We have discovered two metabolically distinct lipoprotein receptors on liver membranes: an apo-B,E(LDL) receptor, which binds to both apo-B-containing LDL and apo-E-containing lipoproteins,and an apo-E specific receptor, which binds lipoproteins containing apo-E but not the apo-B-containing LDL. During the past funding period, we have purified the apo-E receptor from dog liver membranes. The apo-E receptor is immunologically related to the apo-B,E(LDL) receptor, but it is a distinct protein with a molecular weight of 66,000. The hepatic apo-B,E(LDL) receptor is identical to the extrahepatic LDL receptor with an apparent molecular weight of 130,000 on nonreducing SDS-polyacrylamide gels. The objective of this proposal is to determine the structural characteristics of the apo-E receptor and to compare these characteristics with those of the apo-B,E(LDL) receptor.
The first aim of the proposed research is the production of monoclonal antibodies against the apo-E receptor and against the apo-B,E(LDL) receptor. In addition, specific antibodies will also be prepared against synthetic peptides corresponding to different domains of the apo-B,E(LDL) receptor. These peptide-specific antibodies will be used to identify common and/or distinct epitopes between the apo-B,E(LDL) receptor and the apo-E receptor. The second goal of the proposed research is the molecular cloning of the apo-E receptor. A cDNA corresponding to the apo-E receptor will be obtained from a human liver cDNA library. We will initially exploit the apparent homology between the two receptors and use an LDL receptor cDNA probe to screen for the apo-E receptor cDNA. If these initial attempts fail to identify the apo-E receptor clone, we will use information obtained from results of the peptide specific antibodies, and we will prepare oligonucleotide probes against the amino acid sequences common to both the apo-B,E(LDL) and the apo-E receptors. The oligonucleotide probes will then be used for screening of cDNA library for the apo-E receptor gene. The nucleotide sequence of the apo-E receptor cDNA will be determined and will be used to compare the primary structure of the apo-E receptor with that of the apo-B,E(LDL) receptor. The isolated apo-E receptor cDNA will be used to screen a genomic DNA library for the apo-E receptor gene. The apo-E receptor gene will then be transfected into LDL receptor-defective CHO mutants to study the regulation and the function of the apo-E receptor.
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