Mutational analysis of the ligand binding domain of the low density lipoprotein receptor.
The ligand binding domain of the low density lipoprotein (LDL) receptor contains seven imperfect repeats of a 40-amino acid cysteine-rich sequence. Each repeat contains clustered negative charges that have been postulated as ligand-binding sites. The adjacent region of the protein, the growth factor homology region, contains three cysteine-rich repeats (A-C) whose sequence differs from those in the ligand binding domain. To dissect the contribution of these different cysteine-rich repeats to ligand binding, we used oligonucleotide-directed mutagenesis to alter expressible cDNAs for the human LDL receptor which were then introduced into monkey COS cells by transfection. We measured the ability of the mutant receptors to bind LDL, which contains a single protein ligand for the receptor (apoB-100), and beta-migrating very low density lipoprotein (beta-VLDL), which contains apoB-100 plus multiple copies of another ligand (apoE). The results show that repeat 1 is not required for binding of either ligand. Repeats 2 plus 3 and repeats 6 plus 7 are required for maximal binding of LDL, but not beta-VLDL. Repeat 5 is required for binding of both ligands. Repeat A in the growth factor homology region is required for binding of LDL, but not beta-VLDL. Repeat B is not required for ligand binding. These results support a model for the LDL receptor in which various repeats play additive roles in ligand binding, each repeat making a separate contribution to the binding event.
Department of Molecular Genetics, University of Texas Southwestern Medical Center, Dallas 75235.
V
V
Esser
L E
LE
Limbird
M S
MS
Brown
J L
JL
Goldstein
D W
DW
Russell
D000595:Amino Acid Sequence; D053299:Apolipoprotein B-100; D001055:Apolipoproteins B / Q000378:metabolism; D001057:Apolipoproteins E / Q000378:metabolism; D001483:Base Sequence; D001665:Binding Sites; D002872:Chromosome Deletion; D003545:Cysteine; D004247:DNA / Q000378:metabolism; D006801:Humans; D008969:Molecular Sequence Data; D009154:Mutation; D011973:Receptors, LDL / Q000235:genetics* / Q000378:metabolism; D014162:Transfection
D053299:Apolipoprotein B-100; D001055:Apolipoproteins B; D001057:Apolipoproteins E; D011973:Receptors, LDL; D004247:DNA; D003545:Cysteine