This project is concerned with identifying and characterizing structural determinants of GABAA receptor biogenesis. I used flow cytometry to measure surface levels of GABAA receptor subunits on HEK293T cells coexpressing wild-type alpha1 subunits and mutant beta2 subunits containing segmental deletions or point mutations to identify beta2 subunit structural determinants that are important for biogenesis of alpha1beta2 GABAA receptors. I located novel sites in the beta2 subunit N-terminal domain and major M3-M4 cytoplasmic loop that are necessary to attain maximal surface GABAA receptor levels. I used a combination of multiple sequence alignment, glycosidase digestion, brefeldin A treatment and analytic centrifugation and demonstrated that a structural determinant (D450) at the boundary of the beta2 subunit major cytoplasmic loop and M4 transmembrane domain is conserved among all subunits of the Cys-loop superfamily and is required for receptor assembly. Furthermore, using homology modeling and glycosidase digestion, I found that beta2 subunit N-terminal residue, N104, is a glycosylation site that is located on the minus side of the subunit-subunit interaction region and that its N-glycan processing in the Golgi apparatus is affected by the incorporation of gamma2 subunits into alpha1beta2gamma2 pentamers. Together, my studies demonstrated that both beta2 subunit N-termini and M3-M4 cytoplasmic loops contain structural determinants for GABAA receptor biogenesis.
| Identifer | oai:union.ndltd.org:VANDERBILT/oai:VANDERBILTETD:etd-11242008-214043 |
| Date | 01 December 2008 |
| Creators | Lo, Wen-yi |
| Contributors | Robert L. Macdonald, Danny G. Winder, Roger J. Colbran, Jerod Scott Denton |
| Publisher | VANDERBILT |
| Source Sets | Vanderbilt University Theses |
| Language | English |
| Detected Language | English |
| Type | text |
| Format | application/pdf |
| Source | http://etd.library.vanderbilt.edu//available/etd-11242008-214043/ |
| Rights | unrestricted, I hereby certify that, if appropriate, I have obtained and attached hereto a written permission statement from the owner(s) of each third party copyrighted matter to be included in my thesis, dissertation, or project report, allowing distribution as specified below. I certify that the version I submitted is the same as that approved by my advisory committee. I hereby grant to Vanderbilt University or its agents the non-exclusive license to archive and make accessible, under the conditions specified below, my thesis, dissertation, or project report in whole or in part in all forms of media, now or hereafter known. I retain all other ownership rights to the copyright of the thesis, dissertation or project report. I also retain the right to use in future works (such as articles or books) all or part of this thesis, dissertation, or project report. |
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