Cloning, genomic organization and chromosomal localization of the gene encoding the murine sodium-dependent, purine-selective, concentrative nucleoside transporter (CNT2)

A PCR-based strategy was used to isolate a 2653 bp cDNA encoding the mouse sodium-dependent, purine nucleoside selective, concentrative nucleoside transporter (designated mCNT2). The deduced protein sequence exhibits 93 and 80% identity to the previously cloned rat and human sodium-dependent, purine nucleoside selective, nucleoside transporters, respectively. Characterization of 3H-nucleoside uptake by COS-1 cells transiently transfected with the cDNA demonstrated that it encoded a functional nucleoside transport activity with selectivity for purine nucleosides. The cDNA was used to screen a murine (strain 129SvJ/6) genomic library in pBeloBAC11 to identify a clone containing the mCNT2 gene. A PCR strategy was used to identify and sequence the intron-exon boundaries and to determine the approximate sizes of the introns. The mCNT2 gene spans approximately 13.7 kb and is encoded by 15 exons. The gene was mapped to mouse chromosome 2e3 by fluorescence in situ hybridization.     

The human "peripheral-type" benzodiazepine receptor: regional mapping of the gene and characterization of the receptor expressed from cDNA.

A cDNA for the human "peripheral-type" benzodiazepine receptor (PBR) was isolated from a liver cDNA library. The 851-nucleotide probe hybridized with a approximately 1 kb mRNA in Northern blots of RNA extracted from various human tissues and cell lines. The human PBR probe was hybridized to DNA from a somatic cell hybrid mapping panel to determine that the gene maps to chromosome 22. With a regional mapping panel for chromosome 22, we localized the gene within band 22q13.31. The ligand-binding properties of the receptor expressed from the cDNA were examined in transient expression experiments and compared to the endogenous human PBR. The PBR ligand [3H]PK 11195 had high affinity for the expressed receptor in COS-1 cells, but the affinities of a pair of isoquinoline propanamide enantiomers differed remarkably in expressed and endogenous human PBR. These findings reveal that the host cell and/or post-translational modification may have an important influence on PBR function.     

Cloning of the human and murine interleukin-7 receptors: demonstration of a soluble form and homology to a new receptor superfamily

cDNA clones encoding the human and murine interleukin-7 (IL-7) receptor were isolated and expressed in COS-7 cells. Binding of radiolabeled IL-7 to the recombinant IL-7 receptors produced curvilinear Scatchard plots containing high and low affinity classes. These binding properties, as well as the molecular size of the cloned receptor, were comparable to the native forms of the IL-7 receptor. In addition, several cDNA clones were isolated that encode a secreted form of the human IL-7 receptor capable of binding IL-7 in solution. Analysis of the sequence of the IL-7 receptor revealed significant homology in the extracellular domain to several recently cloned cytokine receptors, demonstrating that the IL-7 receptor is a member of a new receptor superfamily.     

The dopamine transporter and cytochrome P45OIID1 (debrisoquine 4-hydroxylase) in brain: resolution and identification of two distinct [3H]GBR-12935 binding proteins

Two [3H]GBR-12935 binding proteins, identified as the dopamine transporter and cytochrome P45OIID1, were solubilized in digitonin from canine striatal membranes, and were resolved following wheat germ agglutinin (WGA)-lectin column chromatography. Protein adsorbed to and specifically eluted from WGA-lectin with N-acetylglucosamine displayed saturable, high affinity (KD approximately 3 nM), and sodium-dependent binding of [3H]GBR-12935, which was inhibited in a concentration-dependent and stereoselective manner by dopamine uptake blockers and substrates with a pharmacological profile indicative of the dopamine uptake site. Protein not adsorbed to WGA-lectin also bound [3H]-GBR-12935 with high affinity (approximately 7 nM), in a sodium-independent manner, and was insensitive to classical dopamine uptake blockers and substrates such as mazindol or dopamine, corresponding to the so-called "piperazine acceptor" site seen in native membranes. [3H]GBR-12935 binding to this latter protein was, however, inhibited by various compounds with a pharmacological profile indicative of a form of cytochrome P450 designated P45OIID1 (debrisoquine/sparteine monooxygenase) with the following rank order of inhibitory potency: GBR-12909 greater than budipine greater than alpha-lobeline greater than quinidine greater than alpha flupenthixol greater than SKF-525A greater than sparteine greater than quinine. Ki values obtained for inhibition of [3H]-GBR-12935 binding to neuronal WGA passthrough fractions by these drugs correlate well with their respective Ki values for liver P45OIID1 activity. Western blotting and immunoprecipitation analysis with rabbit anti-rat P45OIID1 antibody also supported the identity of the mazindol-insensitive [3H]GBR-12935 binding site (or piperazine acceptor site) as P45OIID1. Furthermore, a [3H]GBR-12935 binding protein with pharmacological and immunological characteristics similar to those of P45OIID1 was solubilized from both bovine and human liver membranes, and GBR-12909 was found to be a potent competitive inhibitor (Ki approximately 100 nM) of sparteine monooxygenase activity in human liver microsomes. These data clearly indicate that [3H]GBR-12935 and its analogs display similar affinities for both the dopamine transporter and neuronal P45OIID1, and that this radioligand may be a useful probe of P45OIID1 activity in brain and liver. The exact molecular and functional association (if any) between these two distinct binding protein populations remains to be established; however, it is tempting to speculate that P45OIID1 is involved in the catabolism and processing of neurotransmitters subsequent to their reuptake into target cells.     

Delineating structure-function relationships in the dopamine transporter from natural and engineered Zn2+ binding sites

The dopamine transporter is member of a large family of Na+/Cl- dependent neurotransmitter and amino acid transporters. Little is known about the molecular basis for substrate translocation in this class of transporters as well as their tertiary structure remains elusive. In this report, we provide the first crude insight into the structural organization of the human dopamine transporter (hDAT) based on the identification of an endogenous high affinity Zn2+ binding site followed by engineering of an artificial Zn2+ binding site. By binding to the endogenous site, Zn2+ acts as a potent non-competitive inhibitor of dopamine uptake mediated by the hDAT transiently expressed in COS-7 cells. Systematic mutagenesis of potential Zn2+ coordinating residues lead to the identification of three residues on the predicted extracellular face of the transporter, 193His in the second extracellular loop, 375His at the external end of the putative transmembrane segment (TM) 7, and 396Glu at the external end of TM 8, forming three coordinates in the endogenous Zn2+ binding site. The three residues are separate in the primary structure but their common participation in binding the small Zn(II) ion define their spatial proximity in the tertiary structure of the transporter. Finally, an artificial inhibitory Zn2+ binding site was engineered between TM 7 and TM 8. This binding site both verify the proximity between the two domains as wells as it supports an alpha-helical configuration at the top of TM 8 in the hDAT.     

Cloning and functional expression of ATA1, a subtype of amino acid transporter A, from human placenta

This report describes the primary structure and functional characteristics of human ATA1, a subtype of the amino acid transport system A. The human ATA1 cDNA was isolated from a placental cDNA library. The cDNA codes for a protein of 487 amino acids with 11 putative transmembrane domains. The transporter mRNA ( approximately 9.0 kb) is expressed most prominently in the placenta and heart, but detectable level of expression is evident in other tissues including the brain. When expressed heterologously in mammalian cells, the cloned transporter mediates Na(+)-coupled transport of the system A-specific model substrate alpha-(methylamino)isobutyric acid. The transport process is saturable with a Michaelis-Menten constant of 0. 89 +/- 0.12 mM. The Na(+):amino acid stoichiometry is 1:1 as deduced from the Na(+)-activation kinetics. The transporter is specific for small short-chain neutral amino acids. The gene for the transporter is located on human chromosome 12.     

Presynaptic dopaminergic properties of differentiated mouse embryonic stem cells

This study characterized the presynaptic dopaminergic properties of neuronally differentiated mouse embryonic stem (ES) cells. Approximately 30% of the ES cells expressed tyrosine hydroxylase (TH) immunoreactivity when co-cultured with PA6 cells. These cultures expressed high affinity, sodium-dependent dopamine uptake as well as depolarization-induced and calcium-dependent dopamine release of this transmitter. These and other important dopaminergic genes found expressed in these cultures by RT-PCR included Nurr1, vesicular monoamine transporter 2 (VMAT2), TH, dopamine transporter (DAT), and glial cell line-derived neurotrophic factor (GDNF) receptors c-Ret and GFRalpha1. These results demonstrate that differentiated ES cells have the presynaptic functions for maintaining dopaminergic homeostasis, which may be essential for their long-term use in restoring CNS levels of this transmitter.     

Cloning of the pig renal organic anion transporter 1 (pOAT1)

A pig kidney cDNA library was screened for the porcine ortholog of the multispecific organic anion transporter 1 (pOAT1). Several positive clones were isolated resulting in two alternatively spliced cDNA clones of pOAT1 (pOAT1 and pOAT1A). pOAT1-cDNAs consist of 2126 or 1895 base pairs (EMBL Acc. No. AJ308234 and AJ308235) encoding 547 or 533 amino acid residue proteins with 89, 87, 83 and 81% homology to the human, rabbit, rat, and mouse OAT1, respectively. Heterologous expression of pOAT1 in Xenopus laevis oocytes revealed an apparent K(m) for [3H]PAH of 3.75 +/- 1.6 microM. [3H]PAH uptake mediated by pOAT1 was abolished by 0.5 mM glutarate or 1 mM probenecid. Functional characterization of pOAT1A did not show any affinity for [3H]PAH. In summary, we cloned two alternative splice variants of the pig ortholog of organic anion transporter 1. One splice form (pOAT1) showed typical functional characteristics of organic anion transporter 1, whereas the second form appears not to transport PAH.     

Molecular cloning and expression of a new member of the nerve growth factor receptor family that is characteristic for Hodgkin's disease.

In man, Hodgkin's disease (HD) represents the most frequent lymphoma entity whose pathogenesis is still unknown. In order to contribute to the characterization of the molecular mechanisms of this disease, cDNAs coding for the HD characteristic antigen CD30 were cloned from expression libraries of the human HUT-102 cell line using the monoclonal antibodies Ki-1 and Ber-H2. The open reading frame of the cDNA that can be translated from two mRNA species of 2.6 kb, and 3.8 kb, respectively, predicts a 595 amino acid protein with leader, extracellular, single transmembrane, and intracellular domains. When expressed in COS-1 cells, the cDNA presented properties comparable to native CD30 antigen. The CD30 extracellular domain proved to be homologous to members of the nerve growth factor receptor superfamily. Six cysteine-rich motifs could be recognized within the putative ligand-binding domain.     

Parkin increases dopamine uptake by enhancing the cell surface expression of dopamine transporter

Mutations of parkin, a protein-ubiquitin E3 ligase, are linked to Parkinson's disease (PD). Although a variety of parkin substrates have been identified, none of these is selectively expressed in dopaminergic neurons, whose degeneration plays a critical role in PD. Here we show that parkin significantly increased dopamine uptake in the human dopaminergic neuroblastoma cell line SH-SY5Y. This effect was accompanied by increased V(max) of dopamine uptake and unchanged K(m). Consistent with this, increased binding sites for dopamine transporter (DAT) ligand were observed in SH-SY5Y cells overexpressing parkin. The results were confirmed when parkin was transfected in HEK293 cells stably expressing DAT. In these cells, parkin enhanced the ubiquitination and degradation of DAT, increased its cell surface expression, and augmented dopamine uptake. The effects of parkin were significantly abrogated by its PD-causing mutations. Because the cell surface expression of functional DAT requires its oligomerization, misfolded DAT, induced either by the protein glycosylation inhibitor tunicamycin or by its C-terminal truncation, significantly attenuated cell surface expression of native DAT and reduced dopamine uptake. Expression of parkin, but not its T240R mutant, significantly alleviated these detrimental effects of misfolded DAT. Thus, our studies suggest that parkin increases dopamine uptake by enhancing the ubiquitination and degradation of misfolded DAT, so as to prevent it from interfering with the oligomerization and cell surface expression of native DAT. This function of parkin would enhance the precision of dopaminergic transmission, increase the efficiency of dopamine utilization, and reduce dopamine toxicity on neighboring cells.     

Human dopamine transporter: the first implementation of a combined in silico/in vitro approach revealing the substrate and inhibitor specificities

Parkinson’s disease (PD) is characterized by the loss of dopamine-generating neurons in the substantia nigra and corpus striatum. Current treatments alleviate PD symptoms rather than exerting neuroprotective effect on dopaminergic neurons. New drugs targeting the dopaminergic neurons by specific uptake through the human dopamine transporter (hDAT) could represent a viable strategy for establishing selective neuroprotection. Molecules able to increase the bioactive amount of extracellular dopamine, thereby enhancing and compensating a loss of dopaminergic neurotransmission, and to exert neuroprotective response because of their accumulation in the cytoplasm, are required. By means of homology modeling, molecular docking, and molecular dynamics simulations, we have generated 3D structure models of hDAT in complex with substrate and inhibitors. Our results clearly reveal differences in binding affinity of these compounds to the hDAT in the open and closed conformations, critical for future drug design. The established in silico approach allowed the identification of promising substrate compounds that were subsequently analyzed for their efficiency in inhibiting hDAT-dependent fluorescent substrate uptake, through in vitro live cell imaging experiments. Taken together, our work presents the first implementation of a combined in silico/in vitro approach enabling the selection of promising dopaminergic neuron-specific substrates.