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Regulation of Glutamate Receptor (GLR-1) Under Endoplasmic Reticulum Stress in Caenorhabditis elegans
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Date of Award
Department or Program Affiliation
Master of Science (MS)
Dahlberg, Caroline Lund, 1978-
Rose, Jacqueline K.
Serrano-Moreno, Jose Ramon
Neurons communicate with other cells to elicit outputs that include memory and movement. Cells, including neurons, create proteins every day for specific functions and in particular, neurons produce proteins that enable their communication. Proteins found in cellular membranes are synthesized at the endoplasmic reticulum (ER). However, up to 30% of new proteins are improperly folded and must be removed from the cell. A build-up of misfolded proteins can trigger the Unfolded Protein Response (UPR) which initiates other pathways of protein quality control and can determine the fate of a cell. ER-associated protein degradation (ERAD) is a ubiquitin-dependent process in eukaryotic cells that helps alleviate protein accumulation by breaking down misfolded proteins and targeting them to for degradation at the proteasome. The ERAD system is well described in yeast but is less well studied in multicellular systems. We used Caenorhabditis elegans (C. elegans) as a model organism to examine the trafficking of a single transmembrane protein through the ER under stressed conditions and as part of the ERAD pathway. The glutamate receptor, GLR-1, is a model membrane protein expressed in a subset of interneurons in C. elegans at the ventral nerve cord (VNC) to observe if GLR-1 is a candidate substrate for ERAD. Assessing GLR-1’s accumulation in the absence of ERAD E3 ubiquitin ligases in C. elegans can determine the protein’s regulation in ERAD. Three putative ERAD E3 ligases in C. elegans are: HRD-1, MARC-6, and HRDL-1. Previous results showed that deletion of most of the gene encoding HRDL-1, caused an increased accumulation of GLR-1::GFP in the VNC. HRDL-1 may have a role in regulating GLR-1 degradation at the ER and GLR-1 may be a candidate endogenous substrate for ERAD. In order to understand how ERAD effects GLR-1, we used a chemical stressor, tunicamycin, which blocks ER-dependent glycosylation and initiates the UPR. Tunicamycin treatment induces ER stress, which activates a UPR-reporter construct hsp-4p::GFP in C. elegans. The hsp4p::GFP reporter responds to tunicamycin at lower doses when animals harbor the mutation gene hrdl-1. We used quantitative fluorescence imaging and immunoblotting to determine whether the GLR-1::GFP accumulated due to underlying ER stress caused by the lack of HRDL-1. Contrary to previous results, we found no difference in fluorescently-tagged GLR-1 in the VNC in animals lacking hrdl-1, though overall levels of tagged GLR-1 were increased. GLR-1 with a point mutation that caused ER accumulation was not affected by the loss of hrdl-1. We propose that another ER E3 ligase may be involved in GLR-1’s regulation in the VNC that further investigation of cell bodies may uncover mechanisms of GLR-1 regulation at the ER.
Glutamate, Endoplasmic reticulum, Caenorhabditis elegans, ubiquitin
Western Washington University
Subject – LCSH
Glutamic acid--Receptors; Endoplasmic reticulum; Caenorhabditis elegans; Proteolysis
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Aguilera, Janie, "Regulation of Glutamate Receptor (GLR-1) Under Endoplasmic Reticulum Stress in Caenorhabditis elegans" (2020). WWU Graduate School Collection. 982.