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 b inding by two members of the low density lipoprotein (LDL) receptor fa mily, 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 d ensity lipoprotein (VLDL) receptor cDNA or the LDL receptor cDNA Deter gent-soluble extracts from these and normal Chinese hamster ovary cell s were subjected to sodium dodecyl sulfate-polyacrylamide gel electrop horesis, after which the proteins were transferred to nitrocellulose m embranes and incubated with RAP. When detergent extracts from normal c ells were incubated with RAP, several polypeptides, including a 130-kD a protein, were observed to bind RAP. In cells transfected with the VL DL receptor cDNA, a substantial increase in RAP binding to the 130-kDa polypeptide was noted. This protein was identified as the VLDL recept or by immunoblotting. The VLDL receptor present in detergent extracts from transfected cells bound to RAP-Sepharose, and a K-D of 0.7 nM for the interaction between RAP and the purified VLDL receptor was determ ined using enzyme-linked immunosorbant assay. The purified VLDL recept or bound I-125-labeled VLDL, but not I-125-labeled LDL, and the bindin g of I-125-labeled VLDL was completely inhibited by RAP. Further, RAP inhibited the uptake and degradation of I-125-VLDL by cells overexpres sing the VLDL receptor. Thus the VLDL receptor represents the third me mber of the LDL receptor family whose ligand binding properties are an tagonized by RAP. This suggests a common functional role for RAP in mo dulating ligand binding by members of the LDL receptor family.