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Title page for ETD etd-11242008-214043


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
  • PNGaseF
  • blocking forward trafficking
  • sucrose density gradient
  • 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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