Article ID | Journal | Published Year | Pages | File Type |
---|---|---|---|---|
9886168 | Biochimica et Biophysica Acta (BBA) - General Subjects | 2005 | 13 Pages |
Abstract
The six β-propellers located within the N-terminus of low density lipoprotein receptor-related protein 1 (LRP1) are arranged in two clusters that contain two and four β-propellers, respectively. Working with LRP1 deletion mutants, we found that randomly removing large segments of amino acid sequences did not affect the intracellular trafficking of LRP1 as long as the clustered β-propeller domains were retained. However, deletion mutants with crippled β-propeller clusters invariably exhibited retarded exit from the endoplasmic reticulum (ER). To determine potential functions of the clustered β-propellers, we generated a series of deletion mutants in which the β-propellers were systematically removed from the C-terminal end of the second cluster. The resulting minireceptors, designated LRPβ1-6, β1-5, β1-4, β1-3, and β1-2 containing decreasing numbers of the β-propellers, were stably expressed in LRP1-null CHO cells. Binding/degradation assays with receptor-associated protein or α2-macroglobulin showed that removing one or more β-propellers had little effect on binding or degradation of these ligands. However, minireceptors containing odd number of β-propellers (i.e., LRPβ1-3 and β1-5) showed prolonged retention within the ER and remained endoglycosidase H-sensitive, whereas minireceptors containing even number of β-propellers (i.e., LRPβ1-2, β1-4 and β1-6) exited ER at variable rates. Cell surface biotinylation experiments showed that LRPβ1-3 was absent from the cell surface. Prolonged retention of LRPβ1-3 within the ER was accompanied by increased association with molecular chaperone Grp78/Bip. These results suggest that the clustered β-propellers may play a role in folding and intracellular trafficking of LRP1.
Keywords
EGFPAGELDLRHBSSTCALRP1RAPPNGaseFFBSDMEMpeptide: N-glycosidase FPBSα2MBSADulbecco's modified Eagle MediumHanks balanced salt solutionα2-macroglobulinβ-Propellerbovine serum albumintrichloroacetic acidpolyacrylamide gel electrophoresisEndo Hendoglycosidase HChoChinese Hamster Ovaryfetal bovine serumendoplasmic reticulumepidermal growth factorTraffickingPhosphate-buffered salineReceptor-associated proteinLow density lipoprotein receptor-related protein 1low density lipoprotein receptor
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Authors
Fengcheng Sun, Rita Kohen Avramoglu, Gerard Vassiliou, Robert J. Brown, Kerry W.S. Ko, Ruth McPherson, Zemin Yao,