Expression in vitro of alternatively spliced variants of the messenger RNAfor human apolipoprotein E receptor-2 identified in human tissues by ribonuclease protection assays

The apolipoprotein E receptor-2 (apoER2), also called LR7/8B, is a member o f the low-density lipoprotein (LDL)-receptor family that is expressed in br ain. We have identified mRNA splicing variants in human tissues by ribonucl ease protection assays and found that some variants are preferentially ampl ified by reverse transcription-polymerase chain reaction (RT-PCR). Transcri pts were found that lacked sequences encoding three repeats in the putative ligand-binding domain, the O-linked sugar domain or a novel region in the cytoplasmic domain. When mammalian expression vectors for eight potential p rotein isoforms were transfected into LDL-receptor-deficient Chinese hamste r ovary cells, the proteins were all expressed on the cell surface, as dete cted by immunoblotting of cell extracts with a specific antipeptide antiser um to apoER2 before and after treatment of intact cells with pronase. Altho ugh cells expressing all the variants bound very low-density lipoprotein of beta mobility (P-VLDL), it was with lower affinity and capacity than bindi ng by the LDL-receptor and none was able to degrade P-VLDL. Ligand blotting of cell extracts showed that all variants bound recombinant histidine(6)-t agged receptor-associated protein (His(6)-RAP) with high affinity, although variants lacking exon 5 bound less strongly. The presence of vestiges of t he novel insert in the cytoplasmic domain of apoER2 in the LDL- or VLDL-rec eptor genes was investigated, but nucleotide sequencing showed that no sequ ences homologous to it could be detected in the final intron of these genes .