Kim A. Neve

Dopamine Receptor Signaling

The D1-like (D1, D5) and D2-like (D2, D3, D4) classes of dopamine receptors each has shared signaling properties that contribute to the definition of the receptor class, although some differences among subtypes within a class have been identified. D1-like receptor signaling is mediated chiefly by the heterotrimeric G proteins Gαs and Gαolf, which cause sequential activation of adenylate cyclase, cylic AMP-dependent protein kinase, and the protein phosphatase-1 inhibitor DARPP-32. The increased phosphorylation that results from the combined effects of activating cyclic AMP-dependent protein kinase and inhibiting protein phosphatase 1 regulates the activity of many receptors, enzymes, ion channels, and transcription factors. D1 or a novel D1-like receptor also signals via phospholipase C-dependent and cyclic AMP-independent mobilization of intracellular calcium. D2-like receptor signaling is mediated by the heterotrimeric G proteins Gαi and Gαo. These pertussis toxin-sensitive G proteins regulate some effectors, such as adenylate cyclase, via their Gα subunits, but regulate many more effectors such as ion channels, phospholipases, protein kinases, and receptor tyrosine kinases as a result of the receptor-induced liberation of Gβγ subunits. In addition to interactions between dopamine receptors and G proteins, other protein:protein interactions such as receptor oligomerization or receptor interactions with scaffolding and signal-switching proteins are critical for regulation of dopamine receptor signaling.

Modulation of D2R-NR2B Interactions in Response to Cocaine

Dopamine-glutamate interactions in the neostriatum determine psychostimulant action, but the underlying molecular mechanisms remain elusive. Here we found that dopamine stimulation by cocaine enhances a heteroreceptor complex formation between dopamine D2 receptors (D2R) and NMDA receptor NR2B subunits in the neostriatum in vivo. The D2R-NR2B interaction is direct and occurs in the confined postsynaptic density microdomain of excitatory synapses. The enhanced D2R-NR2B interaction disrupts the association of Ca(2+)/calmodulin-dependent protein kinase II (CaMKII) with NR2B, reduces NR2B phosphorylation at a CaMKII-sensitive site (Ser1303), and inhibits NMDA receptor-mediated currents in medium-sized striatal neurons. Furthermore, the regulated D2R-NR2B interaction is critical for constructing behavioral responsiveness to cocaine. Our findings here uncover a direct and dynamic D2R-NR2B interaction in striatal neurons in vivo. This type of dopamine-glutamate integration at the receptor level may be responsible for synergistically inhibiting the D2R-mediated circuits in the basal ganglia and fulfilling the stimulative effect of psychostimulants.

Plasticity of neostriatal dopamine receptors after nigrostriatal injury: Relationship to recovery of sensorimotor functions and behavioral supersensitivity

Rats given unilateral injections of 6-OHDA along the course of the mesotelencephalic dopaminergic projection show impairments in contralateral sensorimotor functions from which they often recover. Such rats also display an enhanced sensitivity to DA receptor stimulants, e.g. apomorphine, as revealed by contralateral turning, and an increased binding of neuroleptic compounds (e.g. [3H]spiroperidol) to the denervated striatum. This research examines the relationship of these receptor changes to both behavioral supersensitivity and recovery of sensorimotor functions by quantifying the time course of each phenomenon after injury. The supersensitivity to apomorphine and the behavioral recovery developed with a similar time course after injury, being evident within 1.5-3 days and reaching nearly maximal levels by 2 weeks postoperatively. A significant increase in in vivo [3H]spiroperidol binding to the denervated striatum occurred by 4 days postoperatively, and the magnitude of this change increased linearly during the first postoperative month. In contrast, the in vitro binding of this ligand to membranes of the denervated striatum was not increased until 3 weeks after the lesion. The results suggest that a proliferation of DA receptors may contribute to the pharmacological supersensitivity and the recovery of function, and that these early receptor changes may be revealed with greater sensitivity using in vivo binding techniques.

DNA Synthesis and Neuronal Apoptosis Caused by Familial Alzheimer Disease Mutants of the Amyloid Precursor Protein Are Mediated by the p21 Activated Kinase PAK3

Apoptotic pathways and DNA synthesis are activated in neurons in the brains of individuals with Alzheimer disease (AD). However, the signaling mechanisms that mediate these events have not been defined. We show that expression of familial AD (FAD) mutants of the amyloid precursor protein (APP) in primary neurons in culture causes apoptosis and DNA synthesis. Both the apoptosis and the DNA synthesis are mediated by the p21 activated kinase PAK3, a serine-threonine kinase that interacts with APP. A dominant-negative kinase mutant of PAK3 inhibits the neuronal apoptosis and DNA synthesis; this effect is abolished by deletion of the PAK3 APP-binding domain or by coexpression of a peptide representing this binding domain. The involvement of PAK3 specifically in FAD APP-mediated apoptosis rather than in general apoptotic pathways is suggested by the facts that a dominant-positive mutant of PAK3 does not alone cause neuronal apoptosis and that the dominant-negative mutant of PAK3 does not inhibit chemically induced apoptosis. Pertussis toxin, which inactivates the heterotrimeric G-proteins Go and Gi, inhibits the apoptosis and DNA synthesis caused by FAD APP mutants; the apoptosis and DNA synthesis are rescued by coexpression of a pertussis toxin-insensitive Go. FAD APP-mediated DNA synthesis precedes FAD APP-mediated apoptosis in neurons, and inhibition of neuronal entry into the cell cycle inhibits the apoptosis. These data suggest that a normal signaling pathway mediated by the interaction of APP, PAK3, and Go is constitutively activated in neurons by FAD mutations in APP and that this activation causes cell cycle entry and consequent apoptosis.

Contributions of Conserved Serine Residues to the Interactions of Ligands with Dopamine D2 Receptors

Abstract: Four dopamine D2 receptor mutants were constructed, in each of which an alanine residue was substituted for one of four conserved serine residues, i.e., Ser‐193, Ser‐194, Ser‐197, and Ser‐391. Wild‐type and mutant receptors were expressed transiently in COS‐7 cells and stably in C6 glioma cells for analysis of ligand‐receptor interactions. In radioligand binding assays, the affinity of D2 receptors for dopamine was decreased 50‐fold by substitution of alanine for Ser‐193, implicating this residue in the binding of dopamine. Each mutant had smaller decreases in affinity for one or more of the ligands tested, with no apparent relationship between the class of ligand and the pattern of mutation‐induced changes in affinity, except that the potency of agonists was decreased by substitution for Ser‐193. The potency of dopamine for inhibition of adenylyl cyclase was reduced substantially by substitution of alanine for Ser‐193 or Ser‐197. Mutation of Ser‐194 led to a complete loss of efficacy for dopamine and p‐tyramine, which would be consistent with an interaction between Ser‐194 and the p‐hydroxyl substituent of dopamine that is necessary for activation of the receptors to occur. Because mutation of the corresponding residues of β2‐adrenergic receptors has very different consequences, we conclude that although the position of these serine residues is highly conserved among catecholamine receptors, and the residues as a group are important in ligand binding and activation of receptors by agonists, the function of each of the residues considered separately varies among catecholamine receptors.

D2 Dopamine Receptors Stimulate Mitogenesis Through Pertussis Toxin-Sensitive G Proteins and Ras-Involved ERK and SAP/JNK Pathways in Rat C6-D2L Glioma Cells

Abstract: Dopamine D2 receptors are members of the G protein‐coupled receptor superfamily and are expressed on both neurons and astrocytes. Using rat C6 glioma cells stably expressing the rat D2L receptor, we show here that dopamine (DA) can activate both the extracellular signal‐regulated kinase (ERK) and c‐Jun NH2‐terminal kinase (JNK) pathways through a mechanism involving D2 receptor‐G protein complexes and the Ras GTP‐binding protein. Agonist binding to D2 receptors rapidly activated both kinases within 5 min, reached a maximum between 10 and 15 min, and then gradually decreased by 60 min. Maximal activation of both kinases occurred with 100 nM DA, which produced a ninefold enhancement of ERK activity and a threefold enhancement of JNK activity. DA‐induced kinase activation was prevented by either (+)‐butaclamol, a selective D2 receptor antagonist, or pertussis toxin, an uncoupler of G proteins from receptors, but not by (−)‐butaclamol, the inactive isomer of (+)‐butaclamol. Cotransfection of RasN17, a dominant negative Ras mutant, prevented DA‐induced activation of both ERK and JNK. PD098059, a specific MEK1 inhibitor, also blocked ERK activation by DA. Transfection of SEK1(K → R) vector, a dominant negative SEK1 mutant, specifically prevented DA‐induced JNK activation and subsequent c‐Jun phosphorylation without effect on ERK activation. Furthermore, stimulation of D2 receptors promoted [3H]thymidine incorporation with a pattern similar to that for kinase activation. DA mitogenesis was tightly linked to Ras‐dependent mitogen‐activated protein kinase (MAPK) and JNK pathways. Transfection with RasN17 and application of PD098059 blocked DA‐induced DNA synthesis. Transfection with FlagΔ169, a dominant negative c‐Jun mutant, also prevented stimulation of [3H]thymidine incorporation by DA. The demonstration of D2 receptor‐stimulated MAPK pathways may help to understand dopaminergic physiological functions in the CNS.

Increased abundance of alternatively spliced forms of D2 dopamine receptor mRNA after denervation.

The existence of two molecular forms of D2 dopamine receptors suggests that differences in the distribution or regulation of the two forms could be exploited for the pharmacological treatment of disease. Using probes selective for each alternatively spliced variant of D2 receptor mRNA, we determined that both variants were widely distributed in rat brain and pituitary but that the ratio of the forms varied among regions. mRNA for the 444-amino acid-long variant, D2(444), was the most abundant form in pituitary and neostriatum. Intermediate levels of both D2(444) mRNA and the short form, D2(415), were detected in midbrain, and low levels of D2(444) and D2(415) mRNAs were detected in all other regions examined, including hippocampus, cerebellum, and cortex. The D2(444)/D2(415) ratio was generally lower in the regions of low expression than in pituitary and neostriatum. Dopamine-depleting lesions increased the density of D2 receptors in the denervated neostriatum by 29% without altering the affinity of the receptors for [3H]spiperone. The proliferation of receptors appeared to be due to a lesion-induced increase of up to 120% in the abundance of both variants of mRNA in the neostriatum.

Dopamine and extinction: A convergence of theory with fear and reward circuitry

Research on dopamine lies at the intersection of sophisticated theoretical and neurobiological approaches to learning and memory. Dopamine has been shown to be critical for many processes that drive learning and memory, including motivation, prediction error, incentive salience, memory consolidation, and response output. Theories of dopamines function in these processes have, for the most part, been developed from behavioral approaches that examine learning mechanisms in reward-related tasks. A parallel and growing literature indicates that dopamine is involved in fear conditioning and extinction. These studies are consistent with long-standing ideas about appetitive-aversive interactions in learning theory and they speak to the general nature of cellular and molecular processes that underlie behavior. We review the behavioral and neurobiological literature showing a role for dopamine in fear conditioning and extinction. At a cellular level, we review dopamine signaling and receptor pharmacology, cellular and molecular events that follow dopamine receptor activation, and brain systems in which dopamine functions. At a behavioral level, we describe theories of learning and dopamine function that could describe the fundamental rules underlying how dopamine modulates different aspects of learning and memory processes.

Quantitative analysis of [3H]spiroperidol binding to rat forebrain sections: Plasticity of neostriatal dopamine receptors after nigrostriatal injury

The binding of [3H]spiroperidol to rat coronal sections in vitro was investigated using two procedures: swabbing studies, in which the tissue sections are wiped from the microscope slides after incubation in the presence of [3H]spiroperidol, and autoradiographic studies, in which the autoradiographic negatives are analyzed using computer-assisted densitometry. In the swabbing studies, the pharmacological and kinetic properties of butaclamol-displaceable binding were investigated, and the following results suggest that [3H]spiroperidol binds specifically to only a single site within the basal forebrain of tissue sections and that the site is the dopamine D-2 receptor. The pseudo-first order and first order plots for the rate of association to and dissociation from tissue sections appeared to be linear. Dopamine antagonists, such as haloperidol and butaclamol, were much more effective than dopamine agonists or the serotonin S-2 ligand, ketanserin, in inhibiting [3H]spiroperidol binding. The ability of dopamine agonists to inhibit [3H]spiroperidol binding was markedly reduced by the guanine nucleotide, Gpp(NH)p. Saturation analysis of specific [3H]spiroperidol binding revealed a Kd and Bmax of 0.93 nM and 447 fmol/mg protein, and a Hill coefficient of 1.05. The findings are also compatible with the possibility that [3H]spiroperidol binds to several sites that have identical affinities for this ligand. Densitometric studies were used to assess the effect of lesions on [3H]spiroperidol binding in the neostriatum. Intrastriatal injection of kainic acid substantially reduced 1 microM (+)--butaclamol-displaceable binding, indicating that the receptors are in large part on intrinsic striatal neurons. Neostriatal [3H]spiroperidol binding was investigated 7 days after destruction of the mesotelencephalic dopamine system by the ventral tegmental injection of 6-hydroxydopamine. As determined by saturation analysis, the average values for Kd and Bmax were 0.66 nM and 1212 fmol/mg protein in the intact striatum, and 0.82 nM and 1504 fmol/mg in the denervated striatum. The finding of a significant 23.8% increase in receptor density by the end of the first postoperative week, a period during which behavioral supersensitivity to apomorphine increases rapidly, supports the hypothesis that a proliferation of D-2 receptors underlies the behavioral manifestations of denervation supersensitivity.

Molecular Biology of Dopamine Receptors

The molecular biological characterization of the dopamine receptors began with the cloning of the gene and cDNA for the hamster beta-2 adrenergic receptor in 1986 (1), followed closely by the isolation of cDNAs encoding the turkey erythrocyte beta-adrenergic receptor (2) and the porcine ml muscarinic receptor (3). The realization that G protein-coupled receptors form a family of proteins related by primary structure and predicted secondary structural features, a family big enough to include the light-activated receptor rhodopsin, opened up the possibility of exploiting this homology for the molecular cloning of additional members of the gene family. Indeed, all the dopamine receptors have been cloned because of their homology to other dopamine or monoamine receptors.