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INTRODUCTION |
Lipocalins were found to be important extracellular carriers of
lipophilic compounds in vertebrates, invertebrates, plants, and
bacteria (1-4). There is increasing evidence that this group of
proteins is involved in a variety of physiological processes including
retinoid, fatty acid, and pheromone signaling; immunomodulation; inflammation; detoxification; modulation of growth and metabolism; tissue development; apoptosis; and even behavior processes (1, 5-7).
Whereas the structural basis of lipocalin-ligand binding is now well
understood (8), there is a major lack of knowledge regarding the
mechanisms by which lipocalins exert their biological effects. It is
well accepted that many, if not all, of these proteins are able to bind
to specific cell receptors (9), although only two of these receptors
have been identified thus far (10, 11). Due to limited data concerning
the structure of the lipocalin receptors themselves, the molecular
mechanisms beyond this receptor binding are very unclear at the moment.
One hypothesis is that the holo-lipocalin releases its ligand upon
receptor binding, and this ligand diffuses through the cell membrane to
interact with an intracellular fatty acid-binding protein or an
intracellular receptor. There is also some evidence that lipocalins
undergo internalization by receptor-mediated endocytosis. Another
plausible mechanism might be that the lipocalin-receptor interaction
creates a direct signal inducing various physiological processes
(9).
We have recently identified and characterized
LIMR,1 a novel human 57-kDa
cell membrane protein (11), which interacts with Lcn-1, a lipocalin
member produced by a number of secretoric glands and tissues and known
to bind a variety of lipophilic compounds (12-14). Because we found
expression of LIMR in the human NT2 cell line (11), we used an
antisense gene knockout technology to investigate the role of this
receptor in the mechanism of ligand delivery in more detail. The
results of our study clearly demonstrated that LIMR is essential for
cellular internalization of Lcn-1. Thus, it has to be classified as a
novel endocytic receptor. Because sequence and structure analysis
indicated that proteins similar to LIMR are present in several
organisms and at least two closely related orthologues are found in
human and mouse, we suggest LIMR to be the prototype of a new family of
endocytic receptors.
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EXPERIMENTAL PROCEDURES |
Materials--
Human NT2 precursor cells were obtained from
Stratagene (La Jolla, CA), and human T-47D breast cells (ATCC HTB-133)
were obtained from American Type Culture Collection (Manassas, VA).
Cell culture media, fetal bovine serum, and other cell culture
materials were purchased from PAA Laboratories Inc. (Parker Ford, PA).
Cell Culture--
NT2 cells were propagated in Dulbecco's
modified Eagle's medium/Ham's nutrient mixture F-12K supplemented
with 10% fetal bovine serum, 2 mM L-glutamine,
100 units/ml penicillin, and 0.1 mg/ml streptomycin in
75-cm2 culture dishes under 5% CO2 at
37 °C. T-47D cells were grown at 37 °C in RPMI 1640 medium with 2 mM L-glutamine adjusted to contain 1.5 g/liter
sodium bicarbonate, 4.5 g/liter glucose, 10 mM HEPES, and
1.0 mM sodium pyruvate and supplemented with 0.2 unit/ml
bovine insulin and 10% fetal bovine serum.
Vector Construction--
To construct a vector expressing LIMR
antisense RNA, a cDNA fragment corresponding to nucleotides
332-2349 (1917 nucleotides) of LIMR (11) was amplified by PCR using
primers to which XbaI/XhoI restriction enzyme
cutting sites were attached (5'- CTAGCGTCTAGAATGGAAGCACCTGACTAC-3' and
5'- TTTATCTCGAGTCAGGTGGTCCAAAGCCC-3'). The PCR product was gel-purified, digested with XbaI/XhoI restriction
enzymes, and ligated with XbaI/XhoI-digested
vector pOPRSVI/MCS (Stratagene) in an antisense orientation. The
resulting plasmid (pOPRSVI-AS-LIMR) was verified by DNA sequencing. A
religated plasmid backbone pOPRSVI/MCS was used as a transfection control.
Establishment of an Antisense-transfected NT2 Cell
Line--
Stable transfection of NT2 cells was carried out using MBS
Mammalian Transfection Kit (Stratagene) according to the
manufacturer's recommendation. Forty-eight h after transfection, cells
were selected in culture medium containing 400 µg/ml G418.
Twenty-five days after selection, individual G418-resistant colonies
were subcloned. Five subclones of LIMR antisense- and mock-transfected
cells were analyzed by immunoblotting analysis and immunofluorescence
for LIMR expression.
Protein Labeling--
Purified Lcn-1 was labeled with
Na125I (Amersham Biosciences) using IODO-BEAD
Iodination Reagent as described by the supplier (Pierce). To remove
excess Na125I or unincorporated
125I2 from the iodinated protein, dialysis
against phosphate-buffered saline was performed using dialysis
cassettes (Pierce). The proteins had specific activities ranging
from 0.33 to 0.99 GBq/mg.
FITC-labeled Lcn-1 (FITC-Lcn-1) was prepared by incubating purified
Lcn-1 (5 mg/ml) with 5 mg/ml FITC (Sigma-Aldrich) in 200 mM sodium bicarbonate buffer, pH 9.0, for 20 h at
4 °C. Unconjugated FITC was removed by gel filtration on PD-10
columns (Amersham Biosciences).
FITC-Lcn-1 Uptake of Control NT2, AS-NT2, and T-47D Cells
Analyzed by Fluorescence Microscopy--
Cells were grown on
coverslips for 18 h at 37 °C in complete medium (minimal
essential medium containing 10% fetal bovine serum, 2 mM
L-glutamine, 100 units/ml penicillin, 100 µg/ml
streptomycin, and 400 µg/ml G418 for AS-NT2 cells; RPMI 1640 medium
for T-47D cells). The cells were washed once with serum-free medium and incubated for 3 h in serum-free medium at 37 °C. FITC-labeled Lcn-1 was added (final concentration, 50 µg/ml each) and incubated for 1 h at 37 °C. To remove cell surface-bound ligand that had not been internalized, cells were washed twice with phosphate-buffered saline and treated with 50 mM glycine, 150 mM
NaCl, pH 3.0, for 2 min. The cells were washed with Dulbecco's
phosphate-buffered saline, fixed in 100% ice-cold acetone for 5 min,
and analyzed with a ×63 objective on a Carl Zeiss Axioplan2
microscope. The images were resized and saved as 8-bit.tif files by
using the commercially available software MetaMorph® Imaging System
(Universal Imaging Corp., Visitron GmbH, Germany).
Ligand Internalization and Degradation Experiments Using
Radiolabeled Lcn-1--
Cells were seeded into 2.0-cm2
wells and grown to 90% confluence with ~3 × 105
cells attached/cm2. The cells were then washed with
maintenance medium (MEM) and incubated in the same medium containing
125I-labeled Lcn-1 (5 nM) at 4 °C for 2 h. Afterward, cells were washed three times with cold MEM (0.5 ml),
warmed up to 37 °C by the addition of prewarmed MEM, and incubated
at 37 °C for selected intervals. At each time point, the cells were
washed once with ice-cold MEM and treated with a solution containing
trypsin (0.5 mg/ml), proteinase K (0.5 mg/ml), and 5 mM
EDTA in MEM for 15 min at 4 °C to strip cell surface proteins (15,
16). The cell suspension was then centrifuged, and the radioactivity
associated with cell pellets (defining internalized
125I-labeled Lcn-1) was measured. For determination of
degradation rate, cells were incubated with 5 nM
125I-Lcn-1/well, and the culture medium was collected at
various times. The radioactivity appearing in the cell culture medium that was soluble in 10% trichloroacetic acid was corrected for non-cellular-mediated degradation by subtracting the amount of degradation in control wells lacking cells and was taken to represent degraded ligands.
Immunocytochemistry--
Conventional immunohistochemistry was
performed as described previously (11) using LIMR-specific primary
antibodies at a dilution of 1:100 and FITC-labeled secondary antibodies
(DAKO, Copenhagen, Denmark).
Membrane Preparation--
Cell membranes were prepared using the
Mem-Per Mammalian Membrane Protein Extraction Kit (Pierce) according to
the manufacturer's recommendations.
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RESULTS |
Lack of LIMR Production in Antisense-transfected Cells--
After
transfection with pOPRSVI-AS-LIMR or a control vector, NT2 precursor
cells were selected using G418. The presence of the antisense plasmid
in genomic DNA was verified by PCR using plasmid-specific primers (data
not shown). In a first step, the expression of LIMR in
plasmid-transfected and control cells was investigated by Western blot
analysis. As indicated in Fig.
1A, no significant expression
of LIMR could be detected in AS-NT2 cells. To confirm this result, we
performed immunofluorescence analysis using LIMR-specific antibodies.
Whereas the control cells showed a clear and specific staining of the
cell membrane, a highly reduced staining was observed with the
antisense-transfected cells (Fig. 1B). We used ImageQuant
software (Amersham Biosciences) to compare the immunofluorescence
intensity of pOPRSVI-AS-LIMR-transfected cells and control NT2 cells.
We found that the fluorescence intensity of the signal corresponding to
the expression of LIMR was reduced by more than 80% in
antisense-transfected cells relative to the intensity of the LIMR
signal in mock-transfected cells (Fig. 1C).

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Fig. 1.
LIMR antisense expression reduces LIMR
protein expression in NT2 cells. A, equal amounts of
proteins from membrane preparations were separated by SDS-PAGE (10%)
and analyzed by immunoblotting using a LIMR-specific antiserum.
Lane 1, NT2-mock cell membranes; lane 2, AS-NT2
cell membranes. B, LIMR immunostaining of NT2 cells
transfected with basic vector (NT2-mock) or with AS-LIMR. C,
bar graphs showing the reduction of LIMR expression in
AS-NT2-transfected cells, indicated as the mean ± S.E. of
fluorescence intensities. The plotted data are mean values of pixel
intensity derived from analysis with ImageQuant software (Amersham
Biosciences). The LIMR-specific fluorescence intensity in AS-NT2 cells
( ) is 80.56% lower than that in control cells ( ).
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Lcn-1 Accumulation in the Medium of LIMR-deficient Cells--
As
described previously, NT2 precursor cells produce a small amount of
Lcn-1 present in the medium under the conditions used (17). To test
whether there is an effect on the presence of Lcn-1 in the medium of
LIMR-deficient cells, we compared this medium with medium from an equal
amount of wild type or mock-transfected cells by Western blotting. As
shown in Fig. 2, a significant increase in the presence of Lcn-1 was found in the medium of LIMR-deficient cells compared with the controls. This result gave us a first indication that there was an accumulation of Lcn-1 that was
probably associated with a block in endocytosis, similar to that
described for major urinary protein in megalin-deficient mice (10), and that LIMR might be an essential factor for Lcn-1 endocytosis.

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Fig. 2.
Accumulation of Lcn-1 in medium from AS-NT2
cells as detected by Western blot analysis using Lcn-1-specific
antibodies. Cells were grown to 5 × 104
cells/ml, and 25 µl of each medium was subjected to SDS-PAGE (14%)
and immunoblotting. Lane 1, recombinant Lcn-1 protein (2 µg); lane 2, medium from AS-NT2 cells; lane 3,
medium from NT2 cells.
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Inhibition of Lcn-1 Internalization in LIMR-deficient
Cells--
To determine whether LIMR functions to mediate endocytosis
of Lcn-1, we compared the ability of NT2 control cells and
LIMR-deficient cells to internalize Lcn-1. To visualize the uptake,
FITC-Lcn-1 was added to the cultured cells. The cells were fixed and
examined by fluorescence microscopy. As demonstrated in Fig.
3, fluorescence was seen in discrete
granules of the NT2 cells but not in LIMR-deficient cells.

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Fig. 3.
Internalization of FITC-Lcn-1 by NT2
cells. A and C, phase-contrast micrographs
of NT2 cells. B, micrograph of control NT2 cells incubated
with FITC-Lcn-1. D, micrograph of LIMR-deficient AS-NT2
cells. FITC-labeled Lcn-1 was added to a final concentration of 50 µg/ml. Cell surface-bound ligand that had not been internalized was
removed with 50 mM glycine, 150 mM NaCl, pH 3.0 buffer. Control cells incubated with unconjugated FITC showed no
fluorescence (data not shown). Immunofluorescence slides were viewed
with a ×63 objective on a Carl Zeiss Axioplan2 microscope.
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To confirm these results and to study the kinetics of endocytosis,
cells were incubated in the presence of 125I-Lcn-1 for
2 h at 4 °C to allow binding of radiolabeled ligand. After
washing and incubation for various times, the radioactivity associated
with the cell pellet was counted. As indicated in Fig. 4A, the bound radioactivity
was internalized within 2 h in the NT2 control cells. In contrast
to NT2 control cells, no significant uptake of 125I-Lcn-1
could be found in AS-NT2 cells (Fig. 4A), thus demonstrating that LIMR is essential for endocytosis of Lcn-1 and supporting the
result obtained with FITC-Lcn-1.

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Fig. 4.
LIMR antisense-transfected NT2 cells do not
internalize and degrade 125I-Lcn-1. A,
internalization of 125I-Lcn-1 by control NT2 cells ( )
and antisense-transfected NT2 cells ( ). Control NT2 cells and AS-NT2
cells were preincubated in the presence of 125I-labeled
Lcn-1 (5 nM) for 2 h. After washing, cells were warmed
to 37 °C and treated at the time points indicated with
trypsin/proteinase K to remove cell surface-bound radioactivity. The
remainder was collected, and radioactivity was counted. B,
degradation of 125I-Lcn-1 by control NT2 cells ( ) and
antisense-transfected NT2 cells ( ). Control NT2 cells and AS-NT2
cells were incubated with 125I-Lcn-1 (5 nM) for
selected time intervals at 37 °C. At the indicated times, the amount
of degraded radioligand was determined by counting the radioactivity
appearing in the cell culture medium that was soluble in 10%
trichloroacetic acid.
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Uptake of 125I-Lcn-1 in NT2 control cells was followed by
successive degradation, as indicated by the increasing amount of
radioactivity that was secreted into the cell culture medium and
soluble in 10% trichloroacetic acid (Fig. 4B).
Lcn-1 Internalization Is Not Unique to NT2 Cells--
To
investigate whether Lcn-1 internalization is a specific feature of NT2
cells or a more general mechanism, we tested another cell line. Because
it is known that both LIMR and Lcn-1 are expressed in mammary gland
(11, 18), we searched for human breast cells expressing LIMR. It is
evident from immunostaining using LIMR antibodies that human T-47D
cells (19) express LIMR (Fig.
5A). Therefore, this cell line
was tested for uptake of FITC-Lcn-1. Fig. 5D demonstrates
that incubation with FITC-Lcn-1 resulted in an intracellular
immunostaining similar to that obtained with NT2 cells. Thus, it is
evident that Lcn-1 is also internalized by T-47D cells.

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Fig. 5.
LIMR expression and internalization of
FITC-Lcn-1 by human T-47D breast cells. Immunofluorescence showing
T-47D cells incubated with LIMR antiserum (A) and T-47D
cells incubated with only FITC-conjugated secondary antibodies as a
control (B). C, phase-contrast micrograph of
T-47D cells. D, micrograph of T-47D cells incubated with
FITC-Lcn-1. FITC-Lcn-1 incubated cells were treated as described in the
Fig. 3 legend. Control cells incubated with unconjugated FITC showed no
fluorescence (data not shown). Immunofluorescence slides were viewed
with a ×63 objective on a Carl Zeiss Axioplan2 microscope.
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LIMR Is the Prototype of a New Family of Membrane
Proteins--
LIMR was described to be a novel protein with no
significant similarity to known proteins (11). However, we found a
number of thus far uncharacterized genes with statistically significant deduced amino acid similarities (Fig.
6). These include two
proteins from Mus musculus, an uncharacterized
protein (GenBankTM accession number AK003656) with
an amino acid sequence identity of 94.46% and the Lmbr1 protein with
an identity of 57.91% (GenBankTM accession number
AF190665). Dif14 from Homo sapiens (GenBankTM
accession number AF402318), the human counterpart to M. musculus Lmbr1, shows 58.32% identity to LIMR. In addition,
putative proteins from Fugu rubripes (GenBankTM
accession number AF056116), Caenorhabditis elegans
(GenBankTM accession number NM066446), Drosophila
melanogaster (GenBankTM accession number AF132157),
Anopheles gambiae strain PEST (GenBankTM
accession number AAAB01008807), Dictyostelium discoideum
(GenBankTM accession number AC116982), and
Arabidopsis thaliana (GenBankTM accession number
AY035091) were found to show amino acid sequence identities of 56.26%,
28.96%, 42.51%, 42.09%, 20.33% and 9.89%, respectively.
Interestingly, a detailed comparison of the N terminus of LIMR, which
was found by phage-display to be the Lcn-1-interacting domain (11),
with the orthologous human Dif14 and mouse Lmbr1 indicated this region
to be less conserved than the rest of the proteins. Within this region,
there is an amino acid identity of only 12.5% between human LIMR and
human Dif14, whereas the overall identity is 58.32%. A similar result
is also found comparing the mouse LIMR and the orthologous Lmbr1, with
an identity of 37.5% within the N terminus and 57.91% with the entire
protein.


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Fig. 6.
Sequence comparison of LIMR orthologous
proteins. Deduced amino acid sequences were taken from from
GenBankTM. AF260728, H. sapiens LIMR; AK003656,
M. musculus unknown protein; AF402318, H. sapiens Dif14; AF190665, M. musculus Lmbr1;
AF056116, F. rubripes unknown protein; AF132157, D. melanogaster unknown protein; AAAB01008807, A. gambiae
strain PEST unknown protein; NM_066446, C. elegans unknown
protein; AC116982, D. discoideum unknown protein; AY035091,
A. thaliana unknown protein. Identical residues are
gray-shaded, homologous residues are boxed.
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Because LIMR was clearly characterized as a membrane protein (11), we
performed a secondary structure prediction and prediction of putative
transmembrane regions of all the orthologues found using three
different prediction programs (20-24). The results presented in Fig.
7 indicate a striking similarity in the
topography of these proteins. All of them consist of nine transmembrane
regions with an outside N terminus and a C terminus orientated toward the cytoplasm. Most noteworthy, all of these proteins contain a large
central intracellular loop consisting of 90-150 amino acids. Due to
the highly conserved topography and the fact that, at least in human
and mouse, two distinct orthologues are present, we propose this group
of proteins to be a novel family of membrane proteins. Because LIMR,
which is suggested to be the prototype of this protein family, is
essential for endocytosis of Lcn-1, it might be speculated that all of
these proteins function as endocytic receptors.

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Fig. 7.
Putative topography of LIMR orthologous
proteins. The analysis was performed using TopPred 2 (22), the
topology prediction program of membrane proteins at Stockholm
University. Proteins are numbered as described in the Fig. 6
legend.
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DISCUSSION |
A prerequisite for understanding the physiological function of
lipocalins is some knowledge about the mode of delivering the bound
ligands. There is increasing evidence that many lipocalins bind to
specific cell receptors. Receptor binding has been demonstrated for
-1-microglobulin (25, 26), glycodelin (27), insecticyanin (28),
-1-acid glycoprotein (29, 30),
-lactoglobulin (31, 32), and
odorant-binding protein (33). Although this is still a controversial
area, there is considerable evidence that retinol-binding protein (RBP)
binds to its target cells via specific surface receptors (34, 35). In
addition, major urinary protein has been found to bind to megalin (10),
a member of the low density lipoprotein receptor-related protein family
(36). However, with the exception of megalin, none of these cell
receptors has been isolated or characterized on a molecular level. We
have recently isolated a novel human cell membrane protein, LIMR, which
interacts with Lcn-1 (11). Lcn-1 is a lipocalin member produced by a
number of human secretory glands and tissues that is known to bind a variety of ligands, including fatty acids, fatty alcohols, cholesterol, retinol, retinoic acid, phosphatidylcholine, and arachidonic acid and
its peroxidation products (12, 13). Several biological functions have
been proposed for this lipocalin, but its main function seems to be
clearance or detoxification of lipophilic, potentially harmful
compounds (14, 17). We have therefore suggested LIMR to be directly
involved in this process. However, the exact role of LIMR had to be
established. The data presented in this paper clearly demonstrate that
LIMR mediates cellular uptake of Lcn-1, thus it has to be characterized
as a novel endocytic receptor.
In general, several models have been discussed for the ligand transfer
from lipocalins to the cell interior, including releases of the ligand
upon receptor binding and transport or diffusion through the cell
membrane or internalization of the lipocalin-ligand complex by
receptor-mediated endocytosis. Another model suggests that the
lipocalin-receptor interaction creates a direct signal inducing various
physiological processes (37). Most experimental data concerning the
uptake mechanism of lipocalin ligands came from studies on RBP and its
physiological ligand, retinol. Although there are some conflicting
reports, it seems that there is a cell type-specific difference in the
release of retinol from RBP to the cell interior. In retinal pigment
epithelial cells and placental brush-border membranes, it was suggested
that RBP remains external to the cell, with only retinol internalized
(38, 39). Therefore the RBP receptor is supposed to act as a channel
for retinol. By contrast, in hepatocytes and epithelial kidney cells,
RBP was clearly demonstrated to undergo internalization via
receptor-mediated endocytosis and subsequent degradation, thereby
releasing the retinol (40, 41). Megalin seems to be involved in the
epithelial endocytic uptake of RBP (42). Endocytic uptake mediated by
megalin was also demonstrated for major urinary protein (10), a
lipocalin member that binds small natural odorants and is highly
secreted by the liver and filtered by the kidney into the urine of
adult male mice and rats (43).
Although both LIMR and megalin are involved in endocytic uptake of
lipocalins, there is no structural similarity between these proteins.
Whereas megalin has the typical structure of the low density
lipoprotein receptor family consisting of a large extracellular domain
containing different ligand-binding clusters, a single transmembrane
domain, and a short cytoplasmic tail (44), LIMR consists of a short
extracellular domain, nine transmembrane domains interrupted by a large
intracellular loop, and an intermediate-length cytoplasmic tail
(11).
Considering that Lcn-1 binds physiological ligands of a number of
different chemical classes, cellular internalization of the
whole protein-ligand complex would be superior to a mechanism where the
ligand is delivered to a receptor. However, as indicated by the results
obtained with RBP, it seems that internalization of the protein-ligand
complex is also preferred by a lipocalin that is very specific for
binding of retinol. In addition,
-1-acid glycoprotein is another
lipocalin that was found to be endocytosed by a specific receptor (26).
Therefore, internalization of the whole lipocalin-ligand complex seems
to be the preferential mechanism.
Whether LIMR is a specific lipocalin receptor or rather a
multifunctional receptor, similar to megalin, remains to be proven by
additional studies. However, in contrast to megalin, which consists of
a large ligand binding cluster (45), the N-terminal ligand binding
domain of Lcn-1 is small, indicating some specificity. In this context,
the low amino acid similarity within the N-terminal region of LIMR,
which contains the Lcn-1 binding domain, and that of human Dif14
supports this suggestion.
It is novel, but not unexpected, that there are a number of genes
encoding LIMR orthologous proteins in other organisms, including M. musculus, F. rubripes, D. melanogaster, A. gambiae, C. elegans, and
D. disccoideum. Partial cDNA sequences with significant
similarities were also found in expressed sequence tag databases from
Macaca mulatta (GenBankTM accession number
BQ807894), Bos taurus (GenBankTM accession
number BE685341), Sus scrofa (GenBankTM
accession number BE014550), Rattus norvegicus
(GenBankTM accession number BF565677), and
Xenopus laevis (GenBankTM
accession number BQ732574). Most interestingly, in the human, there
is another LIMR orthologous protein called Dif14, encoded on chromosome
7q36 (46), whereas LIMR is encoded on chromosome 12p11 (11). As in
human, in mouse there are also two closely related proteins; one is
highly similar to human LIMR, whereas the other, Lmbr1, is more similar
to human Dif14. Human and mouse Dif14/Lmbr1 is of considerable interest
in genetics because it was suggested to be involved in preaxial
polydactyly, one of the most frequently observed congenital limb
malformations, whereby a disruption of the Dif14/Lmbr1 gene was
speculated to be the basis of this malformation (46, 47). However, more
consistent with the function of an endocytosis receptor, as proposed
here, recent work demonstrated that misexpression of a cis-acting
regulator located within the same respective intron of the Dif14/Lmbr1
gene is the basis for preaxial polydactyly and that the Lmbr1 gene is
incidental to the phenotype (48).
The fact that there are orthologous proteins in one and the same
organism that are encoded by different chromosomes prompted us to
define these proteins as a novel family of putative endocytic receptors. Thus far, this family may be grouped into two branches. According to the amino acid similarities and the deduced dendrogram of
a CLUSTAL-W-PHYLIP analysis, the first group consists of H. sapiens LIMR, M. musculus AK003656, and the putative
proteins from F. rubripes (AF056116), D. melanogaster (AF132157), and C. elegans
(NM066446), whereas the second group consists of H. sapiens
Dif14, M. musculus Lmbr1, and probably the proteins from
A. gambiae strain PEST (AAAB01008807), D. discoideum (AC116982), and A. thaliana (AY035091).