 receptor mutation converting I212 to phenylalanine has been described in patients with a hyperkinetic movement disorder.SIGNIFICANCE STATEMENTS100B is a small calcium-binding protein that modulates signaling by the dopamine D2 receptor. New data suggest that the previous hypothesis about the involvement of an S100B-binding motif is incorrect, and that an important determinant of S100B binding includes a residue that is mutated in patients with a hyperkinetic movement disorder.

The dopamine (DA) D2 receptor (D2R) is an important target for the treatment of neuropsychiatric disorders such as schizophrenia and Parkinson’s disease. However, the development of improved therapeutic strategies has been hampered by our incomplete understanding of this receptor’s downstream signaling processes in vivo and how these relate to the desired and undesired effects of drugs. D2R is a G protein-coupled receptor (GPCR) that activates G protein-dependent as well as non-canonical arrestin-dependent signaling pathways. Whether these effector pathways act alone or in concert to facilitate specific D2R-dependent behaviors is unclear. Here, we report on the development of a D2R mutant that recruits arrestin but is devoid of G protein activity. When expressed virally in “indirect pathway” medium spiny neurons (iMSNs) in the ventral striatum of D2R knockout mice, this mutant restored basal locomotor activity and cocaine-induced locomotor activity in a manner indistinguishable from wild-type D2R, indicating that arrestin recruitment can drive locomotion in the absence of D2R-mediated G protein signaling. In contrast, incentive motivation was enhanced only by wild-type D2R, signifying a dissociation in the mechanisms that underlie distinct D2R-dependent behaviors, and opening the door to more targeted therapeutics.

Dopamine is critical for many processes that drive learning and memory, including motivation, prediction error, incentive salience, memory consolidation, and response output. Theories of dopamine’s function in these processes have, for the most part, been developed from behavioral approaches that examine learning mechanisms in appetitive tasks. A parallel and growing literature indicates that dopamine signaling is involved in consolidation of memories into stable representations in aversive tasks such as fear conditioning. Relatively little is known about how dopamine may modulate memories that form during extinction, when organisms learn that the relation between previously associated events is severed. We investigated whether fear and reward extinction share common mechanisms that could be enhanced with dopamine D1/5 receptor activation. Pharmacological activation of dopamine D1/5 receptors (with SKF 81297) enhanced extinction of both cued and contextual fear. These effects also occurred in the extinction of cocaine-induced conditioned place preference, suggesting that the observed effects on extinction were not specific to a particular type of procedure (aversive or appetitive). A cAMP/PKA biased D1 agonist (SKF 83959) did not affect fear extinction, whereas a broadly efficacious D1 agonist (SKF 83822) promoted fear extinction. Together, these findings show that dopamine D1/5 receptor activation is a target for the enhancement of fear or reward extinction.

Imbalance between the dopamine and serotonin (5-HT) neurotransmitter systems has been implicated in the comorbidity of Parkinson’s disease (PD) and psychiatric disorders. L-DOPA, the leading treatment of PD, facilitates the production and release of dopamine. This study assessed the action of L-DOPA on monoamine synaptic transmission in mouse brain slices. Application of L-DOPA augmented the D2-receptor-mediated inhibitory postsynaptic current (IPSC) in dopamine neurons of the substantia nigra. This augmentation was largely due to dopamine release from 5-HT terminals. Selective optogenetic stimulation of 5-HT terminals evoked dopamine release, producing D2-receptor-mediated IPSCs following treatment with L-DOPA. In the dorsal raphe, L-DOPA produced a long-lasting depression of the 5-HT1A-receptor-mediated IPSC in 5-HT neurons. When D2 receptors were expressed in the dorsal raphe, application of L-DOPA resulted in a D2-receptor-mediated IPSC. Thus, treatment with L-DOPA caused ectopic dopamine release from 5-HT terminals and a loss of 5-HT-mediated synaptic transmission.

Arrestins mediate G protein-coupled receptor desensitization, internalization, and signaling. Dopamine D2and D3receptors have similar structures but distinct characteristics of interaction with arrestins. The goals of this study were to compare arrestin-binding determinants in D2and D3receptors other than phosphorylation sites and to create a D2receptor that is deficient in arrestin binding. We first assessed the ability of purified arrestins to bind to glutathione transferase (GST) fusion proteins containing the receptor third intracellular loops (IC3). Arrestin3 bound to IC3 of both D2and D3receptors, with the affinity and localization of the binding site indistinguishable between the receptor subtypes. Mutagenesis of the GST-IC3 fusion proteins identified an important determinant of the binding of arrestin3 in the N-terminal region of IC3. Alanine mutations of this determinant (IYIV212-215) in the full-length D2receptor generated a signaling-biased receptor with intact ligand binding and G-protein coupling and activation, but deficient in receptor-mediated arrestin3 translocation to the membrane, agonist-induced receptor internalization, and agonist-induced desensitization in human embryonic kidney 293 cells. This mutation also decreased arrestin-dependent activation of extracellular signal-regulated kinases. The finding that nonphosphorylated D2-IC3 and D3-IC3 have similar affinity for arrestin is consistent with previous suggestions that the differential effects of D2and D3receptor activation on membrane translocation of arrestin and receptor internalization are due, at least in part, to differential phosphorylation of the receptors. In addition, these results imply that the sequence IYIV212-215 at the N terminus of IC3 of the D2receptor is a key element of the arrestin binding site.

Dopamine D2and D3receptors are similar subtypes with distinct interactions with arrestins; the D3receptor mediates less agonist-induced translocation of arrestins than the D2receptor. The goals of this study were to compare nonphosphorylated arrestin-binding determinants in the second intracellular domain (IC2) of the D2and D3receptors to identify residues that contribute to the differential binding of arrestin to the subtypes. Arrestin3 bound to glutathione transferase (GST) fusion proteins of the D2receptor IC2 more avidly than to the D3receptor IC2. Mutagenesis of the fusion proteins identified a residue at the C terminus of IC2, Lys149, that was important for the preferential binding of arrestin3 to D2-IC2; arrestin binding to D2-IC2-K149C was greatly decreased compared with wild-type D2-IC2, whereas binding to the reciprocal mutant D3-IC2-C147K was enhanced compared with wild-type D3-IC2. Mutating this lysine in the full-length D2receptor to cysteine decreased the ability of the D2receptor to mediate agonist-induced arrestin3 translocation to the membrane and decreased agonist-induced receptor internalization in human embryonic kidney 293 cells. The reciprocal mutation in the D3receptor increased receptor-mediated translocation of arrestin3 without affecting agonist-induced receptor internalization. G protein-coupled receptor crystal structures suggest that Lys149, at the junction of IC2 and the fourth membrane-spanning helix, has intramolecular interactions that contribute to maintaining an inactive receptor state. It is suggested that the preferential agonist-induced binding of arrestin3 to the D2receptor over the D3receptor is due in part to Lys149, which could be exposed as a result of receptor activation.

Dopamine D2and D3receptors are similar subtypes with distinct interactions with arrestins; the D3receptor mediates less agonist-induced translocation of arrestins than the D2receptor. The goals of this study were to compare nonphosphorylated arrestin-binding determinants in the second intracellular domain (IC2) of the D2and D3receptors to identify residues that contribute to the differential binding of arrestin to the subtypes. Arrestin3 bound to glutathione transferase (GST) fusion proteins of the D2receptor IC2 more avidly than to the D3receptor IC2. Mutagenesis of the fusion proteins identified a residue at the C terminus of IC2, Lys149, that was important for the preferential binding of arrestin3 to D2-IC2; arrestin binding to D2-IC2-K149C was greatly decreased compared with wild-type D2-IC2, whereas binding to the reciprocal mutant D3-IC2-C147K was enhanced compared with wild-type D3-IC2. Mutating this lysine in the full-length D2receptor to cysteine decreased the ability of the D2receptor to mediate agonist-induced arrestin3 translocation to the membrane and decreased agonist-induced receptor internalization in human embryonic kidney 293 cells. The reciprocal mutation in the D3receptor increased receptor-mediated translocation of arrestin3 without affecting agonist-induced receptor internalization. G protein-coupled receptor crystal structures suggest that Lys149, at the junction of IC2 and the fourth membrane-spanning helix, has intramolecular interactions that contribute to maintaining an inactive receptor state. It is suggested that the preferential agonist-induced binding of arrestin3 to the D2receptor over the D3receptor is due in part to Lys149, which could be exposed as a result of receptor activation.

Arrestins mediate G protein-coupled receptor desensitization, internalization, and signaling. Dopamine D2and D3receptors have similar structures but distinct characteristics of interaction with arrestins. The goals of this study were to compare arrestin-binding determinants in D2and D3receptors other than phosphorylation sites and to create a D2receptor that is deficient in arrestin binding. We first assessed the ability of purified arrestins to bind to glutathione transferase (GST) fusion proteins containing the receptor third intracellular loops (IC3). Arrestin3 bound to IC3 of both D2and D3receptors, with the affinity and localization of the binding site indistinguishable between the receptor subtypes. Mutagenesis of the GST-IC3 fusion proteins identified an important determinant of the binding of arrestin3 in the N-terminal region of IC3. Alanine mutations of this determinant (IYIV212-215) in the full-length D2receptor generated a signaling-biased receptor with intact ligand binding and G-protein coupling and activation, but deficient in receptor-mediated arrestin3 translocation to the membrane, agonist-induced receptor internalization, and agonist-induced desensitization in human embryonic kidney 293 cells. This mutation also decreased arrestin-dependent activation of extracellular signal-regulated kinases. The finding that nonphosphorylated D2-IC3 and D3-IC3 have similar affinity for arrestin is consistent with previous suggestions that the differential effects of D2and D3receptor activation on membrane translocation of arrestin and receptor internalization are due, at least in part, to differential phosphorylation of the receptors. In addition, these results imply that the sequence IYIV212-215 at the N terminus of IC3 of the D2receptor is a key element of the arrestin binding site.

S100B is a calcium-binding protein with both extracellular and intracellular regulatory activities in the mammalian brain. We have identified a novel interaction between S100B and the dopamine D2receptor. Our results also suggest that the binding of S100B to the dopamine D2receptor enhances receptor signaling. This conclusion is based on the following observations: 1) S100B and the third cytoplasmic loop of the dopamine D2receptor interact in a bacterial two-hybrid system and in a poly-histidine pull-down assay; 2) immunoprecipitation of the D2receptor also precipitates FLAG-S100B from human embryonic kidney 293 cell homogenates and endogenous S100B from rat neostriatal homogenates; 3) S100B immunoreactivity was detected in cultured neostriatal neurons expressing the D2receptor; 4) a putative S100B binding motif is located at residues 233 to 240 of the D2receptor, toward the amino terminus of the third cytoplasmic loop. D3-IC3, which does not bind S100B, does not contain this motif; and 5) coexpression of S100B in D2receptor-expressing 293 cells selectively increased D2receptor stimulation of extracellular signal-regulated kinases and inhibition of adenylate cyclase.

The defining characteristic of G protein-coupled receptor homologous desensitization is that the receptor must be occupied by an agonist or in an activated conformation that mimics an agonist-induced state. In most instances, the mechanistic basis for this characteristic is the high selectivity of G protein-coupled receptor kinases for the activated receptor. In this issue, Rankin et al. (p. 759) demonstrate that under some conditions, at least, the G protein-coupled receptor kinase GRK4 does not display a preference for the agonist-occupied D1dopamine receptor. Coexpression of GRK4 and the D1receptor in a heterologous system induces phosphorylation of the receptor in the absence of agonist, causing constitutive desensitization and internalization of the receptor. Lacking the normal rapid feedback mechanisms associated with homologous desensitization, a system incorporating constitutively active GRK4 will be prone to dysregulation, perhaps explaining the generally low expression of GRK4. Indeed, considerable evidence suggests that just such dysregulation resulting from mutationally activated GRK4 contributes to the heritable component of human essential hypertension (Physiol Genomics 19 : 223-246, 2004).

To test the hypothesis that pharmacological differentiation between D1and D2dopamine receptors results from interactions of selective ligands with nonconserved residues lining the binding pocket, we mutated amino acid residues in the D2receptor to the corresponding aligned residues in the D1receptor and vice versa and expressed the receptors in human embryonic kidney 293 cells. Determinations of the affinity of the 14 mutant D2receptors and 11 mutant D1receptors for D1- and D2-selective antagonists, and rhodopsin-based homology models of the two receptors, identified two residues whose direct interactions with certain ligands probably contribute to ligand selectivity. The D1receptor mutant W993.28F showed dramatically increased affinity for several D2-selective antagonists, particularly spiperone (225-fold), whereas the D2receptor mutant Y4177.43W had greatly decreased affinity for benzamide ligands such as raclopride (200-fold) and sulpiride (125-fold). The binding of the D1-selective ligand R-(+)-7-chloro-8-hydroxy-3-methyl-1-phenyl-2,3,4,5-tetrahydro-1H-3-benzazepine (SCH23390) was unaffected, indicating that SCH23390 makes little contact with these ancillary pocket residues. Mutation of A/V5.39caused modest but consistent and reciprocal changes in affinity of the receptors for D1and D2-selective ligands, perhaps reflecting altered packing of the interface of helices 5 and 6. We also obtained some evidence that residues in the second extracellular loop contribute to ligand binding. We conclude that additional determinants of D1/D2receptor-selective binding are located either in that loop or in the transmembrane helices but, like residue 5.39, indirectly influence the interactions of selective ligands with conserved residues by altering the shape of the primary and ancillary binding pockets.

To test the hypothesis that pharmacological differentiation between D1 and D2 dopamine receptors results from interactions of selective ligands with nonconserved residues lining the binding pocket, we mutated amino acid residues in the D2 receptor to the corresponding aligned residues in the D1 receptor and vice versa and expressed the receptors in human embryonic kidney 293 cells. Determinations of the affinity of the 14 mutant D2 receptors and 11 mutant D1 receptors for D1- and D2-selective antagonists, and rhodopsin-based homology models of the two receptors, identified two residues whose direct interactions with certain ligands probably contribute to ligand selectivity. The D1 receptor mutant W993.28F showed dramatically increased affinity for several D2-selective antagonists, particularly spiperone (225-fold), whereas the D2 receptor mutant Y4177.43W had greatly decreased affinity for benzamide ligands such as raclopride (200-fold) and sulpiride (125-fold). The binding of the D1-selective ligand R-(+)-7-chloro-8-hydroxy-3-methyl-1-phenyl-2,3,4,5-tetrahydro-1H-3-benzazepine (SCH23390) was unaffected, indicating that SCH23390 makes little contact with these ancillary pocket residues. Mutation of A/V5.39 caused modest but consistent and reciprocal changes in affinity of the receptors for D1 and D2-selective ligands, perhaps reflecting altered packing of the interface of helices 5 and 6. We also obtained some evidence that residues in the second extracellular loop contribute to ligand binding. We conclude that additional determinants of D1/D2 receptor-selective binding are located either in that loop or in the transmembrane helices but, like residue 5.39, indirectly influence the interactions of selective ligands with conserved residues by altering the shape of the primary and ancillary binding pockets.

There is an enormous initiative to establish the genetic basis for disorders of brain function. Unfortunately, genetic intervention is not accomplished easily in the nervous system. One strategy is to engineer and deliver to neurons specialized viral vectors that carry a gene (or genes) of interest, thereby exploiting the natural ability of viruses to insert genetic material into cells. When delivered to brain cells, these vectors cause infected cells to increase the expression of the genes of interest. The ability to deliver genes into neurons in vitro and in vivo with herpes simplex virus (HSV) amplicon vectors has made it possible to carry out exactly these sorts of experiments. This technology has the potential to offer new insights into the etiology of a wide variety of neuropsychiatric disorders. We describe the use of HSV amplicon vectors to study Alzheimer disease, drug addiction, and depression, and discuss the considerations that enter into the use of these vectors both in vitro and in vivo. The HSV amplicon virus is a user-friendly vector for the delivery of genes into neurons that has come of age for the study of brain function.

In this issue of Molecular Pharmacology, Oliveras-Reyes et al. (p. 356) describe the agonist-stimulated formation of a caveolin-dependent signalplex that includes both the angiotensin AT1receptor and the epidermal growth factor receptor, and probably also a number of other signal transduction intermediates. The signalplex is thought to facilitate the action of protein kinases that mediate angiotensin II-induced transactivation of the epidermal growth factor receptor and activation of extracellular signal-regulated kinase, and epidermal growth factor-induced inositol phosphate accumulation and phosphorylation/desensitization of the AT1receptor. This work contributes to an emerging view of the complexity and nonlinearity of signaling via G protein-coupled receptors and receptor tyrosine kinases, and of the importance of membrane compartmentalization to signal transduction.

In this issue of Molecular Pharmacology, Oliveras-Reyes et al. (p. 356) describe the agonist-stimulated formation of a caveolin-dependent signalplex that includes both the angiotensin AT1 receptor and the epidermal growth factor receptor, and probably also a number of other signal transduction intermediates. The signalplex is thought to facilitate the action of protein kinases that mediate angiotensin II-induced transactivation of the epidermal growth factor receptor and activation of extracellular signal-regulated kinase, and epidermal growth factor-induced inositol phosphate accumulation and phosphorylation/desensitization of the AT1 receptor. This work contributes to an emerging view of the complexity and nonlinearity of signaling via G protein-coupled receptors and receptor tyrosine kinases, and of the importance of membrane compartmentalization to signal transduction.

Dopamine D2 receptor interactions with arrestins and arrestin-dependent internalization have been characterized using heterologously expressed D2 receptor and arrestins. The purpose of this study was to investigate D2 receptor interaction with endogenous arrestins. Arrestin2 and arrestin3 in striatal homogenates bound to the third cytoplasmic loop of the D2 receptor, and purified arrestin2 and arrestin3 bound to the second and third loops and C terminus of the D2 receptor, in a glutathione S-transferase pull-down assay. In NS20Y neuroblastoma cells expressing an enhanced green-fluorescent protein-tagged D2 receptor (D2-EGFP), 2-h D2 agonist stimulation enhanced the colocalization of D2-EGFP with endogenous arrestin2 and arrestin3. These results suggest that the D2 receptor has the intrinsic ability to bind both nonvisual arrestins. Agonist treatment of D2-EGFP NS20Y cells induced D2 receptor internalization (36-46%) that was maximal within 20 min, but that was prevented by small interfering RNA-induced depletion of arrestin2 and arrestin3. In neostriatal neurons, 2-h agonist treatment selectively increased the colocalization of the endogenous D2 receptor with arrestin2, whereas receptor colocalization with arrestin3 was reduced. Agonist stimulation caused translocation of arrestin2, but not arrestin3, to the membrane in neurons and selectively enhanced the coimmunoprecipitation of the D2 receptor and arrestin2. All three measures of receptor/arrestin interaction (colocalization, translocation, and coprecipitation) demonstrated selective agonist-induced interaction between the D2 receptor and arrestin2 in neurons.

Dopamine D2 receptor interactions with arrestins and arrestin-dependent internalization have been characterized using heterologously expressed D2 receptor and arrestins. The purpose of this study was to investigate D2 receptor interaction with endogenous arrestins. Arrestin2 and arrestin3 in striatal homogenates bound to the third cytoplasmic loop of the D2 receptor, and purified arrestin2 and arrestin3 bound to the second and third loops and C terminus of the D2 receptor, in a glutathione S-transferase pull-down assay. In NS20Y neuroblastoma cells expressing an enhanced green-fluorescent protein-tagged D2 receptor (D2-EGFP), 2-h D2 agonist stimulation enhanced the colocalization of D2-EGFP with endogenous arrestin2 and arrestin3. These results suggest that the D2 receptor has the intrinsic ability to bind both nonvisual arrestins. Agonist treatment of D2-EGFP NS20Y cells induced D2 receptor internalization (36-46%) that was maximal within 20 min, but that was prevented by small interfering RNA-induced depletion of arrestin2 and arrestin3. In neostriatal neurons, 2-h agonist treatment selectively increased the colocalization of the endogenous D2 receptor with arrestin2, whereas receptor colocalization with arrestin3 was reduced. Agonist stimulation caused translocation of arrestin2, but not arrestin3, to the membrane in neurons and selectively enhanced the coimmunoprecipitation of the D2 receptor and arrestin2. All three measures of receptor/arrestin interaction (colocalization, translocation, and coprecipitation) demonstrated selective agonist-induced interaction between the D2 receptor and arrestin2 in neurons.

The aim of this study was to use pharmacological inhibition of protein kinase A and mutation of potential protein kinase A phosphorylation sites to determine the role of protein kinase A-catalyzed phosphorylation of the dopamine D1receptor in agonist-stimulated desensitization and internalization of the receptor. To facilitate purification and imaging of the D1receptor, we attached a polyhistidine tag to the amino terminus and enhanced green fluorescent protein to the carboxyl terminus of the receptor (D1-EGFP). D1-EGFP was similar to the untagged D1receptor in terms of affinity for agonist and antagonist ligands, coupling to G proteins, and stimulation of cyclic AMP accumulation. D1-EGFP and two mutants in which either Thr268 or Ser380 was replaced with Ala were stably expressed in NS20Y neuroblastoma cells. Pretreatment with the protein kinase A inhibitor H-89 (N-[2-(p-bromocinnamylamino)ethyl]-5-isoquinolinesulfonamide) or substitution of Ala for Thr268 reduced agonist-stimulated phosphorylation of the receptor and resulted in diminished trafficking of the receptor to the perinuclear region of the cell. Substitution of Ala for Thr268 had no effect, however, on agonist-induced receptor sequestration or desensitization of cyclic AMP accumulation. Substitution of Ala for Ser380 had no effect on D1receptor phosphorylation, sequestration, desensitization, or trafficking to the perinuclear region. We conclude that protein kinase A-dependent phosphorylation of the D1receptor on Thr268 regulates a late step in the sorting of the receptor to the perinuclear region of the cell, but that phosphorylation of Thr268 is not required for receptor sequestration or maximal desensitization of cyclic AMP accumulation.

The aim of this study was to use pharmacological inhibition of protein kinase A and mutation of potential protein kinase A phosphorylation sites to determine the role of protein kinase A-catalyzed phosphorylation of the dopamine D(1) receptor in agonist-stimulated desensitization and internalization of the receptor. To facilitate purification and imaging of the D(1) receptor, we attached a polyhistidine tag to the amino terminus and enhanced green fluorescent protein to the carboxyl terminus of the receptor (D(1)-EGFP). D(1)-EGFP was similar to the untagged D(1) receptor in terms of affinity for agonist and antagonist ligands, coupling to G proteins, and stimulation of cyclic AMP accumulation. D(1)-EGFP and two mutants in which either Thr268 or Ser380 was replaced with Ala were stably expressed in NS20Y neuroblastoma cells. Pretreatment with the protein kinase A inhibitor H-89 (N-[2-(p-bromocinnamylamino)ethyl]-5-isoquinolinesulfonamide) or substitution of Ala for Thr268 reduced agonist-stimulated phosphorylation of the receptor and resulted in diminished trafficking of the receptor to the perinuclear region of the cell. Substitution of Ala for Thr268 had no effect, however, on agonist-induced receptor sequestration or desensitization of cyclic AMP accumulation. Substitution of Ala for Ser380 had no effect on D(1) receptor phosphorylation, sequestration, desensitization, or trafficking to the perinuclear region. We conclude that protein kinase A-dependent phosphorylation of the D(1) receptor on Thr268 regulates a late step in the sorting of the receptor to the perinuclear region of the cell, but that phosphorylation of Thr268 is not required for receptor sequestration or maximal desensitization of cyclic AMP accumulation.

Whereas acute stimulation of Gαi/o-coupled receptors inhibits the activity of adenylyl cyclase, a delayed consequence of persistent activation of the receptors is heterologous sensitization, an enhanced responsiveness of adenylyl cyclase to activators such as forskolin or agonists of Gαs-coupled receptors. Gαs-insensitive mutants of adenylyl cyclase type V were used to test the hypothesis that heterologous sensitization requires Gαs-dependent activation of adenylyl cyclase. When adenylyl cyclase was stably expressed in human embryonic kidney (HEK) 293 cells with the D2Ldopamine receptor, basal, forskolin-stimulated, and isoproterenol-stimulated cyclic AMP accumulation were all enhanced by 2-h pretreatment with the D2receptor agonist quinpirole. Transient expression of wild-type adenylyl cyclase and three Gαs-insensitive mutants (F379L, R1021Q, and F1093S) in HEK293 cells stably expressing the D2Lreceptor demonstrated that all three mutants had little or no responsiveness to β-adrenergic receptor-mediated activation of Gαsbut that the mutants retained sensitivity to forskolin and to D2Lreceptor-mediated inhibition. Transiently expressed adenylyl cyclase V was robustly sensitized by 2-h pretreatment with quinpirole. In contrast, the Gαs-insensitive mutants displayed no sensitization of forskolin-stimulated cyclic AMP accumulation, indicating that responsiveness to Gαsis required for the expression of heterologous sensitization.

A homology model of the dopamine D2receptor was constructed based on the crystal structure of rhodopsin. A putative sodium-binding pocket identified in an earlier model (PDB 1I15) was revised. It is now defined by Asn-419 backbone oxygen at the apex of a pyramid and Asp-80, Ser-121, Asn-419, and Ser-420 at each vertex of the planar base. Asn-423 stabilizes this pocket through hydrogen bonds to two of these residues. Highly conserved Asn-52 is positioned near the sodium pocket, where it hydrogen-bonds with Asp-80 and the backbone carbonyl of Ser-420. Mutation of three of these residues, Asn-52 in helix 1, Ser-121 in helix 3, and Ser-420 in helix 7, profoundly altered the properties of the receptor. Mutants in which Asn-52 was replaced with Ala or Leu or Ser-121 was replaced with Leu exhibited no detectable binding of radioligands, although receptor immunoreactivity in the membrane was similar to that in cells expressing the wild-type D2Lreceptor. A mutant in which Asn-52 was replaced with Gln, preserving hydrogen-bonding capability, was similar to D2Lin affinity for ligands and ability to inhibit cAMP accumulation. Mutants in which either Ser-121 or Ser-420 was replaced with Ala or Asn had decreased affinity for agonists (Ser-121), but increased affinity for the antagonists haloperidol and clozapine. Interestingly, the affinity of these Ser-121 and Ser-420 mutants for substituted benzamide antagonists showed little or no dependence on sodium, consistent with our hypothesis that Ser-121 and Ser-420 contribute to the formation of a sodium-binding pocket.

We characterized the effects of drugs on the uptake of [3H]neurotransmitter by and the binding of [125I](3β-(4-iodophenyl)tropane-2β-carboxylic acid methyl ester ([125I]RTI-55) to the recombinant human dopamine (hDAT), serotonin (hSERT), or norepinephrine (hNET) transporters stably expressed in human embryonic kidney 293 cells. RTI-55 had similar affinity for the hDAT and hSERT and lower affinity for hNET (Kd= 1.83, 0.98, and 12.1 nM, respectively). Kinetic analysis of [125I]RTI-55 binding indicated that the dissociation rate (k−1) was significantly lower for hSERT and the association rate (k+1) was significantly lower for hNET compared with the hDAT. The potency of drugs at blocking [3H]neurotransmitter uptake was highly correlated with potency at blocking radioligand binding for hDAT and hSERT. Substrates were more potent at the inhibition of [3H]neurotransmitter uptake than radioligand binding. The potency of drugs was highly correlated between displacement of [3H]nisoxetine (Kd= 6.0 nM) and [125I]RTI-55 from the hNET, suggesting that these radioligands recognize similar sites on the transporter protein. The correlation observed between inhibitory potency for uptake and binding of either ligand at the hNET was lower than correlations between uptake and binding for hDAT and hSERT. The present results indicate that the cocaine analog [125I]RTI-55 has unique binding properties at each of the transporters and that the use of recombinant transporters expressed by a single cell type can provide a powerful screening tool for drugs interacting with biogenic amine transporters, such as possible cocaine antagonists.

Dopamine D2receptors contain a cluster of serine residues in the fifth transmembrane domain that contribute to activation of the receptor as well as to the binding of agonists. We used rat D2Sdopamine receptor mutants, each containing a serine-to-alanine substitution (S193A, S194A, S197A), to investigate the mechanism through which these residues affect activation of the receptor. Activation of the mutant receptor S194A was abolished in an agonist-dependent manner, such that dopamine no longer inhibited cAMP accumulation in C6 glioma cells or activated G protein-regulated K+channels in Xenopus laevisoocytes, whereas the efficacy of several other agonists was unaffected. Dihydrexidine did not inhibit cAMP accumulation at either S193A or S194A. The decreased efficacy of dihydrexidine at S193A and S194A and dopamine at S194A was associated with a decreased ability to detect a GTP-sensitive high affinity binding state for these agonists. The ability of dopamine to stimulate [35S]guanosine-5′-O-(3-thio)triphosphate binding via S194A also was decreased by ∼50%. Finally, constitutive stimulation of [35S]guanosine-5′-O-(3-thio)triphosphate binding and inhibition of adenylate cyclase by the D2Sreceptor was reduced by mutation of either S193 or S194. These data support the existence of multiple active receptor conformations that are differentially sensitive to mutation of serine residues in the fifth-transmembrane domain.

Chimeric D1/D2receptors were constructed to identify structural determinants of drug affinity and efficacy. We previously reported that chimeras that had D1receptor transmembrane domain VII together with amino-terminal sequence from the D2receptor were nonfunctional. D2/D1chimeras were constructed that contained D2receptor sequence at the amino- and carboxyl-terminal ends and D1receptor sequence in the intervening region. Chimeric receptors with D2sequence from transmembrane domain 7 to the carboxyl terminus together with D2receptor sequence from the amino terminus through transmembrane helix 4 (D2[1–4,7]) and 5 (D2[1–5,7]) bound [3H]spiperone with high affinity, consistent with the hypothesis that D2receptor transmembrane domain I or II is incompatible with D1receptor transmembrane domain VII. D2[1–4,7]and D2[1–5,7]had affinities similar to D1and D2receptors for most nonselective dopamine antagonists and had affinities for most of the selective antagonists that were intermediate between those of the parent receptors. D2[1–4,7]and D2[1–5,7]mediated dopamine receptor agonist-induced stimulation and inhibition, respectively, of cAMP accumulation. The more efficient coupling of D2[1–5,7]to inhibition of cAMP accumulation, compared with the coupling of D2[5–7]and D2[3–7], supports the view that multiple D2receptor cytoplasmic domains acting in concert are necessary for receptor activation of Gi. In contrast, D2[1–4,7], which contains only one cytoplasmic loop (the third) from the D1receptor, is capable of activating Gs. D2[1–4,7]exhibited several characteristics of a constitutively active receptor, including enhanced basal (unliganded) stimulation of cAMP accumulation, high affinity for agonists even in the presence of GTP, and blunted agonist-stimulated cAMP accumulation. A number of dopamine receptor antagonists were inverse agonists at D2[1–4,7], inhibiting basal cAMP accumulation. Some of these drugs were also inverse agonists at the D1receptor. Interestingly, several antagonists also potentiated forskolin-stimulated cAMP accumulation via D2[1–5,7]and via the D2receptor, which could reflect inverse agonist inhibition of native constitutive activity of this receptor.

The D2-like dopamine receptors couple to a variety of signal transduction pathways, including inhibition of adenylate cyclase, mitogenesis, and activation of potassium channels. Although these effects are mediated via pertussis toxin-sensitive G proteins, Gi/o, it is likely that some of these effects are influenced by the release of G protein βγ subunits. Type II adenylate cyclase (ACII) is highly regulated by multiple biochemical stimuli, including protein kinase C, forskolin, G protein α subunits, and G protein βγ subunits. The ability of βγ subunits to activate this enzyme in the presence of activated αshas been particularly well characterized. Although stimulation by βγ subunits has been described as conditional on the presence of activated αs, βγ subunits also potentiate ACII activity after activation of protein kinase C. We created stable cell lines expressing ACII and the D2Lreceptor, the D3receptor, or the D4.4receptor. Activation of D2Lor D4.4receptors, but not D3receptors, potentiated β-adrenergic receptor/Gs-stimulated activity of ACII, as measured by the intracellular accumulation of cAMP. Similarly, stimulation of D2Lor D4.4receptors potentiated phorbol ester-stimulated ACII activity in the absence of activated αs, whereas stimulation of D3receptors did not. The effect of D2-like receptor stimulation was blocked by pretreatment with pertussis toxin and by inhibition of protein kinase C. We propose that activation of both D2Land D4.4dopamine receptors potentiated phorbol-12-myristate-13-acetate-stimulated ACII activity through the release of βγ subunits from pertussis toxin-sensitive G proteins. In contrast, the lack of D3receptor-mediated effects suggests that stimulation of D3receptors does not result in an appreciable release of βγ subunits.

Abstract:To determine if catechol-O-methyltransferase (COMT) metabolizes catecholamines within cell lines used for heterologous expression of plasmalemmal transporters and alters the measured characteristics of 3H-substrate transport, the uptake of monoamine transporter substrates was assessed in three cell lines (C6 glioma, L-M fibroblast, and HEK293 cells) that had been transfected with the recombinant human transporters. Uptake and cellular retention of 3H-catecholamines was increased by up to fourfold by two COMT inhibitors, tropolone and Ro 41-0960, with potencies similar to those for inhibition of COMT activity, whereas the uptake of two transporter substrates that are not substrates for COMT, [3H]serotonin and [3H]MPP+, was unaffected. Direct measurement of monoamine substrates by HPLC confirmed that tropolone (1 mM) increased the retention of the catecholamines dopamine and norepinephrine, but not the retention of serotonin in HEK293 cells. Saturation analysis of the uptake of [3H]dopamine by C6 cells expressing the dopamine transporter demonstrated that tropolone (1 mM) decreased the apparent Kmof transport from 0.61 µMto 0.34 µMwithout significantly altering the maximal velocity of transport. These data suggest that endogenous COMT activity in mammalian cells may alter neurotransmitter deposition and thus the apparent kinetic characteristics of transport.

Two forms of antisense technology were used to explore the role of the neuronal growth-associated protein GAP-43 (neuromodulin, B-50, Fl, P-57) in the brain. The first method exploited the ability of dividing cell lines to take up and stably integrate recombinant retrovirus vectors. We transfected PC12 cells with a recombinant expression vector containing a human GAP-43 cDNA in reverse (antisense) orientation. Retrieval and characterization of two stable transfectants, AS1 and AS2, expressing high levels of antisense GAP-43 RNA revealed that the level of GAP-43 protein in these cell lines was reduced relative to controls. in the presence of extraceilular calcium, a depolarizing concentration of K+ failed to stimulate dopamine release from AS1 and AS2 cells although evoked dopamine release from control cells was normal. Similarly, the calcium ionophore A23189 was ineffective in stimulating dopamine release from the cells expressing GAP-43 antisense cRNA even though it evoked dopamine release from control cells. These data suggest that GAP-43 plays an essential role in calcium-dependent neurotransmitter release in PC12 cells. The antisense GAP-43-transfected PC12 cells displayed processes of normal length when treated with NGF on polylysine or laminin substrates. in contrast, primary rat hippocampal cell cultures plated in the presence of GAP-43 antisense oligonucleotides, in a series of experiments exploiting a second antisense technology, were deficient In process outgrowth. This deficiency was dose-dependent. To venture further into the realm of GAP-43 function in the brain, we have designed a procedure to depress levels of GAP-43 protein in cultured hippocampal slices.Copyright 1993, 1999 Academic Press

Drug‐induced efflux of substrates was characterized in C6 rat glioma cells stably expressing a recombinant human dopamine (DA) or serotonin (5‐HT) transporter (C6‐hDAT and C6‐hSERT, respectively). In the absence of Ca2+, these cells spontaneously and rapidly released preloaded [3H]DA or [3H]5‐HT, respectively, but maintained constant levels of [3H]N‐methy‐4‐phenylpyridinium (MPP+) for up to 90 minutes. In C6‐hSERT cells, transporter substrates such as methamphetamine, amphetamine, and dopamine induced relatively rapid release of [3H]MPP+, with t1/2values of approximately 15 minutes, while the tl/2value for serotonin was about 30 minutes. Similar results were obtained with C6‐hDAT cells. Uptake blockers that are not substrates at the transporters had considerably greater t1/2values, as compared to substrates, suggesting different mechanisms for altering transporter function. Dose‐response curves for each drug, conducted at each drug's tl/2, indicated considerable differences in potency (EC50) at stimulating [3H]MPP+release from C6‐hSERT cells [3β‐(4‐iodophenyl)tropane‐2β‐carboxylic acid methyl ester (RTI‐55) > imipramine > 1‐[2‐diphenylmethoxy]ethyl‐4‐(3‐phenylpropyl)‐piperazine (GBR‐12935) threo‐(±)‐methylphenidate > cocaine > mazindol > 2‐β‐carbomethoxy‐3β‐(4‐fluorophenyl)tropane (CFT) > (+)methamphetamine > amphetamine > DA > fenfluramine > norepinephrine (NE) > 5‐HT]. A different rank order of potency was observed for the effects of drugs on [3H]MPP+release from C6‐hDAT cells [imipramine > RTI‐55 > cocaine > mazindol > CFT > GBR‐12935 > threo‐(±)‐methylphenidate > amphetamine > (+)methamphetamine > fenfluramine > DA > NE > 5‐HT]. Based on efficacies for stimulating [3H]MPP+release from C6‐hDAT cells, drugs could be grouped into three categories, with substrates causing release of ∼75% of loaded [3H]MPP+, cocaine analogues causing ∼50% release, and other drugs causing an average release of ∼25% of loaded [3H]MPP+. The results, taken together with results from previous reports, suggest that the transfected cell type contributes to the characteristics of transporter‐mediated release, that drugs interact with different sites on the transporters in the uptake and release process, and that the mechanism of transporter‐mediated release may not be a simple reversal of substrate uptake. Synapse 30:97–106, 1998.© 1998 Wiley‐Liss, Inc.

Dopamine receptors are classified into D1and D2subtypes on the basis of their pharmacological and biochemical characteristics1,2. The D2dopamine receptor has been implicated in the pathophysiology and treatment of movement disorders3, schizophrenia4and drug addiction5. The D2dopamine receptor interacts with guanine nucleotide-binding proteins to induce second messenger systems6,7. Other members of the family of receptors that are coupled to G proteins share a significant similarity in primary amino-acid sequence and exhibit an archetypical topology predicted to consist of seven putative transmembrane domains8. We have taken advantage of the expected nucleotide sequence similarities among members of this gene family to isolate genes coding for new receptors. Using the hamster β2-adrenergic receptor gene as a hybridization probe we have isolated related genes including a cDNA encoding the rat D2dopamine receptor. This receptor has been characterized on the basis of three criteria: the deduced amino-acid sequence which reveals that it is a member of the family of G-protein-coupled receptors; the tissue distribution of the mRNA which parallels that of the D2dopamine receptor; and the pharmacological profile of mouse fibroblast cells transfected with the cDNA.

This overview describes issues that must be considered before attempting to express neural cDNAs in mammalian cells, including the choice of expression vector and cell type. Considerations for introducing recombinant vectors into cells are discussed along with a comparison of achieving stable or transient expression. Finally, the appropriate promoter is crucial and must be chosen to fit the design of the expression system.

The availability of clonal cell lines for norepinephrine, dopamine, and serotonin transporters allows the characterization of drug interactions with transporter recognition sites using radioligands, as well as the characterization of drug effects on selective transporter-mediated uptake and release of substrate. In addition to clonal cell lines, synaptosomes prepared from specific brain regions can be used to conduct these studies without interference by endogenous transporters or binding proteins that are present in other tissues. This unit presents protocols for uptake and release of tritiated substrates using intact cells (either detached or in suspension) or synaptosomes. An HPLC procedure for electrochemical detection of nonradiolabeled substrates is also provided. Time-dependent release can also be measured in assays involving real-time sampling.