THEJOURNAL
OF BIOLOGICAL
CHEMISTRY
0 1994 by The American Society for Biochemistry and Molecular Biology, Inc.
Val. 269,No. 38,Issue of September 23,pp. 23750-23756, 1994
Printed in U.S.A.
Identification of a Specific Binding Protein
for
la,25-DihydroxyvitaminD, in Basal-Lateral Membranesof Chick
Intestinal Epithelium and Relationship to Transcaltachia*
(Received for publication, April 18, 1994, and in revised form, June 27, 1994)
Ilka NemereSS, Murray C. DormanenS, Marion W. Hammondn, William H. Okamuran, and
Anthony W. Norman411
From the Departments of $Biochemistry and of BChemistry and the IDivision of Biomedical Sciences, University of
California, Riverside, California92521
side. White Leghorn cockerels (Lakeview Farms, Lakeview, CA) were
raised 4-5.5 weeks on a commercially available (0.H. Kruse, Ontario,
CA), vitamin D-supplemented diet. On the day of use, chicks were
Since the first suggestion by Nemere and Szego (1, 2) that
ether-anesthetized prior to surgical removal of the duodenal loop to
la,25-dihydroxyvitamin D, (1a,25-(OH),DJ1 may act through
a ice-cold saline. Tissue handling, media, homogenization conditions,and
plasmalemma1 receptor, a large body of evidence has been ac- fractionation procedures for the isolation of basal-lateral membranes
were as described previously(22), except that for preparative purposes,
*This workwas funded by United States Public Health Service the P, fraction (20,000 x g post-nuclear pellet) was resuspended in 50 ml
grants DK 09012-28(toA. W. N.)and DK 16-595(to W. H. 0.).The costs of PercollRto ultimately yield two gradients.
of publication of this article were defrayed in part by the payment of
To remove the Percoll, duplicate basal-lateral membrane fractions
page charges. This article must therefore be hereby marked “aduertise- (fractions 16, 17, and 18) were pooled and diluted to 210 ml with TED
ment” in accordance with 18 U.S.C. Section 1734 solelyto indicate this (10 m~ Tris, 1.5 mM EDTA, 1 mM dithiothreitol) for centrifugation at
fact.
72,000 x g, 1 h. The supernatants were carefully aspirated, and the
5 To whom correspondence should be addressed. ”el.: 909-787-4778; loose, fluffy membrane pellet removed fromthe firmer Percoll pellet. All
Fax:909-787-4784.
membrane fractions within a group were pooled at this point, yielding
The abbreviations used are: la,25-(OH),D3,la,25-dihydroxyvitamin approximately 2 ml of material per group (8-10 mg of proteixdml).
D,;24R,25-(OH),D3, 24R,25-dihydroxyvitamin D,;25-(OH)D,, 25-hyTo solubilize the putative BLM-VDR, membrane protein was addroxyvitamin D,;BLM, basal-lateral membrane; VDR, vitamin D re- justed to 11-13 mg/ml and 1volume combined with 1volume of deterceptor; CHAPS, 3-[(3-cholamidopropyl)dimethylammoniol-l-propanegent (Boehringer-Mannheim)solution to yieldoptimal protein to detersulfonate; CHAPSO, 3-[(3-cholamidopropyl)dimethylammoniol-2gent ratios as reported by others (23) (see below).An equivalent degree
hydroxy-1-propanesulfonate;AT, 25-hydroxy-16-ene-23-yne-vitamin
D,;
BT, la,25S-dihydroxy-22-ene-26,27-dehydrovitamin
D,; JM, la,25-di- of solubilization (approximately 50% of binding activity for [3Hlla,25hydroxy-7-dehydrocholesterol;J N , la,25-dihydroxy-lumistero13;
6-s-cis, (OH),D,) was achieved by either incubation with detergent at 23 “C,15
la,25-(OH),-9,14,19,19,19-pentadeuterio-pre-vitamin
D,;HAP, hy- min, or by homogenization (25 strokes) on ice. In most of the reported
droxylapatite; PAGE, polyacrylamide gelelectrophoresis;ANOVA, anal- work, the latter method was used. Detergent-treated BLM were then
centrifuged at 160,000 x g, 1 h, and the solubilized material in the
ysis of variance.
23750
Downloaded from www.jbc.org by on October 15, 2006
The steroid hormone
la,25-dihydroxyvitamin
D, cumulated to indicate
that the seco-steroid hormone modulates
(la,25-(OH),D3)elicits biologicalresponses
by both certain cellular functions through non-nuclear mechanismsin
genomicandnongenomicpathways.Thisreportdenormal, vitamin D-replete animals (1-18), in addition to the
scribespurificationofareceptorforla,25-(OH),D3
expected regulationof the genome bya nuclear steroid receptor
(VDR) located in the basal-lateralmembrane (BLM) of (19,201. Indirect evidence for
a putative membrane receptor for
vitamin D-replete chick intestinal epithelium, which
is la,25-(OH),D3 was obtained in the chick perfusion system (3)
implicated in the nongenomic stimulation of calcium
where it was reported that treatment of the basal-lateral memtransport (transcaltachia).The BLM-VDRexhibited sat- brane (which is normally exposed to circulating hormones in
urable binding for [‘H]1,25-(OH),D3(K, = 0.72 x lo-’ M, vivo) to exogenous 1,25-(OH)2D, in vitro resulted in a rapidly
B,, = 0.24 pmoYmg of protein).A 4500-fold purification enhanced transportof calcium, whereas exposureof t h e brush
of the BLM-VDRreceptor was achieved.
In addition, satborder to agonist failed to have
a stimulatory effect. Norman et
urable binding was observed for [‘H124R,25-(OH),D3at
al. (21), also using transcaltachiaas a biological response, rephysiologically relevantlevels (K, = 19 x lo-’ M, B,, = 2.4
pmoVmg of protein) to a component apparently distinctported that a dihydroxy analogof pre-vitamin D which did not
bind well to the intestinal nuclear receptor in vitro, potently
from the la,25-(OH),D3BLM-VDR.
the rapid, hormonal stimulation of calcium transport.
induced
A functional correlation between the BLM-VDR and
The results obtained with this “6-s-cis” analog suggested that
transcaltachiawasobservedinthreeexperimental
t h e cell surface receptor might prefera different conformation
situations: (i) vitamin D deficiency, which suppresses
In addition, Liebertranscaltachia, resulted in reduced specific [‘HI la,25- of 1a,25-(OH),D3 than the nuclear receptor.
of a cell surface receptor
(OH),D, binding in the BLM-VDR, relative to corre- herr et al.(14) suggested the necessity
sponding fractions from vitamin D-replete chicks; (ii) for the early actions of la,25-(OH),D3 on phosphoinositidemethe BLM-VDR exhibiteddown-regulation of specific tabolism. The present workis believed tobe the first attempt to
[3H]1a,25-(OH),D, binding following exposure to nonra-identify and biochemically characterize the putative basal-latdioactive la,25-(OH),D3; and(iii) the relative potencies era1membranevitamin
D receptor (BLM-VDR) forla,25of two “6-s-cis”analogs of la,25-(OH),D3 to initiate trans(OH)&.
caltachia and their ability to compete with [’H]la,25(OH),D, for bindingto the BLM-VDR were parallel.The
MATERLALSANDMETHODS
combined results support the existence
of a plasmalemPreparation and Solubilization of Basal-Lateral Membranes-All exmal la,25-(OH),D3 receptor which
is a prime candidate periments employing animals were approvedby the Chancellor’s Comfor signal transductionin transcaltachia.
mittee on Animals in Research at the University of California, River-
A Putative Membrane
Receptor
for
RESULTS
Specific Binding of pH11 a,25-(0H)$, to Basal-Lateral
Membranes-& a first step toidentifying a non-nuclear receptor for 1a,25-(OH),D3, post-nuclear membranes were prepared
from the duodenal mucosaof normal, vitaminD-replete chicks,
fractionated on Percoll gradients (22), and tested for specific
binding of the tritiated metabolite. Fig. 1depicts the results of
these experiments in duplicate gradients.
Reproducible specific
binding wasobserved in fractions of P, previously identified as
BLM by marker enzyme activities (22), but not in lysosomes,
mitochondria, or Golgi membranes. Inmicrosomal membranes,
specific binding was observed in the as yet unidentified fractions 9 and 10, as well as in endoplasmic reticulum (fractions
14 and 15). Basal lateral membranes were chosen for further
studies in the currentwork.
23751
P2 (20,000x g Pellet)
LYSO
800 600
-
400
-
SOMES
1
MITOCHONDRIA
GA
ELM
I
-
200
s:
-200
d
W
n
I
600 -
0
400
g
200
LC
0
Pg (105,000 x g Pellet)
-
BBM EV
ER
t
3.
0
,200
BOTTOM
FRACTION NUMBER
TOP
FIG.1. Analyses of membrane fractions in Percoll gradients
for specific [SH]lcu,26-(OH),D,binding by the HAP assay. Duodenal mucosae from normal chicks (two per group) were homogenized in
250 mM sucrose, 5 mM histidine:imidazole,2 mM EGTA, 10 pg/ml ruthenium red, pH 7.0, and pellet fractions P2 (20,000 x g postnuclear pellet)
and P, (105,000 x g microsomes) wereprepared for further resolution on
Percoll gradients. Two hundred-pl aliquots of each 40-drop fraction
were incubated with 1 nM [3H11,25-(OH)2D,in the absence or presence
of excess nonradioactivehormone (0"C, overnight). Separation of bound
and free metabolites was achieved by addition of hydroxylapatite, and
three washes of the resulting pellet with 0.5%Triton X-100in TED. The
ethanol-extracted fraction was ultimately taken for liquid scintillation
counting. The asterisk (*I denotes those fractions in duplicate gradients
that exhibited a reproducible level of specific binding (average 2 range).
Negative values, where nonspecificbinding was greater than total binding, by definition indicate an absence of specific binding.
Solubilization of the BLM-associated Binding Moiety-The
membrane-associated ligand binding activity was not solubilized by washing pelleted membranes with TED buffer, nor
treatment with highsalt (0.3-0.5M KC1; data not shown). Triton X-100(0.1-0.5%, v/v), completely abolished the specific
seco-steroid binding activity. Incubation of BLM with 10 mM
CHAPS or CHAPSO (final concentration), followed by centrifugation at 160,000 x g , 1h, resulted in6 and 10fmol of specific
[3H11a,25-(OH),D3binding per mg of protein in therespective
supernatant fractions. Thus, levels of CHAPS or CHAPSO
above (but not below) the critical micellar concentration, solubilized the binding activity, suggesting an integral membrane
protein. The binding activity was also solubilized by 25 mM
n-octylglucoside, but not by 0.5 mM n-dodecylglucoside (data
not shown). Therequirement
for detergent solubilization
coupled with the highspecificity for 1a,25-(OH),D3(see below)
further suggested that the bindingcomponent was not thevitamin D-binding protein, the serum transport proteinfor vitamin D metabolites.
Comparison of the HAP and HC10, Procedures for Determination of Specific Binding-The low levels of specific [3H]1a,25-(OH),D3 observed with the HAP assay precluded its use
for saturation analyses. Precipitation
of bound ligand withperchloric acid2 was therefore investigated. Specific binding of
[3Hlla,25-(OH),D3 was linearly dependent on protein (Fig. 2)
A. Alpert, personal communication.
Downloaded from www.jbc.org by on October 15, 2006
supernatant concentrated in aCentricon-10(Amicon, Beverly,MA). The
last step also served to decrease detergent below the critical micellar
concentration, thereby minimizing interference with binding studies.
ChrornatographySolubilized BLM proteins were further fractionated using a Mono Q PC 1.6/5 anion exchange column in conjunction
with the SMART system (Pharmacia Biotech Inc.). Solubilized proteins
were passed through a 0.45-pm filter prior to application to the column
by means of a 2 0 0 4 injection loop. Proteins were eluted with a 0-500
m~ KC1 gradient in 10 mM Tris, 1 mM CHAPSO (Sigma), pH 8.0, according to a library program (see below). For chromatography on a
Superose-12 column (Pharmacia), Mono Q fractions were concentrated
to 50 pl prior to injection and theneluted with 150 mM NaCI, 20mM Tris,
1mM EDTA, 1 mM CHAPSO, pH 8.0.
Analytical Determinations-Specific binding of vitamin D metabolites was assessed in two ways. All incubations contained 200-pl
samples in a Tris buffer (usually supplemented with 1mM CHAPSO for
analyses of solubilized components), and 20 p1of either tritiated metabolite to determine total binding, or 20 pl of tritiated metabolite plus
a 100-fold24R,25-(OH),D3 and 25-(OH)D, or 200-fold la,25-(OH),D3
molar excess of nonradioactive metabolite to determine nonspecific
binding. All metabolites were in 100%ethanol and diluted to givea final
concentration of 10% (v/v) in the assay. Each sample was normally
assayed in triplicate for total binding, and in duplicate for nonspecific
binding. All incubations were conducted at 0 "C overnight prior to separation of bound and free metabolites by one of two methods. The first
method relied on the affinity of the seco-steroid binding moiety for
hydroxylapatite (HAP)and was an adaptation of the nuclear chromatin
HAP assay as described elsewhere (24).
In the second procedure, perchloric acid and carrier protein (bovine
y-globulin, Sigma) were added to each tube to yield final concentrations
of 325 mM HCIO, and 152 pg of y-globulin. The mixture was incubated
on ice for 30min, and the precipitated protein pelleted at top speed in
a microcentrifuge (15 min, 4 "C). The supernatant fractions were decanted, and while in theinverted position, eachtube was swabbed with
a Kimwipe. Rinsing the pellet did not significantly improve the reproducibility of replicate tubes (variability was usually * 10%).The pellets
which contained the seco-steroid binding moiety were solubilized
in the
presence of 500 pl ofTBS (Beckman, Fullerton, CA) and 500 pl of
Betafluor (National Diagnostics,Mannville, NJ) and decanted into scintillation vials, and the microcentrifuge tubes were rinsed with an additional 1 ml of Betafluor. After combining the rinse, the scintillation
mixture was brought to 6 ml, and 20 pl of glacial acetic acid wereadded
to quench chemiluminesence.
Due to the variable levels of detergent in many of the samples,
protein was determined by the Bradford method according to the instructions supplied by the manufacturer of the dye (Bio-Rad)and with
bovine y-globulin as the standard.
Vitamin D metabolites were obtained from the followingsources:
la,25-(OH),D3and 25-(OH),-16ene-23yne-D3(analog AT) were the generous gift ofM. Uskokovic(Hoffmann-LaRoche);la,24-(OH),-24-cyclopropyl-D, (analog BT) was provided byDr. Lisa Binderup ofLeo
Pharmaceuticals (Ballerup, Denmark); [3H11a,25-(OH),D, (lor,25-dihydroxy[23,24(n)-3Hlcholecalciferol) and [3H]25-(OH)D,
(25-hydroxy
[26(27)-methyZ-3Hlcholecalciferol)
werefrom Amersham Corp.; [,HI24R,25-(OH),D8 (24R,25-dihydroxy[6,19,19-3H]cholecalciferol)
was the
generous gift of Kureha Chemical Co., Tokyo, Japan; 1,25-(OH),-7-dehydrocholesterol (analog JM) and 1,25-(OH),-lumistero13(analog JN)
were synthesized as described previously (25).
1 a,25-(0H)$,
A Putative
Membrane
23752
Receptor for la,25-(0H)J13
TABLEI
Distribution of specific ~Hlla,25-(OHI$, binding as judgedby the
HAP (hydroxylapatite) andHCIO, assays
All ligand binding assays were performed in 2 2 0 4 final volume: 10
pl of la,25-(OH),D3(1 nM final concentration) plus 10 pl of ethanol or
nonradioactive seco-steroid (200-fold molar excess, final volume). The
mixtures were incubated overnight (04°C) after which bound and free
ligand were separated by either HAP or HCLO, (see text). Separate
preparations were used for the two different types of assays.
Fraction
Recovery relative to whole
homogenate (HAPassay)
P, (1000x g pellet)
P, (20,000 x g pellet)
S, (20,000 x g supernatant)
55.0 f 8.0"
1.3
0.3"
23.0 f 6.0"
%
0
I
I
I
'
0.032
0.064
0.096
0.128
,
I
Fraction
Recovery relative to Pz
(HCIO, assay)
BLM (basal lateral membranes)
CH S (CHAPSO soluble)
CH P (CHAPSO insoluble)
CENT (Centricon-10concentrated)
29 f llb
34 f 12*
47 f 7b
52 f 25"
1.0
PROTEIN 1 (mg/0.2ml)
within the range of 0-0.06 mg/0.2 ml (r2= 0.97 by linear regression analysis).
Table I presents the results of specific binding analyses by
both assay procedures. When determined by the H A P method,
recovery of specific [3H11a,25-(OH),D, binding was, as expected, greatest in the nuclear fraction (PI, 1000 x g pellet),
with only 1.3 f 0.3% of the total binding in fraction P, (20,000
x g pellet containing BLM). The perchloric acid procedure did
not yield reliable results for determination of whole homogenate and nuclear binding, perhaps due t o the high dilutions
employed. The distribution of specific [3H]la,25-(OH)zD3binding is presented for subfractions of P, (Table I), namely BLM,
CHAPSO-soluble and insoluble fractions, and Centricon-coneentrated fraction (CENT)of soluble material. Homogenization
in
of BLM with CHAPSO yielded an apparent increase specific
binding (CHAPSO soluble plus CHAPSO insoluble). The final
fraction (CENT) indicated a 50% recovery of total P, binding
sites.
A comparison of the specific activity of binding (fmollmg of
protein) obtained by the two methods is also presented inTable
I. Clearly, perchloric acid precipitation detectsa greater level of
specific binding, relative to theHAP assay. To further validate
the HCIO, procedure, the HAP assay was also employed in
many subsequent experiments to determine whether
comparable qualitative results could be obtained (see below).
Sensitivity of the rHlla,25-(OH)J13Binding Moiety to
Proteolysis-In two independent experiments, Centricon fractions of solubilized BLM were diluted to
approximately 2500pg
proteid2.5 ml and incubated in the
presence or absence of 200
pg of proteinase K (37 "C, 30 min). Analysesof specific binding
by the HAP assay yielded values (mean2 range) of 115 0.4 and
2.8 2 0.3 fmols [3H]la,25-(OH),D, bound for native anddigested
samples, respectively; perchloric acid precipitation gave values
of 80 e 40 and 3.4 2 3.4 fmols [3Hlla,25-(OH)zD3bound for
native anddigested samples, respectively. Thus it is concluded
that the membranebinding activity ismost likely a protein.
Saturation Analyses-Using the HC10, method to evaluate
binding, saturation analyses were undertaken for [,Hlla,25(OH),D,, [3H]24R,25-(OH),D3,and I3H125-(OH)D3.Fig. 3 illustrates the results of four such experiments conducted with
Fraction
Specific activity
HAP assay
HC10. assay
~~
fmol bound j mg protein
BLM
CH S
CH P
CENT
9 f 3"
8 f 3"
3 f 4"
4 2 1"
57 f 28'
110 f 56'
87 2 29'
103 26'
Values represent mean + S.E., n = 5.
Values represent mean -c S.E., n = 3.
Values represent mean r S.E., n = 4.
[3H11a,25-(OH),D,. Using a curve-fitting program for a rectangular hyperbola (Graphpad by Inplot), a KD= 0.72 x lo-' M and
B,, = 240 x
mol/mg of protein werecalculated. Bycomparison, the nuclear
receptor has been found to haveKD= 0.5 x
lo-' M and B,, = 200-300 x
mollmg of protein (26).
Similar
saturation
studies
were undertaken with
[3H124R,25-(OH),D3as ligand. This vitamin D metabolite is
present in normal, vitaminD replete chicks a t concentrations
that areapproximately 28-fold higher than those obsemed for
1~~,25-(0H)~D,
(27). As shown in Fig. 4,much higher levels of
24R,25-(OH),D3 were required to achieve saturation, and for
this metabolite, KD= 19 x lo-' M (approximately 28-fold higher
than that observed for l a , 25-(OH),D,), and B,,, = 2.4 x 10"'
mollmg of protein. In three separate experiments, no saturation with r3H]25-(OH)D3could be demonstrated.
Competition Studies with Analogs-The relative competitive
index (RCI), was determinedfor two analogs of la,25-(OH),D3,
namely "AT" and "BT" by the HCIO, procedure (18,24). Unlike
the nuclearreceptor, the RCI for membrane protein binding of
BT was not greater than thatfor la,25-(OH),D3 (which is defined as 100) (18).Moreover, in contrastt o the efficacy in stimulating transcaltachia (AT >> BT), the RCI for AT was less than
that for BT in both solubilized and intact membrane preparations. The averageRCI forfive independent experiments5 S.E.
for BT was 24 2 3.2 (Y-intercept= 1.025 2 0.55 for BT, and 1.065
2 0.22 for la,25-(OH),D3), and for AT 0.70 2 0.39 (Y-intercept =
0.945 -r 0.33). However, in two additional experiments,AT was
found to compete with [3H124R,25-(OH),D3for binding. Thus,
when solubilized BLM were incubated with 16 n~ [3H]24R,25(OH),D3 without or with a 200 M excess of non-radioactive
24R,25-(OH),D3 or analog AT, specific binding was 432 5 and
57 2 4 fmoV0.2 ml, respectively, as determined by the HAP
assay.Normalizing to competition observed with unlabeled
24R,25-(OH),D3,AT exhibited 135 2 5%competition.
In an earliercommunication (21)a 6-s-cis analog was shown
to effectively initiate and sustain transcaltachia.
Another such
Downloaded from www.jbc.org by on October 15, 2006
FIG.2. Protein dependence of specific [SHlla,26-(OH),D, binding to solubilized basal lateral membranes in the perchloric
acid assay. Serial dilutions of sample (200 pl) were incubated overnight (4 "C) with 1 nM [3Hlla,25-(OH),D, without or with a 200-fold
molar excess of nonradioactive ligand as described in the text. The
following day, 325mM HCIO, and 152 pg of y-globulin (final concentrations) were added, the samples mixed and held on ice for 30 min, and
the precipitate was collected by centrifugation. The solubilized pellets
were then analyzed for radioactivity and specific binding calculated as
the difference between total and nonspecific binding.
%
A Putative
Membrane
D
0-0
A-&
z
K O = 0.72
P
Bmax= 0.24 ~ 1 0 "molslmg
~
protein
3
0.00
X
10-9 M
1.00
0.50
0.25
1 cu,25-(OH)p3
Receptor
for
2.00
4.00
3.00
*
(nM)
a
I
I
I
1
1
1
1
1
1
1
1
1
1
1
0
4
8
12
16
20
24
28
32
36
40
TIME (min)
FIG.5. Effect of 300 PM lcu,25-(OH),D,, 1,26-(OH),-lumisterol~,
or
1,25-(OH),-7-dehydrocholesterolon transcaltachia.Duodena from
normal, vitamin D-replete chicks werevascularly perfused with control
media for a 20-min basal phase and then exposedto the indicated
metabolite, analog, or additional control media for a further 40-min
period. The lumen was perfused with medium containing 45Ca(5 pCi/
ml), and the venous effluent was collected. Results are expressed as
counts/min in the treated phase relative to average basal transport.
Values represent mean * S.E. for three to four duodena per group.
0.40
yz
0.30
-
a
m
U
g
0.20
m
Q:
0.10
-
0.00
1.o
2.0
3.0
(ml)
FIG. 6.Fractionationof solubilizedBLM on ananion exchange
column with a 0-500 m~ KC1 gradient in 10 m~ Tris, 1 m~
CHAPSO, pH 8.0. A 2 0 0 4 injection loop was used in conjunction with
a Mono Q column and theSMART system (Pharmacia). One hundred-pl
fractions were collected into 0.5-ml microcentrifugetubes.
The analogs J N and JM werealso tested for their ability t o
compete with [3H11a,25-(OH)2D3
for binding tosolubilized BLM
preparations.Designating competition with 200 n~ la,25FIG.4. 24R,25-(OH)$, saturation analyses of solubilizedBLM. (OH),D, as loo%, incubationof sample with radiolabeled secoSpecific binding was assayed in four independent experiments by persteroid hormone and 200 nM J N resulted in 152% 2 49% comchloric acid precipitation as described in Fig. 3.
petition, whereas200 nM JM produced 66% 2 29% competition.
Thus, the relative abilities
of the analogs to compete with
analog, J N , was subsequently found to be equipotent to la,25- [3H]1a,25-(OH),D, for binding to the BLM-VDR (JN >> JM)
(OH),D, in inducing transcaltachia.
Fig. 5 depicts the results
of closely parallels the abilityof the two analogs to induce trans(4) caltachia (JN>> JM).Incubation of solubilized BLM with 16n~
studies inwhich isolated duodena were vascularly perfused
with control medium or 300 PM test compound. Both la,25[3H]24R,25-(OH),D3and competition with a 200 fold molar ex(OH),D, andanalog JN augmented calcium transport, as cess of either J N or JM demonstrated a lack of competition (8
judged by 45Ca2+in the venous effluent, to 200% of controls, 7%) by either analogcompared to displacementby homologous
whereas JM elicited an increase of only 120% of vehicle con- unlabeled ligand.
trols. A more complete dose-response analysis of these analogs
Anion
Exchange
Chromatography of Solubilized BLM
has appeared elsewhere (25).
Proteins-To further isolate and
purify the la,25-(OH),D3 bind4
8
12
16 3 2
20
28
24
CONCENTRATION 24,25(OH)pDg (nM)
*
Downloaded from www.jbc.org by on October 15, 2006
FIG.3. lcu,25-(OH)$, saturation analysesof solubilized basallateral membranes. Preparation and solubilization of basal-lateral
membranes with CHAPSO were as described in Fig. 2, except that the
membranes were homogenized(25 strokes) on icewith 10 m~ detergent,
final concentration. Solubilized proteins were concentrated in a Centricon-10 celland often stored at -20 "C prior to use. Aliquots werediluted
1/13 with 10 m~ Tris, 1mM CHAPSO, pH 8.0, or TED, 1 mM CHAPSO,
pH 7.4, and incubated with the indicated concentrations of L3H1la,25(OH),D, in theabsence (total binding) or presence of a 200 molar excess
of cold seco-steroid (nonspecific binding). Final assay volume was 220
pl. Bound and free ligand were separated by addition of 0.325 M HC10,
and 150 pg of bovine y-globulin (final concentrations), 30 min incubation on ice, followed by centrifugation in a microcentrifuge. The supernatants were decanted and the inside of the tubes carefully swabbed
twice. The solubilized pellet was analyzed for radioactivity by liquid
scintillation counting. For each concentration tested, 0.25,0.5,1,2, and
4 nM la,25-(OH),D3,the average total andnonspecifc dpm were 10,010,
8873; 15,808, 14,269;27,989,26,360;55,352,52973;
and 106,292,
100187; respectively, for
four independent experiments. Symbols in this
and subsequent figures represent mean values * S.E. unless otherwise
I
Control
300pM 1,25(OH)$3
M 300pm 1.25(OH)2- Lumisteroll
H 300pM 1.25(0H)2-7- Dehydrocholesterol
l-
CONCENTRATION
1,25(OH)2D3
23753
A Putative
Membrane
23754
Receptor for la,25-(0H)p3
200
U
-10
I
%
0"
2
5
16
28
bE L U T E 0 1
1 UNBOUND 4
ELUTED
4
-200
30
I
BLM
k UNBOUNOi
e
31
FRACTION
FIG.7. Specific binding of [3Hlla,25-(OH),D, to column chromatography fractions. Mono Q fractions 2-4 were pooled, as were
5-6, 15-17,28-30,
and 31-32, and analyzed forspecific [3H11~,25(OH),D, binding by the HClO, method in four independent experiments. Values represent mean * S.E. Statistical analyses were by
ANOVA.
20
HAP
I
peo.05
I
-2
+2 -5 +5
-16 +16
- 2 8 + 2 8 -31+31
FRACTION
FIG.9. Comparison of specific [3Hlla,25-(OH)eDg binding to
Mono Q columnchromatographyfractions. Starting materials
were the CENT fractions (see Fig. 8)prepared from vitamin D-deficient
(-, open bars) and -replete (+, stippled bars) chicks. Fractions were
pooled as describe in the legend to Fig. 7 and analyzed by either the
HAP (upper panel)or HCIO, (lower panel) methods (two independent
experiments each). Values in this and the following figure represent
mean * range.
P
'5
T
500
400
3
A
fl
-CENT
FRACTION
-
0
CONTROL MEDIUM
-
650 PM 1.25(OH1+J3
a .r_
0
300
LLE
0 0 200
g&
*
100
-1
a3c"
N O
0
0
FIG.8. Comparison of specific [3H]la,25-(OH)~,
binding to
fractions prepared from vitamin D-deficient (-Pzor -CENT,
open bars) and -replete chick intestine
(+Pz
or +CENT, stippled
bars).Results are presented for fourindependent experiments in which
aliquots of each fraction were analyzed by HAP and HClO, methods.
Values represent mean * S.E. Statistical significance was assessed by
ANOVA.
rn
Analyses of these fractions from four independent experiments
for specific [3Hllu,25-(OH),D3 binding by the HClO, method
gave the results presented in
Fig. 7. Some binding was evident
in the fall-throughfractions 2 and 5, suggesting saturation of
the column. Of the bound and sequentially eluted fractions,
number 16 contained significantly more specific binding than
pooled fractions 28-30 and 31-32. When related to protein
levels, specific binding in Mono Q 16 represented a 150 f 43fold purification over solubilized BLM material.
Dependence of Specific PH]la,,25-(OH)@, Binding on Vitamin D Status-It has been reported that vitaminD deficiency
abolishes the rapid responses to la,25-(OH),D3 in intestinal
cells of chick (3) and rat (17). To test whether thiscould be due
to reduced binding of [3H]1a,25-(OH),D, to a membrane protein, aliquots of the Pz, Centricon-concentrated, and Mono Q
fractions were prepared from the intestinal epitheliumof vitamin D-deficient and -replete chicks. Fig. 8 illustrates the results of four independent experiments inwhich parallel prepa-
u)
-100
7
-200
I
3
-3001
"p*
I
"-I
C - C H P --I
+CH
S"1
FRACTION
FIG.10. Comparison of specific [SHlla,26-(OH)~3
binding to
fractions prepared from normal duodena vascularly perfused
ing protein associated with the BLM, fast protein liquid chro- with control mediumor 650 PM la,25-(OH),D,. Two duodena were
matography on a Mono Q anion exchange column was under- simultaneously perfused for 15 min with either control medium(0.005%
taken. Fig. 6 illustrates a typicalelution profile in which ethanol, 0.125% BSA, final concentrations) in Gey's balanced salt solufractions 2and 5 contain material unbound to thecolumn at pH tion, or the seco-steroid. The first pair of duodena were then held in
ice-cold saline while the second pair were perfused. Scraped mucosae
8.0, fractions 15-17 (Mono Q 16) contain material eluted at were then processed to yield the indicated fractions (defined in Table I),
approximately 75 mM KC1, fractions 28-30 and 31-32 contain and analyzed for [3H]1~,25-(OH),D3
binding by the HCIO, method.
material eluted at 2250 mM and 400 mM KC1, respectively.
Downloaded from www.jbc.org by on October 15, 2006
-
A Putative Membrane Receptor for 1a,25-(OH)p3
A ,
I
23755
I
I
CONDUCTIVITY
r-1
I
I
I
I
I
0.20
-
0.15
-
0.10
-
0.05
-
W
9
3
gm
CT
I
-
I
-
I
I
I
I
I
1
-
a
-"_""
0.00
I
I
8 16 12
I
'
1.o
0.0
B
2%
I
1 1
4
c
MW
x lo-:
I
I
28 FR. NO.
20
xyy-3
97.4 -
97.4
-
66 -
66
-
45-
45
-
31
-
31-
4
5
10
15
3
1
11
6.5
rations of P, and Centricon-concentrated fractionswere
analyzed by the HAP (upper panel) andHClO, (lower panel)
methods.
The Centricon-concentrated fractions were subsequently
chromatographed on a Mono Q column, and two preparations
each were delegated to the determinationof binding by either
the HAP or HClO, method (Fig. 9). Results of the HAP assay
revealed either an absence or inconsistent
presence of specific
binding in fractions 2, 5 , 28, and 31. In Mono Q 16, specific
ligand binding was absent in fractions preparedfrom vitamin
D-deficient chicks, and reproducibly present in those isolated
from normal chick intestine. Results of the HCIO, procedure
revealed a n absence of specific binding in fraction 2, and an
absence of a vitamin D effect in fraction28, although low levels
of binding were present. Fractions 5 , 16, and31 each exhibited
a vitamin D-dependent difference in binding in the averageof
2 experiments. However, binding in fractions+5 and +31 was
highly variable, whereas binding in
fraction +16 was reproducible. These results are consistent with the statistical analyses
presented in Fig. 7.
Down-regulation or Desensitization of Specific pH]la,25(OH)$3 Binding-In each of two independent experiments,
two sets of two duodena were simultaneously perfused through
the vasculature with either control medium or 650 PM la,25(OH),D, for 15min (4).The duodena were then
chilled in saline
processing to yield P, and CHAPSO
for 15 min prior to further
insoluble and soluble fractions. The results of these studies,
illustratedin
Fig. 10, indicatethe
presence of specific
16 Column
Fr. No.
7 fmols/0.2ml
glq;lTn
10 15 16
9 4.5
fmols/0.2ml
5.5
[3H]1a,25-(OH),D, binding, as judged by the HClO, procedure,
in fractions preparedfrom duodena perfused with control medium. In contrast,
binding was severely reducedor abolished in
equivalent fractions preparedfrom duodena perfused with 650
PM la,25-(OH),D3.
Gel Filtration Chromatography ofActiue Mono Q Fractionsspecific binding levels
Mono Q fractions containing the highest
of [3H]1a,25-(OH),D, were concentrated to 50 p1 for chromatography on a Superose-12 column. Eighty-pl fractions were collected with 150 mM NaCl,20 mM "is, 1 mM EDTA, 1 mM
CHAPSO, pH 8.0, as eluent. Fig. 1lA illustrates a representative elution profile in which fractions demonstrating protein
levels above base line were analyzed for specific [3Hlla,25(OH),D, binding by the HAP assay, and further resolved by
SDS-PAGE and silver staining. Gel profiles and specific
[3H]la,25-(OH),D, binding aregiven for two such experiments
in Fig. 11, B and C. The highest bindingactivity in each preparation was found in fraction 10. The gel profile in Fig. 11B
demonstrates a number of proteins, probably co-eluting as micelles, including a triplet at >60 kDa. These triple bandswere
the only major species apparent in Fig. 11C, suggesting one or
more bands is a component of the BLM-VDR.
DISCUSSION
The current report describes for the first time the partial
purification and biochemical characterization of a putative receptor for la,25-(OH),D3 (BLM-VDR) from basal lateral membranes of chick intestinal epithelium, which is involved in the
Downloaded from www.jbc.org by on October 15, 2006
FIG.11. Gel filtration chromatography of BLM-VDRactivity.Mono Q fractions were pooled and concentrated to 50
pl for chromatography on a Superose-12
column with 150 mM NaCI, 20 mM "is, 1
mM EDTA, 1 mM CHAPSO,pH 8.0, as
eluent. Fractions containing protein (A)
two
from
separate
runs
analyzed
were
for
specific [7Hlla,25-(OH),D, binding (fmoV
0.2 ml)andfurtherresolved
on SDSPAGE followed by silver staining.Gel profiles and specific binding for each fraction
are given in B and C.
23756
A Putative
Membrane
Receptor
for
The combined data suggest that there may be a family of
membrane receptors for hormonally active metabolites of vitamin D, including la,25-(OH),D3 and possibly 24R,25-(OH),D3.
Whether similar or disparate signal transduction pathways
are activated by receptor-ligand interactionremainsto
be
determined.
In summary, the present data and related reports (18, 21)
clearly document that la,25-(OH),D3 may generate biological
responses by both a membrane receptor (BLM-VDR) and the
classical nuclear receptor. Thesefindings are in agreement
with other reports concerning the existence of membrane receptors for numerous steroid hormones (cf: Ref. 311, including
estrogen, progesterone, androgens, thyroid hormone, and glucocorticoids.
Acknowledgments-We thank Dr. Thomas Thysseril for expert help
in intestinal perfusions, June Bishop for technical help in analyzing
initial Percoll gradients for binding, Drs. Elizabeth Cowles and Sajeet
Gill for helpful discussions regarding fast protein liquid chromatography, and Marian Herbert for secretarial support.
REFERENCES
1. Nemere, I., and Szego, C. M. (1981) Endocrinology 108, 1450-1462
2. Nemere, I., and Szego, C. M. (1981) Endocrinology 109, 2180-2187
3. Nemere, I., Yoshimoto,Y.,and Norman, A. W. (1984)Endocrinology 115,14761483
4. Nemere, I., and Norman, A. W. (1986) Endocrinology 119, 14061408
5. de Boland, A. R., and Norman, A. W. (1990) Endocrinology 127, 2475-2480
6. de Boland, A. R., and Norman, A. W. (1990) Endocrinology 1 2 7 , 3 9 4 5
7. de Boland, A. R., and Boland, R. L. (1987) Endocrinology 120, 1858-1864
8. Baran, D. T., Sorensen, A. M., Honeyman, T. W.,Ray, R., andHolick,M.F.
(1990) J. Bone Miner. Res. 5, 517-522
9. Barsony, J., and Man, S. J. (1991) Proc. Natl. Acad. Sci. U. S. A. 88, 14361440
10. Ben Nasr, L., Monet, J.-D., and Lucas, P. A. (1988)Endocrinology 123, 17781782
11. Bourdeau,A., Atmani, F., Grosse, B., and Lieberherr, M.(1990)Endocrinology
127,2783-2743
12. Civitelli, R., Kim, Y. S., Gunsten, S . L., Fujimori, A,, Huskey, M., Avioli, L. V.,
and Hruska, K. A. (1990) Endocrinology 127,2253-2262
13. Caffrey, J. M., and Farach-Carson,M.C. (1989) J . Biol. Chem. 264, 2026620274
14. Lieberherr, M., Grosse, B., Duchamhon, P., and Drueke, T. (1989) J. Biol.
Chem. 264,20403-20406
15. Schwartz, Z., Schlader, D. L., Swain, L. D., and Boyan, B. D. (1988) Endocrinology 123,2878-2884
16. Desai, S. S., Appel, M. C., and Baran, D. T. (1986) J. Bone Miner. Res. 1,
497-501
17. Wali, R. K., Baum, C. L., Sitrin, M. D., Bolt, M. J., Dudeja, P. K.,and Brasitus,
T. A. (1992)Am. J. Physiol. 262, G9454953
18. Zhou, L.-X., Nemere, I., andNorman, A. W. (1992) J. Bone Miner. Res. 7,
457463
19. Theofan, G., Nguyen, A. P., and Norman, A. W. (1986) J. Biol. Chem. 261,
16943-16947
20. Lowe, K. E., Maiyar, A. C., and Norman, A. W. (1992) Crit. Reu. Eukaryotic
Gene Expression 2 , 6 6 1 0 9
21. Norman, A. W.,Okamura, W. H., Farach-Carson, M. C., Allewaert, IC,
Branisteanu, D., Nemere, I., Mulralidharan,IC R., and Bouillon, R. (1993)
J. B i d . Chem. 268, 13811-13819
22. Nemere, I., Leathers, V., and Norman,A. W. (1986)J. Bid. Chem. 261,1610616114
23. Simonds, W. F., Koski,G., Streaty, R. A., Hjelmeland,L. M., and Klee, W. (1980)
Proc. Natl. Acad. Sci. U. S. A . 77,46234627
24. Wecksler, W. R., and Norman, A. W. (1979) Anal. Biochem. 92, 3 1 4 3 2 3
25. Dormanen, M. C., Bishop, J. E., Hammond, M.W., Okamura, W. H., Nemere,
I., and Norman, A. W. (1994) Biochem. Biophys. Res. Commun. 201, 394401
26. Wilhelm, F., and Norman, A. W. (1985) J. Biol. Chem. 260, 10087-10092
27. Horst, R. L., Littledike, E. T., Riley, J. L., and Napoli, J. L. (1981) Anal.
Biochem 116, 189-197
28. Henry, H. L., and Norman, A. W. (1978) Science 201,835-857
29. Somjen, D., Binderman, I., and Weisman, Y. (1983) Biochem. J . 214,263-298
30. Yoshimoto, Y., and Norman, A. W. (1986) J. Steroid Biochem. 25,905-909
31. Nemere, I., Zhou, L.-X., and Norman, A. W.(1993) Receptor 3,277-291
Downloaded from www.jbc.org by on October 15, 2006
signal transductionprocess associated withtranscaltachia. The
detergent solubilized BLM-VDR exhibits a specific and saturaM)
ble binding for [3H11a,25-(OH)zD3
with a KD ( = 0.72 x
that is physiologically relevant to the prevailing plasma concentrations of la,25-(OH),D3 (27). The BLM-VDR has been purified 150-fold above basal-lateral membrane levels by anionexchange chromatography. This is approximately 4500-fold
above whole homogenate, based on the observation that the
marker enzyme activity, Na+,K+-ATPase, is purified 30-fold
over whole homogenate. Further separation of active fractions
from anion exchange chromatography by gel filtration, and
analyses of the fractions for specific [3H11a,25-(OH),D3binding,
as well as determination of composition bySDS-PAGE and
silver staining, suggest that theBLM-VDR is composed of 1-3
proteins with molecular weight(s) 2 60,000.
In addition to the substantialprogress made inpurifying the
BLM-VDR, three functional correlations between the binding
activity and transcaltachia have been made. Earlier work (3,
17) has indicated that vitaminD-deficient animals lack a rapid,
non-nuclear response to la,25-(OH),D3. In the present work,
impairedbutnot
abolished specific binding of r3H]la,25(OH),D3 was found inmembrane fractions (P,, solubilized
BLM, and Mono Q fraction 16) prepared from vitamin D-deficient chicks, relative to corresponding fractions from normal
chicks. Plausibleexplanations for the absence of a transcaltachicresponse in vitamin D-deficient chicks include reduced amounts of a membrane receptor, the presence of
an inactive receptor, or a defect inother components of
signal transduction. Wali et al. (17) have arrived at similar
conclusions.
A second line of evidence to support theexistence of a BLMVDR comes from the observation that vascularperfusion with
650 PM of nonradioactive 1a,25-(OH),D3 results in desensitization or down-regulation of specific [3Hlla,25-(OH),D3 binding
(Fig. 10). Although this might be due t o internalization of receptor, binding activity was notobserved in aliquotsof fraction
P,. Nor was redistribution of the membrane receptor to a cytoskeleton-associated compartment (CHAPSO pellet) found.
Alternative explanationsinclude occupancy of the ligand binding site, precluding detection by isotopically labeled seco-steroid, or a conformational change of the membrane receptor
leading to inactivation of binding.
A third line of evidence, derived from analog studies, also
suggests that the BLM-VDR mediates signal transduction in
transcaltachia. Norman et al. (21) have postulated that the
membrane receptor for la,25-(OH),D3 might preferentially
bind the conformationally mobile seco-steroid when the broken
B-ring assumes a closed, “steroid-like” configuration. The postulate is supportedby the current study inwhich the relative
efficacies of two additional 6-s-cis analogs in inducing transto
caltachia areparallelled by the abilityof the analogs compete
with [3H]1a,25-(OH)ZD3for binding to the solubilized BLMVDR.
The metabolite 24R,25-(OH),D3 has been proposed to have
hormonal activity (28), especially in bone (29). Yoshimoto and
Norman (30) have demonstrated that
24R,25-(OH),D3induced
transcaltachia. These observations, coupled with the finding
that 24R,25-(OH),D3 also exhibits saturable bindingt o solubilized BLM with a physiologically relevant affinity, suggest that
this metabolite may play an importantrole in contributing to
calcium homeostasis.
1a,25-(OH)p3