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Administration of a novel and selective small molecule integrin αvβ6 inhibitor, MORF-627, to young cynomolgus monkeys for 28 days resulted in the rapid induction of epithelial proliferative changes in the urinary bladder of 2 animals, in the absence of test agent genotoxicity. Microscopic findings included suburothelial infiltration by irregular nests and/or trabeculae of epithelial cells, variable cytologic atypia, and high mitotic rate, without invasion into the tunica muscularis. Morphologic features and patterns of tumor growth were consistent with a diagnosis of early-stage invasive urothelial carcinoma. Ki67 immunohistochemistry demonstrated diffusely increased epithelial proliferation in the urinary bladder of several monkeys, including those with tumors, and αvβ6 was expressed in some epithelial tissues, including urinary bladder, in monkeys and humans. Spontaneous urothelial carcinomas are extremely unusual in young healthy monkeys, suggesting a direct link of the finding to the test agent. Inhibition of integrin αvβ6 is intended to locally and selectively block transforming growth factor beta (TGF-β) signaling, which is implicated in epithelial proliferative disorders. Subsequent in vitro studies using a panel of integrin αvβ6 inhibitors in human bladder epithelial cells replicated the increased urothelial proliferation observed in monkeys and was reversed through exogenous application of TGF-β. Moreover, analysis of in vivo models of liver and lung fibrosis revealed evidence of epithelial hyperplasia and cell cycle dysregulation in mice treated with integrin αvβ6 or TGF-β receptor I inhibitors. The cumulative evidence suggests a direct link between integrin αvβ6 inhibition and decreased TGF-β signaling in the local bladder environment, with implications for epithelial proliferation and carcinogenesis.

The D1 dopamine receptor is linked to a variety of neuropsychiatric disorders and represents an attractive drug target for the enhancement of cognition in schizophrenia, Alzheimer disease, and other disorders. Positive allosteric modulators (PAMs), with their potential for greater selectivity and larger therapeutic windows, may represent a viable drug development strategy, as orthosteric D1 receptor agonists possess known clinical liabilities. We discovered two structurally distinct D1 receptor PAMs, MLS6585 and MLS1082, via a high-throughput screen of the NIH Molecular Libraries program small-molecule library. Both compounds potentiate dopamine-stimulated G protein- and β-arrestin-mediated signaling and increase the affinity of dopamine for the D1 receptor with low micromolar potencies. Neither compound displayed any intrinsic agonist activity. Both compounds were also found to potentiate the efficacy of partial agonists. We tested maximally effective concentrations of each PAM in combination to determine if the compounds might act at separate or similar sites. In combination, MLS1082 + MLS6585 produced an additive potentiation of dopamine potency beyond that caused by either PAM alone for both β-arrestin recruitment and cAMP accumulation, suggesting diverse sites of action. In addition, MLS6585, but not MLS1082, had additive activity with the previously described D1 receptor PAM “Compound B,” suggesting that MLS1082 and Compound B may share a common binding site. A point mutation (R130Q) in the D1 receptor was found to abrogate MLS1082 activity without affecting that of MLS6585, suggesting this residue may be involved in the binding/activity of MLS1082 but not that of MLS6585. Together, MLS1082 and MLS6585 may serve as important tool compounds for the characterization of diverse allosteric sites on the D1 receptor as well as the development of optimized lead compounds for therapeutic use.

The D1 dopamine receptor is linked to a variety of neuropsychiatric disorders and represents an attractive drug target for the enhancement of cognition in schizophrenia, Alzheimer disease, and other disorders. Positive allosteric modulators (PAMs), with their potential for greater selectivity and larger therapeutic windows, may represent a viable drug development strategy, as orthosteric D1 receptor agonists possess known clinical liabilities. We discovered two structurally distinct D1 receptor PAMs, MLS6585 and MLS1082, via a high-throughput screen of the NIH Molecular Libraries program small-molecule library. Both compounds potentiate dopamine-stimulated G protein- and β-arrestin-mediated signaling and increase the affinity of dopamine for the D1 receptor with low micromolar potencies. Neither compound displayed any intrinsic agonist activity. Both compounds were also found to potentiate the efficacy of partial agonists. We tested maximally effective concentrations of each PAM in combination to determine if the compounds might act at separate or similar sites. In combination, MLS1082 + MLS6585 produced an additive potentiation of dopamine potency beyond that caused by either PAM alone for both β-arrestin recruitment and cAMP accumulation, suggesting diverse sites of action. In addition, MLS6585, but not MLS1082, had additive activity with the previously described D1 receptor PAM “Compound B,” suggesting that MLS1082 and Compound B may share a common binding site. A point mutation (R130Q) in the D1 receptor was found to abrogate MLS1082 activity without affecting that of MLS6585, suggesting this residue may be involved in the binding/activity of MLS1082 but not that of MLS6585. Together, MLS1082 and MLS6585 may serve as important tool compounds for the characterization of diverse allosteric sites on the D1 receptor as well as the development of optimized lead compounds for therapeutic use.

Bacteria artificial chromosome (BAC) transgenic mice expressing the reporter protein enhanced green fluorescent protein (EGFP) under the control of the D1 and D2 dopamine receptor promoters (Drd1-EGFPandDrd2-EGFP) have been widely used to study striatal function and have contributed to our understanding of the physiological and pathological functions of the basal ganglia. These tools were produced and promptly made available to address questions in a cell-specific manner that has transformed the way we frame hypotheses in neuroscience. However, these mice have not been fully characterized until now. We found thatDrd2-EGFPmice display an ∼40% increase in membrane expression of the dopamine D2 receptor (D2R) and a twofold increase in D2R mRNA levels in the striatum when compared with wild-type andDrd1-EGFPmice. D2R overexpression was accompanied by behavioral hypersensitivity to D2R-like agonists, as well as enhanced electrophysiological responses to D2R activation in midbrain dopaminergic neurons. Dopamine (DA) transients evoked by stimulation in the nucleus accumbens showed slower clearance in Drd2-EGFP mice, and cocaine actions on DA clearance were impaired in these mice. Thus, it was not surprising to find thatDrd2-EGFPmice were hyperactive when exposed to a novel environment and locomotion was suppressed by acute cocaine administration. All together, this study demonstrates thatDrd2-EGFPmice overexpress D2R and have altered dopaminergic signaling that fundamentally differentiates them from wild-type andDrd1-EGFPmice.

Spanish-translated Miranda warnings are administered annually to thousands of Hispanic custodial suspects. In examining 121 Spanish translations and their English counterparts from 33 states, the lengths of Miranda warnings were generally comparable but marked differences were observed in the reading levels for individual Miranda components. The adequacy of Miranda translations varies markedly from minor variations to substantive errors. The most serious problems involved the entire omission of Miranda components; several omissions were observed in the Spanish translations for even the basic rights to silence and counsel. More commonly, Miranda discrepancies involved dissimilar content with a substantial trend toward more information in English than Spanish versions. Findings related to the Miranda translations, different word lengths, and varied reading levels are discussed using the totality of circumstances as its framework.

It is well documented that dopamine can increase or decrease the activity of the Na+,K+-ATPase (NKA, sodium pump) in an organ-specific fashion. This regulation can occur, at least partially, via receptor-mediated second messenger activation and can promote NKA insertion or removal from the plasma membrane. Using co-immunoprecipitation and mass spectrometry, we now show that, in both brain and HEK293T cells, D1 and D2 dopamine receptors (DARs) can exist in a complex with the sodium pump. To determine the impact of NKA on DAR function, biological assays were conducted with NKA and DARs co-expressed in HEK293T cells. In this system, expression of NKA dramatically decreased D1 and D2 DAR densities with a concomitant functional decrease in DAR-mediated regulation of cAMP levels. Interestingly, pharmacological inhibition of endogenous or overexpressed NKA enhanced DAR function without altering receptor number or localization. Similarly, DAR function was also augmented by small interfering RNA reduction of the endogenous NKA. These data suggest that, under basal conditions, NKA negatively regulates DAR function via protein-protein interactions. In reciprocal fashion, expression of DARs decreases endogenous NKA function in the absence of dopamine, implicating DAR proteins as regulators of NKA activity. Notably, dopamine stimulation or pertussis toxin inhibition of D2 receptor signaling did not alter NKA activity, indicating that the D2-mediated decrease in NKA function is dependent upon protein-protein interactions rather than signaling molecules. This evidence for reciprocal regulation between DARs and NKA provides a novel control mechanism for both DAR signaling and cellular ion balance.

Annually, more than 1.5 million juvenile offenders are arrested and routinely Mirandized with little consideration regarding the comprehensibility of these warnings. The current investigation examined 122 juvenile Miranda warnings from across the United States regarding their length, reading level, and content. Even more variable than general Miranda warnings, juvenile warnings ranged remarkably from 52 to 526 words; inclusion of Miranda waivers and other material substantially increased these numbers (64 –1,020 words). Flesch-Kincaid reading estimates varied dramatically from Grade 2.2 to postcollege. Differences in content included such critical issues as (a) right to parent/guardian input, (b) specification of free legal services for indigent defendants, and (c) statements of right to counsel in conditional terms. Recommendations for simplified juvenile Miranda warnings are presented.

Miranda warnings are remarkably heterogeneous in their language, length, and content. Past research has focused mostly on individual Miranda warnings. Lacking in generalizability, these studies have limited applicability to both public policy and professional practice. A large-scale survey by R. Rogers et al. [2007b, Law and Human Behavior, 31, 177-192] examined Miranda warnings from across the United States and documented striking differences in the length, content, and reading comprehension. In moving from single jurisdiction studies to nationally representative research, the replication of the Rogers et al. survey is essential. With an additional 385 general Miranda warnings, most of the original findings were confirmed; this replication allows Miranda researchers to use findings based upon nationally-representative warnings for their subsequent research. Beyond reading comprehension, the study makes an original contribution to the understanding of Miranda vocabulary that is often infused with abstruse words and legalistic terms. It provides the first analysis of sentence complexity, which affects both Miranda comprehension and retention. As a result of these analyses, preliminary guidelines are provided for increasing the comprehension and understanding of Miranda warnings.

A conservative estimate is that 695,000 mentally disordered offenders are arrested and Mirandized annually in the United States. Past research has focused almost exclusively on cognitive factors affecting the comprehension of Miranda rights. The current study broadens the scope by including diagnostic variables and by extending the investigation to basic elements of Miranda reasoning. A sample of 107 mentally disordered defendants was administered two research measures, the Miranda Statements Scale (MSS) and Miranda Rights Scale (MRS), in addition to standardized tests. Most defendants lacked good comprehension of all but the simplest (Flesch-Kincaid

As for all proteins, G protein-coupled receptors (GPCRs) undergo synthesis and maturation within the endoplasmic reticulum (ER). The mechanisms involved in the biogenesis and trafficking of GPCRs from the ER to the cell surface are poorly understood, but they may involve interactions with other proteins. We have now identified the ER chaperone protein calnexin as an interacting protein for both D(1) and D(2) dopamine receptors. These protein-protein interactions were confirmed using Western blot analysis and co-immunoprecipitation experiments. To determine the influence of calnexin on receptor expression, we conducted assays in HEK293T cells using a variety of calnexin-modifying conditions. Inhibition of glycosylation either through receptor mutations or treatments with glycosylation inhibitors partially blocks the interactions with calnexin with a resulting decrease in cell surface receptor expression. Confocal fluorescence microscopy reveals the accumulation of D(1)-green fluorescent protein and D(2)-yellow fluorescent protein receptors within internal stores following treatment with calnexin inhibitors. Overexpression of calnexin also results in a marked decrease in both D(1) and D(2) receptor expression. This is likely because of an increase in ER retention because confocal microscopy revealed intracellular clustering of dopamine receptors that were co-localized with an ER marker protein. Additionally, we show that calnexin interacts with the receptors via two distinct mechanisms, glycan-dependent and glycan-independent, which may underlie the multiple effects (ER retention and surface trafficking) of calnexin on receptor expression. Our data suggest that optimal receptor-calnexin interactions critically regulate D(1) and D(2) receptor trafficking and expression at the cell surface, a mechanism likely to be of importance for many GPCRs.

Naturally occurring variation within the human 5-HT2A receptor results in an amino acid substitution in the carboxyl terminus of the receptor. This single nucleotide polymorphism (SNP), encoding a His452Tyr substitution, occurs at a frequency of 9% in the general population. It is noteworthy that this SNP has been linked to attention deficit hyperactivity disorder and has been associated with schizophrenic patients that do not respond to treatment with clozapine. To evaluate functional consequences of this SNP, agonist-stimulated signaling was investigated in NIH3T3 cells stably expressing either wild-type or 452Tyr variant receptors. The 452Tyr variant of the 5-HT2a receptor had reduced ability to activate phospholipases C and D, suggesting that signaling through both Gq and G13 pathways is hindered. This conclusion was supported by assays of G protein coupling, which documented a loss of agonist-induced high affinity binding and a decreased turnover of guanosine 5′-O-(3-[35S]thio)triphosphate after agonist stimulation. Kinetic analysis of time-course data revealed an altered desensitization phenotype, resulting in a blunted signal downstream of receptor activation. This diminished signaling implies that the His452Tyr variant receptor alters physiological responses, possibly contributing to psychiatric disease.

Dopamine is a critically important neurotransmitter in the CNS and peripheral nervous system, with five receptor subtypes mediating its effects. Importantly, D1 receptor (D1R)-selective agents may prove beneficial in the treatment of many neuropsychiatric disorders. In an effort to identify novel selective allosteric modulators of the D1R we used a high throughput screening approach. 380,000 small molecules in the NIH Molecular Libraries Screening Center Network library were screened using a cell line expressing the D1R coupled to G15 resulting in a robust Ca2+ signal upon receptor stimulation. Hit compounds were triaged through secondary functional and radioligand displacement binding assays to determine subtype selectivity and their allosteric versus orthosteric nature. We initially identified 96 putative positive allosteric modulator (PAM) hits that enhanced the EC20 activity of dopamine in the Ca2+ response - 6 of these were subsequently confirmed during triage and were chosen for further characteriza...

Dopamine receptors are rhodopsin-like seven-transmembrane receptors (also called G protein-coupled receptors) that mediate the central and peripheral actions of dopamine. Dopamine receptors are most abundant in pituitary and brain, particularly in the basal forebrain, but are also found in the retina and in peripheral organs such as the kidney. Stimulation of dopamine receptors modulates natriuresis in the kidney, as well as cell division and hormone synthesis and secretion in the pituitary. Brain dopamine receptors regulate movement and locomotion, motivation, and working memory. Five subtypes of mammalian dopamine receptors have been identified that are divided into D1-like (D1, D5) or D2-like (D2, D3, D4) subgroups. The D1-like receptors couple primarily to the Gs family of G proteins (Gs and Golf), whereas the D2-like receptors couple primarily to the Gi/o family. This chapter discusses the molecular pharmacology of the five dopamine receptor subtypes.

Historically, dopamine receptors (DARs) and other G protein-coupled receptors (GPCRs) were believed to be independent signaling units in the plasma membrane, interacting only transiently with G proteins to initiate a downstream signaling cascade. However, in recent years it has become clear that DARs do not function in isolation, but in fact exist as members of macromolecular protein complexes. The DAR protein complex, or the signalplex, consists of a variety of protein interactors that may be transient or stable in nature and that are collectively referred to as dopamine receptor-interacting proteins (DRIPs). Ultimately, the goal of the signalplex is to organize the cellular machinery or signaling components that are critical to the DAR at any given time in an orderly fashion. This higher level of organization around the DAR enables the receptor to process all information, from extracellular ligands or intracellular signaling molecules, and propagate the necessary cellular response in a timely and efficient manner.

Proteomics has evolved from genomic science due to the convergence of advances in protein chemistry, separations, mass spectroscopy, and peptide and protein databases. Where identifying protein-protein interactions was once limited to yeast two-hybrid analyses or empirical data, protein-protein interactions can now be examined in both cells and native tissues by precipitation of the protein complex of interest. Coupling this field to receptor pharmacology has recently allowed for the identification of proteins that differentially and selectively interact with receptors and are integral to their biological effects. It is becoming increasingly apparent that receptors in neurons do not exist as singular independent units, but rather are part of large macromolecular complexes of interacting proteins. It is a primary quest of neuroscience to piece together these interactions and to characterize the regulatory signalplexes of all proteins. This unit presents co-immunoprecipitation-coupled mass spectroscopy as one way of identifying signalplex partners.

Traditionally, high levels of suggestibility have been widely assumed to be linked with diminished Miranda abilities, especially in relationship to the voluntariness of waivers. The current investigation examined suggestibility on the Gudjonsson Suggestibility Scales in a multisite study of pretrial defendants. One important finding was the inapplicability of British norms to American jurisdictions. Moreover, suggestibility appeared unrelated to Miranda comprehension, reasoning, and detainees’ perceptions of police coercion. In testing rival hypotheses, defendants with high compliance had significantly lower Miranda comprehension and ability to reason about exercising Miranda rights than their counterparts with low compliance. Implications of these findings to forensic practice are examined.

Unipolar major depressive disorder (MDD) is a prevalent, disabling condition with multiple genetic and environmental factors impacting disease risk. The diagnosis of MDD relies on a cumulative measure derived from multiple trait dimensions and alone is limited in elucidating MDD genetic determinants. We and others have proposed that MDD may be better dissected using paradigms that assess how specific genes associate with component features of MDD. This within-disease design requires both a well-phenotyped cohort and a robust statistical approach that retains power with multiple tests of genetic association. In the present study, common polymorphic variants of genes related to central monoaminergic and cholinergic pathways that previous studies align with functional change in vitro or depression associations in vivo were genotyped in 110 individuals with unipolar MDD. Subphenotypic characteristics were examined using responses to individual items assessed with the Structured Clinical Interview for Diagnostic and Statistical Manual of Mental Disorders (DSM IV), the 17-item Hamilton Rating Scale for Depression (HAM-D) and the NEO Five Factor Inventory. Multivariate Permutation Testing (MPT) was used to infer genotype—phenotype relationships underlying dimensional findings within clinical categories. MPT analyses show significant associations of the norepinephrine transporter (NET, SLC6A2) -182 T/C (rs2242446) with recurrent depression [odds ratio, OR 5 4.15 (1.91–9.02)], NET -3081 A/T (rs28386840) with increase in appetite [OR 5 3.58 (1.53–8.39)] and the presynaptic choline transporter (CHT, SLC5A7) Ile89Val (rs1013940) with HAM-D-17 total score {i.e. overall depression severity [OR 5 2.74 (1.05–7.18)]}. These relationships illustrate an approach to the elucidation of gene influences on trait components of MDD and with replication, may help identify MDD sub-populations that can benefit from more targeted pharmacotherapy.

In synaptic signaling, the response of an individual neuron is determined by its complement of receptors. As discussed in other chapters in this volume, ligand-gated ion channels mediate fast synaptic transmission between neurons. However, signaling within an individual neuron is mediated by a complex set of interactions that occur primarily as a result of G protein linked signal transduction. The beauty of this system is in its diversity and inherent capacity for amplification and plasticity in the modulation of many different cellular functions. The initial signal sequence in this type of signaling typically is the generation of a second messenger inside the cell that then in turn activates a number of different proteins capable of modifying cellular processes. This chapter will present examples and an in depth discussion of these intracellular signaling pathways beginning with G protein linked signaling, perpetuated by G protein coupled receptor (GPCR) stimulation. Next, ion channel modulation via G proteins as well as direct GPCR interactions with the channels will be discussed. The long list of intracellular downstream effector pathways including cyclic nucleotide signaling, protein kinase signaling and calcium regulated signaling are also described. The final focus of the chapter is protein phosphorylation cascades, a predominant mechanism in many signal transduction schemes. It is through these complex interactions that an individual cell is able to respond to neurotransmitter stimulation and modify many of its cellular processes to contribute to the overall symphony of synaptic signaling. Keywords: G proteins; RGS proteins; Gβγ; GIRK channels; adenylyl cyclase; guanylyl cyclase; cAMP; cGMP; phospholipase C; protein kinase C; phosphorylation; protein kinase; receptor kinase; phosphatase

Miranda warnings enshrine the constitutional rights of custodial suspects against self-incrimination. However, the wording and sentence complexity of Miranda warnings and waivers vary dramatically from jurisdiction to jurisdiction. This study is the first extensive investigation of Miranda warning variations examining 560 Miranda warnings from across the United States. With Flesch-Kincaid reading comprehension as a useful metric, Miranda warnings varied from very simple comprehension (i.e., grade 2.8) to requiring postgraduate education. Miranda warnings are composed of five components (e.g., silence and evidence against you); marked variations were also observed in the comprehensibility of individual components. On average, the Miranda warning component on "continuing rights" requires a reading comprehension level six grades higher than the comparatively simple expression of the right to silence. Similar analyses were conducted on Miranda waivers. The content of these warnings differed on such issues as communicating (a) when access to an attorney would be granted (e.g., 45.9% specified only "during questioning") and (b) explicitly that indigent legal services were free (e.g., 31.8% directly informed suspects). Finally, the study identified representative Miranda components at different levels of reading comprehension as a template for further research.

Naturally occurring variation within the human 5-HT(2A) receptor results in an amino acid substitution in the carboxyl terminus of the receptor. This single nucleotide polymorphism (SNP), encoding a His452Tyr substitution, occurs at a frequency of 9% in the general population. It is noteworthy that this SNP has been linked to attention deficit hyperactivity disorder and has been associated with schizophrenic patients that do not respond to treatment with clozapine. To evaluate functional consequences of this SNP, agonist-stimulated signaling was investigated in NIH3T3 cells stably expressing either wild-type or 452Tyr variant receptors. The 452Tyr variant of the 5-HT(2a) receptor had reduced ability to activate phospholipases C and D, suggesting that signaling through both G(q) and G(13) pathways is hindered. This conclusion was supported by assays of G protein coupling, which documented a loss of agonist-induced high affinity binding and a decreased turnover of guanosine 5'-O-(3-[(35)S]thio)triphosphate after agonist stimulation. Kinetic analysis of time-course data revealed an altered desensitization phenotype, resulting in a blunted signal downstream of receptor activation. This diminished signaling implies that the His452Tyr variant receptor alters physiological responses, possibly contributing to psychiatric disease.

Investigations of functional interactions among axons and glia over the last decade have revealed the extent and complexity of glial-neuronal and glial-glial communication during development, adult function and recovery from injury. These data have profound implications for the understanding of central nervous system (CNS) disorders, which until recently, have been classified as either neuronal or glial diseases. Re-evaluation of the pathological processes in a number of conditions has clearly shown involvement of both neurons and glia in early pathology. In multiple sclerosis (MS), the myelin sheath has traditionally been regarded as the primary target. However, recent evidence has clearly demonstrated axonal damage in new lesions. We have addressed the question of the role of axonal pathology in early MS by using well-characterized murine models for the relapsing-remitting (RR) or the primary progressive (PP) forms of the disease. We performed a histopathological survey of the CNS, following induction of the disease, to determine the timing of appearance, as well as the development of lesions. Then we analysed the relationship between inflammation, demyelination and axonal damage together with responses from astrocytes and microglia in each model from the earliest evidence of inflammation. We found that axonal damage begins well ahead of the appearance of motor symptoms. Pathology appears to be more closely related to the degree of inflammation than to demyelination. We also show that early astrocyte responses and the degree of axonal loss are markedly different in the two models and relate to the severity of pathology. These data support the now widely accepted hypothesis that axonal damage begins early in the disease process, but also suggest modulation of axonal loss and disease progression by the astrocytic response.

IntroductionLipid rafts are specialized membrane microdomains enriched in cholesterol and sphingolipids and are important in the organization of receptor-protein complexes and the regulation of signaling.Objective/aimsGiven the emerging significance of lipids with respect to receptor structure and activation, we investigated the role of lipid rafts and membrane cholesterol on D2 dopamine receptor (DAR) signaling. As the D2 DAR is the molecular target for all antipsychotic drugs, more information about its signaling may help refine therapeutics for schizophrenia.MethodsD2 DAR constructs were expressed in HEK293T cells. Sucrose density fractionation resolved lipid rafts from other membrane components. Methyl-β-cyclodextrin (MCD) was used to deplete membrane cholesterol and to disrupt lipid rafts.ResultsDetergent solubilization followed by sucrose gradient centrifugation resolved lipid rafts from heavier membrane fractions. The D2 DAR was equally distributed amongst both the lipid raft and heavier membrane fractions. Pretreatment with MCD, however, eliminated both lipid raft markers and the D2 DAR from lipid raft fractions, although the receptor was still found in heavier membrane fractions. We also found that MCD treatment abolished D2 DAR-mediated inhibition of cAMP accumulation. In contrast D1 DAR-stimulated cAMP accumulation was unaffected by MCD treatment.ConclusionsOur current results show that the D2 DAR is distributed in multiple membrane microdomains, including cholesterol-rich lipid rafts. We found that extraction of cholesterol disrupted lipid rafts and also an eliminated D2 DAR-mediated signaling. Thus, we hypothesize that lipid rafts are critical for D2 DAR signaling to occur.