SUNDAY, DECEMBER 4- Late Abstracts 1 - Molecular Biology of the ...

SUNDAY
1972
Proteomic Characterization of the Binding Partners of Dopamine Transporter (DAT) and
Functional DAT Mutants.
S. M. Moore 1,2 , A. Rao 2 , A. Sorkin 1 , C. C. Wu 1 ; 1 Cell Biology and Physiology, University of
Pittsburgh, Pittsburgh, PA, 2 Pharmacology, University of Colorado Anschutz Medical Campus,
Aurora, CO
Dopamine transporter (DAT) is a 12-transmembrane domain, integral membrane protein that
acts to terminate the synaptic transmission of the neurotransmitter dopamine. DAT is normally
expressed primarily on the plasma membrane of the cell and is internalized through clathrinmediated
endocytosis. It has been shown that with N-terminal deletion, DAT undergoes
increased internalization through augmented levels of endocytosis. Conversely, a C-terminal
deletion variant of DAT has been shown to reside primarily in the endoplasmic reticulum, with
unknown endocytic effects. The main objective of this project is to utilize mass spectrometry
profiling methods to analyze the binding partners of these different DAT constructs in order to
increase the understanding of DAT trafficking and endocytosis. Initial studies in mice expressing
a full-length hemagglutinin (HA)-tagged DAT construct combined immunoprecipitation (IP) using
an HA antibody with profiling mass spectrometry. These studies resulted in the identification of a
variety of potential DAT binding partners. Some of the identified proteins were previously known
to be DAT binding partners, others were known to be involved in endocytosis, and still others
were known to be involved in synaptic transmission. These mouse studies inspired further
proteomic studies in cell lines containing the aforementioned DAT constructs, due to the
identification of endocytic proteins in the mouse studies along with the difference in endocytic
patterns in the DAT construct containing cell lines. Full length (FL), N-terminally deleted (ΔN),
and C-terminally deleted (ΔC) DAT constructs, dually tagged with YFP and HA, were stably
expressed in porcine aortic endothelial (PAE) cells. These constructs then underwent IP using
either HA or GFP antibodies. After IP the samples were digested with trypsin and analyzed on
either an LTQ linear ion trap mass spectrometer, for profiling experiments, or a TSQ Vantage
triple quadrupole mass spectrometer, for quantitative experiments. In these cell studies, several
proteins were identified that were present in all three samples while others were expressed
differentially between the samples when compared to the parental PAE cell line. Proteins
present in all samples included ubiquitin, calmodulin, and alpha-tubulin. Differentially expressed
proteins included clathrin heavy chain, Rho-GEF 12, and AP2 alpha-1 subunit, among a variety
of others. This differential expression indicates that the endocytic changes that occur with these
deletion mutations are accompanied by changes in the proteins that associate with DAT.
1973
Cell adhesion and survival requires integrin rapid recycling.
N. Waxmonsky 1 , S. Conner 1 ; 1 University of Minnesota, Minneapolis, MN
Integrins are transmembrane heterodimers composed of an α and a β subunit that mediate
attachment between the cell and its environment, which is a requirement for cell survival in
many cell types. Cells maintain adhesion by trafficking integrins to the cell surface through
endosomal sorting pathways; however, the molecular mechanisms governing this process are
undefined. We hypothesized that integrin recycling through the endosomal compartment is
important for maintenance of cell adhesion. In order to test this, we depleted HeLa cells of
factors with known roles in long- and short-loop recycling. Depletion of a long-loop recycling
factor, EHD1 (Eps15 Homology Domain), did not lead to loss of adhesion. However, depletion
of factors that have established roles at the early endosome, AAK1 (Adaptor-Associated Kinase
1) and EHD3, led to cell rounding and an eventual loss of cell adhesion. Defects in integrin
delivery to the cell surface disrupts formation of focal adhesion complexes. We investigated