From the Departments of Medicine,
§ Urology, and
Microbiology and ** Institute of Human
Nutrition, College of Physicians & Surgeons of Columbia University,
New York, New York, 10032 and ¶ Department of Biological
Sciences, New England College of Optometry,
Boston, Massachusetts 02115
Received for publication, June 13, 2000, and in revised form, August 25, 2000
![]() |
ABSTRACT |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Cellular retinol-binding protein, type I (CRBP-I)
and type II (CRBP-II) are the only members of the fatty acid-binding
protein (FABP) family that process intracellular retinol. Heart and
skeletal muscle take up postprandial retinol but express little or no
CRBP-I or CRBP-II. We have identified an intracellular retinol-binding protein in these tissues. The 134-amino acid protein is encoded by a
cDNA that is expressed primarily in heart, muscle and adipose tissue. It shares 57 and 56% sequence identity with CRBP-I and CRBP-II, respectively, but less than 40% with other members of the
FABP family. In situ hybridization demonstrates that the
protein is expressed at least as early as day 10 in developing heart
and muscle tissue of the embryonic mouse. Fluorescence titrations of
purified recombinant protein with retinol isomers indicates binding to
all-trans-, 13-cis-, and
9-cis-retinol, with respective Kd
values of 109, 83, and 130 nM. Retinoic acids
(all-trans-, 13-cis-, and 9-cis-),
retinals (all-trans-, 13-cis-, and
9-cis-), fatty acids (laurate, myristate, palmitate,
oleate, linoleate, arachidonate, and docosahexanoate), or fatty
alcohols (palmityl, petrosenlinyl, and ricinolenyl) fail to bind. The
distinct tissue expression pattern and binding specificity suggest that
we have identified a novel FABP family member, cellular retinol-binding protein, type III.
Retinoids lack appreciable solubility in water and consequently
must be bound to proteins in the aqueous environment of the cell
(1-5). Many retinoid-specific binding proteins have been identified;
some are found solely intracellularly and others only extracellularly
(1-5). Two cytosolic proteins that specifically bind
all-trans-retinol with high affinity have been characterized (1-5). These cytosolic binding proteins for retinol, cellular retinol-binding protein, type I
(CRBP-I)1 and cellular
retinol-binding protein, type II (CRBP-II), belong to a family of small
intracellular proteins known as the fatty acid-binding protein (FABP)
family. Similarly, two cytosolic FABP family members that bind retinoic
acid with high affinity, cellular retinoic acid-binding protein, types
I and II, have been described (1-5).
To date, 12 members of the FABP family have been identified in mammals
(6-10). All are low molecular weight cytosolic proteins that bind
hydrophobic ligands noncovalently (6-10). The three-dimensional structures of FABP family members are highly homologous (2, 6-10).
Their secondary structures comprise 10 Members of the FABP family solubilize and transport their respective
ligands in the cytosol and, in some instances, may regulate the
metabolism of their ligands (2, 6-10). For example, CRBP-I helps to
regulate retinol hemeostasis (2, 5, 13-15). Recent studies of knockout
mice indicate a crucial role for CRBP-I in maintaining normal hepatic
retinol storage. These studies demonstrate that CRBP-I is needed to
facilitate conversion of retinol to retinyl ester, thus slowing
turnover of retinol from the liver (15). Likewise, CRBP-II regulates
intestinal retinoid metabolism, facilitating reduction of retinal to
retinol and the subsequent esterification of retinol to retinyl ester.
The retinyl ester is packaged along with other dietary lipids into
nascent chylomicrons (2, 5, 13, 14). Thus, CBRP-I and CRBP-II promote
retinol uptake from the diet and its storage in the liver.
We recently demonstrated that the amount of postprandial retinol taken
up by heart, skeletal muscle, and adipose tissue is markedly influenced
by the level of lipoprotein lipase expression in the tissue
(16). Overexpression of lipoprotein lipase in heart, muscle, or adipose
tissue increases retinol uptake from chylomicrons or chylomicron
remnants (16). Because neither heart nor skeletal muscle express
CRBP-II (2) and very little if any CRBP-I (1-5, 17, 18), we wondered
whether these tissues might express other intracellular binding
proteins that facilitates retinol uptake, transport, and metabolism. We
now report the identification and characterization of a new member of
the FABP family that binds retinol and is highly expressed in heart,
skeletal muscle, and adipose tissue.
Identification and Sequence Analysis of a cDNA Encoding a
Novel Intracellular Retinol-binding Protein--
We screened sequences
deposited with GenBankTM (National Center for Biotechnology
Information, Bethesda, MD) and identified an expressed sequence tag
clone with a predicted amino acid sequence that has a high degree of
homology to both mouse CRBP-I and CRBP-II. We obtained this clone
(AA466092) through Research Genetics, Inc (Huntsville, AL). After
subcloning the cDNA into pcDNA 3 (Invitrogen, San Diego, CA),
it was sequenced in both directions through the Columbia University
Comprehensive Cancer Center Core DNA Sequencing Facility. Additional
sequencing was performed after the cDNA was subcloned into the
baculovirus expression vector pBlueBac4.5 (Invitrogen) and into the
prokaryotic expression vector pET11A (Novagen, Madison WI) to verify
that the cDNA was incorporated into these vectors in the proper
orientation and with the reading frame intact.
Retinoids, Fatty Acids, and Other
Lipids--
All-trans-retinol was obtained as a gift from
Dr. Christian Eckhoff (Hoffman La Roche Inc., Nutley, NJ).
9-cis-Retinol was synthesized through reduction of
9-cis-retinaldehyde (Sigma) with sodium borohydride
(19, 20) and subsequently purified on a 10% deactivated alumina column
(19, 20). The purity of the 9-cis-retinol was determined by
normal phase HPLC employing procedures that are described in detail
below (19). Isomers of retinoic acid and retinal (9-cis-,
13-cis-, and all-trans),
13-cis-retinol, and sphingosine were purchased from
Sigma. Lauric acid, myristic acid, palmitic acid, docosahexanoic acid,
palmityl alcohol, petrosenlinyl alcohol, and ricinolenyl alcohol were
purchased from NuChek (NuChek Prep Inc., Elysian, MN). Phytanic
acid, oleic acid, arachidonic acid, linolenic acid, and lignoceric
ceramide were purchased from Cayman Chemical Co. (Ann Arbor, MI).
Expression of Recombinant Protein in Sf9 Cells--
The
protein encoded by the cDNA described above was expressed in
Spodoptera frugiperda (Sf9) cells using a baculovirus
expression system (Invitrogen), according to the manufacturer's
protocol. The mouse cDNA was subcloned into the pBlueBac4.5 vector
and transfected together with disabled virus into actively dividing
Sf9 cells. Recombinant virus containing the cDNA insert was
plaque purified. The presence of the cDNA in the recombinant virus
was verified by PCR. The initial low titer viral stock was used to
generate a high titer viral stock. For expression of the protein,
Sf9 cells were plated at a density of 107
cells/150-cm2 flask and infected with recombinant virus at
a multiplicity of infection of 10. Cells were harvested 72 h after
infection, and the washed cell pellet was homogenized in 20 mM potassium phosphate, pH 7.4, containing 1 mM
EDTA, 10 mM Expression of Recombinant Protein in Escherichia coli--
As an
alternative to expressing the cDNA in Sf9 cells, we employed
a prokaryotic expression vector to generate recombinant protein in
E. coli. For this purpose, the cDNA was cloned into a
pET11A expression vector (Novagen, Madison, WI). To clone the cDNA
into the pET11A vector, a NdeI restriction site upstream of
the translation start site and a BamHI restriction site
downstream of the translation termination site were created through PCR
amplification of the cDNA employing primers containing the
specified restriction sites. The sequence of the 5' primer we
employed was 5'-GGGAATTCCATATGCCAGCAGACCTCAGCGGTAC-3' and that of the
3' primer was 5'-CGCGGATCCTCAGGCTCTCTGGAAGGTTTG-3'. Each PCR
amplification reaction contained forward and reverse primers (0.2 µM for each), 0.2 mM of each dNTP, 1.5 mM MgCl2, 2 units of Taq DNA
polymerase (Life Technologies, Inc.), and 5 µl of 10× PCR buffer in
50 µl. The following PCR conditions were employed: initial
denaturation at 95 °C for 10 min, followed by 35 cycles of
denaturation at 95 °C for 45 s, annealing at 55 °C for
45 s, and extension at 72 °C for 1 min in a DNA Thermal Cycler (PerkinElmer Life Sciences). The PCR product was subcloned into pCR II
according to the instructions of the supplier (Invitrogen). Subsequently, the cDNA insert was excised from pCR II and
directionally subcloned into the NdeI and BamHI
restrictions sites of the pET11A vector. Recombinant protein was
expressed in BL23(DE3) E. coli (Novagen). E. coli
containing the pET11A expression vector were grown at 30 °C to an
A660 of 0.6 and expression was induced
with 100 mM
isopropyl-1-thio- Protein Purification--
Recombinant CRBP-III protein expressed
either in Sf9 cells or in E. coli was purified from
the cytosol fraction obtained from Sf9 cell or E. coli homogenates (as described above) using two size exclusion
columns. The cytosol fraction was initially applied to a Bio-Gel P-100
polyacrylamide gel exclusion column (100 × 2.5 cm) (Bio-Rad),
equilibrated in homogenization buffer. Following absorbance at 280 nm
monitored elution of proteins from this column. The recombinant protein
eluted from the Bio-Gel P-100 column in approximately 60-69 ml of
buffer, an elution volume that should contain proteins with masses
ranging from 10 to 20 kDa. The fractions thought to contain the 15-kDa
protein were confirmed by analysis on 15% SDS-PAGE gels. These
fractions were combined and concentrated to 2 ml using Centriplus
concentrators with a molecular weight cut off of 3000 (Millipore,
Bedford, MA). The concentrate of fractions containing the 15-kDa
protein was then applied to a Bio-Gel P-30 sizing column (120 cm × 2.5 cm) (Bio-Rad) equilibrated with homogenization buffer. Again,
protein was followed by absorbance monitored at 280 nm. The presence of
a 15-kDa protein in fractions eluting from the Bio-Gel P-30 column was
identified by analysis on 15% SDS-PAGE gels. These fractions were
combined and concentrated prior to use for the binding assays described below.
Determination of the N-terminal Amino Acid Sequence of the
Purified Recombinant Protein--
To confirm that we had indeed
purified a protein predicted by the CRBP-III cDNA, the N-terminal
sequence of the purified recombinant protein was analyzed. For this
purpose the protein was electrophoresed on a 15% SDS-PAGE gel and
transferred electrophoretically to a polyvinylidene difluoride transfer
membrane (Millipore). The membrane was stained with Coomassie Blue, and
the protein band running at approximately 15 kDa was excised from the
membrane and subjected to automated N-terminal sequencing (21).
N-terminal sequence analysis was performed at the Columbia University
Howard Hughes Protein Chemistry Core Facility.
Total RNA Extraction and Northern Blot Analysis--
Female and
male adult C57Bl/6J mice were sacrificed by CO2 asphyxia,
tissues removed, immediately placed in liquid nitrogen, and stored at
In Situ Hybridization--
In situ hybridization
studies of expression of the cDNA in the embryonic mouse was
performed using digoxigenin-labeled riboprobes essentially as described
in Mendelsohn et al. (23). Similarly, the procedures
we employed to stage the mouse embryos, for their fixation and the
sectioning of embryos are also described in Mendelsohn et
al. (23). For antisense probes, the cDNA in pcDNA3 was
linearized with EcoRI, and antisense transcripts were
generated with T7 polymerase. For preparation of sense probes, the same
cDNA was linearized with HindIII, and sense transcripts
were generated with T3 polymerase.
Isolation of Adipocytes and Stromal Vascular
Cells--
Adipocyte and stromal vascular cell fractions were prepared
from mouse epididymal adipose tissue essentially as described by
Tsutsumi et al. (24) for the rat tissue. For this purpose, epididymal fat pads from five adult C57Bl/6J mice were combined (approximately 1 g of tissue), rinsed in 0.9% NaCl at room
temperature, and minced. The minced tissue was digested with 0.1%
collagenase D (Roche Molecular Biochemicals) in 100 mM
HEPES, pH 7.4, containing 120 mM NaCl, 50 mM
KCl, 5 mM glucose, 1 mM CaCl2, and
1.5% bovine serum albumin for 60 min in a shaking water bath at
37 °C. Undigested tissue was removed by filtering the digest through
a 250-µm nylon mesh. The resulting filtrate was centrifuged at
1000 × g for 10 min at 25 °C. The adipocytes (top
layer) and the stromal vascular cells (pellet) were collected by
aspiration using plastic pipettes. Total RNA was extracted from the two
cell fractions as described above and used for analysis by RT-PCR.
RT-PCR Procedures--
RT-PCR was used to identify the cellular
sites of expression of the novel retinol-binding protein and, for
comparison, CRBP-I in adipose tissue and several other tissues and
primary cell isolates. For this purpose, cDNA was generated from
approximately 1 µg total RNA prepared from primary isolates of mouse
stromal vascular cells and adipocytes using a Superscript
Preamplification Kit (Life Technologies, Inc.). The target cDNAs
were directly amplified by PCR using PCR beads (Amersham Pharmacia
Biotech). The primers used for amplification of the novel
retinol-binding protein were identical to those described above for the
cloning of cDNA into the pET11A prokaryotic expression vector. For
CRBP-I, the primers were designed based on the published mouse cDNA
sequence (GenBankTM accession number X60367). As the
5' primer we employed 5'-GGGAATTCCATATGCCTGTGGACTTCAACGGGTA-3', and as
the 3' primer we used 5'-CGCGGATCCCAGTGTACTTTCTTGAACACT-3'. The
same PCR conditions employed above also were used to amplify CRBP-I
cDNA. The amplified cDNA was electrophoresed on a 1% agarose gel.
Fluorescence Binding Assays--
All fluorescence measurements
were carried out on an Aminco luminescence spectrometer (Spectronic
Unicam, Rochester, NY) equipped with a magnetic stirring unit using a
2-ml fluorescence cuvette. To assess potential binding of
all-trans-retinol, 13-cis-retinol, and
9-cis-retinol to the purified recombinant protein, we
measured the enhancement of retinol fluorescence observed upon binding. For this purpose, the protein solution was excited at 330 nm (bandpass of 4 nm), and retinol emission was measured at 480 nm (bandpass of 4 nm). To assess the potential binding of 9-cis-,
13-cis-, and all-trans-retinoic acid, the
excitation wavelength was set at 360 nm (4 nm bandpass), and emission
was monitored at 470 nm (4 nm bandpass) (25). To assess potential
binding of fatty acids, fatty alcohols, sphingosine, and
ceramide, potential changes in tryptophan fluorescence were monitored
upon excitation at 290 nm and emission at 340 nm (bandpasses of 4 nm)
(25). Concentrated retinoid stocks were prepared by dissolving each
retinoid in absolute ethanol. Stock concentrations were calculated
based on the absorbance of each retinoid at its respective absorbance
maximium (26). Diluted ethanolic solutions prepared from these stocks
were used for binding assays. Fatty acid, fatty alcohol,
sphingosine, and ceramide stocks diluted in ethanol were used to
assess potential binding through changes in tryptophan fluorescence.
For these titrations, purified recombinant protein at a final
concentration of 2.5 µM was dissolved in 150 mM sodium phosphate, pH 7.4, containing 5 mM
KCl and 10 mM HEPES. Retinoids or other lipids were added
in 2 µl (100 pmol/µl) aliquots to an initial assay volume of 2 ml.
To estimate changes in fluorescence arising from unbound retinoids,
identical titration curves were performed for buffer alone.
Calculations were done essentially as described by Cogan et
al. (25).
HPLC Analyses of Retinol Isomers Following Fluorescence Binding
Studies--
Because all-trans-, 13-cis-, and
9-cis-retinol are light sensitive and easily isomerize and
degrade upon exposure to intense light like that employed in the
fluorescent titrations, we determined whether the
all-trans-, 13-cis-, or 9-cis-retinol
remained intact during/following the titrations. Thus, following
titration with these retinol isomers, the solutions were extracted and
subjected to analysis by normal phase HPLC. For this purpose, the
solution was transferred to a 15-ml conical tube; 2 ml of absolute
ethanol was added, and the mixture was vortexed well. Retinoids were
then extracted into 3 ml of hexane. After one backwash against 0.5 ml
of distilled water, the hexane extract was evaporated to dryness under
a gentle stream of N2. The dried extract was immediately reconstituted in 120 µl of hexane and injected to a normal phase HPLC
system (see below). To establish retention times and to assess purity
of these stocks, the diluted retinoid stocks used for titration were
extracted simultaneously with the protein solutions generated in the
fluorescence titrations.
Retinol isomers were separated on 4.6 × 150 mm Supelcosil silica
column (Supelco, Belefonte, PA), preceded by a silica guard column
(Supleco) using hexane/ethyl acetate/n-butanol (96.9:3:0.1 v/v/v) as the mobile phase at a flow rate of 0.8 ml/min. Elution of
retinol isomers were monitored at 325 nm. The identities of each isomer
was based on the absorbance spectrum obtained from an inline Waters 996 Photodiodearray detector (Waters, Milford, MA). Concentrations of
all-trans-, 13-cis-, and 9-cis-retinol in the extracts were calculated based on standard curves relating integrated peak area with known amounts of authentic
all-trans-retinol, 13-cis-retinol, or
9-cis-retinol.
Western Blot Analysis--
Recombinant protein expressed in
E. coli and recombinant rat CRBP-I (27) were analyzed on a
15% SDS-PAGE gradient gel (Bio-Rad), and the protein was transferred
to a polyvinylidene difluoride transfer membrane (Millipore) by
electroblotting. The membrane was blocked in 5% nonfat dry milk in
Tris-buffered saline, pH 7.4, containing 0.1% Tween-20 (TTBS)
overnight. The blocked membrane was washed the next morning three times
with TTBS and incubated with rabbit anti-CRBP-I antibody (28) for
1 h. The blot was visualized using ECL Western blot reagents
(Amersham Pharmacia Biotech) according to the manufacturer's instructions.
Determination of Tissue Retinol Concentrations--
Heart,
epididymal fat, and skeletal muscle (gastronemius) were dissected from
adult male C57Bl/6J mice under yellow light and immediately frozen in
liquid N2 and stored at Identification of a cDNA Encoding a Novel Protein with Sequence
Identity to CRBP-I and CRBP-II--
We sought to isolate intracellular
proteins that bind retinol with high affinity from tissues that contain
little or no CRBP-I or CRBP-II. Searching expressed sequence tags
deposited in GenBankTM, we located a full-length expressed
sequence tag, originally cloned from
mouse mammary tissue. The full sequence for this clone is shown in Fig.
1A.2 The cDNA
encodes a protein consisting of 134 amino acids with a theoretical
isoelectric point of 6.13 and a calculated molar extinction coefficient
of 25,920 (29). The cDNA carries a putative polyadenylation site
166 base pairs downstream of the stop codon.
Based on the deduced amino acid sequence, the cDNA encodes a
previously unidentified member of the intracellular FABP family (Fig.
1B). The alignment of the primary amino acid sequence for the encoded protein with several other members of the FABP family, CRBP-I, CRBP-II, ALBP, and heart-type fatty acid-binding protein is
shown in Fig. 1B. Also shown are elements of the secondary structure characteristic of FABP family members. These elements, 10 stretches of
The three-dimensional structures of several members of the FABP
protein family have been determined (2, 6-10, 30, 31). Several amino
acid residues participate in ligand binding. As shown in Table
I, amino acid residues 4, 40, 108, 126, and 128 (based on the numbering of ALBP) interact with ligand and help confer specificity (6-10, 30-36). At three of these five positions, CRBP-I, CRBP-II, and CRBP-III contain the same amino acids, lysine 40, glutamine 128, and phenylalanine 130 (Table I). The three proteins
differ at residue 4. At position 108, CRBP-III carries histidine
instead of the glutamine found in CRBP-I and CRBP-II. This difference
between CRBP-I, CRBP-II, and CRBP-III might affect ligand specificity
or binding affinity.
Expression of CRBP-III cDNA in Adult Mouse Tissues--
We
examined the pattern of tissue expression for CRBP-III mRNA in
adult mice by Northern blot analysis. Fig.
2A provides a representative
Northern blot that gives the tissue pattern of CRBP-III expression. For
comparison, tissue CRBP-I mRNA levels are also shown in Fig.
2A. A single CRBP-III transcript with an approximate size of
0.7 kilobases was detected in several tissues. The size of the CRBP-III
transcript is similar to that of rat CRBP-I mRNA, although the
tissue expression pattern is quite distinct (Fig. 2A). As
seen in Fig. 2A, CRBP-III mRNA was observed in heart, epididymal fat, and skeletal muscle but not in liver, kidney, brain,
lung, or eyes. In other Northern blots, CRBP-III expression was not
detected in spleen, testes, ovaries, fallopian tubes, or seminal
vesicles (data not shown). In contrast, CRBP-I (Fig. 2A) is
highly expressed in kidney, liver, lung, testis, epididymal fat, and
eyes and, to a lesser extent, in heart. Interestingly, CRBP-III is
highly expressed in two adult tissues, heart and skeletal muscle, that
express little or no CRBP-I (2, 17). Both CRBP-III and CRBP-I are
expressed in adipose tissue (2, 24). Low levels of CRBP-III expression
were also detected in small intestine, an organ that also expresses
CRBP-II (data not shown) (2, 24).
The cellular sites of CRBP-III and CRBP-I expression in adipose
tissue were compared. CRBP-III was expressed in both primary adipocytes
and stromal vascular cell preparations (Fig. 2B). This pattern of expression is different from that of CRBP-I, which is almost
entirely restricted to stromal vascular cells (Ref. 24 and Fig.
2B). CRBP-III expression was unaffected by inactivation of
ob, which increases adipose tissue formation. Northern blot analysis showed that the concentration of CRBP-III mRNA was
equivalent in ob/ob and wild type mice (data not shown).
Because Northern analysis failed to reveal CRBP-III expression in
liver, kidney and testes, we checked for CRBP-III expression in these
three tissues by the more sensitive RT-PCR analysis. Nevertheless, even
by RT-PCR analysis, we were unable to detect CRBP-III expression in
these tissues (Fig. 2B).
Expression of CRBP-III cDNA in the Embryonic Mouse--
The
prominent expression of CRBP-III cDNA in heart and skeletal muscle
of adult mice led us to examine its tissue expression pattern during
organogenesis. Fig. 3 shows the results
of in situ hybridization analysis of muscle tissue in E10
mouse embryos with digoxigenin-labeled antisense riboprobes. At E10 and
at later stages, expression of the CRBP-III cDNA was robust in the
walls of the dorsal aorta, the outflow tract, heart, and somites (Fig. 3, A and C). In heart, expression was highest in
the atrial myocardium and in the ventricular trabeculae (Fig.
3B). At embryonic day 14, expression was localized in the
developing muscle in heart, dorsal aorta, trachea, and bronchi of the
lungs, as well as in skeletal muscle (data not shown). Overall, these
data indicate that CRBP-III is expressed most strongly in developing
heart and in muscle tissue destined to become part of the
cardiovascular and arterial system.
Expression and Purification of Recombinant CRBP-III--
To
characterize the biochemical properties of CRBP-III, we transfected
CRBP-III cDNA into Sf9 cells. CRBP-III was recovered in the
cytosolic fraction. The protein migrated on a 15% SDS-PAGE gel with an
apparent molecular mass of ~15 kDa, in good agreement with the
deduced molecular mass of 15.4 kDa. Purification from the Sf9
cytosol was achieved by fractionation through two successive size
exclusion columns. The first fractionation on Bio-Gel P-100 yielded a
preparation that was approximately 70% homogeneous based on SDS-PAGE
gels (data not shown). After further fractionation on a Biogel P-30
column, a protein that migrated as a single band on a 4-20% SDS-PAGE
gradient gel at the same position as recombinant rat CRBP-I was
detected (Fig. 4A). The
sequence of the 30 N-terminal amino acids of the purified recombinant
protein was identical to that predicted from the cDNA sequence:
PADLSGTWNLLSSDNFEGYMLALGIDFATR. Despite the high degree of sequence
identity between CRBP-III and CRBP-I, there was no cross-reactivity
with rabbit antibodies directed against rat testis CRBP-I (Fig.
4B and Ref. 28).
Ligand Binding Studies--
We measured the binding affinities of
purified recombinant CRBP-III (Fig. 4A) to 23 small
hydrophobic molecules including nine retinoids:
all-trans-retinol, 13-cis-retinol,
9-cis-retinol, all-trans-retinaldehyde,
13-cis-retinaldehyde, 9-cis-retinaldehyde, all-trans-retinoic acid, 13-cis-retinoic acid,
and 9-cis-retinoic acid. In aqueous solvents, retinoids show
little or no fluorescence. However, when bound to protein, retinoid
fluorescence is significantly enhanced (1, 2, 25), permitting a
sensitive assay of binding affinity (1, 2, 25, 32, 33, 36). In
addition, we measured the affinities of fatty acids (phytanic,
octanoic, lauric, myristic, palmitic, oleic, linolenic, arachidonic,
and docosahexanoic acids), fatty alcohols (palmityl, petrosenlinyl, and
ricinolenyl alcohols), spingosine, and lignoceric ceramide to
CRBP-III. For these potential ligands, we monitored changes in CRBP-III
tryptophan fluorescence (32-35). A saturable fluorescence enhancement,
indicative of protein-bound retinoid, was observed only when
all-trans-retinol, 13-cis-retinol or
9-cis-retinol was titrated into the CRBP-III solution (Fig.
5). Mean dissociation constants
determined from multiple binding assays for these three retinol isomers
were 109, 83, and 130 nM, respectively (Table
II). In contrast to retinol isomers, no
fluorescent enhancement was observed with any of the other retinoids
listed above. These data indicate that CRBP-III binds retinol isomers
but not retinaldehydes or retinoic acids. Similarly, because tryptophan
fluorescence did not change upon titration with any of the nine fatty
acids, three fatty alcohols, sphingosine, or lignoceric
ceramide, we conclude that these lipids do not bind CRBP-III. Although
early studies of the FABP protein family members reported dissociation
constants for fatty acid binding to be in the micromolar range (6-10),
more recent studies indicate that these dissociation constants are in
the nanomolar range (36). Thus, it appears that CRBP-III binds
all-trans-, 13-cis-, and 9-cis-retinol
with high affinity but does not bind other possible FABP ligands.
Because 9-cis-retinol can readily isomerize to the
all-trans-isomer, we were concerned that
9-cis-retinol isomerized to all-trans-retinol prior to binding to CRBP-III. To eliminate this possibility, we measured the rate of isomerization of all-trans-retinol and
9-cis-retinol in assay buffer. We found that both
all-trans-retinol and 9-cis-retinol retained
their isomeric configurations during the titration reaction. At the end
of the titration, 99 and 95% of the added all-trans-retinol and 9-cis-retinol were recovered as all-trans-
and 9-cis-retinol, respectively. About 5% of the
9-cis-retinol was isomerized to all-trans-retinol. No isomerization of
13-cis-retinol to either the all-trans-retinol or
9-cis-retinol isomer was detected.
Concentrations of All-trans-retinol in Murine Skeletal Muscle,
Heart, and Epidydimal Fat--
To determine whether the retinol
binding constants for CRBP-III were compatible with tissue retinol
levels, we measured the concentrations of all-trans-retinol
in these tissues by normal phase HPLC. We are not aware of previous
reports in the literature that provide heart and muscle levels of
retinol or retinyl ester, as measured by modern HPLC techniques. Mean
concentrations of all-trans-retinol for mouse heart,
skeletal muscle, and epidydimal fat were 0.37, 0.12, and 2.76 nmol/g,
respectively (Table III). No retinyl
esters were detected in heart or skeletal muscle; however, as
previously reported (24), retinyl esters were detected in adipose
tissue (data not shown). Our low limit of detection for retinyl ester
measurement was less than 0.003 nmol/g. Thus, CRBP-III is expressed in
tissues that contain significant all-trans-retinol concentrations but in two tissues that contain little or no retinyl ester.
In this report, we describe a new member of the intracellular FABP
protein family, CRBP-III. CRBP-III is highly expressed in adult tissues
that express little or no CRBP-I and CRBP-II, viz heart and
muscle. In adipose tissue it is expressed along with CRBP-I. In
contrast, CRBP-III is not expressed in liver, lungs, testis, or kidney,
which express relatively high levels of CRBP-I. CRBP-III is expressed,
albeit it at relatively low levels, in small intestine along with
CRBP-II. Of the FABP proteins, CRBP-III shares the most sequence
identity with CRBP-I and CRBP-II, 57 and 56% identity, respectively.
Like CRBP-I and CRBP-II it is CRBP-III binds all-trans-, 13-cis-, and
9-cis-retinol with relatively high affinity but does not
bind retinoic acid or retinaldehyde isomers, long chain, short chain,
or branched chain fatty acids or fatty alcohols. The amino acid
sequence of CRBP-III is consistent with its binding specificity. The
positively charged lysine 40 helps stabilize binding of retinol to
CRBP-I and CRBP-II through interactions with the polyene chain of the
retinoid (6-10, 30-37). At positions 128 and 130, which also contact
ligand, CRBP-III, like CRBP-I and CRBP-II, carries glutamine and
phenylalanine. FABP family members that primarily bind fatty acids
contain arginine and tyrosine residues at positions 128 and 130, respectively. At position 108, a site critical in determining ligand
specificity, CRBP-III contains a histidine residue; CRBP-I and CRBP-II
carry glutamine, and FABP proteins that bind fatty acids carry arginine (6-10, 30-37). For CRBP-I and CRBP-II, the glutamine residue at position 108 is proposed to stabilize retinol binding through hydrogen
bond formation with the hydroxyl group of the retinol molecule.
Substitution of the glutamine at position of CRBP-I with an arginine
residue decreases the affinity of the mutant CRBP-I for retinol.
Moreover, the same substitution in CRBP-II increases the affinity of
the mutant CRBP-II for both retinoic acid and fatty acids (30-33).
Similarly for intestinal FABP, substitution of the arginine at position
108 with a glutamine increases binding affinity of the mutant
intestinal FABP for all-trans-retinol (35). We propose that
histidine 108 in CRBP-III acts both as a hydrogen bond donor and
hydrogen bond acceptor. Histidine 108 thus could stabilize retinol
binding by participating as a donor in hydrogen bond formation between
the hydroxyl group of the retinol molecule in a manner similar to but
less effective than the interaction between retinol and glutamine 108 of CRBP-I.
CRBP-III binds all-trans-retinol with an apparent
dissociation constant of 109 nM, compared with CRBP-I and
CRBP-II, which bind with apparent dissociation constants in the range
of 10-16 nM (1-5). The weaker binding affinity of
CRBP-III for retinols is consistent with the idea that CRBP-III may
have a different physiological role(s) than CRBP-I or CRBP-II. CRBP-I
and CRBP-II are proposed to play important roles in retinol metabolism,
especially in facilitating retinol esterification (2, 14, 15) and retinol oxidation to retinoic acid (2, 5). CRBP-I knockout mice are
unable to esterify retinol efficiently and have difficulty in
maintaining hepatic retinoid stores (15). On this basis, it has been
suggested that CRBP-I and CRBP-II deliver retinol to the enzyme
lecithin:retinol acyltransferase, which catalyzes retinyl ester
formation (2, 14, 15). CRBP-III cannot play this role in heart or
skeletal muscle, because these tissues lack detectable levels of
retinyl ester (Table III).
Retinoids are needed to maintain the health of the heart and
cardiovascular system in the adult. Experimentally induced myocardial infarction induces mobilization of hepatic retinol and its delivery to
the damaged heart (38). We suggest that CRBP-III mediates retinol
uptake from circulating retinol-RBP and/or helps facilitate retinol
oxidation to retinoic acid within the heart. The precise biochemical
mechanisms responsible for these roles must still be elucidated.
At days 9.5-10.5 during mouse embryogenesis, expression of CRBP-III is
most pronounced in the developing heart and cardiovascular system (Fig.
3). CRBP-I is much more widely distributed at this embryonic stage than
CRBP-III and is expressed highly in liver, gut, tongue, and spinal cord
and other components of the central nervous system (39, 40). At
embryonic day 12.5, CRBP-I is expressed in the epicardial layer of the
developing heart (40). It is well established that retinoids are
critically needed during embryonic heart and cardiovascular system
development. Both vitamin A deficiency and vitamin A excess during
pregnancy cause a wide spectrum of defects including embryonic heart
defects (41). Knockout mice that lack retinaldehyde dehydrogenase type
2 and the ability to synthesize retinoic acid from retinol die at
mid-gestation showing severe heart defects (42). Similarly, knockout
mice lacking functional retinoid X receptor Both CRBP-III and CRBP-I are expressed in adipose tissue (Ref. 24 and
Fig. 2B). Adipose tissue consists of adipocytes and stromal
vascular cells, which include adipocyte precursors, macrophages, fibroblasts, red blood cells and other blood cells (44). Stromal vascular cells account for over 50% of the protein and DNA content of
rat white adipose tissue (44-46). CRBP-III is expressed in both adipocytes and stromal vascular cells (Fig. 2B). The
expression of CRBP-I in adipose tissue is almost entirely limited to
stromal vascular cells (Ref. 24 and Fig. 2B). The distinct
cellular expression patterns of CRBP-III and CRBP-I implies that the
two proteins have different physiological roles within adipose tissue.
Adipose tissue also contains ALBP, a FABP family member that shares
38% amino acid identity with mouse CRBP-III and CRBP-I. ALBP, which
binds unsaturated and saturated fatty acids and retinoic acid but not
retinol, is expressed more highly in adipocytes compared with stromal
vascular cells (11, 47, 48).
One aim of this work was to elucidate the mechanism of tissue retinol
uptake and intracellular processing. We earlier showed a correlation
between lipoprotein lipase expression in heart, skeletal muscle, and
adipose tissue and the amount of retinol taken up by these tissues from
chylomicron-bound retinyl ester (16, 49). We proposed that lipoprotein
lipase facilitates uptake of postprandial retinol by hydrolyzing
retinyl esters (16, 49). In tissues other than heart and skeletal
muscle, intracellular processing of the retinol is thought to be
promoted by CRBP-I (2, 14, 17, 50, 51) or CRBP-II (2, 14, 52). We now
report the identification and characterization of a new intracellular
retinol binding protein, CRBP-III, which may substitute for CRBP-I or
CRBP-II in these tissues. However, definitive evidence for this or
other physiologic actions of CRBP-III remains to be gathered, and this
will be the direction of our future studies.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-sheet domains and 2
-helices that together form a
-barrel structure (6-10). For most
FABP family members, one hydrophobic ligand molecule is bound within
this
-barrel structure (6-10). Certain FABP members bind only fatty
acids, retinol/retinal (e.g. CRBP-I and -II), or retinoic
acid (e.g. cellular retinoic acid-binding protein, types I
and II) with high affinity (6-10), whereas adipocyte lipid-binding protein (ALBP) binds both fatty acids and all-trans-retinoic
acid (11), and myelin P2 protein is reported to bind both
all-trans-retinol and all-trans-retinoic acid and
fatty acids (12).
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-mercaptoethanol, 15% (v/v) glycerol,
0.05% (w/v) sodium azide, 7.5 µM aprotinin, and 0.5 mM phenylmethanesulfonyl fluoride using a Dounce
homogenizer. The homogenate was centrifuged at 8000 × g for 20 min to remove cell debris and the nuclear fraction.
Cytosolic and the crude microsomal fractions were obtained upon
centrifugation at 100,000 × g for 1 h at 4 °C
using a Beckman TC-100 ultracentrifuge (Beckman Coulter, Fullerton,
CA). The presence of recombinant 15-kDa protein in the crude microsomal
and cytosolic fractions was assessed on a 15% SDS-PAGE gel to verify
protein expression.
-D-galactopyranoside for 3 h at
30 °C. After harvesting of the induced bacterial cells by
centrifugation at 10,000 × g for 30 min at 4 °C,
the cell pellet was resuspended in 20 mM potassium
phosphate, pH 7.4, containing 1 mM EDTA, 10 mM
-mercaptoethanol, 15% (v/v) glycerol, 0.05% (w/v) sodium azide, 7.5 µM aprotinin, and 0.5 mM
phenylmethanesulfonyl fluoride and sonicated with three bursts of
30 s duration at half-maximal intensity. During sonication, the
resuspended bacterial pellet was kept on ice. Following sonication, the
soluble fraction was obtained upon ultracentrifugation at 100,000 × g at 4 °C using a Beckman TC-100 ultracentrifuge
(Beckman Coulter, Fullerton, CA).
70 °C until RNA extraction. Total RNA from liver, kidney, heart,
lung, spleen, skeletal muscle (gastronemius), small intestine, ovaries,
fallopian tubes, testes, seminal vesicles, and the ovarian and
epididymal fat depots was extracted using RNAzol (Tel-Test Inc.,
Friendswood, TX). Total RNA (25 µg/lane) was resolved by gel
electrophoresis in 1% agarose containing 0.98 M
formaldehyde (22). For all RNA samples, the ratio of intensities of the
28 and 18 S ribosomal RNA bands after staining with ethidium bromide
was approximately 2. Immediately following electrophoresis, the RNA was
transferred by capillary action to a positively charged Nylon membrane
(Amersham Pharmacia Biotech) and subsequently hybridized at 65 °C
with 32P-labeled probes for the mouse cDNA and with
control probes for rat CRBP-I (22) or glyceraldehyde-3-phosphate
dehydrogenase (CLONTECH, Palo Alto, CA). The probes
were labeled with 32P using a random priming kit (Roche
Molecular Biochemicals). Hybridized membranes were washed at a final
stringency of 0.1× SSC, 1.0% SDS at 65 °C and exposed to Kodak
AR-2 film at
80 °C. An RNA ladder from Ambion (Austin, TX) was
used to estimate transcript sizes.
70 °C until analysis. On the
day of analysis, each tissue sample was weighed, thawed on ice, and
homogenized with a Polytron homogenizer (Brinkmann Instruments,
Westbury, NY) in 2 ml of 150 mM sodium phosphate, pH 7.4, containing 150 mM NaCl. The synthetic retinoid,
all-trans-9-(4-methoxy-2,3,6-trimethylphenyl)-3,7-di-methyl-2,4,6,8-nonatetraen-1-ol (a gift from Dr. Christian Eckhoff, Hoffman La Roche Inc., Nutley, NJ)
was added as internal standard to monitor recovery of the retinoids
during extraction and HPLC analysis. The tissue homogenates were
denatured with equal volume of 100% ethanol, extracted into 3 ml of
hexane and backwashed once with 0.5 ml of distilled water. After
evaporation of the hexane under a gentle stream of N2 to dryness, the samples were reconstituted in hexane and analyzed by
normal phase HPLC essentially as described above (19).
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
View larger version (75K):
[in a new window]
Fig. 1.
A, the nucleotide sequence for the
cDNA clone encoding a new member of the FABP protein family. The
start and the termination codons are underlined.
B, the amino acid sequence deduced from this cDNA. For
comparison, the primary sequence encoded by the cDNA is aligned
with those of four other members of the fatty acid binding
protein family (2, 6, 8): CRBP-I, CRBP-II, ALBP, and heart FABP. The
boxed amino acids designated
A-
J represent 10 stretches of
-sheet structure, and those designated
I
and
II designate stretches of
-helix.
-sheet structure and 2
-helix domains, are also present in the encoded protein sequence (2, 6-10, 30). The amino acid
sequence of this protein shares 57 and 56% identity with murine CRBP-I
and CRBP-II, respectively, 35% identity with heart FABP, and 38% with
ALBP. The protein has a 2-amino acid insertion at amino acid positions
75 and 76 in a turn site between the
E and
F strands (Fig.
1B). Of all FABP proteins only the two cellular
retinol-binding proteins, CRBP-I and CRBP-II, show this two amino acid
insertion. Thus, the protein encoded by this cDNA appears to be
most closely related to CRBP-I and CRBP-II. On the basis of this
similarity and additional properties described below, we term the
protein cellular retinol-binding protein, type III (CRBP-III).
Amino acid residues present in the ligand binding site for members of
the FABP protein family
View larger version (78K):
[in a new window]
Fig. 2.
A, Northern blot analysis of total RNA
isolated from different murine tissues for expression of CRBP-III and
CRBP-I. Tissues examined for expression include liver (L),
kidney (K), heart (H), adipose tissue
(A), testes (T), brain (B), skeletal
muscle (M), and eyes (E). Each lane contains 25 µg of total RNA, and intactness of the RNA was visualized by probing
for GAPDH. B, RT-PCR analysis for expression of CRBP-III and
CRBP-I in isolated mouse adipocytes (AD), stromal-vascular
cells (SV) and in mouse liver (L), kidney
(K), and testes (T). STD, DNA size
markers.
View larger version (108K):
[in a new window]
Fig. 3.
In situ hybridization studies of
expression of CRBP-III in murine fetal tissues at embryonic day
10. A, expression in the E10 embryo. In situ
hybridization analysis of transverse sections probed with antisense
cDNA. Note expression in the dorsal aorta, heart, and somites.
B, higher magnification of the same section shown in
A showing expression in the walls of the atria and in the
ventricular myocardium. C, expression in the walls of the
dorsal aorta and in the myotome. D, expression in the walls
of the atrial myocardium. at, atrium; da,
dorsal aorta; he, heart; ot, outflow tract;
so, somite; v, ventricle; mu,
cardiomyocytes. Magnifications: A, ×40; B,
×100; C, ×100; D, ×400.
View larger version (40K):
[in a new window]
Fig. 4.
A, SDS-PAGE gel of purified recombinant
mouse CRBP-III (lane 1) expressed in Sf9 cells and
partially purified rat testes CRBP-I (lane 3) stained with
Coomassie blue dye. Each lane contains 25 µg of protein. Lane
2 provides mobilities of molecular mass markers. B,
Western blot analysis for recombinant mouse CRBP-III and rat testes
CRBP-I probed with a rabbit antiserum directed against rat testis
CRBP-1. Lane 1, 10 µg of purified recombinant CRBP-III;
lane 2, 6 µg of partially purified CRBP-I; lane
3, 3 µg of partially purified CRBP-I; lane 4, 5 µg
of purified recombinant CRBP-III.
View larger version (10K):
[in a new window]
Fig. 5.
Representative fluorescence titration curves
for the titration of recombinant apo-CRBP-III with
all-trans-retinol (A),
13-cis-retinol (B), and
9-cis-retinol (C). The
excitation wavelength was 330 nm (using a 4 nm bandpass) and the
emission wavelength 480 nm (using a 4 nm bandpass). Each titration was
carried out in 2 ml of buffer alone (closed circles) and 2 ml of apo-CRBP-III in the same buffer (open circles) (see
"Experimental Procedures" for details). The CRBP-III concentration
for this experiment was 2.5 µM.
Kd values for binding of different retinol isomers to the
purified recombinant murine CRBP-III
All-trans-retinol concentrations in mouse heart, skeletal muscle,
and epidydimal fat
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
40% identical with other members of
the family (1-5). It shares predicted structural motifs with the
CRBP-I and CRBP-II proteins. Like CRBP-I and CRBP-II, CRBP-III carries
a 2-amino acid insertion between
E and
F sheets and a lysine
residue at position 40 (Fig. 1B) that is not found in other
members of the FABP family (6-10, 30-37).
develop severe
cardiovascular defects and die by embryonic day 16 (43). A major cause
of the embryonic lethality associated with the retinoid X receptor
knockout has been attributed to impaired cardiovascular system development (43). Interestingly, CRBP-III, retinaldehyde dehydrogenase and retinoid X receptor
are colocalized in the myocardium
ventricules of the developing mouse heart at embryonic days 9.5-10.5
(42, 43). Considering both the temporal and spatial pattern of
expression of CRBP-III and these other proteins in the embryonic heart,
we speculate that CRBP-III plays a role in maintaining normal retinoic acid homeostasis in the developing heart and cardiovascular system.
![]() |
ACKNOWLEDGEMENT |
---|
Helpful advice from Dr. Judith Storch regarding the ligand binding studies is gratefully acknowledged.
![]() |
FOOTNOTES |
---|
* This work was supported by National Institutes of Health Grant R01 DK52444.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
To whom correspondence should be addressed: Dept. of Medicine,
Columbia University, 701 W 168th St., New York, NY 10032. Tel.: 212-305-5429; Fax: 212-305-5384; E-mail: WSB2@columbia.edu.
Published, JBC Papers in Press, October 5, 2000, DOI 10.1074/jbc.M005118200
2 The GenBankTM sequence for this cDNA contains several errors.
![]() |
ABBREVIATIONS |
---|
The abbreviations used are: CRBP-I, cellular retinol-binding protein, type I; CRBP-II, cellular retinol-binding protein, type II; CRBP-III, cellular retinol-binding protein, type III; FABP, fatty acid-binding protein; HPLC, high performance liquid chromatography; ALBP, adipocyte lipid-binding protein; PCR, polymerase chain reaction; RT, reverse transcription; PAGE, polyacrylamide gel electrophoresis.
![]() |
REFERENCES |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
1. | Noy, N. (1999) in Retinoids: The Biochemical and Molecular Basis of Vitamin A and Retinoid Action: Handbook of Experimental Pharmacology (Nau, H. , and Blaner, W. S., eds), Vol. 139 , pp. 3-30, Springer Verlag, Heidelberg |
2. | Ong, D. E., Newcomer, M. E., and Chytil, F. (1994) in The Retinoids: Biology, Chemistry, and Medicine (Sporn, M. B. , Roberts, A. B. , and Goodman, D. S., eds), 2nd Ed. , pp. 283-318, Raven Press Ltd., New York |
3. |
Newcomer, M. E.
(1995)
FASEB J.
9,
229-239 |
4. | Newcomer, M. E., Jamison, R. S., and Ong, D. E. (1998) Subcell. Biochem. 30, 53-80[Medline] [Order article via Infotrieve] |
5. | Blaner, W. S., Piantedosi, R., Sykes, A., and Vogel, S. (1999) in Retinoids: The Biochemical and Molecular Basis of Vitamin A and Retinoid Action: Handbook of Experimental Pharmacology (Nau, H. , and Blaner, W. S., eds), Vol. 139 , pp. 117-149, Springer Verlag, Heidelberg |
6. | Veerkamp, J. H., Peeters, R. A., and Maatman, R. G. H. J. (1991) Biochim. Biophys. Acta 1081, 1-24[Medline] [Order article via Infotrieve] |
7. | Veerkamp, J. H., van Kuppevelt, T. H. M. S. M., Maatman, R. G. H. J., and Prinsen, C. F. M. (1993) Prostagl. Leukot. Essent. Fatty Acids 49, 887-906[Medline] [Order article via Infotrieve] |
8. | Banaszak, L., Winter, N., Xu, Z., Bernlohr, D. L., Cowan, S., and Jones, T. A. (1994) Adv. Protein Chem. 45, 89-151[Medline] [Order article via Infotrieve] |
9. | Glatz, J. F. C., and van der Vusse, G. J. (1996) Prog. Lipid Res. 3, 243-282 |
10. | Glatz, J. F. C., van Breda, E., and van der Vusse, G. J. (1998) in Skeletal Muscle Metabolism in Exercise and Diabetes (Ricter, E. L., ed) , pp. 207-218, Plenum Press, New York |
11. | Baxa, C. A., Sha, R. S., Buelt, M. K., Smith, A. J., Vatarese, V., Chinander, L. L., Boundy, K. L, and Bernlohr, D. A. (1989) Biochemistry 28, 8683-8690[Medline] [Order article via Infotrieve] |
12. | Uyemura, K., Yoshimura, K., Suzuki, M., and Kitamura, K. (1984) Neurochem. Res. 28, 8683-8690 |
13. | Blaner, WS, and Olson, JA. (1994) in The Retinoids: Biology, Chemistry, and Medicine (Sporn, M. B. , Roberts, A. B. , and Goodman, D. S., eds), 2nd Ed. , pp. 229-256, Raven Press, Ltd., New York |
14. | Vogel, S., Gamble, M. V., and Blaner, W. S. (1999) in Retinoids: The Biochemical and Molecular Basis of Vitamin A and Retinoid Action: Handbook of Experimental Pharmacology (Nau, H. , and Blaner, W. S., eds), Vol. 139 , pp. 31-96, Springer Verlag, Heidelberg |
15. |
Ghyselinck, N. B.,
Bavik, C.,
Sapin, V.,
Mark, M.,
Bonnier, D.,
Hindelang, C.,
Dierich, A.,
Nilsson, C. B.,
Hakansson, H.,
Sauvant, P.,
Azais-Braesco, V.,
Frasson, M.,
Picaud, S.,
and Chambon, P.
(1999)
EMBO J.
18,
4903-4914 |
16. |
Van Bennekum, A. M.,
Kako, Y.,
Weinstock, P. H.,
Harrison, E. H.,
Deckelbaum, R. J.,
Goldberg, I. J.,
and Blaner, W. S.
(1999)
J. Lipid Res.
40,
565-574 |
17. |
Ong, D. E.,
Crow, J. A.,
and Chytil, F.
(1982)
J. Biol. Chem.
257,
13385-13389 |
18. |
Levin, M. S.,
Li, E.,
Ong, D. E.,
and Gordon, J. I.
(1987)
J. Biol. Chem.
262,
7118-7124 |
19. |
Mertz, J. R.,
Shang, E.,
Piantedosi, R.,
Wei, S.,
Wolgemuth, D. J.,
and Blaner, W. S.
(1997)
J. Biol. Chem.
272,
11744-11749 |
20. |
Blaner, W. S.,
Das, S. R.,
Gouras, P.,
and Flood, M. T.
(1987)
J. Biol. Chem.
262,
53-58 |
21. | Ferrara, P., Rosenfeld, J., Guillemot, J. C., and Capdeville, J. (1993) in Techniques in Protein Chemistry IV (Angeletti, R. H., ed) , pp. 379-387, Academic Press, San Diego |
22. | Rajan, N., Blaner, W. S., Soprano, D. R., Suhara, A., and Goodman, D. S. (1990) J. Lipid Res. 31, 821-829[Abstract] |
23. |
Mendelsohn, C.,
Batourina, K.,
Fung, S.,
Gilbert, T.,
and Dodd, J.
(1999)
Development
126,
1139-1148 |
24. |
Tsutsumi, C.,
Okuno, M.,
Tannous, L.,
Piantedosi, R.,
Allan, M.,
Goodman, D. S.,
and Blaner, W. S.
(1992)
J. Biol. Chem.
267,
1805-1810 |
25. | Cogan, U., Kopelman, M., Mokady, S., and Shinitzky, M. (1976) Eur. J. Biochem. 65, 71-78[Abstract] |
26. | Furr, H. C., Barua, A. B., and Olson, J. A. (1994) in The Retinoids, Biology, Chemistry, and Medicine (Sporn, M. B. , Roberts, A. B. , and Goodman, D. S., eds), 2nd Ed. , pp. 179-209, Raven Press Ltd., New York |
27. |
Levin, M. S.,
Locke, B.,
Yang, N.-C. C.,
Li, E.,
and Gordon, J. I.
(1988)
J. Biol. Chem.
263,
17715-17723 |
28. | Kato, M., Kato, K., and Goodman, D. S. (1984) J. Cell Biol. 98, 1696-1704[Abstract] |
29. | Gill, S. C., and Hippel, P. H. (1989) Anal. Biochem. 182, 319-326[Medline] [Order article via Infotrieve] |
30. | Cowan, S. W., Newcomer, M. E., and Jones, T. A. (1993) J. Mol. Biol. 230, 1225-1246[CrossRef][Medline] [Order article via Infotrieve] |
31. | Winter, N. S., Brantt, J. M., and Banaszak, L. J. (1993) J. Mol. Biol. 230, 1247-1259[CrossRef][Medline] [Order article via Infotrieve] |
32. |
Strump, D. G.,
Lloyd, R. S.,
and Chytil, F.
(1991)
J. Biol. Chem.
266,
4622-4630 |
33. |
Cheng, L.,
Qian, S-J.,
Rothschild, C.,
d'Avignon, A.,
Lefkowith, J. B.,
Gordon, J. I.,
and Li, E.
(1991)
J. Biol. Chem.
266,
24404-24412 |
34. | Bernlohr, D. A., Simpson, M. A., Hertzel, A. V., and Banaszak, L. J. (1997) Annu. Rev. Nutr. 17, 277-303[CrossRef][Medline] [Order article via Infotrieve] |
35. | Jakoby, M. G., Miller, K. R., Toner, J. J., Bauman, A., Cheng, L., Li, E., and Cistola, D. P. (1993) Biochemistry 32, 872-878[Medline] [Order article via Infotrieve] |
36. |
Richieri, G. V.,
Ogata, R. T.,
and Kleinfeld, A. M.
(1994)
J. Biol. Chem.
269,
23918-23930 |
37. |
MacDonald, P. N.,
and Ong, D. E.
(1987)
J. Biol. Chem.
262,
10550-10556 |
38. | Palace, V. P., Hill, M. F., Khaper, N., and Singal, P. K. (1999) Free Radic. Biol. Med. 26, 1501-1507[CrossRef][Medline] [Order article via Infotrieve] |
39. | Perez-Castro, A. V., Toth-Rogler, L. E., Wei, L. N., and Nguyen-Huu, M. C. (1989) Proc. Natl. Acad. Sci. U. S. A. 86, 8813-8817[Abstract] |
40. | Dollé, P., Ruberte, E., Leroy, P., Morriss-Kay, G., and Chambon, P. (1990) Development 110, 1133-1151[Abstract] |
41. | Wilson, J. G., and Warkany, J. (1949) Am. J. Anat. 85, 113-155 |
42. | Neiderreither, K., Subbarayan, V., Dolle, P., and Chambon, P. (1999) Nat. Genet. 21, 444-448[CrossRef][Medline] [Order article via Infotrieve] |
43. |
Subbarayan, V.,
Mark, M.,
Messadeq, N.,
Rustin, P.,
Chambon, P.,
and Kastner, P.
(2000)
J. Clin. Invest.
105,
387-394 |
44. | Hauseman, G. J. (1985) in New Perspectives in Adipose Tissue (Cryer, A. , and Van, R. L. R., eds) , pp. 1-21, Butterworths, Boston, MA |
45. |
Rodbell, M.
(1964)
J. Biol. Chem.
239,
375-380 |
46. | Kirtland, J., and Gurr, M. I. (1979) Int. J. Obesity 3, 15-55[Medline] [Order article via Infotrieve] |
47. |
Matarese, V.,
and Bernlohr, D. A.
(1988)
J. Biol. Chem.
263,
14544-14551 |
48. |
Xu, Z.,
Bernlohr, D. A.,
and Banaszak, L. J.
(1993)
J. Biol. Chem.
268,
7874-7885 |
49. |
Blaner, W. S.,
Obunike, J. C.,
Kurlandsky, S. B.,
Al-Haideri, M.,
Piantedosi, R.,
Deckelbaum, R. J.,
and Goldberg, I. J.
(1994)
J. Biol. Chem.
269,
16559-16565 |
50. | Kato, M., Blaner, W. S., Mertz, J. R., Das, K., Kato, K., and Goodman, D. S. (1985) J. Biol. Chem. 260, 4832-4838[Abstract] |
51. |
Eriksson, U.,
Das, K.,
Busch, C.,
Nordlinger, H.,
Rask, L.,
Sundelin, J.,
Sallstrom, J.,
and Peterson, P. A.
(1984)
J. Biol. Chem.
259,
13464-13470 |
52. | Li, E., Demmer, L. A., Sweetser, D. A., Ong, D. E., and Gordon, J. I. (1986) Proc. Natl. Acad. Sci. U. S. A. 83, 5779-5783[Abstract] |