Direct coupling of oligomerization and oligomerization-driven endocytosis of the dopamine transporter to its conformational mechanics and activity.

Dopamine transporter (DAT) mediates the reuptake of synaptically released dopamine, and thus controls the duration and intensity of dopamine neurotransmission. Mammalian DAT has been observed to form oligomers, although the mechanisms of oligomerization and its role in DAT activity and trafficking remain largely unknown. We discovered a series of small molecule compounds that stabilize trimers and induce high-order oligomers of DAT and concomitantly promote its clathrin-independent endocytosis. Using a combination of chemical cross-linking, fluorescence resonance energy transfer microscopy, antibody-uptake endocytosis assay, live-cell lattice light sheet microscopy, ligand binding and substrate transport kinetics analyses, and molecular modeling and simulations, we investigated molecular basis of DAT oligomerization and endocytosis induced by these compounds. Our study showed that small molecule-induced DAT oligomerization and endocytosis are favored by the inward-facing DAT conformation and involve interactions of four hydrophobic residues at the interface between transmembrane (TM) helices TM4 and TM9. Surprisingly, a corresponding quadruple DAT mutant displays altered dopamine transport kinetics and increased cocaine-analog binding. The latter is shown to originate from an increased preference for outward-facing conformation and inward-to-outward transition. Taken together, our results demonstrate a direct coupling between conformational dynamics of DAT, functional activity of the transporter, and its oligomerization leading to endocytosis. The high specificity of such coupling for DAT makes the TM4-9 hub a new target for pharmacological modulation of DAT activity and subcellular localization.

Flotillins regulate membrane mobility of the dopamine transporter but are not required for its protein kinase C dependent endocytosis.

Flotillins were proposed to mediate clathrin-independent endocytosis, and recently, flotillin-1 was implicated in the protein kinase C (PKC)-triggered endocytosis of the dopamine transporter (DAT). Since endocytosis of DAT was previously shown to be clathrin-mediated, we re-examined the role of clathrin coat proteins and flotillin in DAT endocytosis using DAT tagged with the hemagglutinin epitope (HA) in the extracellular loop and a quantitative HA antibody uptake assay. Depletion of flotillin-1, flotillin-2 or both flotillins together by small interfering RNAs (siRNAs) did not inhibit PKC-dependent internalization and degradation of HA-DAT. In contrast, siRNAs to clathrin heavy chain and µ2 subunit of clathrin adaptor complex AP-2 as well as a dynamin inhibitor Dyngo-4A significantly decreased PKC-dependent endocytosis of HA-DAT. Similarly, endocytosis and degradation of DAT that is not epitope-tagged were highly sensitive to the clathrin siRNAs and dynamin inhibition but were not affected by flotillin knockdown. Very little co-localization of DAT with flotillins was observed in cells ectopically expressing DAT and in cultured mouse dopaminergic neurons. Depletion of flotillins increased diffusion rates of HA-DAT in the plasma membrane, suggesting that flotillin-organized microdomains may regulate the lateral mobility of DAT. We propose that clathrin-mediated endocytosis is the major pathway of PKC-dependent internalization of DAT, and that flotillins may modulate functional association of DAT with plasma membrane rafts rather than mediate DAT endocytosis.

Improved precision of proteomic measurements in immunoprecipitation based purifications using relative quantitation.

Mass spectrometry coupled immunoprecipitation (MS-IP) studies are useful in identifying and quantitating potential binding partners of a target protein. However, they are often conducted without appropriate loading controls. Western blots are often used to analyze loading controls, yet there are limitations to their usefulness as analytical tools. One remedy for this is the use of selected reaction monitoring (SRM), where the areas under the curve (AUCs) of peptides from a protein of interest can be normalized to those from the constant regions of the immunoglobulins used for the IP. Using this normalization method, significant changes in relative peptide abundance were observed between samples when there appeared to be an unequal load based on immunoglobulin peptide abundance.

Protein kinase C-dependent ubiquitination and clathrin-mediated endocytosis of the cationic amino acid transporter CAT-1.

Cationic amino acid transporter 1 (CAT-1) is responsible for the bulk of the uptake of cationic amino acids in most mammalian cells. Activation of protein kinase C (PKC) leads to down-regulation of the cell surface CAT-1. To examine the mechanisms of PKC-induced down-regulation of CAT-1, a functional mutant of CAT-1 (CAT-1-HA-GFP) was generated in which a hemagglutinin antigen (HA) epitope tag was introduced into the second extracellular loop and GFP was attached to the carboxyl terminus. CAT-1-HA-GFP was stably expressed in porcine aorthic endothelial and human epithelial kidney (HEK) 293 cells. Using the HA antibody internalization assay we have demonstrated that PKC-dependent endocytosis was strongly inhibited by siRNA depletion of clathrin heavy chain, indicating that CAT-1-HA-GFP internalization requires clathrin-coated pits. Internalized CAT-1-HA-GFP was accumulated in early, recycling, and late endosomes. PKC activation also resulted in ubiquitination of CAT-1. CAT-1 ubiquitination and endocytosis in phorbol ester-stimulated porcine aorthic endothelial and HEK293 cells were inhibited by siRNA knockdown of NEDD4-2 and NEDD4-1 E3 ubiquitin ligases, respectively. In contrast, ubiquitination and endocytosis of the dopamine transporter was dependent on NEDD4-2 in all cell types tested. Altogether, our data suggest that ubiquitination mediated by NEDD4-2 or NEDD4-1 leading to clathrin-mediated endocytosis is the common mode of regulation of various transporter proteins by PKC.

Negative regulation of dopamine transporter endocytosis by membrane-proximal N-terminal residues.

The plasma membrane dopamine transporter (DAT) takes extracellular dopamine back up into dopaminergic neurons. Although the number of DATs at the cell surface is regulated by endocytosis and recycling, the molecular mechanisms that control this endocytic trafficking of DAT are not defined. To map the sequence motifs that are involved in constitutive DAT endocytosis, mutagenesis of human DAT tagged with yellow fluorescent protein (YFP) and an extracellular HA epitope was performed. Removal of the entire N terminus of DAT resulted in accumulation of the resulting DAT mutant (YFP-HA-DeltaN-DAT) in early and recycling endosomes in HeLa and PAE cells, and in primary rat mesencephalic-striatal neuronal cocultures. This endosomal accumulation was due to rapid constitutive internalization of YFP-HA-DeltaN-DAT by the clathrin-dependent pathway. Small deletions and multialanine substitutions in the N terminus revealed two molecular determinants within the membrane proximal residues 60-65 that are important for preventing rapid internalization of DAT. First, mutations of Arg60 or Trp63, leading to disruption of the "outward facing" DAT conformation, correlated with an increased pool of mobile DATs in the plasma membrane and accelerated constitutive internalization of the DAT mutants. Second, mutation of Lys65 also correlated with elevated endocytosis. While none of these mutations alone recapitulated the marked endocytic phenotype of YFP-HA-DeltaN-DAT, simultaneous elimination of both the outward conformation of DAT and Lys65 resulted in DAT mutants that were rapidly internalized. Thus, our studies reveal a new link between DAT endocytosis and conformation-dependent uptake activity that represents a novel mode for regulating DAT function.

Three ubiquitin conjugation sites in the amino terminus of the dopamine transporter mediate protein kinase C-dependent endocytosis of the transporter.

Dopamine levels in the brain are controlled by the plasma membrane dopamine transporter (DAT). The amount of DAT at the cell surface is determined by the relative rates of its internalization and recycling. Activation of protein kinase C (PKC) leads to acceleration of DAT endocytosis. We have recently demonstrated that PKC activation also results in ubiquitylation of DAT. To directly address the role of DAT ubiquitylation, lysine residues in DAT were mutated. Mutations of each lysine individually did not affect ubiquitylation and endocytosis of DAT. By contrast, ubiquitylation of mutants carrying multiple lysine substitutions was reduced in cells treated with phorbol ester to the levels detected in nonstimulated cells. Altogether, mutagenesis data suggested that Lys19, Lys27, and Lys35 clustered in the DAT amino-terminus are the major ubiquitin-conjugation sites. The data are consistent with the model whereby at any given time only one of the lysines in DAT is conjugated with a short ubiquitin chain. Importantly, cell surface biotinylation, immunofluorescence and down-regulation experiments revealed that PKC-dependent internalization of multilysine mutants was essentially abolished. These data provide the first evidence that the ubiquitin moieties conjugated to DAT may serve as a molecular interface of the transporter interaction with the endocytic machinery.

Trimerization of dopamine transporter triggered by AIM-100 binding: Molecular mechanism and effect of mutations.

Recent work demonstrated the propensity of dopamine transporters (DATs) to form trimers or higher oligomers, enhanced upon binding a furopyrimidine, AIM-100. AIM-100 binding promotes DAT endocytosis and thereby moderates dopaminergic transmission. Despite the neurobiological significance of these events, the molecular mechanisms that underlie the stabilization of DAT trimer and the key interactions that modulate the trimerization of DAT, and not serotonin transporter SERT, remain unclear. In the present study, we determined three structural models, termed trimer-W238, -C306 and -Y303, for possible trimerization of DATs . To this aim, we used structural data resolved for DAT and its structural homologs that share the LeuT fold, advanced computational modeling and simulations, site-directed mutagenesis experiments and live-cell imaging assays. The models are in accord with the versatility of LeuT fold to stabilize dimeric or higher order constructs. Selected residues show a high propensity to occupy interfacial regions. Among them, D231-W238 in the extracellular loop EL2, including the intersubunit salt-bridge forming pair D231/D232-R237 (not present in SERT) (in trimer-W238), the loop EL3 (trimers-C306 and -Y303), and W497 on the intracellularly exposed IL5 loop (trimer-C306) and its spatial neighbors (e.g. K525) near the C-terminus are computationally predicted and experimentally confirmed to play important roles in enabling the correct folding and/or oligomerization of DATs in the presence of AIM-100. The study suggests the possibility of controlling the effective transport of dopamine by altering the oligomerization state of DAT upon small molecule binding, as a possible intervention strategy to modulate dopaminergic signaling. This article is part of the issue entitled 'Special Issue on Neurotransmitter Transporters'.

Small molecule induced oligomerization, clustering and clathrin-independent endocytosis of the dopamine transporter.

Clathrin-independent endocytosis (CIE) mediates internalization of many transmembrane proteins but the mechanisms of cargo recruitment during CIE are poorly understood. We found that the cell-permeable furopyrimidine AIM-100 promotes dramatic oligomerization, clustering and CIE of human and mouse dopamine transporters (DAT), but not of their close homologues, norepinephrine and serotonin transporters. All effects of AIM-100 on DAT and the occupancy of substrate binding sites in the transporter were mutually exclusive, suggesting that AIM-100 may act by binding to DAT. Surprisingly, AIM-100-induced DAT endocytosis was independent of dynamin, cholesterol-rich microdomains and actin cytoskeleton, implying that a novel endocytic mechanism is involved. AIM-100 stimulated trafficking of internalized DAT was also unusual: DAT accumulated in early endosomes without significant recycling or degradation. We propose that AIM-100 augments DAT oligomerization through an allosteric mechanism associated with the DAT conformational state, and that oligomerization-triggered clustering leads to a coat-independent endocytosis and subsequent endosomal retention of DAT.

RNA interference screen reveals an essential role of Nedd4-2 in dopamine transporter ubiquitination and endocytosis.

The function of the dopamine transporter (DAT) to terminate dopamine neurotransmission is regulated by endocytic trafficking of DAT. To elucidate the mechanisms of DAT endocytosis, we generated a fully functional mutant of the human DAT in which a hemagglutinin epitope (HA) was incorporated into the second extracellular loop. The endocytosis assay, based on the uptake of an HA antibody, was designed to study constitutive- and protein kinase C (PKC)-dependent internalization of HA-DAT expressed in non-neuronal cells and rat dopaminergic neurons. Large-scale RNA interference analysis of PKC-dependent endocytosis of HA-DAT revealed the essential and specific role of an E3 ubiquitin ligase, Nedd4-2 (neural precursor cell expressed, developmentally downregulated 4-2), as well as the involvement of adaptor proteins present in clathrin-coated pits, such as epsin, Eps15 (epidermal growth factor pathway substrate clone 15), and Eps15R (Eps15-related protein). Depletion of Nedd4-2 resulted in a dramatic reduction of PKC-dependent ubiquitination of DAT. Endogenous Nedd4-2, epsin, and Eps15 were coimmunoprecipitated with heterologously expressed human HA-DAT and endogenous DAT isolated from rat striatum. A new mechanistic model of DAT endocytosis is proposed whereby the PKC-induced ubiquitination of DAT mediated by Nedd4-2 leads to interaction of DAT with adaptor proteins in coated pits and acceleration of DAT endocytosis.

Calmodulin regulates intracellular trafficking of epidermal growth factor receptor and the MAPK signaling pathway.

The epidermal growth factor receptor (EGFR) is a member of the tyrosine kinase receptor family involved in signal transduction and the regulation of cellular proliferation and differentiation. It is also a calmodulin-binding protein. To examine the role of calmodulin in the regulation of EGFR, the effect of calmodulin antagonist, W-13, on the intracellular trafficking of EGFR and the MAPK signaling pathway was analyzed. W-13 did not alter the internalization of EGFR but inhibited its recycling and degradation, thus causing the accumulation of EGF and EGFR in enlarged early endosomal structures. In addition, we demonstrated that W-13 stimulated the tyrosine phosphorylation of EGFR and consequent recruitment of Shc adaptor protein with EGFR, presumably through inhibition of the calmodulin-dependent protein kinase II (CaM kinase II). W-13-mediated EGFR phosphorylation was blocked by metalloprotease inhibitor, BB94, indicating a possible involvement of shedding in this process. However, MAPK activity was decreased by W-13; dissection of this signaling pathway showed that W-13 specifically interferes with Raf-1 activity. These data are consistent with the regulation of EGFR by calmodulin at several steps of the receptor signaling and trafficking pathways.

Key Roles of Size and Crystallinity of Nanosized Iron Hydr(oxides) Stabilized by Humic Substances in Iron Bioavailability to Plants.

Availability of Fe in soil to plants is closely related to the presence of humic substances (HS). Still, the systematic data on applicability of iron-based nanomaterials stabilized with HS as a source for plant nutrition are missing. The goal of our study was to establish a connection between properties of iron-based materials stabilized by HS and their bioavailability to plants. We have prepared two samples of leonardite HS-stabilized iron-based materials with substantially different properties using the reported protocols and studied their physical chemical state in relation to iron uptake and other biological effects. We used Mössbauer spectroscopy, XRD, SAXS, and TEM to conclude on iron speciation, size, and crystallinity. One material (Fe-HA) consisted of polynuclear iron(III) (hydr)oxide complexes, so-called ferric polymers, distributed in HS matrix. These complexes are composed of predominantly amorphous small-size components (<5 nm) with inclusions of larger crystalline particles (the mean size of (11 ± 4) nm). The other material was composed of well-crystalline feroxyhyte (δ'-FeOOH) NPs with mean transverse sizes of (35 ± 20) nm stabilized by small amounts of HS. Bioavailability studies were conducted on wheat plants under conditions of iron deficiency. The uptake studies have shown that small and amorphous ferric polymers were readily translocated into the leaves on the level of Fe-EDTA, whereas relatively large and crystalline feroxyhyte NPs were mostly sorbed on the roots. The obtained data are consistent with the size exclusion limits of cell wall pores (5-20 nm). Both samples demonstrated distinct beneficial effects with respect to photosynthetic activity and lipid biosynthesis. The obtained results might be of use for production of iron-based nanomaterials stabilized by HS with the tailored iron availability to plants. They can be applied as the only source for iron nutrition as well as in combination with the other elements, for example, for industrial production of "nanofortified" macrofertilizers (NPK).

Enhanced ubiquitylation and accelerated degradation of the dopamine transporter mediated by protein kinase C.

Dopamine transporter (DAT) localization in dopaminergic neurons plays an important role in regulating dopamine signaling. However, the mechanisms of DAT trafficking that control DAT localization are still poorly understood. To gain insight into these mechanisms, human DAT was purified in large amounts using a two-step affinity chromatography procedure from untreated HeLa cells or cells treated with phorbol 12-myristate 13-acetate (PMA). Mass spectrometric analysis of purified DAT complexes revealed the presence of several proteins, among which ubiquitin was particularly abundant in the PMA-treated sample. Western blotting of highly purified DAT protein confirmed constitutive ubiquitylation of DAT and a dramatic increase in DAT ubiquitylation in cells treated with PMA. This increase was blocked by pretreatment with the protein kinase C (PKC) inhibitor bis-indolylmaleimide. DAT ubiquitylation by ectopically expressed ubiquitin was demonstrated in cells transiently transfected with yellow fluorescent protein-tagged ubiquitin. In addition, fluorescence resonance energy transfer was detected between cyan fluorescent protein-tagged DAT and yellow fluorescent protein-tagged ubiquitin, indicative of DAT-ubiquitin conjugation. Interestingly, the largest fluorescence resonance energy transfer signals were observed in endosomes. Ubiquitylated DAT was detected in the plasma membrane using cell surface biotinylation as well as in intracellular compartments, suggesting that ubiquitylation begins at the plasma membrane and is maintained in endosomes. In both porcine aortic endothelial and HeLa cells, where PKC-dependent DAT ubiquitylation was observed, PKC activation resulted in rapid degradation of DAT (t12 = 1-2 h). Altogether, these data suggest that PKC-induced DAT ubiquitylation may target DAT to lysosomal degradation.

Constitutive and protein kinase C-induced internalization of the dopamine transporter is mediated by a clathrin-dependent mechanism.

The amount of dopamine transporter (DAT) present at the cell surface is rapidly regulated by the rates of DAT internalization to endosomes and DAT recycling back to the plasma membrane. The re-distribution of the transporter from the cell surface to endosomes was induced by phorbol ester activation of protein kinase C in porcine aortic endothelial cells stably expressing the human DAT. Inhibition of DAT recycling with the carboxylic ionophore monensin also caused significant accumulation of DAT in early endosomes and a concomitant loss of DAT from the cell surface, due to protein kinase C-independent constitutive internalization of DAT in the absence of recycling. Such monensin-induced relocation of DAT to endosomes was therefore utilized as a measure of the constitutive internalization of DAT. Knock-down of clathrin heavy chain or dynamin II by small interfering RNAs dramatically inhibited both constitutive and protein kinase C-mediated internalization of DAT. In contrast, neither monensin-dependent nor phorbol ester-induced re-distribution of DAT were affected by inhibitors of endocytosis through cholesterol-rich membrane microdomains. Mutational analysis revealed the potential importance of amino acid residues 587-597 in DAT internalization. Altogether, the data suggest that both constitutive and protein kinase C-mediated internalization of DAT utilize the clathrin-dependent endocytic pathway, but likely involve unconventional mechanisms.

Effect of tyrosine kinase inhibitors on clathrin-coated pit recruitment and internalization of epidermal growth factor receptor.

Several inhibitors of epidermal growth factor receptor (EGFR) kinase and Src family kinases (SFK) were employed to study the role of these kinases in EGFR internalization through clathrin-coated pits. The EGFR kinase-specific compound PD158780 substantially diminished EGFR internalization. PP2, an inhibitor of SFK, had a moderate effect on EGFR internalization in several types of cells, including cells lacking SFK, indicating that the inhibition of endocytosis by PP2 is mediated by kinases other than SFK. In contrast, SU6656, a more specific inhibitor of SFK, did not affect EGFR internalization. To examine what stage of internalization requires receptor kinase activity, we established a quantitative assay based on three-dimensional fluorescence microscopy that measures co-localization of an EGF-rhodamine conjugate and a fluorescently tagged clathrin adaptor protein complex, AP-2. Interestingly, recruitment of EGFR into coated pits did not require physiological temperature because the maximal accumulation of EGFR in coated pits was observed at 4 degrees C. Pretreatment of the cells with PD158780 prevented EGFR recruitment into coated pits, whereas the inhibitor did not block the internalization of receptors that had first been allowed to enter the coated pits at 4 degrees C. These data demonstrate that the activation of receptor kinase is essential for the initial, coated pit recruitment step of endocytosis.

Multiple molecular determinants in the carboxyl terminus regulate dopamine transporter export from endoplasmic reticulum.

The plasma membrane dopamine transporter (DAT) has an essential role in terminating dopaminergic neurotransmission by reuptake of dopamine into the presynaptic neurons. Therefore, the amount of DAT at the cell surface is a critical determinant of DAT function. In this study, we examined the role of the carboxyl terminus of DAT in trafficking of the transporter through the biosynthetic pathway to the plasma membrane. Live cell fluorescence microscopy and cell surface biotinylation were used to study the effects of systematic deletions and alanine substitutions in the carboxyl terminus on DAT localization. It was found that alanine substitutions of Lys-590 and Asp-600 significantly delayed the delivery of DAT to the plasma membrane because of retention of DAT in the endoplasmic reticulum (ER). Most surprising, mutation of Gly-585 to alanine completely blocked the exit of DAT from the ER and surface expression of the transporter. The effect of these three mutations on ER export of DAT was demonstrated in porcine aortic endothelial cells and the immortalized neuronal cell line 1RB3AN27. In primary cultures of rat embryonic midbrain neurons, DAT G585A, K590A, and D600A mutants were restricted to the cell soma and did not traffic to the dendrites or axonal processes. These data are consistent with the model whereby the local conformation and/or intramolecular interactions of the sequences of the DAT carboxyl terminus proximal to the last transmembrane domain are essential for the ER export of the transporter.

Oligomerization of dopamine transporters visualized in living cells by fluorescence resonance energy transfer microscopy.

To examine the oligomeric state and trafficking of the dopamine transporter (DAT) in different compartments of living cells, human DAT was fused to yellow (YFP) or cyan fluorescent protein (CFP). YFP-DAT and CFP-DAT were transiently and stably expressed in porcine aortic endothelial (PAE) cells, human embryonic kidney (HEK) 293 cells, and an immortalized dopaminergic cell line 1RB3AN27. Fluorescence microscopic imaging of cells co-expressing YFP-DAT and CFP-DAT revealed fluorescence resonance energy transfer (FRET) between CFP and YFP, which is consistent with an intermolecular interaction of DAT fusion proteins. FRET signals were detected between CFP- and YFP-DAT located at the plasma membrane and in intracellular membrane compartments. Phorbol esters or amphetamine induced the endocytosis of YFP/CFP-DAT to early and recycling endosomes, identified by Rab5, Rab11, Hrs and EEA.1 proteins. Interestingly, however, DAT was mainly excluded from Rab5- and Hrs-containing microdomains within the endosomes. The strongest FRET signals were measured in endosomes, indicative of efficient oligomerization of internalized DAT. The intermolecular DAT interactions were confirmed by co-immunoprecipitation. A DAT mutant that was retained in the endoplasmic reticulum (ER) after biosynthesis was used to show that DAT is oligomeric in the ER. Moreover, co-expression of an ER-retained DAT mutant and wild-type DAT resulted in the retention of wild-type DAT in the ER. These data suggest that DAT oligomers are formed in the ER and then are constitutively maintained both at the cell surface and during trafficking between the plasma membrane and endosomes.