Molecular Mechanisms of Endocytosis: Trafficking and Functional Requirements for the Transferrin Receptor, Small Interfering RNAs and Dopamine Transporter: A Dissertation

Endocytosis is an essential function of eukaryotic cells, providing crucial nutrients and playing key roles in interactions of the plasma membrane with the environment. The classical view of the endocytic pathway, where vesicles from the plasma membrane fuse with a homogenous population of early endosomes from which cargo is sorted, has recently been challenged by the finding of multiple subpopulations of endosomes. These subpopulations vary in their content of phosphatidylinositol 3- phosphate (PI3P) and Rab binding proteins. The role of these endosomal subpopulations is unclear, as is the role of multiple PI3P effectors, which are ubiquitously expressed and highly conserved. One possibility is that the different subpopulations represent stages in the maturation of the endocytic pathway. Alternatively, endosome subpopulations may be specialized for different functions, such as preferential trafficking of specific endocytosed cargo. To determine whether specific receptors are targeted to distinct populations of endosomes, we have built a platform for total internal reflection fluorescence (TIRF) microscopy coupled with structured illumination capabilities named TESM (TIRF Epifluorescence Structured light Microscope.) In this study, TESM, along with standard biochemical and molecular biological tools, was used to analyze the dynamic distribution of two highly conserved Rab5 and PI3P effectors, EEA1 and Rabenosyn-5, and systematically study the trafficking of transferrin. Rabenosyn-5 is necessary for proper expression of the transferrin receptor as well as internalization and recycling of transferrin-transferrin receptor complexes. Results of combining TIRF with structured light Epifluorescence (SLE) indicate that the endogenous populations of EEA1 and Rabenoysn-5 are both distinct and partially overlapping. The application of antisense oligonucleotides as potential therapeutic agents requires effective methods for their delivery to the cytoplasm of target cells. In collaboration with RXi Pharmaceuticals we show the efficient cellular uptake of the antisense oligonucleotide sd-rxRNA® in the absence of delivery vehicle or protein carrier. In this study TIRF, SLE, and biochemical approaches were utilized to determine whether sd-rxRNA traffics and functions along specific endosomal pathways. Sd-rxRNA was found to traffic along the degradative pathway and require EEA1 to functionally silence its target. These new findings will help define the cellular pathways involved in RNA silencing. Neurotransmitter reuptake and reuse by neurotransmitter transport proteins is fundamental to transmitter homeostasis and synaptic signaling. In order to understand how trafficking regulates transporters in the brain and how this system may be disregulated in monoamine-related pathologies, the transporter internalization signals and their molecular partners must be defined. We utilized a yeast two-hybrid system to identify proteins that interact with the dopamine transporter (DAT) endocytic signal. The small, membrane associated, GTPase Rin was determined to specifically and functionally interact with the DAT endocytic signal, regulating constitutive and protein kinase C (PKC) – stimulated DAT endocytosis. The results presented in this study provide new insights into functions and components of endocytosis and enhance the understanding of endocytic organization

Insertion of Tetracysteine Motifs into Dopamine Transporter Extracellular Domains

The neuronal dopamine transporter (DAT) is a major determinant of extracellular dopamine (DA) levels and is the primary target for a variety of addictive and therapeutic psychoactive drugs. DAT is acutely regulated by protein kinase C (PKC) activation and amphetamine exposure, both of which modulate DAT surface expression by endocytic trafficking. In order to use live imaging approaches to study DAT endocytosis, methods are needed to exclusively label the DAT surface pool. The use of membrane impermeant, sulfonated biarsenic dyes holds potential as one such approach, and requires introduction of an extracellular tetracysteine motif (tetraCys; CCPGCC) to facilitate dye binding. In the current study, we took advantage of intrinsic proline-glycine (Pro-Gly) dipeptides encoded in predicted DAT extracellular domains to introduce tetraCys motifs into DAT extracellular loops 2, 3, and 4. [3H]DA uptake studies, surface biotinylation and fluorescence microscopy in PC12 cells indicate that tetraCys insertion into the DAT second extracellular loop results in a functional transporter that maintains PKC-mediated downregulation. Introduction of tetraCys into extracellular loops 3 and 4 yielded DATs with severely compromised function that failed to mature and traffic to the cell surface. This is the first demonstration of successful introduction of a tetracysteine motif into a DAT extracellular domain, and may hold promise for use of biarsenic dyes in live DAT imaging studies

The Plasma Membrane-Associated GTPase Rin Interacts with the Dopamine Transporter and Is Required for Protein Kinase C-Regulated Dopamine Transporter Trafficking

Dopaminergic signaling and plasticity are essential to numerous CNS functions and pathologies, including movement, cognition, and addiction. The amphetamine- and cocaine-sensitive dopamine (DA) transporter (DAT) tightly controls extracellular DA concentrations and half-life. DAT function and surface expression are not static but are dynamically modulated by membrane trafficking. We recently demonstrated that the DAT C terminus encodes a PKC-sensitive internalization signal that also suppresses basal DAT endocytosis. However, the cellular machinery governing regulated DAT trafficking is not well defined. In work presented here, we identified the Ras-like GTPase, Rin (for Ras-like in neurons) (Rit2), as a protein that interacts with the DAT C-terminal endocytic signal. Yeast two-hybrid, GST pull down and FRET studies establish that DAT and Rin directly interact, and colocalization studies reveal that DAT/Rin associations occur primarily in lipid raft microdomains. Coimmunoprecipitations demonstrate that PKC activation regulates Rin association with DAT. Perturbation of Rin function with GTPase mutants and shRNA-mediated Rin knockdown reveals that Rin is critical for PKC-mediated DAT internalization and functional downregulation. These results establish that Rin is a DAT-interacting protein that is required for PKC-regulated DAT trafficking. Moreover, this work suggests that Rin participates in regulated endocytosis

Amphetamine-induced decreases in dopamine transporter surface expression are protein kinase C-independent

Amphetamine (AMPH) is a potent dopamine (DA) transporter (DAT) inhibitor that markedly increases extracellular DA levels. In addition to its actions as a DAT antagonist, acute AMPH exposure induces DAT losses from the plasma membrane, implicating transporter-specific membrane trafficking in amphetamine\u27s actions. Despite reports that AMPH modulates DAT surface expression, the trafficking mechanisms leading to this effect are currently not defined. We recently reported that DAT residues 587-596 play an integral role in constitutive and protein kinase C (PKC)-accelerated DAT internalization. In the current study, we tested whether the structural determinants required for PKC-stimulated DAT internalization are necessary for AMPH-induced DAT sequestration. Acute amphetamine exposure increased DAT endocytic rates, but DAT carboxy terminal residues 587-590, which are required for PKC-stimulated internalization, were not required for AMPH-accelerated DAT endocytosis. AMPH decreased DAT endocytic recycling, but did not modulate transferrin receptor recycling, suggesting that AMPH does not globally diminish endocytic recycling. Finally, treatment with a PKC inhibitor demonstrated that AMPH-induced DAT losses from the plasma membrane were not dependent upon PKC activity. These results suggest that the mechanisms responsible for AMPH-mediated DAT internalization are independent from those governing PKC-sensitive DAT endocytosis

DA uptake and PKC-mediated downregulation are preserved when a tetracysteine motif is introduced into EL2, but not EL3 or EL4.

<p><i>DA uptake assay</i>. Cells were transfected as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0009113#s2" target="_blank"><i>Methods</i></a> and [³H]DA uptake was assessed 48 hours post transfection. <b>A.</b> Introduction of tetracysteine motifs decreases DA uptake. Data are expressed as % wildtype activity ±S.E.M. *Significantly different from wildtype (p<.01, One-way ANOVA with Tukey's multiple comparison test, n = 3), **Significantly different from EL2-CCPGCC DAT (p<.001, One-way ANOVA with Tukey's multiple comparison test, n = 3). <b>B.</b> EL2-CCPGCC DAT is acutely downregulated by PKC activation, whereas EL3- and EL4-CCPGCC are not. Cells were treated with either vehicle or 100 nM PMA, 30 min, 37°C and [³H]DA uptake was measured. Data are expressed as % vehicle-treated DA uptake ±S.E.M. for each construct. *Significantly different from vehicle treated (p<.05, Student's t test, n = 3), **significantly different from vehicle treated (p<.02, Student's t test, n = 3).</p

The presence of a tetracysteine in DAT EL2 does not alter DAT maturation or surface expression.

<p><i>Surface biotinylation assay</i>. Cells were transfected with the indicated DAT constructs and cell surface proteins were biotinylated and isolated from intracellular proteins as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0009113#s2" target="_blank"><i>Methods</i></a>. <b>A.</b> Representative immunoblot displaying surface (S) and intracellular (I) wildtype and EL2-CCPGCC DAT. Mature (90 kDa) and immature (56 kDa) species are indicated. <b>B.</b> Averaged data. Data are expressed as %total mature DAT on cell surface ±S.E.M. (n = 4). <b>C.</b> Representative immunoblot displaying surface (S) and intracellular (I) EL3-CCPGCC and EL4-CCPGCC DAT. Total lysates are displayed in the far right hand lanes. Note that no mature protein is detected. Immature species (56 kDa) are indicated.</p

Schematic of DAT with target sites for tetracysteine mutagenesis.

<p><i>Top:</i> DAT model. Asteriks indicate extracellular Pro-Gly residues targeted for tetracysteine mutagenesis. <i>Bottom:</i> DAT sequences spanning across the Pro-Gly tetracysteine target sites. Pro-Gly dipeptides are highlighted in the shaded boxes.</p

Cellular distribution of tetracysteine mutant DATs.

<p>EL2-CCPGCC DAT is robustly expressed at the cell surface and internalizes in response to PKC activation. <i>Immunofluorescence microscopy</i>. Cells were transfected with the indicated constructs and were fixed and stained with rat anti-DAT antibodies as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0009113#s2" target="_blank"><i>Methods</i></a>. A representative panel of deconvolved images is presented. A single plane through each cell center is shown. <b>A</b>. Effect of PKC activation on EL2-CCPGCC DAT. Prior to fixation, cells were treated with either vehicle or 1 µM PMA (37°C, 30 min). <b>B</b>. Cellular distribution of EL3-CCPGCC and EL4-CCPGCC DAT. Note the lack of either protein at the cell surface.</p

Dopamine transporter endocytic determinants: carboxy terminal residues critical for basal and PKC-stimulated internalization

Dopamine (DA) reuptake terminates dopaminergic neurotransmission and is mediated by DA transporters (DATs). Acute protein kinase C (PKC) activation accelerates DAT internalization rates, thereby reducing DAT surface expression. Basal DAT endocytosis and PKC-stimulated DAT functional downregulation rely on residues within the 587-596 region, although whether PKC-induced DAT downregulation reflects transporter endocytosis mechanisms linked to those controlling basal endocytosis rates is unknown. Here, we define residues governing basal and PKC-stimulated DAT endocytosis. Alanine substituting DAT residues 587-590 1) abolished PKC stimulation of DAT endocytosis, and 2) markedly accelerated basal DAT internalization, comparable to that of wildtype DAT during PKC activation. Accelerated basal DAT internalization relied specifically on residues 588-590, which are highly conserved among SLC6 neurotransmitter transporters. Our results support a model whereby residues within the 587-590 stretch may serve as a locus for a PKC-sensitive braking mechanism that tempers basal DAT internalization rates

Rabenosyn-5 Defines the Fate of the Transferrin Receptor Following Clathrin-mediated Endocytosis

Cell surface receptors and other proteins internalize through diverse mechanisms at the plasma membrane and are sorted to different destinations. Different subpopulations of early endosomes have been described, raising the question of whether different internalization mechanisms deliver cargo into different subsets of early endosomes. To address this fundamental question, we developed a microscopy platform to detect the precise position of endosomes relative to the plasma membrane during the uptake of ligands. Axial resolution is maximized by concurrently applied total internal reflection fluorescence and epifluorescence-structured light. We found that transferrin receptors are delivered selectively from clathrin-coated pits on the plasma membrane into a specific subpopulation of endosomes enriched in the multivalent Rab GTPase and phosphoinositide-binding protein Rabenosyn-5. Depletion of Rabenosyn-5, but not of other early endosomal proteins such as early endosome antigen 1, resulted in impaired transferrin uptake and lysosomal degradation of transferrin receptors. These studies reveal a critical role for Rabenosyn-5 in determining the fate of transferrin receptors internalized by clathrin-mediated endocytosis and, more broadly, a mechanism whereby the delivery of cargo from the plasma membrane into specific early endosome subpopulations is required for its appropriate intracellular traffic