title: Mutant Lrp1 knock-in mice generated by recombinase-mediated cassette exchange reveal differential importance of the NPXY motifs in the intracellular domain of LRP1 for normal fetal development.
abstract: Lrp1 knock-in mice carrying either a wild-type allele or three different mutated alleles encoding the multifunctional endocytic receptor LRP1 were generated by recombinase-mediated cassette exchange (RMCE). Reinsertion by RMCE of a wild-type allele led to a normal pattern and level of gene expression and a completely normal phenotype, indicating that the RMCE procedure itself is neutral with respect to the function of the gene locus. In contrast, reinsertion of mutated LRP1 alleles carrying either inactivating mutations in the proximal NPXY motif (NPTY-->AATA) of the cytoplasmic domain or in the furin cleavage site (RHRR-->AHAA) caused distinctive liver phenotypes: respectively, either a late fetal destruction of the organ causing perinatal death or a selective enlargement of von-Kupffer cell lysosomes reminiscent of a mild lysosomal storage without an apparent negative effect on animal survival. Notably, mutation of the distal NPXY motif overlapping with an YXXL motif (NPVYATL-->AAVAATL) did not cause any obvious pathological effect. The mutations showed no effect on the LRP1 expression level; however, as expected, the proteolytic maturation of LRP1 into its two subunits was significantly impaired, although not completely abolished, in the furin cleavage mutant. These data demonstrate that RMCE is a reliable and efficient approach to generate multiple mutant knock-in alleles for in vivo functional analysis of individual domains or motifs of large multidomain proteins. Its application in Lrp1 reveals dramatically variant phenotypes, of which further characterization will definitively contribute to our understanding of the biology of this multifunctional receptor.
authors.affiliation: Experimental Mouse Genetics, Center for Human Genetics, KU Leuven and Flanders Interuniversity Institute for Biotechnology, Herestraat 49, bus 602, B-3000 Leuven, Belgium. anton.roebroek@med.kuleuven.be
authors.forename: Anton J M
authors.initials: AJ
authors.lastname: Roebroek
authors.forename: Sara
authors.initials: S
authors.lastname: Reekmans
authors.forename: Annick
authors.initials: A
authors.lastname: Lauwers
authors.forename: Nathalie
authors.initials: N
authors.lastname: Feyaerts
authors.forename: Liesbet
authors.initials: L
authors.lastname: Smeijers
authors.forename: Dieter
authors.initials: D
authors.lastname: Hartmann
mesh_terms: D000483:Alleles; D020816:Amino Acid Motifs; D000818:Animals; D002460:Cell Line; D020964:Embryo Loss; D047109:Fetal Development / Q000502:physiology*; D045683:Furin / Q000378:metabolism; D015870:Gene Expression; D007728:Kupffer Cells / Q000378:metabolism / Q000473:pathology; D008099:Liver / Q000378:metabolism / Q000473:pathology; D026503:Low Density Lipoprotein Receptor-Related Protein-1; D008247:Lysosomes / Q000378:metabolism / Q000473:pathology; D051379:Mice; D008817:Mice, Mutant Strains; D016254:Mutagenesis, Insertional; D009154:Mutation; D010641:Phenotype; D021122:Protein Subunits / Q000235:genetics / Q000378:metabolism; D011973:Receptors, LDL / Q000235:genetics / Q000378:metabolism*; D045522:Recombinases / Q000235:genetics*; D025521:Tumor Suppressor Proteins / Q000235:genetics / Q000378:metabolism*
chemical_list: D026503:Low Density Lipoprotein Receptor-Related Protein-1; C486645:Lrp1 protein, mouse; D021122:Protein Subunits; D011973:Receptors, LDL; D045522:Recombinases; D025521:Tumor Suppressor Proteins; D045683:Furin