THE JOURNAL OF BIOLOGICAL CHEMISTRY© 2003 by The American Society for Biochemistry and Molecular Biology, Inc.

Vol. 278, No. 15, Issue of April 11, pp. 12613–12617, 2003
Printed in U.S.A.

FEEL-1 and FEEL-2 Are Endocytic Receptors for Advanced
Glycation End Products*
Received for publication, October 4, 2002, and in revised form, December 2, 2002
Published, JBC Papers in Press, December 6, 2002, DOI 10.1074/jbc.M210211200

Yoshiaki Tamura‡§, Hideki Adachi§¶, Jun-ichi Osuga‡, Ken Ohashi‡, Naoya Yahagi‡,
Motohiro Sekiya‡, Hiroaki Okazaki‡, Sachiko Tomita‡, Yoko Iizuka‡, Hitoshi Shimano储,
Ryozo Nagai**, Satoshi Kimura‡‡, Masafumi Tsujimoto¶, and Shun Ishibashi‡§§¶¶
From the ‡Departments of Metabolic Diseases, **Cardiovascular Medicine, and ‡‡Infectious Diseases, Faculty of Medicine,
University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo, 113-8655 Japan; the ¶Laboratory of Cellular Biochemistry,
The Institute of Physical and Chemical Research (RIKEN), Wako-shi, Saitama, 351-0198 Japan; the 储Departments ofMetabolism, Endocrinology, and Atherosclerosis, Institute of Clinical Medicine, University of Tsukuba, 1-1-1 Tennodai,
Tsukuba, Ibaraki, 305-8575 Japan; and the §§Department of Internal Medicine, Jichi Medical School,
3311-1 Yakushiji, Minamikawachi-machi, Kawachi-gun, Tochigi, 329-0498 Japan

Advanced glycation end products (AGEs) are nonenzymatically glycosylated proteins, which accumulate
in vascular tissues in aging and diabetes. Receptors for
AGEs include scavenger receptors, which recognize
acetylated low density lipoproteins (Ac-LDL) such as
scavenger receptor class AI/AII (SR-A), cell surface glycoprotein CD36, scavenger receptor class B type I (SRBI), and lectin-like oxidized low density lipoprotein
receptor-1. The broad ligand repertoire of these receptors as well as the diversity of the receptors for AGEs
have prompted us to examine whether AGEs are also
recognized by the novel scavenger receptors, which wehave recently isolated from a cDNA library preparedfrom human umbilical vein endothelial cells, such as
the scavenger receptor expressed by endothelial cells-I(SREC-I); the fasciclin EGF-like, laminin-type EGFlike, and link domain-containing scavenger receptor-1(FEEL-1); and its paralogous protein, FEEL-2. At 4 °C,
125
I-AGE-bovine serum albumin (BSA) exhibited highaffinity specific binding to Chinese hamster ovary(CHO) cells overexpressing FEEL-1 (CHO-FEEL-1) and
FEEL-2 (CHO-FEEL-2) with Kd of 2.55 and 1.68 ␮g/ml,
respectively, but not to CHO cells expressing SREC
(CHO-SREC) and parent CHO cells. At 37 °C, 125I-AGEBSA was taken up and degraded by CHO-FEEL-1 andCHO-FEEL-2 cells but not by CHO-SREC and parent
CHO cells. Thus, the ability to bind Ac-LDL is not necessarily a prerequisite to bind AGEs. The 125I-AGE-BSA
binding to CHO-FEEL-1 and CHO-FEEL-2 cells was effectively inhibited by Ac-LDL and polyanionic SR-A
inhibitors such as fucoidan, polyinosinic acids, anddextran sulfate but not by native LDL, oxidized LDL,or HDL. FEEL-1, which is expressed by the liver andvascular tissues, may recognize AGEs, thereby contributing to the development of diabetic vascular complications and atherosclerosis.

* The costs of publication of this article were defrayed in part by thepayment of page charges. This article must therefore be hereby marked“advertisement” in accordance with 18 U.S.C. Section 1734 solely toindicate this fact.
§ Both authors contributed equally to this work.
¶¶ To whom correspondence should be addressed: Dept. of Internal
Medicine, Division of Endocrinology and Metabolism, Jichi Medical
School, 3311-1 Yakushiji, Minamikawachi-machi, Kawachi-gun,Tochigi 329-0498, Japan. Tel.: 81-285-58-7355; Fax: 81-285-40-6035;
E-mail: ishibash@jichi.ac.jp.
This paper is available on line at http://www.jbc.org

Advanced glycation end products (AGEs)1 are generated bynonenzymatic glycosylation of proteins or lipids after prolonged
exposure to glucose (1). AGEs elicit a wide variety of cellularresponses including induction of growth factors and cytokines(2), adhesion molecules (3), oxidant stress (4), and chemotaxis(5). These proinflammatory responses are implicated to contribute to the development of pathologies associated with aging, diabetes mellitus, and Alzheimer’s disease (6). Indeed,
AGEs were shown to be present in atherosclerotic lesions (7)and diabetic kidney (8).
The AGE-elicited proinflammatory reactions are mediatedby its receptors or binding proteins, which include the receptor
for advanced glycation end product (RAGE) (9, 10), OST-48(ARE-R1)/80K-H (AGE-R2)/galectin-3 (AGE-R3) (11), scavenger receptor class AI/AII (SR-A) (12), scavenger receptor
class B type I (SR-BI) (13), cell surface glycoprotein CD36 (14),lectin-like oxidized low density lipoprotein receptor-1 (15), lactoferin (16), and lysozyme (16).
The broad ligand repertories of these AGE-binding proteins
as well as the diversity of receptors for AGEs have prompted us
to examine whether AGEs are recognized by novel members ofscavenger receptors that Adachi et al. (17, 18) have recently
cloned from a cDNA library prepared from human umbilicalvein endothelial cells as receptors for acetylated low density
lipoproteins (Ac-LDL), such as the scavenger receptor expressed by endothelial cells-I (SREC-I) (17) and the fasciclin
EGF-like, laminin-type EGF-like, and link domain-containing
scavenger receptor-1 (FEEL-1) (18). These receptors are structurally unrelated to other scavenger receptors. SREC is a protein of 830 amino acids with five epidermal growth factor-like
cysteine pattern signatures. FEEL-1 is a protein of 2570 aminoacids including 7 fasciclins, 16 EGF-like, 2 laminin-type EGFlike, and 1 link domain near the transmembrane region.
FEEL-2 is a paralogous gene of FEEL-1 whose amino acidsequence is ⬃40% identical to FEEL-1. Quantitative PCR analyses showed that both FEEL-1 and FEEL-2 are expressed in
1
The abbreviations used are: AGEs, advanced glycation end products; LDL, low density lipoprotein; Ac-LDL, acetylated low density
lipoprotein; Ox-LDL, oxidized low density lipoprotein; HDL, high density lipoprotein; SR-A, scavenger receptor class AI/AII; SR-BI, scavenger receptor class B type I; SREC, scavenger receptor expressed byendothelial cells; EGF, epidermal growth receptor; FEEL-1, fasciclin
EGF-like, laminin-type EGF-like, and link domain-containing scavenger receptor-1; RAGE, receptor for advanced glycation end product;
BSA, bovine serum albumin; poly I, polyinosinic acid; CHO, Chinese
hamster ovary; HUVECs, human umbilical endothelial cells; PBS,
phosphate-buffered saline.

12613

12614

FEEL-1 and FEEL-2 as AGE Receptors

FIG. 1. Northern blot analysis of FEEL-1, FEEL-2, and SREC intransfected CHO cells. 3 ␮g of poly(A)⫹ RNA was subjected to Northern blot analysis with cDNA probe for FEEL-1, FEEL-2, and SREC.
36B4 was used as a molecular reference.

the spleen and lymph node, whereas only FEEL-1 is detectablein CD14⫹-mononuclear cells and vascular endothelial celllines (18).
Here we show that FEEL-1 and FEEL-2, but not SREC, areendocytic receptors for AGEs. Because FEEL-1 is expressed by
the liver, macrophages, and endothelial cells in an amountcomparable with other receptors for AGEs, FEEL-1 may play asignificant role in the elimination of AGEs from the circulation
as well as in the development of diabetic vascular complications and atherosclerosis.
EXPERIMENTAL PROCEDURES

Materials—Ham’s F-12 medium, Dulbecco’s modified Eagle medium(DMEM), penicillin G, streptomycin sulfate, G418, and TriZOL werepurchased from Invitrogen. Bovine serum albumin (BSA), MOPC21(mouse IgG), fucoidan, polyinosinic acid (poly I), and dextran sulfate
were purchased from Sigma. Heparin sodium salt was purchased from
Mitsubishi Pharma (Osaka, Japan). Glucose 6-phosphate was purchased from Oriental Yeast (Tokyo, Japan). Endothelial cell growth
supplement was purchased from BD Biosciences. Oligotex-dT30TM waspurchased from Roche Molecular Biochemicals. Hybond N was purchased from Amersham Biosciences. Na-125I was purchased from Daiichi Chemical (Osaka, Japan). IodogenTM and BCA protein assay reagent kit were purchased from Pierce. Human umbilical vein endothelial
cells (HUVECs) were purchased from Kurabo (Tokyo, Japan). A murine
monoclonal antibody against human FEEL-1 (FE-1-1) was described
previously (18). cDNA probes for murine FEEL-1, FEEL-2 SREC,
RAGE, galectin-3, SR-A, CD36, and SR-B1 were prepared by reverse
transcriptase-PCR using primers designed based on the reported nucleotide sequences.
Ligand Preparation and Radiolabeling—AGE-BSA was prepared as
described previously (19). 600 mg of BSA was incubated with 50 mM
glucose 6-phosphate in 10 ml of sterile sodium phosphate buffer orphosphate-buffered saline (PBS) for 10 weeks at 37 °C and dialyzed
overnight against PBS. AGE-specific fluorescence was measured at 450
nm after excitation at 360 nm at a concentration of 1 mg/ml with afluorescence spectrometer (Biolumin, Amersham Biosciences). The
AGE-BSA exhibited ⬃38-fold higher fluorescence intensity than BSA.
AGE-BSA was labeled with 125I by using Iodogen according to the
manufacturer’s instruction. Protein concentrations were determined by
BCA protein assay reagent kit. LDL (d ⫽ 1.019 –1.063 g/ml) and highdensity lipoprotein (HDL) (d ⫽ 1.063–1.21 g/ml) were prepared by
stepwise ultracentrifugation from plasma obtained from healthy volunteers. LDL was acetylated with acetic anhydrate as described previously (20) and oxidized by incubation in a buffer containing 5 ␮M CuSO4
for 16 h at 37 °C (21). The lipoproteins were dialyzed against a buffer
containing 10 mM sodium phosphate, pH 7.4, and 150 mM NaCl.
Cell Culture—CHO cells overexpressing human FEEL-1 (CHOFEEL-1), human FEEL-2 (CHO-FEEL-2), and human SREC (CHOSREC) were obtained as described previously (17, 18). These cells weremaintained at 37 °C with 5% (v/v) CO2 in medium A (Ham’s F-12
supplemented with 100 units/ml penicillin and 100 units/ml streptomycin) containing 10% (v/v) fetal calf serum and 0.2 mg/ml G418 (medium
B). Untransfected CHO cells designated as CHO-Wild were maintained
in medium B without G418. The four lines of CHO cells were cultured
for 2 days to confluence in 12-well plates and used for the following
experiments. Thioglycolate-elicited mouse peritoneal macrophages
were prepared as described previously (22) and maintained in DMEM
containing 10% (v/v) fetal calf serum. HUVECs were cultured withDMEM containing 20% fetal calf serum, 30 ␮g/ml endothelial cell

FIG. 2. Binding of 125I-AGE-BSA to CHO-FEEL-1 (A), CHOFEEL-2 (B), CHO-SREC (C), and parent CHO cells (D). Cells wereincubated with the indicated concentrations of 125I-AGE-BSA for 2 h at
4 °C in the presence (open triangle) or absence (open circles) of 20-foldexcess amounts of unlabeled AGE-BSA in triplicate wells. Specificbinding (closed circle) was determined by subtracting nonspecific binding from total binding. Error bars represent mean ⫾ S.D. Insets show
the Scatchard analysis of the specific binding curve.
growth supplement, and 10 IU/ml heparin. Confluent dishes were used
for the studies within the 15 passages.
Northern Blot Analysis—Total RNA was prepared by TriZOL.
Poly(A)⫹ RNA was purified using Oligotex-dT30 and subjected to 1%(w/v) agarose gel electrophoresis in the presence of formalin. The fractionated RNA was transferred to Hybond N, hybridized to 32P-labeled
cDNA probes, and analyzed by BAS2000 (FUJI XEROX, Tokyo, Japan).
The expression levels of each gene were compared between variousorgans after adjusting to the expression level of 36B4.
Binding of 125I-AGE-BSA at 4 °C—After washing twice with PBS,
the confluent cells were incubated with 0.5 ml of medium C (medium A
supplemented with 3% (w/v) BSA) containing the indicated concentrations of iodinated AGE-BSA with or without 20-fold excess of unlabeled
AGE-BSA for 2 h at 4 °C. After washing three times with ice-cold PBS
containing 2 mg/ml BSA and three times further with PBS, the cells
were dissolved with 0.1 N NaOH and the cell-bound radioactivity andcellular proteins were determined.
Uptake and Degradation of 125I-AGE-BSA—One hour prior to thestudy, the media of the confluent cells were changed to medium C. The
media were replaced with medium C containing the indicated concentrations of 125I-AGE-BSA with or without 20-fold excess of unlabeled
AGE-BSA and incubated for 6 h at 37 °C. The amounts of 125I-AGE-BSA
either degraded by or associated with the cells were measured using
trichloroacetic acid and AgNO3 as described previously (22). The differences of the means were compared by Student’s t test.
RESULTS

Expression of FEEL-1, FEEL-2, and SREC in Transfected
CHO Cells—Northern blot analyses were performed to compare the mRNA expression of FEEL-1, FEEL-2, and SREC in
CHO-FEEL-1, CHO-FEEL-2, and CHO-SREC cells, respectively (Fig. 1). CHO-FEEL-1 and CHO-SREC expressed comparable amounts of the respective mRNA, which was 2-foldhigher than the mRNA of FEEL-2 mRNA in CHO-FEEL-2
cells.
Binding of 125I-AGE-BSA to CHO-FEEL-1, CHO-FEEL-2,
and CHO-SREC Cells—125I-AGE-BSA bound to both CHOFEEL-1 and CHO-FEEL-2 cells in a saturable manner at 4 °C
(Fig. 2, A and B). Scatchard analysis (insets) showed the presence of single binding site for AGE-BSA with an apparent Kd of
2.55 and 1.68 ␮g/ml for FEEL-1 and FEEL-2, respectively.

FEEL-1 and FEEL-2 as AGE Receptors

12615

FIG. 3. Cellular uptake (A–D) and degradation (E–H) of 125I-AGE-BSA by CHO-FEEL-1 (A and E), CHO-FEEL-2 (B and F), CHOSREC (C and G), and parent CHO cells (D and H). Cells were incubated with the indicated concentrations of 125I-AGE-BSA for 6 h at 37 °C
in the presence (open triangles) or absence (open circles) of excess amounts of unlabeled AGE-BSA in triplicate wells. Cellular uptake (A–D) anddegradation (E–H) of 125I-AGE-BSA were determined. Specific uptake or degradation (closed circles) was determined by subtracting nonspecificvalues from total values. Error bars represent mean ⫾ S.D.

FIG. 4. Competitive inhibition of the 125I-AGE-BSA binding to
CHO-FEEL-1 (A and C) or CHO-FEEL-2 cells (B and D). A and B,
cells were incubated with 2 ␮g/ml 125I-AGE-BSA for 2 h at 4 °C in thepresence of indicated dose of AGE-BSA (closed circles), LDL (closed
triangles), Ac-LDL (open circles), Ox-LDL (open triangles), and HDL(open squares) in triplicate wells. C and D, cells were incubated with 2
␮g/ml 125I-AGE-BSA for 2 h at 4 °C in the presence of indicated dose of
fucoidan (closed circles), poly I (closed triangles), dextran sulfate (opencircles), and heparin (open triangles) in triplicate wells. Error bars
represent mean ⫾ S.D.

CHO-FEEL-2 cells bound ⬃17 times larger amounts of 125IAGE-BSA than CHO-FEEL-1 cells (Bmax ⫽ 315.5 versus 18.9
ng/mg). On the other hand, the specific 125I-AGE-BSA binding
to CHO-SREC cells was 30% of that to CHO-FEEL-1 cells withlower affinity (Fig. 2C). Untransfected CHO cells bound negligible amounts 125I-AGE-BSA (Fig. 2D).
Cellular Uptake and Degradation of 125I-AGE-BSA by CHOFEEL-1, CHO-FEEL-2, and CHO-SREC Cells—To examine
whether the binding is associated with cellular uptake anddegradation of 125I-AGE-BSA, we incubated the cells with 125IAGE-BSA at 37 °C and determined the amounts of 125I-AGE-

BSA bound (Fig. 3, A–D) and degraded by the cells (Fig. 3,
E–H). Significant amounts of 125I-AGE-BSA were taken up anddegraded by both CHO-FEEL-1 and CHO-FEEL-2 cells but notby CHO-SREC and untransfected CHO cells. The activity waslarger in CHO-FEEL-2 cells than in CHO-FEEL-1 cells. These
results indicate that FEEL-1 and FEEL-2 but not SREC are
endocytic receptors for AGE-BSA.
Ligand Specificity of 125I-AGE-BSA Binding to CHO-FEEL-1
or CHO-FEEL-2 Cells—To characterize the ligand specificity
for the AGE-BSA binding site of the FEEL-1 and FEEL-2, we
performed a competition study. First, we examined whether
the binding of 125I-AGE-BSA is inhibited by lipoproteins (Fig.
4, A and B). The binding of 125I-AGE-BSA to CHO-FEEL-1 (Fig.
4A) and CHO-FEEL-2 (Fig. 4B) cells was inhibited by Ac-LDL
but not by LDL and HDL. It is noteworthy that Ac-LDL inhibited the binding more potently than AGE-BSA itself in CHOFEEL-1 cells, whereas AGE-BSA inhibited the binding morepotently than Ac-LDL in CHO-FEEL-2 cells. Table I showsconcentrations required to inhibit the binding by 50% (IC50).
Ox-LDL partially inhibited the binding only in CHO-FEEL-1
cells (⬃40% reduction at 100 ␮g/ml). We next examined theinhibitory effect of various materials known as SR-A inhibitors(Fig. 4, C and D). Fucoidan, poly I, and dextran sulfate inhibited the 125I-AGE-BSA binding almost completely in both CHOFEEL-1 (Fig. 4C) and CHO-FEEL-2 cells (Fig. 4D). Heparin
was also effective in suppressing the binding, but the effect was
weaker than the other three compounds (Table I).
Inhibition of Uptake and Degradation of 125I-AGE-BSA by
CHO-FEEL-1 Cells by Anti-FEEL-1 Antibody—We determinedthe effects of anti-FEEL-1 monoclonal antibody (FE-1-1) as
well as other compounds on the endocytic uptake and degradation of 125I-AGE-BSA by CHO-FEEL-1 cells (Fig. 5). We havepreviously reported that FE-1-1 effectively suppressed thecellular uptake of Ac-LDL in CHO-FEEL-1 cells (18). AGEBSA and Ac-LDL effectively inhibited the cellular uptake anddegradation of 125I-AGE-BSA by CHO-FEEL-1 cells. No inhibitory effect was observed with 100 ␮g/ml native LDL,Ox-LDL, HDL, and 30 ␮g/ml control IgG. FE-1-1 inhibited
both cellular uptake and degradation of 125I-AGE-BSA by 72
and 48%, respectively. The extent of inhibition by the antibody was slightly smaller than that by AGE-BSA or Ac-LDL
but was almost the same as observed in inhibition study forcellular uptake of Ac-LDL (18).

12616

FEEL-1 and FEEL-2 as AGE Receptors

TABLE I
Concentrations of various lipoproteins and SR-A inhibitors to125
inhibit the binding of
I-AGE-BSA to CHO-FEEL-1 andCHO-FEEL-2 cells (IC50)
CHO-FEEL-1 and CHO-FEEL-2 cells were incubated with 2 ␮g/ml
125
I-AGE-BSA for 2 h at 4 °C with AGE-BSA, LDL, Ac-LDL, Ox-LDL,
HDL, fucoidan, polyinosinic acid (poly I), dextran sulfate, or heparin at
the concentrations of 0, 10, 50, or 100 ␮g/ml. The IC50 value were
calculated and shown for competitors that inhibited the binding of
125
I-AGE-BSA ⬎50% at 100 ␮g/ml. Values in parentheses are IC50 inmolar concentrations, which were calculated based on the assumptionthat molecular masses of AGE-BSA, AcLDL, fucoidan, poly I, dextran
sulfate, and heparin are 66, 549, 66.4, 200⬃500, 500, and 5⬃20 kDa,
respectively.
Competitors

CHO-FEEL-1

CHO-FEEL-2

␮g/ml (nM)

AGE-BSAAcLDL
FucoidanPolyinosinic acid
Dextran sulfate
Heparin22.2 (337)⬍5.9 (11)⬍5.4 (81)⬍5.3 (11⬃26)⬍5.5 (11)8.9 (443⬃1,770)⬍6.9 (104)16.8 (31)⬍5.0 (76)⬍5.0 (10⬃25)⬍5.1 (10)7.2 (358⬃1,434)

FIG. 6. Tissue distribution of FEEL-1 and FEEL-2 in comparison with other receptors for AGEs. 10 ␮g of total RNA was extracted from liver, kidney, heart, lung, brain, spleen, testis, white
adipose tissue (WAT), and aorta of mice and subjected to Northern blot
analysis with cDNA probe for FEEL-1, FEEL-2, RAGE, galectin-3,
SR-A, CD36, and SR-B1. 36B4 was used as a loading control.

FIG. 7. Expression of FEEL-1 in HUVEC and mouse peritoneal
macrophages. 10 ␮g of total RNA from HUVEC and 2 ␮g of poly(A)⫹RNA from mouse peritoneal macrophages were subjected to Northern blot analysis. The expression levels of FEEL-1 were comparedwith those of galectin-3 in HUVEC and SR-A in mouse peritoneal
macrophages.

cular Endothelial Cells—We further performed Northern blot
analysis to evaluate the expression of FEEL-1 in mouse peritoneal macrophages and HUVECs (Fig. 7). Mouse peritoneal
macrophages and HUVECs expressed FEEL-1 to a degree thatwas comparable with galectin-3 and SR-A, respectively.
DISCUSSION

FIG. 5. Competitive inhibition of the uptake (A) and degradation (B) of 125I-AGE-BSA by CHO-FEEL-1 cells. Cells were incubated with 2 ␮g/ml 125I-AGE-BSA for 6 h at 37 °C in the presence of 100
␮g/ml AGE-BSA, LDL, Ac-LDL, Ox-LDL, HDL, or 30 ␮g/ml FE-1-1
antibody or control IgG in triplicate wells. Cellular uptake (A) anddegradation (B) of 125I-AGE-BSA were determined. Error bars represent mean ⫾ S.D. *, p ⬍ 0.05 versus control values.

Tissue Distribution of the Expression of FEEL-1 and FEEL2—To estimate the contribution of FEEL-1 and FEEL-2 to theendocytosis of AGEs in various tissues in comparison withother receptors for AGEs, we performed Northern blot analyses(Fig. 6). FEEL-1 was expressed in a wide variety of organs in
the following order: liver ⬎ lung ⫽ heart ⫽ white adipose
tissue ⬎ aorta ⫽ kidney ⬎ spleen. FEEL-2 whose expression
was much lower than that of FEEL-1 was detectable only in theliver and spleen. In contrast to organs like lung, spleen, andwhite adipose tissue in which RAGE, galectin-3, and CD36
were most highly expressed, respectively, organs like heart,
liver, and aorta appeared to express significant amounts of
FEEL-1 whose expression level was as comparable as that ofother receptors for AGEs.
Expression of FEEL-1 and FEEL-2 in Macrophages and Vas-

We have recently reported the cloning of novel scavenger
receptors expressed on vascular endothelial cells by expression
cloning strategy using 1,1⬘-dioctadecyl-3,3,3⬘,3⬘-tetramethylindocarbo-cyanine perchlorate (DiI)-labeled Ac-LDL as the ligand, such as SREC (17), FEEL-1, and its paralogous gene,
FEEL-2 (18). Structures of these receptors are unique andunrelated to other scavenger receptors. Although the precise
functions of these receptors are currently unknown, in vitro
studies have suggested their involvement in cellular interaction and host defense. For example, both FEEL receptors bindto Gram-negative and Gram-positive bacteria (18). Furthermore, anti-FEEL-1 antibody inhibited the in vitro vasculartube formation (18). On the other hand, SREC-I and its isoform, SREC-II, showed a strong heterophilic trans-interaction
through their extracellular EGF-like repeat domains (23).
This study has first revealed that FEEL-1 and FEEL-2 areendocytic receptors for AGEs, implicating the involvement ofthese receptors in the pathologies of aging or diabetes. Some ofother receptors for AGEs are involved in these pathologies. For
example, Park et al. (24) have shown that intravenous administration of the soluble extracellular domain of RAGE, which
was originally cloned from bovine pulmonary endothelial cells,
efficiently suppressed diabetic atherosclerosis in apolipoprotein E-deficient mice. Second, Pugliese et al. (25) have shownthat diabetic glomerulopathy was rather accelerated in micelacking galectin-3, a critical component of OST-48/80-K-H/galectin-3 complex (11) that is ubiquitously expressed in mammalian cells, suggesting its protective role. Third, Suzuki et al.
(26) and Matsumoto et al. (27) observed that peritoneal macro-FEEL-1 and FEEL-2 as AGE Receptors
phages obtained from SR-A knock-out mice had the reduced
capacity for endocytic degradation of AGE-BSA, which was
30% of that in wild-type cells. However, liver sinusoidal endothelial cells obtained from SR-A knock-out mice had capacity
for endocytic degradation of AGE-BSA, which was indistinguishable from that of wild-type liver sinusoidal endothelial
cells (27). These results suggest that SR-A is the primaryendocytic receptor for AGE-BSA in macrophages, whereas
other receptors mediate the uptake of AGEs in endothelial
cells.
Although FEEL-1 and FEEL-2 are structurally unrelated to
SR-A, their ligand specificity is similar to that of SR-A. CHOFEEL-1 cells bind not only Ac-LDL but also Ox-LDL to a lesser
degree (18). The binding was competitively inhibited by SR-A
inhibitors, such as maleyl-BSA and dextran sulfate (18). Furthermore, both FEEL-1 and FEEL-2 bind AGE-BSA (Figs. 1
and 2) as does SR-A (12). Ac-LDL and negatively charged
compounds such as fucoidan, dextran sulfate, and poly I competitively inhibited the binding of AGE-BSA to CHO-FEEL-1
or CHO-FEEL-2 cells (Figs. 4 and 5) as is the case for SR-A (12).
These results suggest that AGE-BSA and Ac-LDL share a same
binding site on the receptors. Importantly, the binding affinityof AGE-BSA to FEEL-1 and FEEL-2 cells appeared to exceedthat to other receptors for AGEs. Calculated Kd was 25 nM for
FFEL-2; 39 nM for FEEL-1; 85 nM for CD36 (14); 100 nM for
RAGE (9); 126 nM for SR-BI (13); 148 nM for lectin-like oxidized
low density lipoprotein receptor-1 (15); and 350 nM for galectin3(11), assuming that molecular mass of AGE-BSA is 66 kDa. The
binding activity of CHO-FEEL-1 cells was ⬃20-fold lower thanthat of CHO-FEEL-2 cells. A similar although less significant
difference in the binding activity between the isoforms wasobserved for Ac-LDL (18). Together with the fact that themRNA expression of FEEL-2 in CHO-FEEL-2 cells was lower
than that of FEEL-1 in CHO-FEEL-1, these results indicatethat a significant proportion of the binding sites for AGEs on
FEEL-1 was inactive or masked in CHO-FEEL-1 cells. Thispossibility warrants further investigation.
In many organs, the mRNA expression levels of FEEL-1 werehigher than those of FEEL-2 (Fig. 6). Mouse peritoneal macrophages and HUVECs also expressed FEEL-1 to a degree comparable with other receptors for AGEs (Fig. 7). Furthermore,
FEEL-2 mRNA was barely detectable in several vascular endothelial cell lines or in monocyte/macrophages at nonstimulated conditions (18). Taken together, we propose that FEEL-1
and possibly FEEL-2 serve as functional endocytic receptors for
AGEs in vivo. This hypothesis needs to be verified by further
studies, such as gene targeting, that are currently in progressin our laboratory. As suggested by the presence in the aorta(Fig. 6), macrophages, and endothelial cells (Fig. 7) (18),
FEEL-1 may scavenge AGEs accumulated in vascular tissues
in diabetes and aging, thereby directly contributing to thedevelopment of diabetic vascular complications and atherosclerosis. Because Smedsrød et al. (28) reported that ⬎90% of
intravenously injected AGE-BSA was distributed in the liver in
rats, it is also possible that FEEL-1, which was expressed in
the liver (Fig. 6), may be involved in the elimination of AGEs
from the circulation.
In conclusion, we demonstrate that FEEL-1 and FEEL-2, but
not SREC-I, function as endocytic receptors for AGEs when

12617

overexpressed in CHO cells. Although further studies will beneeded to determine the role of these two receptors in vivo, they
may play a pivotal role in the pathogenesis of diabetic vascularcomplications and could be the promising target molecules fortheir prevention.
Addendum—FEEL-1 is identical to stabilin-1 (hyaluronan-scavenger
receptor; HA/S-R) first identified as MS-1 antigen (29). AGE uptake byliver sinusoidal endothelial cells is partially inhibited by an antibody
against HA/S-R (30).
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