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 (NPTY3AATA) of the cytoplasmic domain or in the furin cleavage site (RHRR3AHAA) 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 (NPVYATL3AAVAATL) 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.

Polarized Traffic of LRP1 Involves AP1B and SNX17 Operating on Y-dependent Sorting Motifs in Different Pathways

“…We could anticipate, in accordance with recent data (Roebroek et al, 2006), that natural mutants affecting those critical sorting motifs that we have defined, especially the proximal NPxY, would be relatively unviable.…”

Recombinase-mediated cassette exchange reveals the selective use of Gq/G11-dependent and -independent endothelin 1/endothelin type A receptor signaling in pharyngeal arch development

“…Indeed, the efficiency of RMCE-mediated knock-in recombination was very high in our screening (44 to 87%), compared with conventional homologous recombination. This is similar to or higher than the efficiencies previously reported for RMCE using the Cre-lox system (Araki et al, 2006;Liu et al, 2006;Toledo et al, 2006) or the Flp-FRT system (Cesari et al, 2004;Roebroek et al, 2006). In our RMCE procedure, we replaced a 1.0 kb genomic sequence with knock-in sequences containing mutant lox sites and an FRT-flanked Puro.…”

Structural and Functional Consequences of Tyrosine Phosphorylation in the LRP1 Cytoplasmic Domain

“…The distal NPXY 4507 motif has been shown to bind signaling proteins (10,16,25), and the proximal NPXY 4473 motif has been shown to be involved in receptor recycling and demonstrated a strong phenotype in knock-in mice (7,8). Binding of Shc and Dab1 has been localized to the NPXY 4507 motif and a few surrounding residues using peptides as the pull-down bait (4,9).…”

“…Mutational studies identified the YXXL motif (which overlaps with Tyr 4507 ) as critical for LRP1 receptor endocytosis (6). Recently, the membrane proximal NPXY motif has been identified as the binding site for Snx17 (sorting nexin 17), and knock-in experiments in mice showed a strong phenotype upon mutation of this site (7,8). Mutation of Tyr 4473 to alanine, which abolishes Snx17 binding, resulted in impaired receptor recycling and reduced amounts of the mature form of LRP1 on the cell surface (7).…”