Department of 1 Nutrition and Food Sciences and the Biotechnology Center; 3 Department of Biology, Utah State University, Logan, Utah 84322-8700; and 2 Bioorganic and Protein Chemistry, Vitamin D Laboratory, Boston University School of Medicine, Boston, Massachusetts 02118
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ABSTRACT |
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Antisera were raised against the NH2-terminus of the putative basal lateral membrane (BLM) receptor for 1,25-dihydroxyvitamin D3 [1,25(OH)2D3; BLM-VDR]. In Western analyses of BLM proteins, antibody (Ab) 099 was monospecific for a 64.5-kDa band. A protein of 64.5 kDa was also labeled by the affinity ligand [14C]1,25(OH)2D3-bromoacetate; label was diminished in the presence of excess unlabeled secosteroid. The monoclonal antibody against the nuclear VDR (9A7) failed to detect an appropriate band in BLM fractions. Preincubation of isolated intestinal cells with Ab 099, but not 9A7, affected the following two 1,25(OH)2D3-mediated signal transduction events: augmented intracellular calcium and protein kinase C activity. Subcellular distribution of Ab 099 reactivity by Western analyses and fluorescence microscopy revealed the highest concentrations in BLM followed by the endoplasmic reticulum. Exposure of isolated intestinal cells to 1,25(OH)2D3 for 10 s or vascular perfusion of duodena for 5 min resulted in a time-dependent increase in nuclear localization of the BLM-VDR antigen, as judged by electron microscopy, whereas 24,25-dihydroxyvitamin D3 failed to increase antigenic labeling in nuclei. Densitometric quantitation of Western blots of subcellular fractions prepared from isolated intestinal cells treated with vehicle or 1,25(OH)2D3 confirmed a hormone-induced increase of putative BLM-VDR in the nucleus. It is concluded that a novel cell surface binding protein for 1,25(OH)2D3 has been identified.
steroids; rapid effects; signal transduction; membrane receptor; calcium
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INTRODUCTION |
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MEMBRANE RECOGNITION SITES for estrogen were first reported 20 years ago (28). Since then, cell surface "receptors" have been documented for every class of steroid hormone (16, 26), although experimental evidence for a direct action on membrane lipids (26) has not been found. For 1,25-dihydroxyvitamin D3 [1,25- (OH)2D3], a basal lateral membrane receptor (BLM-VDR) has been implicated in mediating rapid enhancement of intestinal calcium or phosphate transport (11, 14, 17, 19, 20, 24), activation of phospholipase-associated signaling pathways (1, 3, 8, 13, 31, 34), and opening of calcium channels (4-7, 32, 37). Unanswered questions regarding the BLM-VDR are whether it is related to the nuclear/cytoplasmic receptor (9, 12, 27), which has been proposed to "dock" at the inside of the plasma membrane (12), and whether ligand binding induces internalization or recompartmentalization. To begin addressing these questions, a synthetic peptide corresponding to the first 20 amino acids of the putative membrane receptor (20) was synthesized and used for production of antisera in rabbits. The current report confirms the receptor-like nature of the antigen, its localization on the plasmalemma and Golgi/endoplasmic reticulum (ER) elements, the ability of antisera to alter 1,25(OH)2D3-mediated signal transduction events, and detection of apparent ligand-induced redistribution to the nucleus.
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MATERIALS AND METHODS |
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Preparation of subcellular fractions
. Duodenal mucosae from vitamin D-replete white leghorn
cockerels were fractionated as described previously (18). The procedure involves a combination of differential- and Percoll-gradient
centrifugation; enrichment, recovery, and distribution of marker
enzymes are described in detail elsewhere (18). Differential
centrifugation fractions are P1 (nuclei and brush-border
membranes), P2 (lysosomes, mitochondria, Golgi, and BLM),
and S2 (cytosol and microsomes). Percoll gradients of
P2 have been characterized for marker enzyme distribution
as described elsewhere (18). Percoll was removed by dilution with homogenization medium and centrifugation (20). Protein was estimated by
using the Bradford reagent (Bio-Rad, Hercules, CA) against bovine
-globulin as standard (Sigma Chemical, St. Louis, MO).
PAGE and Western analyses . Appropriate fractions were resolved on 8% denaturing gels and were stained with either Coomassie brilliant blue to detect protein bands or used for Western analyses as follows: gels (containing colored molecular weight standards; Bio-Rad) were blotted on polyvinylidene difluoride (PVDF) membranes (Immobilon-P; Millipore, Bedford, MA) using the Hoeffer tank transfer system (10 V, 13 h at 4°C), and Western analyses were performed according to the Millipore protocol. For initial characterization, 60 µg of BLM protein/well were used. No reaction product was visible with normal rabbit serum (NRS); antisera produced in three rabbits against the NH2-terminal peptide sequence of the putative receptor revealed that Ab 099 and Ab 593 were monospecific for a protein of molecular weight 64,500, whereas Ab 980 recognized additional high-molecular-weight bands (22). Rabbit polyclonal antibody (PA1-711) to the COOH-terminal end of the human VDR (amino acids 395-413) and rat monoclonal antibody (9A7) to amino acids 89-105 of the VDR were purchased from Affinity Bioreagents (Golden, CO) and were used at a 1:100 dilution or at 2 µg/ml, respectively.
Ligand binding assays. For affinity labeling, 100 µg of BLM protein and 100 µl of 10 mM Tris, 1.5 mM EDTA, and 1 mM dithiothreitol, pH 7.4 (TED buffer; see Ref. 20), were incubated in microfuge tubes with 20 µl of an ethanolic solution containing 0.13 nmol of [14C]1,25(OH)2D3-bromoacetate (29) in the absence or presence of a 200-fold molar excess of 1,25(OH)2D3 (overnight, 0°C). One milliliter of TED buffer was then added, and the mixture was centrifuged (15,000 rpm, 3 min). After the supernatant was decanted, the pellet was washed one time and then was resuspended in SDS-PAGE sample buffer for resolution on an 8% gel. The Coomassie-stained, dried gel was exposed to X-ray film (Kodak X-OMAT AR) for 1 mo.
Loading of isolated intestinal cells with fura 2. Intestinal cells isolated by citrate chelation (18) were harvested by low-speed centrifugation and were resuspended in divalent cation-free PBS containing 100 µM EGTA, 5.55 mM glucose, 0.32 mM sodium pyruvate, 3 mg/ml BSA, 0.02% Pluronic F-127, and 2 µM fura 2-AM (Molecular Probes, Eugene, OR), as described elsewhere (36). Cells were incubated on ice for a 20-min loading period, diluted fourfold with PBS, collected by centrifugation, and resuspended in divalent cation-free PBS. Intracellular calcium was determined by ratio changes of fluorescence intensity at an emission wavelength of 510 nm when excited at 340 and 380 nm. A camera control switched the filter wheel between 340 and 380 nm. Images of cells were collected with a ×20 fluorescence objective for transmission by a video camera to a PC 486 computer. Images were processed, saved, and displayed on the monitor in pseudocolor. For each condition tested, six regions of ~100 cells each were monitored.
Determination of protein kinase C activity.
Time course studies with isolated intestinal cells indicated
that optimal responsiveness to hormone occurred within 5 min (19). In
subsequent studies, ~106 cells were incubated with
control medium (0.125% BSA in bicarbonate-free Gey's balanced salt
solution; GBSS) containing NRS or Ab 099 (1:500 dilution, final
concentration) for 5 min at 23°C. Cells were then treated with
vehicle or hormone [130 pM 1,25(OH)2D3 or
65 pM bovine parathyroid hormone (PTH)-(134); Sigma] for an
additional 5 min. Cells were pelleted at low speed and homogenized in
20 mM Tris, pH 7.5, containing 0.5 mM EDTA, 0.5 mM EGTA, 0.5% Triton
X-100, and 25 µg/ml aprotinin and leupeptin. The solubilized extracts were analyzed for the ability to phosphorylate exogenous substrate (50 µM myelin basic protein) in the presence or absence of a specific protein kinase C (PKC) inhibitor, corresponding to the psuedosubstrate region PKC-(19
36). The reaction mixture also contained 20 mM Tris, pH
7.5, containing 1 mM CaCl2, 20 mM MgCl2, 20 µM ATP, and 25 µCi/ml [32P]ATP (New England
Nuclear) according to directions supplied with kits purchased from
GIBCO-BRL Life Technologies (Waverly, MA). Samples were incubated at
30°C for 5 min, and 25 µl were spotted on phosphocellulose disks.
The disks were then washed two times in 1% phosphoric acid and two
times in water before determination of retained radioactivity by liquid
scintillation counting. The assay conditions did not discriminate
between PKC isozymes.
Immunofluorescence and electron microscopy. For immunofluorescence microscopy, segments of distal duodenal loop from two chicks were fixed (0.25% glutaraldehyde, 4% paraformaldehyde in PBS), rinsed, and embedded in optimum-cutting temperature compound (VWR, McGaw Park, IL) for cryosectioning (21). Sections were incubated overnight (4°C) with NRS, Ab 099, Ab 593, or Ab 099 preabsorbed with BLM (each at 1:1,000, final dilution in 0.1% BSA-PBS); secondary antibody was tetramethylrhodamine B isothiocyanate- or Texas red-conjugated goat anti-rabbit antibodies for 30 min at 23°C (1:100 final dilution; Jackson Immunoresearch, West Grove, PA).
For electron microscopic analyses of ligand-induced receptor redistribution, isolated intestinal cells were initially chosen to allow rapid processing of material. Intestinal cells from three chick duodenal loops were isolated by citrate chelation (18), pelleted at 500 g for 5 min (4°C), and resuspended in GBSS (pH 7.3) lacking glucose and bicarbonate and modified to contain 0.9 mM CaCl2. Cells were then treated with either vehicle (0.1% vol/vol ethanol, final concentration) or 650 pM 1,25(OH)2D3 (final concentration) and were swirled for 10 s before removal of an aliquot to fixative (4% paraformaldehyde in 0.1 M sodium cacodylate, pH 7.1) for 4 h. Cell pellets were dehydrated, infiltrated, and embedded in LR White for preparation of thin sections. After being exposed to blocking buffer (0.5% fish gelatin, 0.5% normal goat serum in 20 mM Tris, pH 7.4; 15 min), nonspecific staining controls from the hormone-treated sample were incubated with buffer (50 mM Tris, 150 mM NaCl, pH 7.4), whereas sections from both the control group and hormone-treated group were incubated with Ab 099 (1/500) in buffer overnight (4°C). After six washes in buffer, sections were labeled with 5 nm gold-conjugated goat anti-rabbit antibody (1:50 dilution in buffer) for 2 h (23°C). A final six washes were performed with reagent-grade water before enhancement with silver reagent and counterstaining with uranyl acetate and lead citrate. Representative nuclei were photographed under single-blind conditions, in which specimens were identified as treatment A, B, or C. Labeling densities were determined by overlaying a transparent film over the photograph with a 1 µm section delimited. Silver-enhanced gold particles were then counted. Alternatively, duodena were vascularly perfused (18) for 5 min with either 6.5 nM 24,25-dihydroxyvitamin D3 [24,25(OH)2D3] or 650 pM 1,25(OH)2D3 before removal of a segment from the adjoining pancreas, slitting of the tissue, and immersion in fixative. Thick (16 µm) sections were then prepared for preembed labeling (21).Electron spectrographic imaging technique. Immunolabeled sections were silver enhanced by immersion in silver enhancing solution (catalog no. sekl15; BB International) for 1 h. Ultrathin sections were cut on a Leica Ultracut E (Leica, Deerfield, IL), counterstained with uranyl acetate and lead citrate (30) for 2 and 4 min, respectively, analyzed with conventional transmission electron microscopic imaging, and recorded on Kodak film SO-163 (Eastman Kodak, Rochester, NY) with the Zeiss 902CEM transmission electron microscope (LEO, Thornwood, NY). Electron spectrographic images were also generated with the Zeiss 902CEM as digital images. Images were collected at the silver edge of 430 eV loss and the adjacent background of 355 eV loss. The background images were subtracted from the corresponding silver edge images, with the resultant being the elemental map for silver in the section. The elemental map was statistically analyzed and false colorized according to McManus et al. (16). Pixels three SD above the mean background are yellow; pixels four SD above the mean background are green; pixels five and six SD above the mean background are light blue; pixels seven to nine SD above the mean background are dark blue; and pixels ten SD or greater above the mean background are black.
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RESULTS |
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In the first series of experiments, the antibody was characterized with regard to specificity and protein dependence of Western analyses.
Western analyses and affinity labeling.
Figure 1 demonstrates the protein
dependence of Western analyses with Ab 099 (lanes in middle)
and shows that only one band (mol wt 64,500) was recognized at the
concentrations tested. The affinity reagent
[14C]1,25(OH)2D3-bromoacetate
(29) labeled a band of equivalent molecular weight (second from
right), which was reduced by excess unlabeled
1,25(OH)2D3 (lane on right). The
substantial degree of competition shown in Fig. 1 was not always the
case; in other experiments, the label was reduced by ~50%. Although
a number of other bands were faintly labeled by affinity reagent in
these overnight incubation protocols (Fig. 1), these bands were
completely absent when shorter incubation times (90 min) were used.
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Effect of 1,25(OH)2D3 and Ab 099 on
intracellular calcium.
Figure 2 depicts the results of
fura 2 fluorescence intensity (expressed as a ratio of emission and
excitation wavelengths; see Ref. 36) in intestinal cells incubated in
the absence of calcium (A) or the presence of 1 mM
extracellular calcium (B and C). As reported elsewhere
(7), the absence of extracellular calcium abolished the augmentation of
intracellular calcium by 1,25(OH)2D3. In Fig.
2, A-C, the average response of several hundred cells is
depicted. In the absence of extracellular calcium, intracellular fluorescence of fura 2 declined over time, with no noticeable increase
elicited by addition of 130 pM secosteroid (Fig. 2A).
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Effect of 1,25(OH)2D3 and Ab 099 on PKC
activity.
The secosteroid hormone has also been shown to activate the PKC
signal transduction pathway (8, 33, 34), perhaps in part as a
consequence of calcium mobilization. In time course studies with
isolated intestinal cells, 130 pM 1,25(OH)2D3
was found to increase PKC activity 5 min after hormone and protein kinase A (PKA) activity 7 min after hormone (19). PKC activity in
intestinal cells was then tested in the absence or presence of a 5-min
preincubation with Ab 099. As a control, PTH-mediated stimulation of
PKA activity was monitored under equivalent incubation conditions. As
revealed in Fig. 4A, cells
incubated with control medium or Ab 099 in the absence of hormone
exhibited a low level of activity. Addition of 130 pM
1,25(OH)2D3 resulted in a twofold increase in
activity (P < 0.05, relative to controls) that was abolished
by preincubation with Ab 099 (P < 0.005, relative to hormone
alone; Fig. 4A). In contrast, 9A7 had no effect on
1,25(OH)2D3-stimulated PKC activity. The
average values
(pmol · min1 · mg
protein
1) ± range for replicate experiments were
93 ± 11 for controls, 98 ± 9 for controls plus 9A7, 199 ± 28 for
1,25(OH)2D3-treated cells, and 211 ± 26 for
hormone plus 9A7.
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Subcellular distribution of antigen.
Studies on the subcellular distribution of the BLM-VDR antigen
were undertaken by Western analyses. In four independent experiments, 15 µg of protein from fraction P1 (nuclei and brush
borders), supernatant fraction S2 (microsomes and cytosol),
lysosomes, mitochondria, Golgi, and BLM were loaded in individual
lanes. Figure 5 shows a representative
Coomassie-stained gel (A) and Western blot with Ab 099 (B), and the results of densitometric analyses of the
Western blots are shown in Fig. 5C. Only low amounts of the
BLM-VDR antigen were found (average ± SE) in P1 (26 ± 4%), S2 (31 ± 6%), lysosomes (21 ± 7%), and
mitochondria (36 ± 6%), whereas the Golgi contained larger amounts
(64 ± 12%) compared with BLM (set to 100%). For comparison, the
relative distribution of the BLM marker enzyme activity,
Na+-K+-ATPase, in P1,
S2, lysosomes, mitochondria, and Golgi, respectively, was
17, 17, 20, 44, and 52% compared with BLM (set to 100%). Golgi and
BLM contained 7-10% of Percoll gradient acid phosphatase activity (a lysosomal marker enzyme) and 4-5% of gradient succinate
dehydrogenase activity (a mitochondrial marker enzyme). Distribution of
the putative membrane receptor antigen parallels earlier findings that
BLM and Golgi/ER fractions from the postnuclear 20,000-g pellet
contained reproducible specific binding of
[3H]1,25(OH)2D3 (20).
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Immunofluorescence microscopy.
To confirm and extend the observations obtained from Western
analyses, immunofluorescence microscopy of fixed frozen sections of
chick duodena was undertaken. Use of standard epifluorescence or
confocal microscopy of 4-µm sections revealed only basal lateral staining with Ab 099 or Ab 593 in epithelium separated from the villus
core (data not shown). Confocal microscopy of whole villi permeabilized
with either 0.1% Triton X-100 or lysophosphatidylcholine revealed only
lateral staining with Ab 099 (data not shown). In comparison, confocal
microscopy of 16-µm sections revealed basal membrane staining and
labeling of structures that may be ER/Golgi or lateral membranes from
lower cell layers (Fig. 6B). Nuclei and brush borders were devoid of label. Incubation of 16-µm sections with preabsorbed antisera resulted in a complete absence of
immunofluorescence (Fig. 6A)
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Electron microscopy. To investigate the possibility of ligand-induced redistribution of the BLM-VDR, isolated intestinal epithelial cells were treated with 0.1% ethanol (final concentration) or with 650 pM 1,25- (OH)2D3 for 10 s, after which 500 µl of cells were removed from each suspension and placed in fixative (4 h, 23°C). Samples were then prepared for electron microscopy.
With the use of postembed labeling with Ab 099 as primary antisera, followed by gold-conjugated secondary antibody, concentrated areas of electron-dense particles were not observed in controls, most likely due to decreased antigenicity caused by the plastic embedding media. However, nuclei from hormone-treated cells were found to contain a relative increase in label. Figure 7 displays pseudocolored labeling densities from representative nuclei selected under single-blind conditions. Averaging the number of gold particles per square micrometer for three nuclei (±SD) in the nonspecific staining group (prepared from hormone-treated cells) yielded 28 ± 5. When corrected for background, nuclei of freshly isolated cells had 53 ± 16 gold particles/µm2 (not shown), vehicle controls had 40 ± 6, and nuclei of cells treated with 1,25(OH)2D3 had 122 ± 17 (P < 0.05, relative to controls). These data indicate that a low level of antigen exists in nuclei of intestinal cells from vitamin D-replete chicks that is not visible at the light microscopic level (Fig. 6). However, treatment of intact cells with secosteroid hormone for very brief periods results in the apparent ligand-induced translocation of the BLM-VDR to the nucleus.
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Western analyses of subcellular fractions after hormone treatment.
Increased nuclear labeling after hormone treatment of isolated
cells might be attributable also to increased antibody binding to the
liganded form of the BLM-VDR. In an attempt to resolve these
possibilities, Western analyses were performed with Ab 099 on
subcellular fractions prepared from isolated intestinal epithelial cells treated with vehicle or 650 pM
1,25(OH)2D3 for 10 s. Although visual
inspection of the blots revealed small changes, densitometric analyses
revealed that they were consistent changes. The results presented in
Table 1 indicate a significant
hormone-mediated increase in antigenicity to Ab 099 in P1
(containing nuclei) and significant decreases in lysosomes and BLM
prepared from cells exposed to 1,25(OH)2D3,
relative to corresponding controls. Increasing the
1,25(OH)2D3 treatment period to 5 min further
augmented BLM-VDR antigenicity in P1 relative to controls
(Table 1).
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DISCUSSION |
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The secosteroid hormone 1,25(OH)2D3 has been known for many years to stimulate 45Ca uptake in isolated rat intestinal cells (24) and transport in perfused chick duodena (19, 20, 25). The physiological importance of these findings has been controversial, because these phenomena are difficult to observe in vivo (2). However, this may be either completely or in part due to the suppressive effect of endogenous 24,25(OH)2D3 levels (19). Indeed, 24-hydroxylase knock- out mice, in which the suppressive hormone is absent, have been reported to be hypercalcemic (10). Thus calcium homeostasis is more complicated than was once thought, and studies on the rapid actions of 1,25(OH)2D3 are warranted.
The current work provides further characterization of a receptor-like protein for 1,25(OH)2D3 identified in BLM of chick intestinal epithelium (20). Affinity labeling of the 65-kDa integral membrane protein strengthens the candidacy of this binding moiety as a cell surface receptor. Treatment of isolated intestinal cells with either 1,25(OH)2D3 or Ab 099 dramatically increased calcium oscillations, as judged by fura 2 fluorescence. These results may be due to a conformational change produced by antibody binding to the putative receptor, with subsequent activation of a coupled calcium channel (6). Enterocytes pretreated with Ab 099 did not exhibit additional oscillations in intracellular calcium when cells were subsequently exposed to 130 pM 1,25(OH)2D3. At present, these results can only be interpreted as a lack of additivity between secosteroid and antibody-induced oscillations.
Clear-cut antibody-mediated abolition of
1,25(OH)2D3 signal transduction was evident in
the PKC pathway. These results confirm the observations in chondrocytes
(23). In contrast, interaction of PTH with its receptor to stimulate
PKA activity, or estradiol 17 with its receptor to simulate PKC
activity, was unaffected by the presence of antibody to the putative
1,25(OH)2D3 hormone membrane receptor. The dual
observations that Ab 099 activated calcium channels yet blocked
secosteroid-enhanced PKC activity are difficult to explain without
further research into signal transduction. However, it may indicate
that the two pathways can be separated and may lead to different
physiological end points. Preliminary studies with the calcium channel
blocker nifedipine support this hypothesis. The specificity of the
effect of Ab 099 on receptor-coupled calcium channel activation was
supported by the inability of the Ab 9A7 to produce similar effects.
Earlier work (20) indicated that, within the 20,000-g pellet fraction, [3H]1,25(OH)2D3 exhibited reproducible, specific binding only in BLM fractions and ER/Golgi elements. With the use of Western analyses of the same subcellular fractions, the present study likewise reveals that BLM and Golgi fractions were enriched in the candidate receptor protein. Thus the binding activity parallels the distribution of the BLM-VDR antigen.
Although the BLM-VDR antigen may be related to the nuclear receptor in the ligand-binding domain, since both molecules manifest an equivalent affinity for 1,25(OH)2D3 (20), several experimental findings suggest that the receptor proteins are different molecular entities. It is evident from epifluorescence studies of immunologically labeled sections that the receptor is not merely "docking" at the cytoplasmic surface as suggested by Kim et al. (12). Specific staining along the BLM in fixed frozen sections of duodena was observed only when the lamina propria was separated from the epithelium. A cytoplasmic localization would allow the observation of labeling even in tissue sections where the BLM was in contact with the substratum. These results, together with the finding that the receptor activity requires detergent for solubilization (20), suggest that the protein is integral to the membrane rather than peripheral. Confocal microscopy with Ab 099 confirmed a basal lateral localization and an absence of brush-border and nuclear labeling at the light level. With the use of 16-µm sections (~3 cell layers thick), immunofluorescent staining was also observed in what could either be intracellular membranes or lateral membranes of underlying cells. Subcellular distribution of antigen in Western analyses paralleled distribution of the BLM marker enzyme activity Na+-K+-ATPase. The presence of antigen in Golgi fractions could thus either be due to contamination with BLM, or it could represent a vesicular compartment for export or retrieval of the putative BLM-VDR.
In addition, Western analyses with the antinuclear VDR receptor indicate an absence of the classical receptor in membrane preparations.
In the presence of hormone, the BLM-VDR antigen is apparently capable of recompartmentalization. On Western analyses, the BLM-VDR is increased in crude nuclei after a brief (10-s) exposure of isolated cells to 1,25(OH)2D3, with a concomitant decrease in antigen in the BLM fraction. This may represent endocytosis of the membrane antigen and delivery to the nucleus. A 5-min exposure of intestinal cells to secosteroid further increases Ab 099 antigenicity in the subsequently prepared low-speed pellet.
A far larger hormone-stimulated increase in nuclear localization of the BLM-VDR was observed with electron microscopy, using either isolated cells (10-s treatment) or vascularly perfused duodena (5-min treatment). The lower sensitivity of the biochemical procedure is undoubtedly due in part to redistribution of antigen during homogenization. A similar occurrence was observed for the loss of calbindin-D28k from lysosomes (21). Electron microscopy of isolated intestinal cells did not, however, allow quantitation of plasmalemmal BLM-VDR in the presence or absence of hormone due to the randomness of the sectioning angle through unaligned cells and loss of antigenicity due to plastic embedding before labeling. The observation that ligand induces the apparent translocation of the BLM-VDR to the nucleus within 10 s of exposure to 1,25(OH)2D3 has several interesting ramifications. It has been widely believed that steroid hormones, due to their lipophilic properties, simply diffuse through membranes. However, as pointed out elsewhere (17, 26), this theory fails to explain why the steroid would leave such a preferred environment. Ligand-induced translocation of the membrane receptor to the nucleus is perhaps a more efficient means of hormone delivery. The swiftness of such delivery (10 s) further indicates that a nonnuclear signaling system must be operational within this time frame to be valid mediators of the so-called rapid effects. A number of second messengers, such as hormone- and receptor-mediated opening of calcium channels (4; Fig. 2 of the present work) and activation of phospholipase C-initiated systems (13, 34), appear to occur within a suitable time frame.
Studies are currently underway to clone and characterize the cDNA for the putative membrane receptor.
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ACKNOWLEDGEMENTS |
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Peptide synthesis and polyclonal antibody production were services provided by the Biotechnology Center, Utah State University. Dr. Milan Uskokovic (Hoffmann-LaRoche, Nutley, NJ) generously provided the 1,25(OH)2D3, and Kureha Chemical (Tokyo, Japan) generously provided 24,25(OH)2D3.
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FOOTNOTES |
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This work was supported in part by the Agricultural Experiment Station (approved as journal paper no. 5038), intramural grant FL-95633, NRI Competitive Grants Program/United States Department of Agriculture award number 98-35200-6466 to I. Nemere, and National Institute of Diabetes and Digestive and Kidney Diseases Grant DK-47418 to R. Ray.
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. §1734 solely to indicate this fact.
Address for reprint requests and other correspondence: I. Nemere, Dept. of Nutrition and Food Sciences, Utah State University, Logan, UT 84322-8700.
Received 28 September 1999; accepted in final form 13 December 1999.
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