Rickets and Osteomalacia

The site of cocaine binding on the dopamine transporter (DAT) was investigated using the photoactivatable irreversible cocaine analog [125 I]3␤-(p-chlorophenyl)tropane-2␤-carboxylic acid, 4-azido-3-iodophenylethyl ester ([ 125 I]RTI 82). The incorporation site of this compound was mapped to transmembrane domains (TMs) 4 -6 using epitope-specific immunoprecipitation of trypsin fragments and further localized using cyanogen bromide (CNBr), which hydrolyzes proteins on the C-terminal side of methionine residues. CNBr hydrolysis of The dopamine transporter (DAT)2 is a neuronal protein that clears dopamine (DA) from the synapse via Na ϩ /Cl Ϫ -dependent active transport. DA reuptake is necessary for appropriate control of dopaminergic function and may be dysregulated in disorders such as Parkinson disease and drug abuse (1). Cocaine and many other compounds bind to DAT and inhibit DA reuptake, resulting in increased transmitter levels believed to mediate drug reinforcement (2). Despite extensive pharmacological investigation of transport inhibitors, the molecular mechanisms by which they are recognized by DAT and exert their effects remain largely unknown.DAT and the related norepinephrine and serotonin transporters (NET and SERT) are integral membrane proteins that consist of 12 transmembrane domains (TMs), connecting intracellular (IL) and extracellular (EL) loops, and intracellular N and C termini (3). Substrate translocation is thought to occur by an alternating access mechanism that sequentially exposes extracellular and intracellular portions of the substrate permeation pathway to opposite sides of the membrane (4). The three-dimensional structures of these proteins are unknown, although they are recently proposed to be similar to that of the homologous Aquifex aeolicus leucine transporter, LeuT Aa (5). The substrate binding site on LeuT Aa is composed of residues from TMs 1, 3, 6, and 8, and some analogous sites have been identified in DAT, NET, and SERT (6, 7).LeuT Aa , however, is not cocaine-sensitive, and its structure does not indicate how cocaine or other uptake blockers might interact at monoamine transporters. Cocaine binding to DAT is reduced by mutagenesis of many amino acids throughout the primary sequence (6, 7), but in most cases it is not clear if these effects are due to disruption of specific ligand interaction sites or to conformational alterations that indirectly impact binding. Thus the structure of the inhibitor binding site on DAT and the identities of the residues that dock cocaine and other blockers

Section: Materials-the Amino Precursor For [mentioning

confidence: 99%

Localization of Cocaine Analog [125I]RTI 82 Irreversible Binding to Transmembrane Domain 6 of the Dopamine Transporter

Vaughan

Sakrikar

Parnas

et al. 2007

“…Aminopeptidase and Carboxypeptidase Treatment-DATs from 32 PO 4 -labeled striatal synaptosomes or striatal slices were immunoprecipitated, gel-purified on 8% SDS-PAGE gels, and electroeluted as described previously (21). Aliquots of the electroeluted samples were treated with 1-100 g/ml of aminopeptidase 1 or carboxypeptidase Y prepared in 50 mM Tris-HCl, pH 8.0, for 1 h at 22°C, followed by addition of SDS-PAGE sample buffer and electrophoresis and autoradiography on 8% SDS-PAGE gels.…”

Section: I]deep and [mentioning

confidence: 99%

“…Immunoprecipitation of Trypsin Fragments-Our initial attempts to map phosphorylation sites utilized procedures that have been successful in identifying sites of photoaffinity label attachment (18,21). For these experiments 32 PO 4 -labeled DATs prepared from OA/OAG-treated slices were proteolyzed with trypsin, which cleaves at lysine and arginine residues, and the digests were immunoprecipitated with antibodies 16 and 18 specific for N-and C-terminal domain epitopes (Fig.…”

Section: Characterization Of Dat Phosphorylation In Striatal Slices-mentioning

confidence: 99%

“…4, right two lanes). The 45-kDa fragment (arrow a) contains epitope 16 in the N-terminal tail and extends through extracellular loop 2 (EL2), whereas the 32-kDa fragment (arrow b) begins in EL2 and extends through epitope 18 in the C-terminal tail (18,21). The 14-kDa fragment (arrow c) is a product of the 45-kDa fragment that retains epitope 16 but terminates in IL1.…”

Section: Characterization Of Dat Phosphorylation In Striatal Slices-mentioning

confidence: 99%

See 1 more Smart Citation

Dopamine Transporters Are Phosphorylated on N-terminal Serines in Rat Striatum

Foster

Pananusorn²,

Vaughan

2002

134

171

Dopamine transporters (DATs) are neuronal phosphoproteins that clear dopamine from the synaptic cleft. Activation of protein kinase C (PKC) and inhibition of protein phosphatases by okadaic acid (OA) increase phosphorylation of DAT and lead to concomitant reduction in DAT activity and cell surface expression. Numerous potential sites for phosphorylation are present on DAT, but the sites utilized and their relationship to transport regulation are currently unknown. We used peptide mapping and epitope-specific immunoprecipitation to identify the region of DAT that undergoes phosphorylation in rat striatal tissue. Phosphoamino acid analysis revealed that basal and stimulated samples were phosphorylated primarily on serine. Digestion of 32 PO 4 -labeled DAT with trypsin and immunoprecipitation with N-or C-terminal specific antisera failed to isolate phosphopeptide fragments corresponding to photoaffinity-labeled fragments that contain all internal interhelical loops. However, digestion of 32 PO 4 -labeled DAT with endoproteinase asp-N and immunoprecipitation with an N-terminal antiserum extracted two phosphopeptide fragments from both basal and PKC/ OA-stimulated samples, demonstrating that the N-terminal cytoplasmic tail is a major site of phosphorylation. Aminopeptidase treatment of PKC-and/or OA-stimulated DAT cleaved essentially all 32 PO 4 label without proteolysis extending past transmembrane domains 1 and 2, providing further evidence that most phosphorylation sites are near the N terminus and not in intracellular loops or C-terminal domains. In situ proteolysis of the N-terminal tail indicates that the majority of stimulated phosphorylation sites are N-terminal to an antibody epitope at residues 42-59. Two-dimensional analysis of purified protein produced three tryptic phosphopeptides that may result from phosphorylation of multiple sites, but the fragments did not co-migrate with synthetic tryptic peptides phosphorylated at serines 2 and 4. These results indicate that most or all of the basal and stimulated phosphorylation of DAT in striatal tissue occurs on one or more residues in a group of six serines clustered near the distal end of the cytoplasmic N terminus.The concentration of dopamine in the synaptic and extrasynaptic space is controlled to a great extent by the action of dopamine transporters (DATs), 1 which utilize the energy of Na ϩ and Cl Ϫ ionic gradients to drive reuptake of the neurotransmitter into the presynaptic cell (1). DAT belongs to a family of transporters including those for norepinephrine and serotonin (SERT) that constitutes the major sites of action for tricyclic antidepressants and psychostimulants (2). Other neurotransmitter reuptake carriers include plasma membrane transporters for glycine (Glyt1) and ␥-aminobutyric acid (GAT1), and the synaptic vesicle monoamine and acetylcholine transporters, vesicular monoamine transporter and vesicular acetylcholine transporter (3, 4). These proteins are believed to have a similar structure of 12 transmembrane-spanning domains, cytoplasmica...

“…All Na ϩ /Cl Ϫ -dependent carriers are believed to share a common topology characterized by the presence of 12 transmembrane segments connected by alternating extracellular and intracellular loops with an intracellular location of the N and C terminus (3,4,11). Despite intense efforts, including many mutagenesis studies (12)(13)(14)(15)(16)(17)(18) and studies using photoaffinity labeling (19), surprisingly little is known about the binding site for cocaine-like substances in the monoamine transporters. Although cysteine-scanning mutagenesis of transmembrane segment 3 in the SERT has suggested that two residues (Ile-172 and Tyr-176) in the middle of the transmembrane segment could be in close proximity to the cocaine binding site (20), no direct contact sites have been established between cocaine and specific transporter residues.…”

mentioning

confidence: 99%

Biophysical Characterization of the Cocaine Binding Pocket in the Serotonin Transporter Using a Fluorescent Cocaine Analogue as a Molecular Reporter

Rasmussen¹,

Carroll²,

Maresch³

et al. 2001

Cocaine is one of the most widely abused psychostimulants, causing major medical and socioeconomic problems (1). Currently, there is no effective treatment against cocaine addiction available; therefore, clarifying the molecular mechanisms underlying the psychostimulatory effects and addictive properties of cocaine should prove critical for potential development of future therapeutic strategies. Cocaine and related drugs act by inhibiting clearance of released monoamine neurotransmitters from the synaptic cleft (2-4). This clearance of monoamines occurs via three distinct but highly homologous monoamine transporters, the serotonin transporter (SERT), 1 the dopamine transporter (DAT), and the norepinephrine transporter (NET) (2-4). Cocaine binds with high affinity to all three transporters and is generally believed to act as a competitive blocker of substrate translocation (4, 5). Several studies have provided evidence that inhibition of the DAT is the predominant mechanism behind the stimulatory effects and addictive properties of cocaine (6 -8). However, this hypothesis has been challenged by recent studies on mice in which the DAT gene has been deleted (1). Despite the absence of the DAT gene, it was surprisingly observed that these mice self-administered cocaine, indicating a possible important role of also the SERT and NET (9, 10).The SERT belongs together with DAT and NET to a family of Na ϩ /Cl Ϫ -dependent solute carriers that are characterized functionally by their dependence on the presence of Na ϩ and Cl Ϫ in the extracellular fluid (3, 11). All Na ϩ /Cl Ϫ -dependent carriers are believed to share a common topology characterized by the presence of 12 transmembrane segments connected by alternating extracellular and intracellular loops with an intracellular location of the N and C terminus (3, 4, 11). Despite intense efforts, including many mutagenesis studies (12-18) and studies using photoaffinity labeling (19), surprisingly little is known about the binding site for cocaine-like substances in the monoamine transporters. Although cysteine-scanning mutagenesis of transmembrane segment 3 in the SERT has suggested that two residues in the middle of the transmembrane segment could be in close proximity to the cocaine binding site (20), no direct contact sites have been established between cocaine and specific transporter residues.