Furin is an endoprotease of the family of mammalian proprotein convertases and is involved in the activation of a large variety of regulatory proteins by cleavage at basic motifs. A large number of substrates have been attributed to furin on the basis of in vitro and ex vivo data. However, no physiological substrates have been confirmed directly in a mammalian model system, and early embryonic lethality of a furin knock-out mouse model has precluded in vivo verification of most candidate substrates. Here, we report the generation and characterization of an interferon inducible Mx-Cre/loxP furin knock-out mouse model. Induction resulted in near-complete ablation of the floxed fur exon in liver.In sharp contrast with the general furin knock-out mouse model, no obvious adverse effects were observed in the transgenic mice after induction. Histological analysis of the liver did not reveal any overt deviations from normal morphology. Analysis of candidate substrates in liver revealed complete redundancy for the processing of the insulin receptor. Variable degrees of redundancy were observed for the processing of albumin, ␣ 5 integrin, lipoprotein receptor-related protein, vitronectin and ␣ 1 -microglobulin/bikunin. None of the tested substrates displayed a complete block of processing. The absence of a severe phenotype raises the possibility of using furin as a local therapeutic target in the treatment of pathologies like cancer and viral infections, although the observed redundancy may require combination therapy or the development of a more broad spectrum convertase inhibitor.

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.

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Objective-The purpose of this study was to determine the significance of the intracellular NPxYxxL motif of LRP1 for the atheroprotective role of this multifunctional receptor. Methods and Results-LRP1 knock-in mice carrying an inactivating mutation in the NPxYxxL motif were crossed with LDLR-deficient mice, a model for atherosclerosis. In this LDLR Ϫ/Ϫ background the mutated mice showed a more atherogenic lipoprotein profile, which was associated with a decreased clearance of postprandial lipids because of a compromised endocytosis rate and reduced lipase activity. On an atherogenic diet LRP1 mutant mice revealed a 50% increased development of atherosclerosis. This aggravation was accompanied by an increase in smooth muscle cell (SMC) and collagen content and apoptotic cells in the lesions. The mutation showed, however, a limited impact on basal PDGFR-␤ expression and signaling and the antimigratory property of apoE on PDGF-BB-stimulated SMCs. Additionally, levels of LRP1 atherogenic ligands, like MMP2, t-PA, FVIII, and the inflammatory ligand TNF-␣ showed to be significantly elevated.Conclusion-These findings demonstrate that the NPxYxxL motif is essential for the atheroprotective role of LRP1. This motif is relevant for normal control of lipid metabolism and of atherogenic and inflammatory ligands, but has no pronounced effect on regulating PDGF-BB/PDGFR-␤ signaling in SMCs. Key Words: atherosclerosis Ⅲ growth factors Ⅲ immune system Ⅲ lipases Ⅲ lipids T he large endocytic receptor low-density lipoprotein (LDL) receptor-related protein 1 (LRP1) is a multifunctional protein that binds multiple extracellular ligands including apolipoprotein E (apoE) containing lipoproteins, lipoprotein lipase, complexes of proteinases-proteinase-inhibitors, hormones, matrix proteins, and growth factors like plateletderived growth factor (PDGF; reviewed by Herz 1 ). This 600 kDa receptor is proteolytically cleaved by a furin-like endoprotease into 2 subunits of 515 kDa and 85 kDa. The very large extracellular ␣-subunit contains the ligand-binding domains and is noncovalent associated with the smaller ␤-subunit, containing an extracellular part, the membrane spanning domain and the cytoplasmatic or intracellular domain (LRP1-ICD).In several studies using conditional inactivation of LRP1 in mice, the receptor has been associated with a clear role in the pathogenesis of atherosclerosis. In the liver it has been shown that the receptor is important for the removal of atherogenic lipoproteins and other proatherogenic ligands from the circulation. 2,3 Boucher et al, moreover, showed that LRP1 has a cholesterol-independent role in atherosclerosis by modulating the activity and cellular localization of the PDGF receptor-␤ (PDGFR-␤) in vascular smooth muscle cells (SMCs). 4 Finally, 2 studies argue that LRP1 in macrophages has an effect on atherosclerosis through the modulation of the extracellular matrix and inflammatory responses. 5,6 In all these studies, the overall function of LRP1 in a specific cell type was disrupted. These app...