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Amyloid precursor protein in unique cholesterol-rich microdomains different from caveolae-like domains
H Hayashi1, T Mizuno, M Michikawa
1Department of Dementia Research, National Institute for Longevity Sciences, 36-3 Gengo, Morioka, Obu, Japan.
Abstract:
To determine the localization of the amyloid precursor protein (APP) on the cellular membrane, we performed membrane fractionation of cultured cells including that of Madin-Darby canine kidney (MDCK) and P19 cells transfected with human APP cDNA, non-transfected SH-SY5Y cells, and rat cerebral cortices. In MDCK cells, APP was exclusively present in abundance in the supernatant following solubilization of the plasma membranes using Triton X-100, and in high-density fractions of sucrose density gradient fractionation (SDGF) following Triton X-100 solubilization of whole cellular membranes. Caveolin-1 was not cofractionated with APP. In experiments using P19 cells and rat cerebral cortices, we detected two isoforms of APP. The APP with the apparently lower molecular weight (immature type) coexisted in abundance with integrin in the high-density fractions, whereas the APP with the apparently higher molecular weight (mature type) was recovered predominantly in the low-density fractions with cholesterol and GM1 gangliosides, the concentrations of which were higher than those in the bulk plasma membranes, but lower than those in caveolae-like domains (CLDs), following SDGF of Triton X-100-solubilized cellular membranes. The results of this study suggest the following; first, APP is not present in abundance in caveolae or CLDs, but is in unique cholesterol-rich microdomains; second, the targeting of APP to these unique microdomains may be linked to the maturation of APP in some cells.
Insights
Amyloid precursor protein (APP) localizes to unique cholesterol-rich microdomains, not caveolae, on cell membranes. APP targeting to these domains may correlate with its maturation process in certain cells.
Area of Science:
- Cell Biology
- Neuroscience
- Biochemistry
Background:
- The cellular membrane localization of amyloid precursor protein (APP) is crucial for understanding its function and role in neurological processes.
- Previous studies have suggested potential associations of APP with membrane microdomains, but precise localization remains debated.
Purpose of the Study:
- To investigate the precise localization of amyloid precursor protein (APP) within cellular membranes.
- To determine if APP is associated with caveolae or other specific membrane microdomains.
Main Methods:
- Utilized membrane fractionation techniques on cultured cells (MDCK, P19, SH-SY5Y) and rat cerebral cortices.
- Employed Triton X-100 solubilization and sucrose density gradient fractionation (SDGF) to separate membrane components.
- Analyzed protein co-fractionation with APP, cholesterol, and GM1 gangliosides.
Main Results:
- APP was found in unique cholesterol-rich microdomains, distinct from caveolae or caveolae-like domains (CLDs).
- Two APP isoforms were identified: an immature type co-localizing with integrin and a mature type associated with cholesterol and GM1 gangliosides.
- Caveolin-1 did not co-fractionate with APP, further differentiating APP's localization from caveolae.
Conclusions:
- Amyloid precursor protein (APP) resides in specific cholesterol-rich microdomains, not abundant in caveolae.
- APP's targeting to these unique microdomains may be linked to its post-translational modification and maturation in certain cell types.