Amyloid precursor protein in unique cholesterol-rich microdomains different from caveolae-like domains

To determine the localization of the amyloid precursor protein (APP) on the cellular membrane, we performed membrane fractionation of cultured cells i ncluding that of Madin-Darby canine kidney (MDCK) and P19 cells transfected with human APP cDNA, non-transfected SH-SY5Y cells, and rat cerebral corti ces. In MDCK cells, APP was exclusively present in abundance in the superna tant following solubilization of the plasma membranes using Triton X-100, a nd in high-density fractions of sucrose density gradient fractionation (SDG F) following Triton X-100 solubilization of whole cellular membranes. Caveo lin-1 was not cofractionated with APP. In experiments using P19 cells and r at cerebral cortices, we detected two isoforms of APP. The APP with the app arently 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 l ow-density fractions with cholesterol and GM1 gangliosides, the concentrati ons 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 foll owing; first, APP is not present in abundance in caveolae or CLDs, but is i n unique cholesterol-rich microdomains; second, the targeting of APP to the se unique microdomains may be linked to the maturation of APP in some cells . (C) 2000 Elsevier Science B.V. All rights reserved.