title: Rapid characterization of disease-causing mutations in the low density lipoprotein receptor (LDL-R) gene by overexpression in COS cells.
abstract: To characterize disease-causing mutations in the low density lipoprotein receptor (LDL-R) gene, COS cells are transfected with the mutant gene in an EBV-based expression vector and characterized by flow cytometry. Using antibodies against the LDL-receptor the amount of receptor protein on the cell surface is quantitated. The receptor activity is measured by incubating the cells with fluorescence labeled LDL (Dil-labelled LDL) at 37 degrees C and 4 degrees C. The transfected cells stained with anti-LDL-R antibodies can also be analysed by immunofluorescence microscopy allowing the study of the intracellular location of variants of the receptor. To evaluate these methods, we are analyzing four previously well-characterized LDL-R mutations, belonging to each of the classes 2 to 5. Preliminary data show that mutant genes belonging to class 3 and 4A give rise to receptor protein on the cell surface, but impaired LDL uptake, while mutant receptors belonging to class 2A and 5 can only be detected intracellularly. Expression of the class 2A mutation results in an ER staining pattern, whereas the class 5 mutation gives rise to an intracellular staining compatible with localization in the endosomal/lysosomal compartments. We conclude that this system is useful for a rapid functional analysis of newly discovered mutations in the LDL-R gene.
authors.affiliation: Institute of Human Genetics, Aarhus University, Denmark.
authors.forename: T G
authors.initials: TG
authors.lastname: Jensen
authors.forename: B S
authors.initials: BS
authors.lastname: Andresen
authors.forename: H K
authors.initials: HK
authors.lastname: Jensen
authors.forename: L G
authors.initials: LG
authors.lastname: Jensen
authors.forename: F
authors.initials: F
authors.lastname: Heath
authors.forename: S
authors.initials: S
authors.lastname: Pedersen
authors.forename: V
authors.initials: V
authors.lastname: Nielsen
authors.forename: U B
authors.initials: UB
authors.lastname: Jensen
authors.forename: T B
authors.initials: TB
authors.lastname: Lund
authors.forename: N
authors.initials: N
authors.lastname: Gregersen
authors.forename: S
authors.initials: S
authors.lastname: Kølvraa
authors.forename: L
authors.initials: L
authors.lastname: Bolund
mesh_terms: D002460:Cell Line; D004252:DNA Mutational Analysis; D005434:Flow Cytometry; D006801:Humans; D006938:Hyperlipoproteinemia Type II / Q000235:genetics*; D008856:Microscopy, Fluorescence; D016296:Mutagenesis; D016133:Polymerase Chain Reaction; D011973:Receptors, LDL / Q000235:genetics*; D014162:Transfection / Q000235:genetics
chemical_list: D011973:Receptors, LDL