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Structural determinants of GABAA receptor biogenesis

dc.creatorLo, Wen-yi
dc.description.abstractThis 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.
dc.subjectblocking forward trafficking
dc.subjectsucrose density gradient
dc.subjectendo H
dc.titleStructural determinants of GABAA receptor biogenesis
dc.contributor.committeeMemberRobert L. Macdonald
dc.contributor.committeeMemberRoger J. Colbran
dc.contributor.committeeMemberJerod Scott Denton
dc.type.materialtext University
dc.contributor.committeeChairDanny G. Winder

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