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Title: An investigation into the binding and signalling properties of group I metabotropic glutamate receptors expressed recombinantly in mammalian cell lines
Author: Selkirk, Julie Victoria
Awarding Body: University of Leicester
Current Institution: University of Leicester
Date of Award: 2001
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This Thesis investigates the G protein coupling profile of group I mGluRs using a novel 35S -GTPyS binding with subsequent Gcc subunit-specific immunoprecipitation protocol. This approach allows the quantitation of activation of individual G proteins or groups of G proteins to be assessed. Using this assay, it was revealed that mGlulct receptors couple to different endogenous sub-populations of G proteins when expressed recombinantly in model cell backgrounds. Thus mGlula receptors expressed stably in BHK cells couple to both PTx-sensitive and PTx-resistant G protein sub-types, yet when expressed inducibly in CHO cells couple solely to PTx-resistant Gctq/n G proteins. One consequence of the PTx-sensitive component of signalling by mGluRlot in BHK cells is to regulate PLC in a negative manner, the potency and efficacy of agonists increasing following pre-treatment of the cells with this toxin. Sequestration of GPy subunits using a PARK C-terminal minigene had no effect on mGluRla-mediated responses and thus the negative regulation of PLC appears to be mediated by the Get subunit of Gj/0 G proteins. The basis for this additional coupling of mGluRla in BHK cells could not conclusively be attributed to differences in receptor or G protein expression levels. Using the same approach, an interaction of mGluR5a could not be observed with either Gi/0 or Gq/ii G proteins when expressed stably in HEK-293 cells or inducibly in CHO cells. Despite this apparent lack of G protein coupling however, an accumulation of inositol phosphates was measurable, as was an elevation of Ca2+ j. The binding characteristics of the novel group I mGluR radioligand 3H -quisqualic acid were assessed at both mGlul and mGlu5 receptors, and also with respect to a soluble form of human mGluRl, truncated just prior to the first transmembrane domain, and secreted by CHO cells. The noticeably higher affinity of 3H -quisqualic acid for this truncated receptor compared to that reported for the rat receptor produced in insect Sf9 cells, prompted studies to investigate whether TV-linked glycosylation was important for agonist binding and subsequent signalling. Prevention of TV-linked glycosylation either by treatment with tunicamycin, or by site-directed mutagenesis, resulted in reduced specific 3H -quisqualic acid binding to mGlula receptors, and also in a reduction of cell surface receptor expression. This suggests that TV-linked glycosylation of mGlula is important for receptor expression and function.
Supervisor: Not available Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID:  DOI: Not available