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TitleThe role of phosphorylation and ubiquitination in the regulation of glutamate receptor trafficking in caenorhabditis elegans
NameKramer, Lawrence B. (author), Rongo, Christopher (chair), Padgett, Richard (internal member), Firestein, Bonnie (internal member), Driscoll, Monica (outside member), Rutgers University, Graduate School - New Brunswick,
Degree Date2011
Date Created2011
SubjectCell and Developmental Biology,
Caenorhabditis elegans,
Phosphorylation,
Cell receptors
DescriptionThe regulation of AMPA-type glutamate receptor (AMPAR) trafficking is a key mechanism by which neurons regulate synaptic strength and plasticity. AMPAR trafficking is modulated through a combination of receptor phosphorylation, ubiquitination, endocytosis, and recycling, yet the in vivo significance of factors
involved in these processes are just beginning to be uncovered. Here we identify the ubiquitin-conjugating enzyme variant UEV-1 as a regulator of AMPAR
trafficking in vivo, and in a related project find a possible link between AMPAR phosphorylation and ubiquitination-driven internalization. We identified mutations in uev-1 in a genetic screen for mutants with altered trafficking of the AMPAR subunit GLR-1 in C. elegans interneurons. Loss of uev-1 activity results in elongated accretions of GLR-1 in neuron cell bodies and along the ventral cord
neurites. Mutants also have a corresponding behavioral defect – a decrease in spontaneous reversals in locomotion – consistent with diminished GLR-1 function. We provide evidence that GLR-1 accumulates at RAB-10-containing endosomes in uev-1 mutants. UEV-1 homologs in other species bind to the
ubiquitin-conjugating enzyme Ubc13 to create K63-linked polyubiquitin chains on substrate proteins. We find that whereas UEV-1 can interact with C. elegans
UBC-13, global levels of K63-linked ubiquitination throughout nematodes appear to be unaffected in uev-1 mutants, even though UEV-1 is broadly expressed in
most tissues. Nevertheless, we find that mutations in ubc-13 result in accumulations of GLR-1 in a similar pattern to that found in uev-1 mutants. Our results suggest that UEV-1 may regulate a small subset of K63-linked ubiquitination events in nematodes, at least one of which is critical in regulating GLR-1 trafficking. In another project, we used a reverse genetics approach to
study the regulation of phosphorylation state on the trafficking of GLR-1. We found that mutation of a single GLR-1 serine (S891) to alanine is sufficient to
strongly reduce GLR-1 puncta in the ventral nerve cord. We also determined that this effect is precluded if the C-terminal portion of GLR-1 is mutated such that it can no longer be ubiquitinated and endocytosed. This suggests that S891 phosphorylation may regulate endocytosis and/or ubiquitination and subsequent
degradation of GLR-1. This may provide a link between ubiquitination and phosphorylation in the trafficking of GLR-1, with possible implications for similar
links in mammalian AMPAR trafficking.
involved in these processes are just beginning to be uncovered. Here we identify the ubiquitin-conjugating enzyme variant UEV-1 as a regulator of AMPAR
trafficking in vivo, and in a related project find a possible link between AMPAR phosphorylation and ubiquitination-driven internalization. We identified mutations in uev-1 in a genetic screen for mutants with altered trafficking of the AMPAR subunit GLR-1 in C. elegans interneurons. Loss of uev-1 activity results in elongated accretions of GLR-1 in neuron cell bodies and along the ventral cord
neurites. Mutants also have a corresponding behavioral defect – a decrease in spontaneous reversals in locomotion – consistent with diminished GLR-1 function. We provide evidence that GLR-1 accumulates at RAB-10-containing endosomes in uev-1 mutants. UEV-1 homologs in other species bind to the
ubiquitin-conjugating enzyme Ubc13 to create K63-linked polyubiquitin chains on substrate proteins. We find that whereas UEV-1 can interact with C. elegans
UBC-13, global levels of K63-linked ubiquitination throughout nematodes appear to be unaffected in uev-1 mutants, even though UEV-1 is broadly expressed in
most tissues. Nevertheless, we find that mutations in ubc-13 result in accumulations of GLR-1 in a similar pattern to that found in uev-1 mutants. Our results suggest that UEV-1 may regulate a small subset of K63-linked ubiquitination events in nematodes, at least one of which is critical in regulating GLR-1 trafficking. In another project, we used a reverse genetics approach to
study the regulation of phosphorylation state on the trafficking of GLR-1. We found that mutation of a single GLR-1 serine (S891) to alanine is sufficient to
strongly reduce GLR-1 puncta in the ventral nerve cord. We also determined that this effect is precluded if the C-terminal portion of GLR-1 is mutated such that it can no longer be ubiquitinated and endocytosed. This suggests that S891 phosphorylation may regulate endocytosis and/or ubiquitination and subsequent
degradation of GLR-1. This may provide a link between ubiquitination and phosphorylation in the trafficking of GLR-1, with possible implications for similar
links in mammalian AMPAR trafficking.
NotePh.D.
NoteIncludes bibliographical references
NoteIncludes vita
Noteby Lawrence B. Kramer
Genretheses
Persistent URLhttp://hdl.rutgers.edu/1782.1/rucore10001600001.ETD.000057606
Languageeng
CollectionGraduate School - New Brunswick Electronic Theses and Dissertations
Organization NameRutgers, The State University of New Jersey
RightsThe author owns the copyright to this work.