Introduction Cobalamin (vitamin B 12 ) is a coenzyme for the enzymes of intermediate metabolism, methionine synthase, and methylmalonyl-CoA mutase, and deficiency of the vitamin leads to potentially lethal manifestations such as megaloblastic anemia and severe combined degeneration of the central nervous system. Cobalamin deficiency, which is one of the most common vitamin-deficiency diseases, is most often due to failure at a step in the complicated and highly specific gastrointestinal uptake mechanisms for dietary cobalamin rather than an insufficient supply from food. 1 Intrinsic factor (IF) is a glycoprotein produced in the gastric epithelium. It tightly binds to cobalamin in the gastrointestestinal tract, and in the distal small intestine the IF-cobalamin complex is recognized by cubilin, a multiligand apical membrane protein that participates in endocytosis of the complex. 2,3 IF is subsequently degraded in enterocyte lysosomes, and cobalamin is secreted into plasma in complex with transcobalamin-II. 4 Cubilin is a large membrane protein (460 kDa) with a unique set of extracellular protein modules comprising 8 tandem epidermal growth factor domains followed by 27 tandem CUB domains (initially found in complement components C1r/C1s, Uegf, and bone morphogenic protein-1) harboring the IF-cobalamin binding site (CUB domains 5-8). 5,6 Although cubilin has no apparent transmembrane segment or cytoplasmic tail, several studies have shown that binding of IF-cobalamin to cubilin leads to endocytosis of the ligand and recycling of the receptor. 2,3 Besides expression and function in the intestine, cubilin has many-fold higher expression in the apical membrane of kidney proximal tubule and rodent yolk sac epithelial cells. 2,3,7,8 Consistent with this pattern of expression, cubilin is involved in reabsorption of several specific nutrient-carrying proteins from renal glomerular filtrate, including albumin, 9 transferrin, 10 vitamin D-binding protein, 11 and apolipoprotein AI, 12,13 and cubilin has a crucial but not-yet-defined role in early embryonic development of rodents. 14 Evidence to date indicates that the mechanism of cubilin-mediated endocytosis is the same in the various cubilin-expressing epithelia. 2 Accordingly, IF-cobalamin is effectively endocytosed in a cubilin-dependent manner in the proximal tubule 15 and yolk sac, 16 and because these tissues have higher density of cubilin in apical membranes and less luminal proteolytic activity than intestine, they have been the preferred tissues for studying cubilin function. 2,8,[14][15][16] However, it should be noted that ligands other than IF-cobalamin are likely to be physiologically more important in the kidney and yolk sac because little or no gastric IF circulates in plasma. An Inside Blood analysis of this article appears in the front of this issue.Reprints: Søren K. Moestrup, Institute of Medical Biochemistry, University of Aarhus, 8000 Aarhus C, Denmark; e-mail: skm@biobase.dk.The publication costs of this article were defrayed in part by page charge ...

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IntroductionAdult-onset gastrointestinal malabsorption of the essential micronutrient, cobalamin (vitamin B 12 ), leads to potentially lethal manifestations as diverse as megaloblastic anemia and neutropenia, degeneration of spinal cord nerve tracts, and dementia. 1 In the very young, signs of cobalamin malabsorption may also include growth retardation or loss of developmental milestones or both. ImerslundGräsbeck syndrome (I-GS; also called megaloblastic anemia 1, OMIM no. 261100) is an autosomal recessive disorder characterized by selective malabsorption of cobalamin in the intestine and, most often, of specific low-molecular-weight proteins in renal proximal tubules. 2,3 Patients with I-GS typically present at 2 to 4 years of age with signs of cobalamin deficiency and proteinuria. More than 200 human cases and familial clusters have been reported in Finland, Norway, the Middle East, and Northern Africa. 1 Various null and missense mutations of the cubilin (CUBN) 4 and amnionless (AMN) genes 5 have been implicated in I-GS kindreds, but both loci have been excluded in some. 6 In addition, a number of patients suffering from gastric intrinsic factor (IF) deficiency have been misdiagnosed as having I-GS. 7 Cubilin is a 460-kDa multiligand receptor protein that mediates endocytosis of the IF-cobalamin complex from distal small intestinal chyme and of several proteins from glomerular filtrate in renal proximal tubules. 8 AMN is an approximately 48-kDa apical membrane protein also expressed in intestinal and proximal tubule epithelia but whose function is poorly defined. 5,9 Recent studies suggested that cubilin and AMN function as subunits of a receptor complex, now called "cubam." 10 However, no studies have directly assessed the effect of CUBN or AMN mutations on cubam expression in patients with I-GS because these proteins are expressed in inaccessible tissues, and the clinical disease is easily treated. Unfortunately, the AMN knockout mouse exhibits an embryonic lethal phenotype. 9 Canine I-GS was derived originally from purebred giant schnauzers (GSs) as a naturally occurring animal model of the human disorder [11][12][13] and has contributed significantly to understanding of its molecular biologic basis as well as aspects of cubilin function. 14-17 CUBN was excluded from the GS disease locus, 18 and the disorder was recently mapped to an approximately 4-Mb interval predicted to harbor AMN. 19 To define the molecular basis of the canine I-GS model, we sought AMN mutations segregating in the GS kindred and an unrelated kindred of Australian shepherd (AS) dogs and investigated in vivo AMN and cubilin expression in affected dogs. Additionally, comparison of these results to expression of mutant canine AMN in a heterologous mammalian-cell transfection system validates the cell-culture system for ex vivo Materials and methods AnimalsDogs were handled according to principles and protocols approved by the MSU All University Committee for Animal Use and Care. MSU University Laboratory Animal Resources housed dogs of...