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Both insect and mammalian life cycle stages of Leishmania mexicana take up glucose and express all three isoforms encoded by the LmGT glucose transporter gene family. To evaluate glucose transporter function in intact parasites, a null mutant line has been created by targeted disruption of the LmGT locus that encompasses the LmGT1, LmGT2, and LmGT3 genes. This [DELTA]lmgt null mutant exhibited no detectable glucose transport activity. The growth rate of the [DELTA]lmgt knockout in the promastigote stage was reduced to a rate comparable with that of WT cells grown in the absence of glucose. [DELTA]lmgt cells also exhibited dramatically reduced infectivity to macrophages, demonstrating that expression of LmGT isoforms is essential for viability of amastigotes. Furthermore, WT L. mexicana were not able to grow as axenic culture form amastigotes if glucose was withdrawn from the medium, implying that glucose is an essential nutrient in this life cycle stage. Expression of either LmGT2 or LmGT3, but not of LmGT1, in [DELTA]lmgt null mutants significantly restored growth as promastigotes, but only LmGT3 expression substantially rescued amastigote growth in macrophages. Subcellular localization of the three isoforms was investigated in [DELTA]lmgt cells expressing individual LmGT isoforms. Using anti-LmGT antiserum and GFP-tagged LmGT fusion proteins, LmGT2 and LmGT3 were localized to the cell body, whereas LmGT1 was localized specifically to the flagellum. These results establish that each glucose transporter isoform has distinct biological functions in the parasite.

Background: We describe a 4‐generation Dutch pedigree with a unique dominantly inherited clinical phenotype of a combined progressive chorea and cervical dystonia carrying a novel heterozygous dopamine D2 receptor (DRD2) variant. Objectives: The objective of this study was to identify the genetic cause of the disease and to further investigate the functional consequences of the genetic variant. Methods: After detailed clinical and neurological examination, whole‐exome sequencing was performed. Because a novel variant in the DRD2 gene was found as the likely causative gene defect in our pedigree, we sequenced the DRD2 gene in a cohort of 121 Huntington‐like cases with unknown genetic cause (Germany). Moreover, functional characterization of the DRD2 variant included arrestin recruitment, G protein activation, and G protein‐mediated inhibition of adenylyl cyclase determined in a cell model, and G protein‐regulated inward‐rectifying potassium channels measured in midbrain slices of mice. Result: We identified a novel heterozygous variant c.634A > T, p.Ile212Phe in exon 5 of DRD2 that cosegregated with the clinical phenotype. Screening of the German cohort did not reveal additional putative disease‐causing variants. We demonstrated that the D2S/L‐I212F receptor exhibited increased agonist potency and constitutive activation of G proteins in human embryonic kidney 239 cells as well as significantly reduced arrestin3 recruitment. We further showed that the D2S‐I212F receptor exhibited aberrant receptor function in mouse midbrain slices. Conclusions: Our results support an association between the novel p.Ile212Phe variant in DRD2, its modified D2 receptor activity, and the hyperkinetic movement disorder reported in the 4‐generation pedigree. © 2020 The Authors. Movement Disorders published by Wiley Periodicals LLC on behalf of International Parkinson and Movement Disorder Society. [ABSTRACT FROM AUTHOR]

In Leishmania mexicana parasites, a unique glucose transporter, LmxGT1, is selectively targeted to the flagellar membrane, suggesting a possible sensory role that is often associated with ciliary membrane proteins. Expression of LmxGT1 is down-regulated ~20-fold by increasing cell density but is up-regulated ~50-fold by depleting glucose from the medium, and the permease is strongly down-regulated when flagellated insect-stage promastigotes invade mammalian macrophages and transform into intracellular amastigotes. Regulation of LmxGT1 expression by glucose and during the lifecycle operates at the level of protein stability. Significantly, a Δlmxgt1 null mutant, grown in abundant glucose, undergoes catastrophic loss of viability when parasites deplete glucose from the medium, a property not exhibited by wild-type or add-back lines. These results suggest that LmxGT1 may function as a glucose sensor that allows parasites to enter the stationary phase when they deplete glucose and that in the absence of this sensor, parasites do not maintain viability when they run out of glucose. However, alternate roles for LmxGT1 in monitoring glucose availability are considered. The absence of known sensory receptors with defined ligands and biologic functions in Leishmania and related kinetoplastid parasites underscores the potential significance of these observations. [ABSTRACT FROM AUTHOR]

Many of the cilia- and flagella-specific integral membrane proteins identified to date function to sense the extracellular milieu, and there is considerable interest in defining pathways for targeting such proteins to these sensory organelles. The flagellar glucose transporter of Leishmania mexicana, LmxGT1, is targeted selectively to the flagellar membrane, whereas two other isoforms, LmxGT2 and LmxGT3, are targeted to the pellicular plasma membrane of the cell body. To define the flagellar targeting signal, deletions and point mutations were generated in the N-terminal hydrophilic domain of LmxGT1, which mediates flagellar localization. Three amino acids, N95-P96- M97, serve critical roles in flagellar targeting, resulting in strong mistargeting phenotypes when mutagenized. However, to facilitate flagellar targeting of other non-flagellar membrane proteins, it was necessary to attach a larger region surrounding the NPM motif containing amino acids 81-113. Molecular modeling suggests that this region might present the critical NPM residues at the surface of the N-terminal domain. It is likely that the NPM motif is recognized by currently unknown protein-binding partners that mediate flagellar targeting of membrane-associated proteins. [ABSTRACT FROM AUTHOR]

Glucose is considered essential for erythrocytic stages of the malaria parasite, Plasmodium falciparum. Importance of sugar and its permease for hepatic and sexual stages of Plasmodium, however, remains elusive. Moreover, increasing global resistance to current antimalarials necessitates the search for novel drugs. Here, we reveal that hexose transporter 1 (HT1) of Plasmodium berghei can transport glucose (Km~87 μM), mannose (Ki~93 μM), fructose (Ki~0.54 mM), and galactose (Ki~5 mM) in Leishmania mexicana mutant and Xenopus laevis; and, therefore, is functionally equivalent to HT1 of P. falciparum (Glc, Km~175 μM; Man, Ki~ μ276M; Fru, Ki~1.25 mM; Gal, Ki5.86mM). Notably, a glucose analog, C3361, attenuated hepatic (IC50~15 μM) and ookinete development of P. berghei. The PbHT1 could be ablated during intraerythrocytic stages only by concurrent complementation with PbHT1-HA or PfHT1. Together; these results signify that PbHT1 and glucose are required for the entire life cycle of P. berghei. Accordingly, PbHT1 is expressed in the plasma membrane during all parasite stages. To permit a high-throughput screening of PfHT1 inhibitors and their subsequent in vivo assessment, we have generated Saccharomyces cerevisiae mutant expressing codon-optimized PfHT1, and a PfHT1-dependent Δpbht1 parasite strain. This work provides a platform to facilitate the development of drugs against malaria, and it suggests a disease-control aspect by reducing parasite transmission. [ABSTRACT FROM AUTHOR]

A glucose transporter null mutant of the parasitic protozoan Leishmania mexicana, in which three linked glucose transporter genes have been deleted by targeted gene replacement, is unable to replicate as amastigote forms within phagolysomes of mammalian host macrophages and is avirulent. Spontaneous suppressors of the null mutant have been isolated that partially restore replication of parasites within macrophages. These suppressor mutants have amplified the gene for an alternative hexose transporter, the LmGT4 permease (previously called the D2 permease), on a circular extrachromosomal element, and they overexpress LmGT4 mRNA and protein. The suppressors have also regained the ability to transport hexoses, and they have reverted other phenotypes of the null mutant exhibiting enhanced resistance to oxidative killing, heat shock and starvation for nutrients, as well as augmented levels of the storage carbohydrate β-mannan, increased cell size and increased growth as insect stage promastigotes compared with the unsuppressed mutant. Complementation of the null mutant with the LmGT4 gene on a multicopy episomal expression vector also reverted these phenotypes, confirming that suppression results from amplification of the LmGT4 gene. These results underscore the importance of hexose transporters for the infectious stage of the parasite life cycle. [ABSTRACT FROM AUTHOR]

R4511 --> A novel DRD2 variant with a mutation in exon 5 of DRD2(c.634A>T; p.I212F)has been identified in a Dutch family. Ile212 is at the N‐terminus of the D2 receptor (D2R) 3rd cytoplasmic loop. D2‐I212F is associated with a novel hyperkinetic movement disorder characterized by progressive chorea and cervical dystonia. We hypothesized that determining the effect of the mutation on D2R function might explain how D2‐I212F causes the movement disorder. Molecular dynamics (MD) simulations of human D2R models suggested that the presence of Phe212 in D2R separates the side chains of Arg132, at the cytoplasmic end of the 3rd transmembrane domain (TM3), and Glu368, at the cytoplasmic end of TM6, spontaneously breaking the ionic lock in the inactive D2‐I212F. This ionic lock contributes to maintaining unliganded receptors in an inactive conformation, and it is broken in agonist‐activated receptors. Conversely, preventing formation of the lock frequently creates a constitutively active receptor. In vitro and ex vivo studies supported the hypothesis that D2‐I212F is a constitutively active receptor: We expressed D2L/S‐I212F in HEK293 cells and assayed activation of Gαi1 and GαoA by bioluminescence resonance energy transfer. We observed increased quinpirole potency to activate Gαi1 by D2L/S‐I212F (2.8 or 3.5 nM for D2L or D2S, respectively) compared to D2L/S‐WT (19 or 27 nM) whereas quinpirole potency for activating GαoA was not substantially changed by the I212F mutation. On the other hand, D2L/S‐I212F exhibited increased basal activation of both Gαi/o proteins compared to D2L/S‐WT (set as 0%); basal GαoA activation by D2L/S‐I212F (43 or 57% of maximal stimulation, respectively) was significantly higher than D2L/S‐I212F‐mediated basal Gαi1 activation (25% and 35%, respectively). We then used AAV to express Cre recombinase‐regulated D2S‐WT or D2S‐I212F in dopamine neurons of D2 autoreceptor knockout mice and characterized D2R activation of G protein‐regulated inward‐rectifying potassium channels (GIRKs). When CyHQ‐sulpiride (5 μM) was circulated over a midbrain slice, photolytic release of sulpiride produced a small inhibition of a tonic GIRK current in cells expressing D2‐WT (‐9 pA), and a much larger inhibition in cells expressing D2‐I212F (‐62 pA). The enhanced inhibition of tonic current by the mutant receptor could reflect constitutive activation of G proteins or a heightened sensitivity of the mutant to dopamine. To distinguish between these possibilities, midbrain slices were treated with reserpine to deplete endogenous dopamine. This treatment abolished the response to sulpiride in cells expressing D2‐WT and greatly decreased the response in cells expressing D2‐I212F, indicating that most of the tonic current was due to endogenous dopamine to which D2‐I212F may be more sensitive, but that D2‐I212F also displayed some constitutive activity in the presumed absence of dopamine. Thus, our in silico, in vitro, and ex vivo studies all indicate that the mutation enhances constitutive and agonist‐induced activation of G proteins by D2R [ABSTRACT FROM AUTHOR]

A dopamine D2 receptor mutation was recently identified in a family with a novel hyperkinetic movement disorder. That allelic variant D2-I 212 F 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-I 212 F variant. Drd2 I212F mice 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 Drd2 I212F allele. Electrically evoked inhibitory postsynaptic conductances in midbrain dopamine neurons and striatum from Drd2 I212F mice, 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 Drd2 I212F mice 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 G α o versus G α i , dopamine had similar potencies in those two brain regions of Drd2 I212F mice. Repeated cocaine treatment, which decreases dopamine potency in the nucleus accumbens of Drd2 +/+ mice, had no effect on dopamine potency in Drd2 I212F mice. The results demonstrate the pathogenicity of the D2-I 212 F mutation and the utility of this mouse model for investigating the role of pathogenic DRD2 variants in early-onset hyperkinetic movement disorders. SIGNIFICANCE STATEMENT: The first dopamine receptor mutation to cause a movement disorder, D2-I 212 F, was recently identified. The mutation makes receptor activation of G protein-mediated signaling more efficient. To confirm the pathogenesis of D2-I 212 F, 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., (U.S. Government work not protected by U.S. copyright.)

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 STATEMENT: S100B 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., (U.S. Government work not protected by U.S. copyright.)