The LDL receptor: oligonucleotide-directed mutagenesis of the cytoplasmic domain

Mutations at many different sites in the gene for the low density lipoprotein (LDL) receptor can cause the disease familial hypercholesterolaemia. A particularly interesting class of mutations includes those producing 'internalization-defective' receptors-receptors which are expressed on the cell surface and which bind LDL normally, but which fail to cluster in coated pits. This defect was first observed in fibroblasts from patient J.D. Cloning and sequencing of the terminal exons of J.D.'s internalization-defective LDL receptor gene revealed a single point mutation which caused the substitution of cysteine for tyrosine at residue 807. On the basis of this observation, we have used techniques of oligonucleotide-directed mutagenesis to make an extensive series of mutations in the full-length LDL receptor cDNA. Stable cell-lines expressing these mutant receptors have been analysed for receptor function. Of 13 different amino acids expressed at residue 807, only the aromatics tyrosine, phenylalanine and tryptophan allowed rapid internalization. Position 807 seems to be a particularly sensitive site, since neither substitution of a cysteine for residue 806 or 808 nor deletion of two triplets downstream had any effect on receptor internalization. In addition, a series of truncations localize the signals for internalization to the membrane-proximal 22 amino acid residues.