The low-density lipoprotein receptor-related protein-1 associates transiently with lipid rafts.
The low-density lipoprotein receptor-related protein-1 (LRP-1) is a multifunctional receptor that undergoes constitutive endocytosis and recycling. To identify LRP-1 in lipid rafts, we biotin-labeled cells using a membrane-impermeable reagent and prepared Triton X-100 fractions. Raft-associated proteins were identified in streptavidin affinity-precipitates of the Triton X-100-insoluble fraction. PDGF beta-receptor was identified exclusively in lipid rafts, whereas transferrin receptor was excluded. LRP-1 distributed partially into rafts in murine embryonic fibroblasts (MEFs) and HT 1080 cells, but not in smooth muscle cells and CHO cells. LRP-1 partitioning into rafts was not altered by ligands, including alpha2-macroglobulin, platelet-derived growth factor-BB, and receptor-associated protein (RAP). To examine LRP-1 trafficking between membrane microdomains, we developed a novel method based on biotinylation and detergent fractionation. Association of LRP-1 with rafts was transient; by 15 min, nearly all of the LRP-1 that was initially raft-associated exited this compartment. LRP-1 in the Triton X-100-soluble fraction, which excludes lipid rafts, demonstrated complex kinetics, with phases reflecting import from rafts, endocytosis, and recycling. Potassium depletion blocked LRP-1 endocytosis but did not inhibit trafficking of LRP-1 from rafts into detergent-soluble microdomains. Our data support a model in which LRP-1 transiently associates with rafts but does not form a stable pool. Fluid movement of LRP-1 between microdomains may facilitate its function in promoting the endocytosis of other plasma membrane proteins, such as the urokinase receptor, which localizes in lipid rafts.
Wu
Lihua
L
Department of Pathology, University of California San Diego, La Jolla, California 92093, USA.
Gonias
Steven L
SL
D000818:Animals; D000077214:Becaplermin; D016466:CHO Cells; D002462:Cell Membrane / Q000378:metabolism; D002478:Cells, Cultured; D002499:Centrifugation, Density Gradient; D002966:Clathrin / Q000737:chemistry; D006224:Cricetinae; D003902:Detergents / Q000494:pharmacology; D004705:Endocytosis; D005347:Fibroblasts / Q000378:metabolism; D006801:Humans; D007700:Kinetics; D008024:Ligands; D008055:Lipids / Q000737:chemistry; D026503:Low Density Lipoprotein Receptor-Related Protein-1 / Q000378:metabolism*; D021962:Membrane Microdomains; D051379:Mice; D032389:Myocytes, Smooth Muscle / Q000378:metabolism; D017830:Octoxynol / Q000494:pharmacology; D010982:Platelet-Derived Growth Factor / Q000378:metabolism; D020574:Proto-Oncogene Proteins c-sis; D020797:Receptor, Platelet-Derived Growth Factor beta / Q000378:metabolism; D011990:Receptors, Transferrin / Q000378:metabolism; D013347:Subcellular Fractions / Q000378:metabolism; D013997:Time Factors; D014461:Ultracentrifugation; D000511:alpha-Macroglobulins / Q000378:metabolism
D002966:Clathrin; D003902:Detergents; D008024:Ligands; D008055:Lipids; D026503:Low Density Lipoprotein Receptor-Related Protein-1; D010982:Platelet-Derived Growth Factor; D020574:Proto-Oncogene Proteins c-sis; D011990:Receptors, Transferrin; D000511:alpha-Macroglobulins; D000077214:Becaplermin; D017830:Octoxynol; D020797:Receptor, Platelet-Derived Growth Factor beta