As much as 29.3% of GLUT4, 45.5% of GLUT1, 58.5% of clathrin, and 17.2% of insulin receptor immunoreactivities in rat adipocyte plasma membranes (PM) were found to be insoluble upon 1% Triton extraction at basal state. Bv insulin treatment the
distributions of insoluble insoluble fraction were changed by 16.2% of GLUT4. 48.5% of GLUT1, 65.3% of clathrin and 31.0% of insulin receptor, repectively. SDS-PAGF revealed that the Triton-insoluble PM fraction contains a number of protein
species
including 110, 80, 70, 50 30-33 and 15 KDa polyeptides. When PM was prewashed with alkaline buffer and 0.5M Tris buffer, which removed most of extrinsic membrane proteins includine clathrin and AP-2 form PM< virtually all of the GLUT4 and GLUT1
in
PM
became soluble in 1% Triton. Subcellular fractionation followed by Western blot indicated that AP-2 distribute to 4.8% at PM/NM, 25.7% at HDM, 38.9% at LDM and 30.6% at cytosol, respectively An insulin treatment which increased GLUT4 content in
PM
by
1.5 fold increased the AP-2 content in PM nearly 2.3 fold with a concomitant decrease in cytosol AP-2contents. These findings suggest that subpopulations of GLUT4 and insulin receptor in the plasma membrane of adipocytes are in specific
association
with
extrinsic proteins, possibly clathrin and/or AP-2, and this association may play a key role in the translocation mechanism of GLUT4 in rat epididymal adipocytes.
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