Different combinations of cysteine-rich repeats mediate binding of low density lipoprotein receptor to two different proteins.
Seven imperfect repeats of a 40-amino acid cysteine-rich sequence constitute the ligand binding domain of the low density lipoprotein (LDL) receptor. To assess the contribution of each repeat, three site-directed mutations were made individually in each repeat: 1) deletion of the repeat, 2) substitution of a conserved isoleucine with aspartic acid, and 3) substitution of a conserved aspartic acid with tyrosine. cDNAs containing these mutations were transfected into simian COS cells and assayed for their ability to bind LDL, which contains a 500-kDa protein ligand (apoB-100), and beta-migrating very low density lipoprotein (beta-VLDL), which contains multiple copies of a 33-kDa ligand (apoE). The results showed that binding of the two ligands required different combinations of repeats. LDL binding required repeats 3-7; deletion of any one of these repeats markedly reduced LDL binding. In contrast, beta-migrating very low density lipoprotein binding was insensitive to the loss of any single repeat with the important exception of repeat 5, whose loss reduced binding by 60%. The same effects were obtained when each of the repeats was altered by either of the two substitution mutations. The current findings suggest that a multiplicity of cysteine-rich repeats may allow a single protein to bind several different protein ligands by employing different combinations of repeats.
Russell
D W
DW
Department of Molecular Genetics, University of Texas Southwestern Medical Center, Dallas 75235.
Brown
M S
MS
Goldstein
J L
JL
D000595:Amino Acid Sequence; D000818:Animals; D002460:Cell Line; D003545:Cysteine; D004247:DNA / Q000235:genetics; D005822:Genetic Vectors; D006801:Humans; D007700:Kinetics; D008024:Ligands; D009154:Mutation; D010957:Plasmids; D011973:Receptors, LDL / Q000235:genetics* / Q000378:metabolism; D012091:Repetitive Sequences, Nucleic Acid; D012689:Sequence Homology, Nucleic Acid; D014162:Transfection
D008024:Ligands; D011973:Receptors, LDL; D004247:DNA; D003545:Cysteine