| Type of Document |
Dissertation |
| Author |
Lo, Wen-yi
|
| URN |
etd-11242008-214043 |
| Title |
STRUCTURAL DETERMINANTS OF GABAA RECEPTOR BIOGENESIS |
| Degree |
PhD |
| Department |
Neuroscience |
| Advisory Committee |
| Advisor Name |
Title |
| Danny G. Winder |
Committee Chair |
| Jerod Scott Denton |
Committee Member |
| Robert L. Macdonald |
Committee Member |
| Roger J. Colbran |
Committee Member |
|
| Keywords |
- sucrose density gradient
- blocking forward trafficking
- PNGaseF
- endo H
|
| Date of Defense |
2008-08-13 |
| Availability |
unrestricted |
Abstract
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.
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| Files |
| Filename |
Size |
Approximate Download Time
(Hours:Minutes:Seconds)
|
| 28.8 Modem |
56K Modem |
ISDN (64 Kb) |
ISDN (128 Kb) |
Higher-speed Access |
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Wenyi_Lo_Dissertation.pdf |
3.41 Mb |
00:15:47 |
00:08:07 |
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