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PLENARY PAPER

The functional cobalamin (vitamin B12)–intrinsic factor receptor is a novel

complex of cubilin and amnionless
John C. Fyfe, Mette Madsen, Peter Højrup, Erik I. Christensen, Stephan M. Tanner, Albert de la Chapelle, Qianchuan He,
and Søren K. Moestrup

Imerslund-Gräsbeck syndrome (I-GS,
megaloblastic anemia 1) is an autosomal
recessive disorder characterized by intes-
tinal cobalamin (vitamin B12) malabsorp-
tion and proteinuria. I-GS–causing muta-
tions are found in either of 2 genes
encoding the epithelial proteins: cubilin
and amnionless (AMN). Cubilin recog-
nizes intrinsic factor (IF)–cobalamin and
various other proteins to be endocytosed
in the intestine and kidney, respectively,
whereas the function of AMN is unknown.
Here we show that cubilin and AMN colo-
calize in the endocytic apparatus of polar-
ized epithelial cells and copurify as a tight
complex during IF-cobalamin affinity and
nondenaturing gel filtration chromatogra-
phy. In transfected cells expressing either
AMN or a truncated IF-cobalamin–binding
cubilin construct, neither protein alone
conferred ligand endocytosis. In cubilin
transfectants, cubilin accumulated in early
biosynthetic compartments. However, in
cells cotransfected with AMN and the
cubilin construct, cubilin trafficked to the
cell surface and endosomes, and the cells
exhibited IF-cobalamin endocytosis and
lysosomal degradation of IF. These data
indicate that cubilin and AMN are sub-
units of a novel cubilin/AMN (cubam) com-
plex, where AMN binds to the amino-
terminal third of cubilin and directs
subcellular localization and endocytosis
of cubilin with its ligand. Therefore, muta-
tions affecting either of the 2 proteins
may abrogate function of the cubam com-
plex and cause IG-S. (Blood. 2004;103:
1573-1579)
© 2004 by The American Society of Hematology

Introduction
Cobalamin (vitamin B12) is a coenzyme for the enzymes of
intermediate metabolism, methionine synthase, and methylmalo-
nyl-CoA mutase, and deficiency of the vitamin leads to poten-
tially 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 expres-
sion 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 apolipopro-
tein 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 tubule15 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.2,8,14-16 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.

From the Department of Microbiology and Molecular Genetics, Michigan State
University, East Lansing; Institute of Medical Biochemistry and the Department
of Cell Biology, Institute of Anatomy, University of Aarhus, Denmark;
Department of Biochemistry and Molecular Biology, Odense University,
Denmark; and the Human Cancer Genetics Program, Comprehensive Cancer
Center, The Ohio State University, Columbus.
Submitted August 19, 2003; accepted October 10, 2003. Prepublished online
as Blood First Edition Paper, October 23, 2003; DOI 10.1182/blood-2003-08-
2852.
Supported in part by grants from the The Danish Medical Research Council,
The Novo Nordisk Foundation, National Institutes of Health (DK064161), the
National Cancer Institute (CA16058), The Plasmid Foundation, and the
Carlsberg Foundation.
J.C.F. and M.M. contributed equally to this work.
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
payment. Therefore, and solely to indicate this fact, this article is hereby
marked ‘‘advertisement’’ in accordance with 18 U.S.C. section 1734.
© 2004 by The American Society of Hematology

BLOOD, 1 MARCH 2004 䡠 VOLUME 103, NUMBER 5 1573

 From www.bloodjournal.org by guest on August 30, 2018. For personal use only.
1574 FYFE et al
Cobalamin deficiency is most often caused by decreased
production or function of IF due to acquired autoimmune disease of
the gastric mucosa (pernicious anemia), but selective malabsorp-
tion of the vitamin may also be due to inherited defects of the
various components in the cobalamin uptake system. Imerslund-
Gräsbeck syndrome (I-GS; aka megaloblastic anemia 1, OMIM no.
261 100) is an autosomal recessive disorder characterized by
selective malabsorption of cobalamin despite normal production
and function of IF.17,18 Proteinuria is often an additional sign of the
disorder.17,18 More than 200 human cases have been reported with
familial clusters in Finland, Norway, the Middle East, and North
Africa.19 The same disorder has also been identified in a dog
kindred.20 Genetic and functional studies provide evidence that
abnormal function of cubilin causes the disease.21-23 For instance,
the majority of the Finnish patients with I-GS are homozygous for a
missense mutation21 that reduces IF-cobalamin binding,23 and
canine I-GS cases have abnormal processing and deficient apical
membrane expression of cubilin.20 However, extensive genetic
analyses of Norwegian and Middle Eastern kindreds24 as well as of
canine I-GS cases25 have disclosed that the disease displays locus
heterogeneity. Very recently, Tanner et al24 demonstrated that
mutations in the amnionless (AMN) gene also cause I-GS, and
canine I-GS shows highly significant linkage to the same locus.26
AMN was initially identified by random mutagenesis27 as a
protein essential for amnion and primitive streak formation in mice.
AMN is a transmembrane 45- to 50-kDa protein of polarized
epithelia.24,27 The function of AMN is otherwise unknown, but it is
highly expressed in cubilin-expressing tissues, including the kid-
ney, intestine, and mouse visceral yolk sac.24,27 Their coincident
tissue distribution combined with the observation that inherited
abnormalities in AMN and cubilin have similar phenotypic conse-
quences14,20,24,27 suggest a functional linkage between these 2 gene
products. In the present study we investigated the possibility that
the 2 proteins interact. Here we present evidence that cubilin and
AMN form a tightly bound complex early in the biosynthetic
pathway that is essential for apical membrane localization and
endocytic functions previously ascribed to cubilin alone.
Materials and methods
Antibodies
Two anti-AMN peptide antibodies were raised in rabbits by immunization
with peptides comprising the human AMN amino acid residues 165 to 179
of the extracellular domain or residues 415 to 430 in the cytoplasmic tail.
Both were tested by immunoblot of kidney cortex proteins and were
demonstrated to be AMN specific; neither cross reacted with purified
cubilin or megalin. A previously described22 rabbit polyclonal antihuman
cubilin antiserum demonstrated not to cross react with megalin or AMN
was used on immunoblots and immunohistochemical and immunoelectron
microscopic examination of kidney proximal tubule cells. Anti-myc anti-
body purchased from Invitrogen (Taastrup, Denmark) was used for
detection of the AMN-myc construct expressed in Chinese hamster ovary
(CHO) cells, and a previously described5 rabbit polyclonal antirat cubilin
antiserum was used in immunoblots of products from CHO cells expressing
a rat cubilin construct. In cases where CHO cells were doubly stained for
immunofluorescent microscopy and the available anti-IF sera was of rabbit
origin, a previously described mouse monoclonal anticubilin antibody14,28
was used. A sheep antimegalin antibody9 recognizing rodent, canine, and
human megalin was used for immunoblotting.
Immunostaining of human kidney cortex
Cryosections of normal human kidney cortex (uninvolved sections from
resected renal carcinoma) were incubated with a rabbit anti-AMN peptide
BLOOD, 1 MARCH 2004 䡠 VOLUME 103, NUMBER 5
(415-430) serum (1:1000 dilution) or polyclonal antihuman cubilin anti-
body. Sections were subsequently incubated with goat antirabbit immuno-
globulin G (IgG) conjugated to horseradish peroxidase for light microscopy
or 10-nm gold particles for electron microscopy as previously.15
Biochemical analysis of the cubilin/AMN complex
Purification of the cubilin/AMN complex from human kidney by affinity
chromatography was carried out as described previously.22 Briefly, total
membrane and supernatant fractions were prepared from homogenates of
postmortem kidney cortex. Triton X-100 (1%) was added to solubilized
proteins in the membrane fraction. Each fraction was applied to an
IF-cobalamin column, and the columns were washed extensively in
phosphate-buffered saline (PBS), pH 7.4, containing 1 mM CaCl2 and 0.6%
3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate (CHAPS).
Column binding proteins were eluted in several fractions of PBS, pH 5,
containing 5 mM ethylenediamine tetraacetic acid (EDTA) and 0.6%
CHAPS. Immunoblots were prepared on polyvinylidene difluoride (PVDF)
membrane after sodium dodecyl sulfate–polyacrylamide gel electrophore-
sis (SDS-PAGE) on 4% to 16% or 10% gels and incubated with rabbit
antihuman cubilin serum diluted 1:1000 or rabbit anti-AMN extracellular
domain peptide serum diluted 1:500. Secondary antibody was antirabbit
IgG–alkaline phosphatase conjugate, and proteins were detected with
P-nitroblue tetrazolium/5-bromo-4-chloro-3-indolyl phosphate. Amino-
terminal sequencing of electroblotted AMN was performed as previously
described.22
For gel filtration analysis of IF-cobalamin affinity-purified proteins,
fractions containing cubilin were combined and concentrated by centrifuga-
tion through a 10 000-kDa cutoff membrane. A 25-mL Superose 6 column
(Amersham Biosciences, Hillerod, Denmark) was equilibrated, loaded, and
eluted variously in pH 7.4 PBS, pH 5 PBS containing 5 mM EDTA, or pH
7.4 PBS containing 0.2% SDS, 2 M urea, or 6 M urea. Proteins in column
fractions were separated by SDS-PAGE and immunoblotted as above. AMN
oligosaccharide analysis was carried out by endoglycosidase H (endo H;
Roche, Mannheim, Germany) and peptide N-glycosidase F (PNGase F;
Roche) digestion of IF-cobalamin affinity purified proteins and SDS-PAGE
as described previously.20 Gel slices of proteins separated by SDS-PAGE
were subjected to in-gel tryptic digestion followed by MALDI mass
spectrometric peptide mapping as described.29,30
Establishment and characterization of stable AMN and cubilin
transfectant cells
The cDNA encoding amino acids 1 to 1389 of rat cubilin was ligated into
the XbaI and HindIII sites of the expression vector pcDNA3.1/Zeo(-)
(Invitrogen), and stable, single-transfectant, Zeocin-resistant CHO cell
clones were established as described.6 Establishment of stable, double-
transfected CHO clones expressing both the truncated cubilin construct and
myc-labeled human AMN was carried out by transfection with a previously
described cDNA plasmid24 encoding AMN and selection with 1 mg/mL
Geneticin (Invitrogen). For establishment of stable, single-transfected CHO
clones expressing AMN-myc, the AMN-myc insert was ligated into the
HindIII and XhoI sites of the pcDNA3.1/Zeo(⫹) expression vector (Invitro-
gen), and CHO cells were processed as above.
Analysis of expression products and IF-cobalamin
endocytosis in CHO cells
Expression products of all clones were analyzed by immunoblotting of
growth medium and cell lysates. In other experiments, porcine IF-
cobalamin coupled to cyanogen bromid (CNBr)–activated Sepharose 4B
beads (Amersham Biosciences), as previously described,15 was incubated
with cell lysates of single- and double-transfected CHO cells expressing the
cubilin construct of amino acid residues 1-1389. Following precipitation of
the recombinant cubilin products, the affinity beads were suspended in 50
mM Na phosphate buffer, pH 5.5, containing 0.01% SDS or in 20 mM Na
phosphate buffer, pH 7.2, containing 10 mM EDTA and 0.5% Triton X-100
and incubated overnight at 37°C with endo H or at 30°C with PNGase F,
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BLOOD, 1 MARCH 2004 䡠 VOLUME 103, NUMBER 5 NOVEL COMPLEX OF CUBILIN AND AMNIONLESS 1575

respectively. No endogenous cubilin or megalin expression was detected in

the nontransfected CHO cells by immunoblotting.
Endocytosis of human IF-cobalamin in cubilin-transfected, AMN-myc–
transfected, cubilin/AMN-myc–double transfected, and mock-transfected
CHO cells was analyzed as described for other ligands.12,31 Briefly,
triplicates of cells grown to confluence in 24-well plates (0.08-0.13 mg
protein/well) were incubated for 4 hours at 37°C with 125I-labeled human
IF-cobalamin with or without added chloroquine and leupeptin. The
medium was removed, and soluble fragments of degraded IF in the medium
were separated from intact radiolabel by addition of 12.5% trichloroacetic
acid (TCA) and centrifugation. The washed cell layers were lysed with 0.5
M NaOH, and radioactivity of all fractions was determined. In this assay,
degradation of ligand is represented by the cell-mediated increase in
TCA-soluble radioactivity in the medium, and ligand uptake is represented
by cell-associated radioactivity plus cell-mediated degradation.

Confocal immunofluorescence and immunoelectron

microscopy of CHO cells

Immunofluorescent analyses of CHO cells expressing cubilin, AMN, and

cubilin/AMN to determine localization of these proteins were performed on
fixed permeabilized cells (fixed 30 minutes at room temperature in 4%
formaldehyde in 10 mM PBS and washed in 10 mM PBS, pH 7.4, 0.05%
Triton X-100) for intracellular distribution or on living nonpermeabilized
cells to visualize proteins on the extracellular surface. Fixed and permeabil-
ized cells were incubated for 1 hour at room temperature variously with
polyclonal antirat cubilin serum, monoclonal antihuman cubilin antibody,
and the anti-myc antibody each diluted to 10 ␮g/mL in washing buffer. For
surface staining, living cells were incubated for 90 minutes at 4°C with
polyclonal antirat cubilin antiserum diluted to 10 ␮g/mL in growth medium,
washed, and then fixed for 1 hour at 4°C. To visualize IF-cobalamin uptake
and intracellular distribution of ligand, living cells were incubated for 30
minutes or 1 hour at 37°C with IF-cobalamin diluted to 40 ␮g/mL in growth
medium, then fixed and incubated as above with rabbit polyclonal
antiporcine IF serum and mouse monoclonal antihuman cubilin antibody.
Finally, the cells were incubated for 1 hour at room temperature with the
fluorescence-labeled secondary antibodies Alexa Fluor 488 goat antirabbit
IgG and/or Alexa Fluor 594 goat antimouse IgG (Molecular Probes,
Eugene, OR), each diluted 1:200. Stained cells were examined by confocal
fluorescence microscopy using a laser scanning confocal unit (LSM510;
Carl Zeiss, Jena, Germany) attached to an Axiovert (Carl Zeiss) microscope.
For electron microscopy, cells were fixed for 30 minutes with 2%
formaldehyde in 0.1 M Na cacodylate buffer, pH 7.4. Cells were removed
from the plastic surface with a rubber policeman in the same buffer
containing 1% gelatin, pelleted, embedded in 12% gelatin, and finally
infiltrated with 2.3 M sucrose in PBS for 30 minutes before freezing in
liquid nitrogen. Ultrathin cryosections (70-90 nm) were prepared with an
FCS Reichert Ultracut S cryoultramicrotome (Leica, Vienna, Austria) at
about ⫺100°C and collected on 200 mesh Ni grids. The sections were
incubated overnight with a rabbit polyclonal antirat cubilin antiserum
diluted 1:1000, prior to 2 hours of incubation with a 10-nm gold
particle–labeled goat antirabbit antibody (BioCell, Cardiff, United King-
dom). All incubations were performed at 4°C. The sections were finally
contrasted with methylcellulose containing 0.3% uranyl acetate and studied
in a Philips CM100 electron microscope (FEI Denmark, Glostrup, Denmark).
Results
In accordance with the hypothesis that cubilin and AMN interact in
polarized epithelia, light and electron microscopy of immuno-
stained human kidney cortex showed indistinguishable localization
of the 2 membrane proteins in the apical membrane and endocytic
apparatus of renal proximal tubule cells (Figure 1A). To identify
possible physical interactions between cubilin and AMN, we
analyzed the material isolated from an IF-cobalamin affinity matrix
loaded with detergent-solubilized renal cortex membranes. SDS-
Figure 1. Identification of AMN as a protein colocalizing with and binding to
cubilin. (A) Top panels: Immuno-peroxidase staining of AMN (left) and cubilin (right)
in human kidney proximal tubules. Bottom panels: Immunoelectron staining with
10-nm gold particles of AMN (left) and cubilin (right) in the dense apical vesicles and
plasma membrane of proximal tubules known to function in membrane receptor
recycling.32 Original magnification, ⫻ 1000 (top panels) and ⫻ 46 000 (bottom
panels). (B) Detergent-solubilized human kidney cortex membrane proteins were
purified by IF-cobalamin affinity chromatography, separated on a 4% to 16%
SDS-PAGE gel (lane 1, Coomassie stain), and subjected to anti-AMN immunoblot
(lane 2). Also shown are anti-AMN immunoblots of IF-cobalamin–binding proteins
from the supernatant fraction of kidney membranes prior to detergent solubilization
(lane 3), human urine (lane 4), and kidney cortex membranes before (lane 5) and
after (lane 6) PNGase-F digestion. (C) Anticubilin and anti-AMN immunoblots of
Superose-6 gel filtration fractions of human kidney cortex IF-cobalamin–binding
proteins eluted in PBS, pH 7.4, with 2 M or 6 M urea. Cubilin and AMN coeluted in
fractions 9 and 10 in 2 M urea but separated in 6 M urea, where AMN elutes in
fractions 16 and 17.
PAGE of the affinity-purified material showed several bands in
addition to the abundant cubilin band (Figure 1B, lane 1). By
immunoblotting, AMN was identified in 2 major bands of approxi-
mately 35 kDa and approximately 45 kDa (Figure 1B, lane 2), and
both had approximately 3 kDa of Asn-linked oligosaccharides as
demonstrated by PNGase F digestion (Figure 1B, lanes 5 and 6).
MALDI mass spectrometric peptide mapping of in-gel tryptic
digests identified AMN peptides 110-119, 120-134, 139-151,
152-162, 172-186, 176-182, and 274-293 in both the 35-kDa and
45-kDa AMN bands and additional peptides 302-323 and 327-347
in the 45-kDa band. Furthermore, mass spectrometry identified
minor-intensity AMN bands of 40 kDa, 42 kDa, and 49 kDa as well
as IgG, ␣-actinin, and 2 mitochondrial proteins. Due to their
expression patterns, the relationship of these non-AMN proteins to
cubilin was not further pursued.
The peptides identified by mass spectrometry in the 35-kDa
AMN product (Figure 1B) and identification of the amino-terminal
sequence of Val-Ser-Lys-Leu in the same protein indicate that it
represents the extracellular domain of AMN cleaved near the
membrane. Consistent with being a protein with no membrane
anchorage, the same AMN form was also identified as a soluble
molecule in the supernatant fraction of human kidney membranes
prior to detergent solubilization (Figure 1B, lane 3).
 From www.bloodjournal.org by guest on August 30, 2018. For personal use only.
1576 FYFE et al
Figure 2. Effect of AMN on cubilin expression and processing in CHO cells.
(A) Cubilin (amino acids 1-1389) and AMN (full length plus myc epitope) constructs
encoded by plasmid cDNA used for stable transfection of CHO cells. The position of
the IF-cobalamin (IF-cbl) binding site (CUB domains 5-8)6 is indicated and the
truncated CUB domains 9-27 are shown by dashed lines. (B) Immunoblots of cubilin
and AMN in cell lysates (lanes 1-3) and endo H digestion of IF-cobalamin affinity-
purified cubilin from cells transfected with cubilin, AMN, and cubilin/AMN cDNA. Gel
mobility shifts indicate that cubilin oligosaccharides in single transfectants are endo H
sensitive (compare lanes 4 and 6), but the bulk of cubilin oligosaccharides in
cubilin/AMN double transfectants are endo H resistant (compare lanes 5 and 7).
Cubilin and AMN coeluted in proportional amounts from the
affinity column, and gel filtration of the eluted material showed that
cubilin and AMN coelute as a strongly bound molecular complex
that required denaturing conditions (6 M urea or 0.2% SDS) to
separate (Figure 1C). Furthermore, in contrast to all other known
cubilin ligands,2,3 the binding of AMN and cubilin was not calcium
dependent, as indicated by coelution of the proteins in the presence
of EDTA. We conclude from this set of analyses that AMN strongly
binds cubilin in a region that is not sterically hindered by
BLOOD, 1 MARCH 2004 䡠 VOLUME 103, NUMBER 5
IF-cobalamin,6 and that cubilin bound to AMN truncated above the
transmembrane segment is easily shed from membranes, as was
previously observed for cubilin.5 While some of the soluble
truncated 35-kDa AMN isolated from postmortem human kidney
may be due to cleavage during autolysis, we also identified the
35-kDa AMN form together with cubilin in human urine (Figure
1B, lane 4), indicating that at least some AMN truncation occurs
in vivo.
In order to investigate whether AMN is involved in cubilin
processing and transport to the plasma membrane and endocytosis
of cubilin-ligand complexes, we transfected CHO cells with cubilin
and AMN cDNA plasmid constructs. Because we could not obtain
a stable transfectant expressing the entire cubilin molecule, we
expressed a “mini-cubilin” protein (amino acids 1-1389) from
which the 19 carboxy-terminal CUB domains, suggested to be
involved in binding other ligands,2,6 had been deleted (Figure 2A).
This construct contains the 100-residue amino-terminal region
previously demonstrated to be required for cellular retention of
cubilin,6 the cluster of 8 epidermal growth factor repeats and CUB
domains 1-8 harboring the IF-cobalamin binding site (CUBs 5-8).6
In CHO cells transfected with only the cubilin construct, immuno-
confocal and immunoelectron microscopic examination revealed
that the cubilin construct accumulated in the Golgi and other
vesicular structures close the Golgi (Figure 3A,C, left panels), but
no expression was seen in endosome-like structures or on the cell
surface (Figure 3B, left panel). Although retained intracellularly,
the cubilin construct was capable of binding to an IF-cobalamin
Sepharose matrix (Figure 2B, lane 4). However, concordant with
absent cell-surface expression of cubilin, these cells did not
effectively endocytose and degrade 125I-labeled IF-cobalamin (Fig-
ure 4). Additionally, Asn-linked oligosaccharides of the expressed
cubilin construct were endo H sensitive, as seen by increased gel
mobility (Figure 2B, compare lanes 4 and 6), thus indicating lack of
oligosaccharide maturation in Golgi.
In distinct contrast, cotransfection of the “mini-cubilin” transfec-
tant cells with cDNA encoding full-length AMN had a striking
Figure 3. Cubilin and AMN localization in single and
doubly transfected CHO cells. (A) Confocal immunoflu-
oresence microscopy of AMN and cubilin in permeabil-
ized cells demonstrating colocalization of AMN and cubi-
lin. Original magnification, ⫻ 630. (B) Surface staining of
cubilin in cubilin single and cubilin/AMN double transfec-
tants demonstrating absent surface expression in the
cubilin single transfectants and punctuate surface expres-
sion in cubilin/AMN double transfectants. Original magni-
fication, ⫻ 1000. (C) Immunogold staining of cubilin in
cubilin single and cubilin/AMN double transfectants exam-
ined by electron microscopy demonstrating cubilin in the
endocytic apparatus of double transfectants. Original
magnification, ⫻ 46 000.
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BLOOD, 1 MARCH 2004 䡠 VOLUME 103, NUMBER 5
Figure 4. Effect of AMN on endocytosis of IF-cobalamin in cubilin-expressing
CHO cells. (A) Endocytosis of 125I–IF-cobalamin after 4 hours at 37°C in cubilin and
AMN single transfectants and in cubilin/AMN double transfectants, in the absence
and presence of the lysosomal enzyme inhibitors chloroquine and leupeptin.
Degradation (f) represents the cell-mediated increase in TCA-soluble radioactivity in
the medium, and uptake (u) represents cell-associated radioactivity plus cell-
mediated degradation. Error bars indicate SD. (B) Confocal immunofluoresence
microscopy of the transfected cells incubated with IF-cobalamin at 37°C. Only the
double cubilin/AMN transfectants take up IF-cobalamin as seen by vesicular staining
of IF. Original magnification, ⫻ 630.
effect on processing and the endocytic function of the cubi-
lin construct.
First, 2 IF-cobalamin binding forms of cubilin were observed
(Figure 2B, lane 5), and most of the cubilin expressed was a higher
molecular weight form hardly seen in the cubilin single transfec-
tants. In contrast to the cubilin expressed in cubilin single
transfectants, the major cubilin protein in the cubilin/AMN double
transfectants showed no gel mobility shift when digested with endo
H (Figure 2B, compare lanes 5 and 7), indicating that this larger
cubilin form had undergone Golgi-mediated processing of Asn-
linked oligosaccharides. PNGase F treatment reduced all forms of
cubilin in the single and the double transfectants to the same size as
the endo H–treated cubilin from single transfectants, indicating that
the different sizes of cubilin in the double transfectants were due to
differences of Asn-linked oligosaccharide processing (not shown).
Second, the location of cubilin in cubilin/AMN double transfec-
tants changed from the exclusively intracellular locations observed
in the cubilin single transfectants to also include the plasma
membrane (Figure 3). Confocal examination of cell-surface immu-
nostained cubilin/AMN double transfectants was seen as a strong
dotlike staining, concordant with expression of cubilin in cell-
surface clusters (Figure 3B, right panel). Immunoelectron micros-
copy showed that cubilin in the double transfectants was expressed
in coated and noncoated endosome-like vesicles as well as in
coated invaginations and noncoated areas of the plasma membrane
(Figure 3C, right panels).
Third, the coexpression of AMN in the cubilin transfectants
conferred to the cells the ability to endocytose IF-cobalamin. The
uptake of 125I-labeled IF-cobalamin by double transfectants was
NOVEL COMPLEX OF CUBILIN AND AMNIONLESS 1577
Figure 5. Multiple species amino acid alignment of the AMN cytoplasmic
domain. Alignment of human (GenBank accession no. NP 112205), mouse (acces-
sion no. NP 291081), rat (accession no. XP 234547), and dog (accession no. AY
368152) AMN sequences (single-letter amino acid code) was performed by web-
based software.33 Residues highlighted in black are identical in all 4 species. Two
conserved copies of the internalization signal FXNPXF are underlined.
accompanied by degradation of ligand and appearance of TCA-
soluble radioactivity in the medium (Figure 4A). Inhibition of
lysosomal enzyme activity by concurrent treatment of cells with
chloroquine and leupeptin strongly inhibited degradation of the
125I–IF-cobalamin complex in a manner compatible with lysosomal
degradation of the endocytosed ligand. Compared with double
transfectants, the uptake of 125I–IF-cobalamin was considerably
less in the single transfectants where no degradation of the ligand
was measured. Endocytosis and accumulation of IF-cobalamin in
endosome- and lysosome-like structures was also seen in immuno-
stained double transfectants but not in cubilin single transfectants
as examined by confocal microscopy (Figure 4B).
The indication of a novel mechanism by which AMN confers
endocytic function to cubilin/AMN complexes was also supported
by alignment of the AMN amino acid sequences of human, dog, rat,
and mouse (Figure 5) that revealed 2 highly conserved copies of the
sequence Phe-X-Asn-Pro-X-Phe in the cytoplasmic domain. This
sequence conforms to the well-characterized AP-2 adaptor protein-
binding signal (Phe-X-Asn-Pro-X-Tyr/Phe)34 for ligand-indepen-
dent internalization via clathrin-coated pits that was first recog-
nized in the low-density lipoprotein receptor family of endocytic
receptors.35
Discussion
The data presented here suggest a model (Figure 6) of an essential
partnership of cubilin with AMN in a functional complex for
endocytosis of IF-cobalamin and other ligands. In this model,
cubilin contributes ligand-binding regions, and AMN contributes
Figure 6. A model of AMN and cubilin assembly in the biosynthetic pathway and
recycling in the endocytic apparatus of polarized epithelial cells. The depicted
model is consistent with data presented here and current paradigms of endocytic
receptor expression. This depiction does not show that AMN is sensitive to cleavage
close to the transmembrane segment leading to partial shedding of cubilin/AMN to
the extracellular fluid.
 From www.bloodjournal.org by guest on August 30, 2018. For personal use only.

1578 FYFE et al BLOOD, 1 MARCH 2004 䡠 VOLUME 103, NUMBER 5

structures required for membrane anchorage, biosynthetic process-
ing, and trafficking to the plasma membrane, as well as putative
signal sequences for endocytosis and receptor recycling. The endo
H sensitivity of Asn-linked oligosaccharides and apparent Golgi
accumulation of a functional “mini-cubilin” construct in AMN-
negative cells indicates that the initial cubilin/AMN interaction
occurs early in the biosynthetic pathway, most likely in the rough
endoplasmic reticulum. Furthermore, the functionality in relation
to IF-cobalamin endocytosis of the highly truncated cubilin con-
struct missing CUB domains 9-27 indicates that AMN binds to a
cubilin site amino-terminal of the IF-cobalamin site in CUB
domain 5-86 (Figure 2A).
In addition to providing a rationale for the recently discovered
I-GS–causing AMN mutations in humans, the present model of
cubilin/AMN interdependence is also consistent with previous in
vivo observations in dogs with inherited selective intestinal IF-
cobalamin malabsorption and proteinuria (canine I-GS). Those
studies explicitly demonstrated that I-GS–affected dogs exhibit
failed surface expression of cubilin in intestinal and renal proximal
tubule epithelial cells and renal cubilin that is retained in an early
biosynthetic compartment in an incompletely folded form ex-
hibiting immature glycosylation.7,20 It was further demonstrated
that the defects of cubilin expression were not due to a cubilin
mutation,25 and recent genetic analysis has mapped canine I-GS to
the AMN locus.26
A mechanistic explanation of cubilin endocytic function has
been sought ever since transmembrane and cytoplasmic domains
were found lacking in the structure of cubilin deduced from cDNA
sequence. Previously, it was suggested that megalin, an endocytic
receptor of the LDL receptor family, might assist the trafficking of
cubilin.5 This suggestion was based on the several observations that
megalin strongly colocalizes5 and is coregulated36 with cubilin, that
megalin-deficient mice have markedly decreased renal cubilin
expression and uptake of cubilin ligands, and that antibodies
against megalin reduce the uptake of some cubilin ligands.10,11
However, since the transfected cells of the present study do not
express detectable megalin, it seems that the cubilin/AMN complex
can function independently of megalin. The observed effects2,3,10,11
of omitting or reducing megalin function in the kidney or in
cultured cells may be due to overlap in the cubilin and megalin
ligand repertoires9,11 and/or to indirect effects of megalin on cubilin
function. The latter is actually indicated by the fact that megalin-
deficient kidney proximal tubules have far fewer endocytic vesicles
than normal tubules, indicating that the endocytic activity is
reduced overall.37 Despite these considerations, our data do not
exclude the possibility that megalin interacts with the cubilin/AMN
complex or certain cubilin ligands, and further studies are needed
to define what may be a functional relationship rather than simple
correlations.
In conclusion, we have discovered that AMN controls the
trafficking of cubilin, which is a “passive” partner of AMN for
binding carriers of vitamins and other nutrients to be endocytosed
by polarized epithelial cells. The novel cubilin/AMN complex,
which we propose to designate the cubam complex, is the
functional receptor essential for cobalamin uptake, renal protein
reabsorption, and early rodent embryogenesis. Since cubilin has
also been detected in epithelia of other organs such as the lung and
genital system,38 the function of the receptor complex may have
even wider implications. Furthermore, it is intriguing to speculate
that AMN may be involved in the processing and transport of other
membrane proteins or endocytosis of directly bound ligands.
Finally, the functional interdependence of cubilin and AMN now
fully explains why IG-S is caused by genetic defects in either the
cubilin or AMN genes.
Acknowledgments
Our deep thanks go to K. Lassen for her technical assistance and
her invaluable input to improve the methods used. Furthermore, we
thank G. Biller, I. Kristoffersen, and G. Ratz for technical
assistance, E. Nexø for human and porcine IF and anti-IF antibody,
P. Verroust for anticubilin antibody, J. Gburek for assistance with
microscopy, and J. A. Fyfe for fruitful discussion. The coauthor
Mette Madsen has previously published under the name Mette
Kristiansen.

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2004 103: 1573-1579

doi:10.1182/blood-2003-08-2852 originally published online
October 23, 2003

The functional cobalamin (vitamin B12)−intrinsic factor receptor is a

novel complex of cubilin and amnionless
John C. Fyfe, Mette Madsen, Peter Højrup, Erik I. Christensen, Stephan M. Tanner, Albert de la
Chapelle, Qianchuan He and Søren K. Moestrup

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