Expression in vitro of alternatively spliced variants of the messenger RNA for 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 of the low-density lipoprotein (LDL)-receptor family that is expressed in brain. We have identified mRNA splicing variants in human tissues by ribonuclease protection assays and found that some variants are preferentially amplified by reverse transcription-polymerase chain reaction (RT-PCR). Transcripts 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 protein isoforms were transfected into LDL-receptor-deficient Chinese hamster ovary cells, the proteins were all expressed on the cell surface, as detected by immunoblotting of cell extracts with a specific antipeptide antiserum to apoER2 before and after treatment of intact cells with pronase. Although cells expressing all the variants bound very low-density lipoprotein of beta mobility (beta-VLDL), it was with lower affinity and capacity than binding by the LDL-receptor and none was able to degrade beta-VLDL. Ligand blotting of cell extracts showed that all variants bound recombinant histidine6-tagged receptor-associated protein (His6-RAP) with high affinity, although variants lacking exon 5 bound less strongly. The presence of vestiges of the novel insert in the cytoplasmic domain of apoER2 in the LDL- or VLDL-receptor genes was investigated, but nucleotide sequencing showed that no sequences homologous to it could be detected in the final intron of these genes.
Sun
X M
XM
Lipoprotein Team, MRC Clinical Sciences Centre, Imperial College School of Medicine, Hammersmith Hospital, London, UK.
Soutar
A K
AK
D017398:Alternative Splicing; D000818:Animals; D001483:Base Sequence; D015153:Blotting, Western; D016466:CHO Cells; D006224:Cricetinae; D017931:DNA Primers; D018076:DNA, Complementary; D006801:Humans; D026502:LDL-Receptor Related Proteins; D012333:RNA, Messenger / Q000235:genetics*; D011973:Receptors, LDL / Q000235:genetics; D018110:Receptors, Lipoprotein / Q000235:genetics* / Q000378:metabolism; D012260:Ribonucleases / Q000378:metabolism
D017931:DNA Primers; D018076:DNA, Complementary; D026502:LDL-Receptor Related Proteins; D012333:RNA, Messenger; D011973:Receptors, LDL; D018110:Receptors, Lipoprotein; C036053:VLDL receptor; C098773:low density lipoprotein receptor-related protein 8; D012260:Ribonucleases