SEARCH · PubMed Health

Results for “spatial domain identification”

Explore indexed PubMed citations for clinical trials, systematic reviews and public health research. Read source abstracts and follow each citation to its original PubMed record.

Quote a phrase for an exact phrase match. Source license links do not imply unrestricted reuse.

At least 145 records · Page 8Linked to original sources

PubMed2004

Tbx5 and Tbx4 transcription factors interact with a new chicken PDZ-LIM protein in limb and heart development.

The T-domain transcription factors, Tbx5 and Tbx4, play important roles in vertebrate limb and heart development. To identify interacting and potential Tbx-regulating proteins, we performed a yeast two-hybrid screen with the C-terminal domain of Tbx5 as bait. We identified a new PDZ-LIM protein composed of one N-terminal PDZ and three C-terminal LIM domains, which we named chicken LMP-4. Among the Tbx2, 3, 4, 5 subfamily, we observed exclusive interaction with Tbx5 and Tbx4 proteins. Tbx3 nor Tbx2 can substitute for LMP-4 binding. While chicken LMP-4 associates with Tbx5 or Tbx4, it uses distinct LIM domains to bind to the individual proteins. Subcellular co-localization of LMP-4 and Tbx proteins supports the protein interaction and reveals interference of LMP-4 with Tbx protein distribution, tethering the transcription factors to the cytoskeleton. The protein-protein interaction indicates regulation of Tbx function at the level of transcription factor nuclear localization. During chicken limb and heart development, Tbx5/LMP-4 and Tbx4/LMP-4 are tightly co-expressed in a temporal and spatial manner, suggesting that they operate in the same pathway. Surprisingly, chicken LMP-4 expression domains outside those of Tbx5 in the heart led to the discovery of Tbx4 expression in the outflow tract and the right ventricle of this organ. The Tbx4-expressing cells coincide with those of the recently discovered secondary anterior heart-forming field. The discrete posterior or anterior expression domains in the heart and the exclusive fore- or hindlimb expression of Tbx5 and Tbx4, respectively, suggest common pathways in the heart and limbs. The identification of a new Tbx5/4-specific binding factor further suggests a novel mechanism for Tbx transcription factor regulation in development and disease.

Amino Acid Sequence↗

PubMed1990

Mouse genetics meets molecular biology at Cold Spring Harbor. Mouse Molecular Genetics sponsored by the Cold Spring Harbor Laboratory, Cold Spring Harbor, NY, USA, August 29-September 2, 1990.

As stated earlier, the digestion of roughly 100 short talks delivered within a few days and containing very condensed information is, even for experts, quite demanding. Sometimes one felt in danger of becoming "psyched out" by all the experiments described; perhaps smaller group discussions dealing with experiments that have not worked, as well as the elimination of redundancies in the presentations, would have added to the flavor of the meeting. But what is the "take home message" from the 1990 CSH Mouse Meeting? With all the described breakthroughs, has mouse molecular genetics and development arrived at a turning point? I am inclined to answer this question with a "yes" on the basis of the following considerations: The elegant studies of development in Drosophila and Caenorhabditis elegans were made possible by the power of genetics with the use of developmental mutations. These studies taught us that the sequential activation of a hierarchy of regulatory genes dictates the temporal and spatial patterns of expression of proteins that define cell phenotypes and the body plan, and thereby control development. With the identification of important regulatory genes responsible for many classical as well as experimentally induced mouse mutations, in conjunction with traditional transgenic studies and the power of deleting and altering genes via ES cell chimeras, the study of mouse development has now gained an important new dimension. It is feasible that the consequences of subtle but precise genetic changes, such as the modification of regulatory elements or DNA-binding domains, can be studied in the whole organism by use of ES cells.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗

PubMed1995

Dipole modelling of MEG rhythms in time and frequency domains.

Local generators of spontaneous brain rhythms can be identified from the measured magnetic field pattern both in the time and frequency domains when the sources are dipolar. The dipole assumption is most efficient when only a few sources contribute to the signal at one time or frequency. We have quantified the effects of different filters and spectral transformation sizes in the analysis of spontaneous magnetic activity measured simultaneously over the whole head to determine the optimal values for maximum identification probability of localized (dipolar) activity. The criteria employed were (i) the percentage of dipolar sources, (ii) the goodness-of-fit of the dipole model, (iii) the confidence volumes and (iv) spatial distributions of the source sites, and (v) the effective spectral resolving power of the various Fast Fourier transform (FFT) sizes. The systematic changes of (i-v) suggested the use of 3-5 Hz passbands around the major spectral peaks and FFT lengths of 2-3 s for extraction of the maximum amount of information from this data set. The most successful choices of filter passband and spectral transformation length for source localization simultaneously provide estimates for the inherent spectral fluctuation of cortical rhythms (0.5 Hz) and for the activation lifetime of individual sources (0.3 s).

Adult↗

PubMed2005

Antigenic and immunogenic investigation of B-cell epitopes in the nucleocapsid protein of peste des petits ruminants virus.

Attempts were made to identify and map epitopes on the nucleocapsid (N) protein of peste des petits ruminants virus (PPRV) (Nigeria75/1 strain) using seven monoclonal antibodies (MAbs) and deletion mutants. At least four antigenic domains (A-I, A-II, C-I, and C-II) were identified using the MAbs. Domains A-I (MAb 33-4) and A-II (MAbs 38-4, P-3H12, and P-13A9) were determined to be located on the amino-terminal half (amino acids [aa] 1 to 262), and domains C-I (P-14C6) and C-II (P-9H10 and P-11A6) were within the carboxy-terminal region (aa 448 to 521). Nonreciprocal competition between A-II MAbs and MAbs to C-I and C-II domains was observed, indicating that they may be exposed on the surface of the N protein and spatially overlap each other. Blocking or competitive enzyme-linked immunosorbent assay studies using PPRV serum antibodies revealed that epitopes on the domains A-II and C-II were immunodominant, whereas those on the domains A-I and C-I were not. The competition between MAb and rinderpest virus (RPV) serum antibodies raised against RPV strain LATC was found in two epitopes (P-3H12 and P-13A9) on the domain A-II, indicating that these epitopes may cause cross-reactivity between PPRV and RPV. Identification of immunodominant but PPRV-specific epitopes and domains will provide the foundation in designing an N-protein-based diagnostic immunoassay for PPRV.

Animals↗

PubMed1993

Expression of tie-2, a member of a novel family of receptor tyrosine kinases, in the endothelial cell lineage.

We are interested in the molecular mechanisms that are involved in the development of the vascular system. In order to respond to morphogenetic and mitogenic signals, endothelial cells must express appropriate receptors. To characterize endothelial cell-specific receptors, we have concentrated on receptor tyrosine kinases, because several lines of evidence suggested the importance of controlled phosphotyrosine levels in endothelial cells. A strategy based on PCR amplification using degenerate oligonucleotides and mouse brain capillaries as mRNA source, led to the identification of a novel receptor tyrosine kinase, which we designated tie-2. In situ hybridization using a tie-2-specific probe revealed an interesting spatial and temporal expression pattern. The gene was expressed specifically in the endothelial lineage. tie-2 transcripts were present in endothelial cell precursors (angioblasts) and also in endothelial cells of sprouting blood vessels throughout development and in all organs and tissues so far examined. tie-2 was down-regulated in the adult. Because of the unusual combination of immunoglobulin, EGF-like and fibronectin type III domains in the extracellular portion of tie-2 which is shared by TEK and tie, these molecules may be considered members of a new family of receptor tyrosine kinases. Signal transduction via this new class of tyrosine kinases could lead to a better understanding of the molecular mechanisms of blood vessel formation.

Amino Acid Sequence↗

PubMed1999

Identification, sequence, and mapping of the mouse multiple PDZ domain protein gene, Mpdz.

The PDZ domain gained its name from the three proteins that were first seen to have homology by virtue of these domains, the mammalian postsynaptic density protein, PSD-95, the Drosophila discs-large septate junction protein, DLG, and the mammalian epithelial tight-junction protein zona occludens, ZO-1. Over 50 PDZ domain-containing genes have been recognized so far from almost any organism subjected to sequencing, including mammals, nematodes, yeast, plants, and bacteria. The domain consists of an approximately 90-amino-acid-residue unit, which is often repeated in the protein. The majority of residues form a conserved spatial structure while a few amino acids in critical positions confer protein binding specificity. A subgroup of PDZ domains have been shown to recognize a short carboxy-terminal amino acid motif, T/SXV (Ser/Thr-X-Val-COO-), where X is any amino acid. We have identified and completely sequenced a gene, Mpdz, that encodes a mouse protein containing 13 such domains. We have also mapped the gene to a series of overlapping deletions on mouse chromosome 4 and can therefore determine that its function is not essential for embryonic development or neonatal survival.

Animals↗

PubMed1992

A truncated laminin chain homologous to the B2 chain: structure, spatial expression, and chromosomal assignment.

We describe the identification of a novel laminin chain. Overlapping clones were isolated from a human fibrosarcoma HT1080 cell cDNA library spanning a total of 5,200 bp. A second set of clones contained an alternative 3' end sequence giving a total of 4,316 bp. The longer sequence contained an open reading frame for a 1,193-residue-long polypeptide. The alternative sequence was shortened at the carboxyl-terminal end coding for a 1,111-residue-long polypeptide. The amino acid sequence contained 21 amino acids of a putative signal peptide and 1,172 residues or alternatively 1,090 residues of a sequence with five distinct domains homologous to domains I-V in laminin chains. Comparison of the amino acid sequences showed that the novel laminin chain is homologous to the laminin B2 chain. However, the structure of the novel laminin chain isolated here differs significantly from that of the B2 chain in that it has no domain VI and domains V, IV, and III are shorter, resulting in a truncated laminin chain. The alternative sequence had a shortened domain I/II. In accordance with the current nomenclature, the chain characterized here is termed B2t. Calculation of possible chain interactions of laminin chains with the B2t chain domain I/II indicated that the B2t chain can replace the B2 chain in some laminin molecules. The gene for the laminin B2t chain (LAMB2T) was localized to chromosome 1q25-q31 in close proximity to the laminin B2 chain gene. Northern analysis showed that the B2t chain is expressed in several human fetal tissues but differently from the laminin B1 and B2 chains. By in situ hybridization expression of the B2t chain was localized to specific epithelial cells in skin, lung, and kidney as opposed to a general epithelial and endothelial cell expression of the laminin B2 chain in the same tissues.

Amino Acid Sequence↗

PubMed1995

Dynamics of bacteriophage T4 DNA polymerase function: identification of amino acid residues that affect switching between polymerase and 3' --> 5' exonuclease activities.

Many DNA polymerases are multifunctional with the ability to replicate DNA as well as to proofread misincorporated nucleotides. Since polymerase and 3'--> 5' exonuclease activities appear to reside in spatially distinct active centers, there must be some mechanism for coordinating replication with proofreading and for transferring DNA between the two active centers. We have designed a genetic selection scheme to isolate bacteriophage T4 mutant DNA polymerases that are defective in "switching" between polymerase and exonuclease activities. Amino acid residues that affected active-site-switching were identified in four regions of the T4 DNA polymerase: two regions in the proposed exonuclease domain. Representative mutant DNA polymerases from each region were purified for biochemical studies. We propose that amino acid substitutions identified by mutational analysis affect critical contacts between T4 DNA polymerase and DNA that are required for transfer of DNA between the polymerase and exonuclease active centers.

Amino Acids↗

PubMed2006

The role of protein kinase A and A-kinase anchoring proteins in modulating T-cell activation: progress and future directions.

Protein kinase A (PKA) is a broad-specificity serine/threonine protein kinase whose spatial and temporal regulation is maintained through interactions with A-kinase anchoring proteins (AKAPs). Subcellular localization of AKAPs through unique targeting domains provides a mechanism by which PKA can respond to localized microdomains of cyclic AMP (cAMP) and phosphorylate nearby substrates. For nearly 40 years, cAMP has been known to be a potent modulator of the immune system. cAMP levels are regulated by G-protein-coupled receptors, adenylyl cyclases (AC), and phosphodiesterases (PDEs). This review discusses recent progress made in the discovery of PKA substrates in T lymphocytes and in the identification of AKAPs in T lymphocytes. Because PKA is activated by cAMP, generation and maintenance of cAMP in T cells is also discussed. These findings are framed in the context of understanding the complexity of cAMP and, thus, PKA signaling and are intended to provide the reader with an overview of current literature, as well as an awareness of questions and concerns to consider.

Adaptor Proteins, Signal Transducing↗

PubMed2002

Patchy interspecific sequence similarities efficiently identify positive cis-regulatory elements in the sea urchin.

We demonstrate that interspecific sequence conservation can provide a systematic guide to the identification of functional cis-regulatory elements within a large expanse of genomic DNA. The test was carried out on the otx gene of Strongylocentrotus purpuratus. This gene plays a major role in the gene regulatory network that underlies endomesoderm specification in the embryo. The cis-regulatory organization of the otx gene is expected to be complex, because the gene has three different start sites (X. Li, C.-K. Chuang, C.-A. Mao, L. M. Angerer, and W. H. Klein, 1997, Dev. Biol. 187, 253-266), and it is expressed in many different spatial domains of the embryo. BAC recombinants containing the otx gene were isolated from Strongylocentrotus purpuratus and Lytechinus variegatus libraries, and the ordered sequence of these BACs was obtained and annotated. Sixty kilobases of DNA flanking the gene, and included in the BAC sequence from both species, were scanned computationally for short conserved sequence elements. For this purpose, we used a newly constructed software package assembled in our laboratory, "FamilyRelations." This tool allows detection of sequence similarities above a chosen criterion within sliding windows set at 20-50 bp. Seventeen partially conserved regions, most a few hundred base pairs long, were amplified from the S. purpuratus BAC DNA by PCR, inserted in an expression vector driving a CAT reporter, and tested for cis-regulatory activity by injection into fertilized S. purpuratus eggs. The regulatory activity of these constructs was assessed by whole-mount in situ hybridization (WMISH) using a probe against CAT mRNA. Of the 17 constructs, 11 constructs displayed spatially restricted regulatory activity, and 6 were inactive in this test. The domains within which the cis-regulatory constructs were expressed are approximately consistent with results from a WMISH study on otx expression in the embryo, in which we used probes specific for the mRNAs generated from each of the three transcription start sites. Four separate cis-regulatory elements that specifically produce endomesodermal expression were identified, as well as ubiquitously active elements, and ectoderm-specific elements. We confirm predictions from other work with respect to target sites for specific transcription factors within the elements that express in the endoderm.

Animals↗

PubMed2000

A distal Schwann cell-specific enhancer mediates axonal regulation of the Oct-6 transcription factor during peripheral nerve development and regeneration.

The POU domain transcription factor Oct-6 is a major regulator of Schwann cell differentiation and myelination. During nerve development and regeneration, expression of Oct-6 is under the control of axonal signals. Identification of the cis-acting elements necessary for Oct-6 gene regulation is an important step in deciphering the complex signalling between Schwann cells and axons governing myelination. Here we show that a fragment distal to the Oct-6 gene, containing two DNase I-hypersensitive sites, acts as the Oct-6 Schwann cell-specific enhancer (SCE). The SCE is sufficient to drive spatially and temporally correct expression, during both normal peripheral nerve development and regeneration. We further demonstrate that a tagged version of Oct-6, driven by the SCE, rescues the peripheral nerve phenotype of Oct-6-deficient mice. Thus, our isolation and characterization of the Oct-6 SCE provides the first description of a cis-acting genetic element that responds to converging signalling pathways to drive myelination in the peripheral nervous system.

Animals↗

PubMed2005

On the Euclidean distance of images.

We present a new Euclidean distance for images, which we call IMage Euclidean Distance (IMED). Unlike the traditional Euclidean distance, IMED takes into account the spatial relationships of pixels. Therefore, it is robust to small perturbation of images. We argue that IMED is the only intuitively reasonable Euclidean distance for images. IMED is then applied to image recognition. The key advantage of this distance measure is that it can be embedded in most image classification techniques such as SVM, LDA, and PCA. The embedding is rather efficient by involving a transformation referred to as Standardizing Transform (ST). We show that ST is a transform domain smoothing. Using the Face Recognition Technology (FERET) database and two state-of-the-art face identification algorithms, we demonstrate a consistent performance improvement of the algorithms embedded with the new metric over their original versions.

Algorithms↗

PubMed1996

Methods in quantitative image analysis.

The main steps of image analysis are image capturing, image storage (compression), correcting imaging defects (e.g. non-uniform illumination, electronic-noise, glare effect), image enhancement, segmentation of objects in the image and image measurements. Digitisation is made by a camera. The most modern types include a frame-grabber, converting the analog-to-digital signal into digital (numerical) information. The numerical information consists of the grey values describing the brightness of every point within the image, named a pixel. The information is stored in bits. Eight bits are summarised in one byte. Therefore, grey values can have a value between 0 and 256 (2(8)). The human eye seems to be quite content with a display of 5-bit images (corresponding to 64 different grey values). In a digitised image, the pixel grey values can vary within regions that are uniform in the original scene: the image is noisy. The noise is mainly manifested in the background of the image. For an optimal discrimination between different objects or features in an image, uniformity of illumination in the whole image is required. These defects can be minimised by shading correction [subtraction of a background (white) image from the original image, pixel per pixel, or division of the original image by the background image]. The brightness of an image represented by its grey values can be analysed for every single pixel or for a group of pixels. The most frequently used pixel-based image descriptors are optical density, integrated optical density, the histogram of the grey values, mean grey value and entropy. The distribution of the grey values existing within an image is one of the most important characteristics of the image. However, the histogram gives no information about the texture of the image. The simplest way to improve the contrast of an image is to expand the brightness scale by spreading the histogram out to the full available range. Rules for transforming the grey value histogram of an existing image (input image) into a new grey value histogram (output image) are most quickly handled by a look-up table (LUT). The histogram of an image can be influenced by gain, offset and gamma of the camera. Gain defines the voltage range, offset defines the reference voltage and gamma the slope of the regression line between the light intensity and the voltage of the camera. A very important descriptor of neighbourhood relations in an image is the co-occurrence matrix. The distance between the pixels (original pixel and its neighbouring pixel) can influence the various parameters calculated from the co-occurrence matrix. The main goals of image enhancement are elimination of surface roughness in an image (smoothing), correction of defects (e.g. noise), extraction of edges, identification of points, strengthening texture elements and improving contrast. In enhancement, two types of operations can be distinguished: pixel-based (point operations) and neighbourhood-based (matrix operations). The most important pixel-based operations are linear stretching of grey values, application of pre-stored LUTs and histogram equalisation. The neighbourhood-based operations work with so-called filters. These are organising elements with an original or initial point in their centre. Filters can be used to accentuate or to suppress specific structures within the image. Filters can work either in the spatial or in the frequency domain. The method used for analysing alterations of grey value intensities in the frequency domain is the Hartley transform. Filter operations in the spatial domain can be based on averaging or ranking the grey values occurring in the organising element. The most important filters, which are usually applied, are the Gaussian filter and the Laplace filter (both averaging filters), and the median filter, the top hat filter and the range operator (all ranking filters). Segmentation of objects is traditionally based on threshold grey values. (AB

Algorithms↗

PubMed1998

Evidence that the fourth ligand to the [2Fe-2S] cluster in animal ferrochelatase is a cysteine. Characterization of the enzyme from Drosophila melanogaster.

In a previous study, site-directed mutagenesis experiments identified three of the four ligands to the [2Fe-2S] cluster in animal ferrochelatase as conserved cysteines in the COOH-terminal extension, Cys-403, Cys-406, and Cys-411 in human ferrochelatase (Crouse, B. R., Sellers, V. M., Finnegan, M. G., Dailey, H. A. & Johnson, M. K. (1996) Biochemistry 35, 16222-16229). The nature of the fourth ligand was left unresolved, and spectroscopic studies raised the possibility of one noncysteinyl, oxygenic ligand. In this work, we report two lines of evidence that strongly suggest the fourth ligand is a cysteine residue. Cysteine at position 196 in human recombinant ferrochelatase when changed to a serine results in an inactive enzyme that is lacking the [2Fe-2S] cluster. Furthermore, whole cell EPR studies demonstrate that in the C196S mutant the cluster fails to assemble. Additionally, the cloning and expression of Drosophila melanogaster ferrochelatase has allowed the identification, by EPR and UV-visible spectroscopy, of a [2Fe-2S]2+ cluster with properties analogous to those of animal ferrochelatases. The observation that Drosophila ferrochelatase contains only four conserved cysteines at positions 196, 403, 406, and 411, is in accord with the proposal that these residues function as cluster ligands. In the case of the ferrochelatase iron-sulfur cluster ligands, NH2-Cys-X206-Cys-X2-Cys-X4-Cys-COOH, the position distant from other ligands may lead to a spatial positioning of the cluster near the enzyme active site or at the interface of two domains, thereby explaining the loss of enzyme activity that accompanies cluster degradation and reinforcing the idea that the cluster functions as a regulatory switch.

Amino Acid Sequence↗

PubMed1990

Synthetic peptides of Venezuelan equine encephalomyelitis virus E2 glycoprotein. I. Immunogenic analysis and identification of a protective peptide.

Fourteen peptides representing 67% of the extramembranal domain of the Venezuelan equine encephalomyelititis (VEE) virus E2 glycoprotein were synthesized and analyzed to determine their antigenic, immunogenic, and protective capacities. Thirteen of 14 peptides elicited antibody for the homologous peptide. Thirteen peptides elicited antiviral antibody that recognized either the Trinidad (TRD) strain of VEE virus or the TC-83 vaccine derivative, or both. Two peptides, VE2pep01(TC-83) and VE2pep01(TRD), protected significant numbers of mice from TRD virus challenge. The majority of the peptides were reactive with antisera from mice immunized with the various subtypes of VEE virus. A competition assay using antipeptide antibodies to block virus binding of anti-VEE virus monoclonal antibodies corroborated previous studies on the spatial relationship of E2 epitopes and provided evidence for a spatial overlap of the E2 amino terminus with a domain composed of residues 180-210.

Amino Acid Sequence↗

PubMed1994

Mutational mapping of functional residues in tissue factor: identification of factor VII recognition determinants in both structural modules of the predicted cytokine receptor homology domain.

Alanine scanning mutagenesis of tissue factor, the initiating receptor and cofactor molecule for the coagulation pathways, was used to define residue side chains with functional contributions. Approximately half of the residues were exchanged, and several stretches of functional residues throughout the entire extracellular domain were identified which contributed to overall coagulant function. Mutants were further characterized with respect to their affinity for binding of ligand, providing evidence that identified functional sequence spans are involved in ligand interaction. The tissue factor extracellular domain is suggested to adopt the folding pattern of the cytokine receptor homology unit, which is typically composed of two seven-beta-strand modules. Evaluation of the mutational analysis within this structural context suggests that functionally important residues are spatially proximate and clustered at the boundary of the predicted beta-strand modules. Residues contributing to ligand binding by tissue factor were identified in positions corresponding to ligand interactive residues in the growth hormone receptor and contact residues of other cytokine receptors, consistent with a conserved structural region for ligand interaction throughout the cytokine receptor family.

Alanine↗

PubMed2002

Molecular phenotype of Fragile X syndrome: FMRP, FXRPs, and protein targets.

Fragile X syndrome (FraX) is one of the most prevalent genetic causes of mental retardation. FraX is associated with an unstable expansion of a polymorphism within the 5' untranslated region of the FMR1 gene. The main consequence of this mutation is a reduction in the levels of the gene product (FMRP). FMRP is an RNA-binding protein with multiple spliced variants (isoforms) and high levels of expression in a variety of tissues, including neurons. In the latter cells, it is localized not only to the perikaryon but also to dendrites and dendritic spines. FMRP belongs to a family of proteins that includes the Fragile X Related Proteins or FXRPs. FXRPs share high homology in their functional domains with FMRP, and also associate with mRNA and components of the protein synthesis apparatus. However, FXRPs do not have the same temporo-spatial pattern of distribution (and other properties) of FMRP. Immunochemical assays have confirmed that a functionally uncompensated FMRP deficit is the essence of the FraX molecular phenotype. Here, we report our preliminary study on FXRPs levels in leukocytes from FraX males. By immunoblotting, we found that a marked reduction in FMRP levels is associated with a modest increase in FXR1P and no changes in FXR2P levels. The consequences of this reduced FMRP expression on protein synthesis, in other words, the identification of FMRP targets, can be studied by different molecular approaches including protein interaction and proteomics methods. By two-dimensional gel electrophoresis, we showed that in FraX leukocytes there is a defect in acetylation that involves prominently the regulatory protein annexin-1. Extension of current studies of the molecular phenotype to more brain-relevant tissue samples, a wider range of proteomics-based methods, and correlative analyses of FMRP homologues and FMRP targets with multiple behavioral measures, will greatly expand our understanding of FraX pathogenesis and it will help to develop and monitor new therapeutic strategies.

Animals↗

PubMed2001

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.

Animals↗