Grants and Contracts Details
Description
Sitosterolemia is a genetic disorder caused by mutations in ABCG5 (G5) or ABCG8 (G8) that results in
hyperabsorption of dietary sterols and a failure to excrete sterols into bile. In humans and mouse models of
this disease, a key feature is diet responsiveness to cholesterol. In addition, mice deficient in G5 and G8 fail to
excrete cholesterol into bile. Thus, G5 and G8 playa critical role in reducing the absorption of dietary sterols
and the excretion of both dietary and endogenously synthesized cholesterol. G5 and G8 are ATP binding
cassette half-transporters that must dimerize in order to traffic beyond the endoplasmic reticulum (ER) and
function. The G5/G8 heterodimer is localized on the apical surface of enterocytes (lumenal) and hepatocytes
(canalicular) in polarized cells and in mice. The transport of the G5G8 complex to the cell surface can be
divided into a three step process: 1) dimer formation, 2) escape from the ER quality control machinery and
sorting into COPII vesicles, and 3) trafficking to the apical surface. The molecular determinants for G5/G8
heterodimer formation and trafficking are not known. The specific aims of this proposal are to 1) determine the
motif(s) required for dimerization of G5 and G8, 2) determine the residues within G5 and G8 monitored by the
ER quality control system and COPII sorting machinery, and 3) determine the motif that confers apical targeting
of the G5/G8 sterol transporter. Chimeras between G2 and G8 will be used to broadly define domains within
G8 that are necessary for dimerization, trafficking and apical targeting of the G5/G8 sterol transporter.
Additional chimeras and site-directed mutagenesis studies will be utilized to further define these domains.
Chimeras and mutants will be expressed in CHO cells and analyzed by endoglycosidases,
immunoprecipitation, sedimentation gradients, immunofluorescence microscopy, cell fractionation and native
gel electrophoresis in order to determine the domains within this complex that mediate the proper folding and
trafficking of this sterol transporter to the apical surface.
Status | Finished |
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Effective start/end date | 1/1/05 → 12/31/05 |
Funding
- American Heart Association: $65,000.00
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