The 39-kDa receptor-associated protein regulates ligand binding by the very low density lipoprotein receptor.
A 39-kDa receptor associated protein (RAP) binds and inhibits ligand binding by two members of the low density lipoprotein (LDL) receptor family, gp330 and low density lipoprotein receptor-related protein/alpha 2-macroglobulin receptor. To determine if additional members of the LDL receptor family may interact with RAP, Chinese hamster ovary cells were transfected with plasmids directing expression of the very low density lipoprotein (VLDL) receptor cDNA or the LDL receptor cDNA. Detergent-soluble extracts from these and normal Chinese hamster ovary cells were subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis, after which the proteins were transferred to nitrocellulose membranes and incubated with RAP. When detergent extracts from normal cells were incubated with RAP, several polypeptides, including a 130-kDa protein, were observed to bind RAP. In cells transfected with the VLDL receptor cDNA, a substantial increase in RAP binding to the 130-kDa polypeptide was noted. This protein was identified as the VLDL receptor by immunoblotting. The VLDL receptor present in detergent extracts from transfected cells bound to RAP-Sepharose, and a KD of 0.7 nM for the interaction between RAP and the purified VLDL receptor was determined using enzyme-linked immunosorbent assay. The purified VLDL receptor bound 125I-labeled VLDL, but not 125I-labeled LDL, and the binding of 125I-labeled VLDL was completely inhibited by RAP. Further, RAP inhibited the uptake and degradation of 125I-VLDL by cells overexpressing the VLDL receptor. Thus the VLDL receptor represents the third member of the LDL receptor family whose ligand binding properties are antagonized by RAP. This suggests a common functional role for RAP in modulating ligand binding by members of the LDL receptor family.
Battey
F D
FD
Holland Laboratory, Department of Biochemistry, American Red Cross, Rockville, Maryland 20855.
Gåfvels
M E
ME
FitzGerald
D J
DJ
Argraves
W S
WS
Chappell
D A
DA
Strauss
J F
JF
Strickland
D K
DK
D000595:Amino Acid Sequence; D000818:Animals; D015153:Blotting, Western; D002352:Carrier Proteins / Q000378:metabolism*; D006224:Cricetinae; D003412:Cricetulus; D018076:DNA, Complementary; D006023:Glycoproteins / Q000378:metabolism*; D006801:Humans; D007457:Iodine Radioisotopes; D026563:LDL-Receptor Related Protein-Associated Protein; D008024:Ligands; D008079:Lipoproteins, VLDL / Q000378:metabolism*; D008565:Membrane Proteins / Q000378:metabolism; D008969:Molecular Sequence Data; D010455:Peptides / Q000378:metabolism; D011973:Receptors, LDL / Q000235:genetics / Q000378:metabolism*; D012685:Sepharose / Q000378:metabolism; D014162:Transfection
D002352:Carrier Proteins; D018076:DNA, Complementary; D006023:Glycoproteins; D007457:Iodine Radioisotopes; D026563:LDL-Receptor Related Protein-Associated Protein; D008024:Ligands; D008079:Lipoproteins, VLDL; D008565:Membrane Proteins; D010455:Peptides; D011973:Receptors, LDL; C036053:VLDL receptor; D012685:Sepharose