First cysteine-rich repeat in ligand-binding domain of low density lipoprotein receptor binds Ca2+ and monoclonal antibodies, but not lipoproteins.
The ligand binding domain of the low density lipoprotein receptor consists of seven cysteine-rich repeats of approximately 40 amino acids each. These repeats, which are located at the NH2 terminus of the protein, are homologous to sequences in complement components C8 and C9. To determine the role of the first repeat (amino acids 2-42), we prepared two plasmids containing expressible low density lipoprotein receptor cDNAs. The first plasmid, p delta R1, lacks only the nucleotides encoding the first repeat. It produced a receptor that bound and internalized lipoproteins and recycled to the cell surface with the same efficiency as the normal receptor. This deleted receptor failed to bind two monoclonal antibodies, IgG-C7 and IgG-15C8, which were shown previously to react with the ligand-binding domain. The second plasmid, pR1, encodes a markedly truncated protein whose extracellular domain consists of the first repeat joined to the transmembrane and cytoplasmic domains. This protein bound the two monoclonal antibodies with the same affinity as the normal receptor, but failed to bind lipoproteins. Binding of IgG-15C8 to the normal receptor and the pR1-encoded protein was Ca2+-dependent, indicating that the first repeat binds Ca2+. We conclude that repeats 2-6 in the ligand-binding domain are sufficient for binding lipoproteins and that the first repeat is highly immunogenic, but is not required for lipoprotein binding.
van Driel
I R
IR
Department of Molecular Genetics, University of Texas Health Science Center, Southwestern Medical School, Dallas 75235.
Goldstein
J L
JL
Südhof
T C
TC
Brown
M S
MS
D000911:Antibodies, Monoclonal; D002118:Calcium / Q000378:metabolism*; D002872:Chromosome Deletion; D003545:Cysteine / Q000032:analysis*; D006801:Humans; D007163:Immunosorbent Techniques; D008079:Lipoproteins, VLDL / Q000378:metabolism; D011973:Receptors, LDL / Q000378:metabolism*; D012091:Repetitive Sequences, Nucleic Acid; D014162:Transfection
D000911:Antibodies, Monoclonal; D008079:Lipoproteins, VLDL; D011973:Receptors, LDL; D003545:Cysteine; D002118:Calcium