Immunological Identification and Functional Quantitation of Retinoic ...

10 downloads 0 Views 3MB Size Report
mal mammalian development and adult organ function (1-3). In most tissues, the biolo~cally active form of retinol is retinoic acid. Retinoic acid is synthesized ...
Val. 269, No.

THBJOURNAL OF BIOLOGICAL C m m 0 1994 by The American Society for Biochemistry and Molecular Biology, IOC

Issue of August 12,pp. 2 ~ 2 ~ 2 ~ ~ 5 , 1 9 9 p Printed in U.S.A.

Immunological Identificationand Functional Quantitationof Retinoic Acid and Retinoid X Receptor Proteins in Human Skin* (Received for publication,January 11, 1994, and in revised form, April 14, 1994)

Gary J. Fisherf, Harvinder S.Talwar, Jia-Ha0 Xiao, Subhash C. Datta, AmbatiP, Reddy, Marie-Pierre Gaubl, Chcile Rochette-EglyO,Pierre ChambonO, and John J.Voorhees From the Department ofllermatology, Universityof Michigan, Ann Arbor Michigan 48109 and the 9Laboratoire De Genetique Moleculaire De EucaryoteslCNRS, U184 IINSEEM, Faculte de Medecine, Strasbourg, 67085 France We have determined protein levels of total and indi- target gene transcription, is believed to be the predominant -PI y) andretinoid mechanism through which all-trans- and 9-cis-retinoic acid vidualnuclearretinoicacid X (RXR-a,-@, -y) receptors by ligand binding, Western regulate biological functions (13-16). analysis, and gel shift assays, in adult human skin, a Nuclear retinoic acid receptors comprise two families, each major retinoid-responsive tissue. Total RARS and RX&, encoded by three genes. Initially, three nuclear receptors, measured by direct binding of specific ligands, were 0.24 termed retinoic acid receptor-a (RAR-a),' RAR-p, and RAR-y, d 0.01 fmoUpg (la = 13) and 1.26 t 0.08 fm0Upg (la = 7), were discovered, which boundand were activated by all-transrespectively. These values calculated on an average per retinoic acid (14,17-21). This was followed by identification of cell basis were1790 RARsfcell and 9400 RKRaeell. Sim- a second family of nuclear receptors, termed retinoid X recepilar results were obtained with competitive ligand bindtors-ar (RXR-a),RXR-p, and RXR-y (9,14,22-24). RXIh bound ing assays. RAR-a,-#$ and -y were each specifically im- and were activated by 9-cis-retinoic acid (5, 6). 9~~s-Retinoic munoprecipitated,and their levelsdeterminedby acid binds to both RGRS and RxR9 with similar high aEnity, ligand binding assays of supernatantsand Western andwhereas all-trans-retinoic acid binds with high affinity t o ysis of precipitates. RAR-y was the most abundant, repR A R s , but only very weakly to RXRs (25). 12-14% of resenting 87% of RAR protein. The remaining Studies on the regulation of expression of RARs and in RAR protein was RAR-a.No RAR-p was detected. Similar ~ ~ o p ~ c i p i t a tstudies i o n revealed that RXR-ar cells and tissues have, for the most part, examined levels and represented W o of RXR protein expressed in human location of transcripts by Northern analysis (9, 17-21,26-28) skin. No RXR-P or RXR-y proteins were detected by and in situ hybridization (24,29-351, respectively. These studWestern blot. Supershift gel retardation with antibodiesies have demonstrated different levels and distribution of indihave led to RARs detected probe-RAR-a and probe-M-y com- vidual RARs and RXRs in adult and fetal tissues and plexes in a 1 to 4 ratio. No probe-RARP complex was to the hypothesis that each RAR and RXR possesses functional detected. With antibodies to both Uy and RXR, a dou- specificity (14, 36, 37). RAR proteins have been detected in ble supershifted complex was formed, indicating that HL60 cells (38), embryonal carcinoma cell lines, mouse emRAR-ylRxR heterodimers bound to the probe. These bryos, and some mouse tissues (39-41). In embryonal carciofRMts are five times noma cells, retinoic acid induction of RAR-pZ mRNA was cordata demonstrate 1)protein levels greater thanRARs, 2) relative protein levels of RAR and related with increased W - p 2 protein levels (40). To date, RXR family members are compatible with their previ- however, there hasnot been a systematic analysis of the relaously described relative mRNA levels, and 3) R X R d tive levels of each RAR and RXR protein in any single tissue. RAR-y heterodimers are the major retinoid receptors Recent evidence indicates that RARs and RXRs bind to DNA that have the potential to regulate transcription of tar- and regulate gene transcription as a heterodimeric complex get genes,in adult human skin. (Refs. 7,14,16,22,42,43 and references therein). Thus, knowledge of the relative levels of RAR and RXR proteins is of critical importance to understanding theirfunction in vivo,since it isa Retinol (vitamin A) is anessential nutrient required for nor- major determinant in theformation of heterodimers. mal mammalian development and adult organ function (1-3). Human skin undergoes continual self-renewal, and retinoic In most tissues, the biolo~cally active form of retinol is retinoic acid is believed to be a key participant in regulating the balance acid. Retinoic acid is synthesized fkom retinol within tissues between cell proliferation and cell daerentiation. Several studlocally where it acts (4).Two biologically active stereoisomers of ies from this and otherlaboratories have described expression retinoic acid have been identified, all-trans-retinoic acid, and of transcripts for RARs and RXRs in normal human skin (9, 9-cis-retinoic acid (5-9). Both all-trans- and 9-cis-retinoic acid 44-47). The predominant RAR transcript is RAR-y, with minibind to specific nuclear receptors, members of the steroid/ mal, but detectable RAR-a and undetectable RAR-p. The prethyroid hormone nuclear receptor s u p e ~ ~ i lwhich y , function dominant RXR transcript is=-CY,with m i n i m ~ ~ detectable y as ~igand-dependentscriptio ion factors (10-22). Activation of RXR-p and undetectable RXR-y. However, whether transcript nuclear retinoic acid receptors, with resultant modulation of levels accurately reflect receptor protein levels is unknown. Therefore, we have utilized specific monoclonaland polyclonal * Supported in part by the Babcock Endowment for Dermatological antibodies, in combination with threeseparate biochemical Research. The costa of publication of this article were defrayedin part methods, to determine levels of RAR and RXR proteins in adult by the payment of page charges. This article must therefore be hereby human skin. marked "advertisement" in accordancewith 18 U.S.C.Section 1734 solely to indicate this fact. $ lb whom correspondence should be addressed Dept. of Dermatolom,Kresge I, R6558,University of Michigan,Ann Arbor, MI 48109."el.: The abbreviations usedare: FUR, retinoic acid receptor;RXR, reti3 1 3 - 7 ~ ~ - Fax: 0 ~ 7313-747-0076. ~ noid X receptor; W E , retinoic acid response element.

-

20629

20630

R4R and RXR Human Skin Proteins in E ~ E R I ~ PROCEDURES E ~ T ~

~ateria~~-[~H~9-cis-Retinoic acid and unlabeled 9-cis-retinoic acid were generously provided by P. F. Sorter, J. F. Grippo, and A. A. Levin, Hoffman LaRoche, Nutley, NJ. rHICD367 and unlabeled CD367 were kindly provided byDr. Braham Shroot, Centre International DeRecherches, Sophia-Antipolis, Valbonne,France. SRI 11237 (81104-BASF) was provided by B. Janssen, BASF-Aktiengesellschaft, D6700, Ludwigshafen, Germany. SRI 11237 was originally prepared by M. I. Dawson (48). Monoclonal antibody to RXR-p was kindly provided by K. Ozato, National Institutes of Child Health and Human Development, Bethesda, MD. Procurement of Human Skin Biopsies-Keratome biopsies were obtained from healthy adult human volunteers as described previously (49). Biopsiescontained primarily epidermis with residual amounts of cell poor dermis. Specifically, these biopsies contain -95% keratinocytes and =5% other cell types (primarily epidermal melanocytes and Langerhans cells and an occasional endothelial cell, fibroblast, or dermal dendritic cell). Therefore, the data in this paper predominantiy pertain to retinoid receptors in epidermal keratinocytes, and our use of the term “human skin” should be interpreted accordingly.Allprocedures involving human subjects received prior approval by the University of Michigan Institutional Review Board, and all subjects provided written informed consent. Preparation ofNuclear Extracts from Human Skin-Individual keratome biopsies were placed in 0.25% trypsin, 0.1% EDTA for 40 min at 37 “C. Trypsinization was stopped by addition of Dulbecco’s modified Eagle’smedium containing 10% fetal bovine serum, and cellswere released from the tissue by scraping. Released cells were pipetted up and down several times to form a single cell suspension and passed through a nylon filter to remove residual tissue. The remaining procedures were performeda t 4 “C. Cells from three to six keratome biopsies were pooled (approximately 3 x 10’ cells), washed three times in phosphate-buffered saline, and resuspended in 20 mM Tris (pH 8.01, 20 miu NaCI, 6 mM MgCl,, 0.2% Triton X-100, 1 mM dithiothreitol, 200 mM sucrose, 1 mM phenylmethylsulfonyl fluoride, 0.02mg/ml leupeptin, 0.02 mg/mI pepstatin A, at a concentration of 2 x lo7 celldml. Nuclear extracts containing retinoid receptors were prepared as described previously (50). Extracts were aliquoted and stored at -70 “C prior to use. Extracts from each pool of keratomes represents a sample number f n ) of one. Expression of Retinoid Receptor Proteins-Human RAR-a,RAR-p, RAR-y, and RXR-a! were expressed in Sf9 insect cells using the Baculovirus expression system as described previously (51). Following transmM fection, cells were lysedin buffer containing 20 mM Tris (pH 8.0),130 NaCl, 1mM CaCl,, 10% glycerol,1% Nonidet P-40, 2 mM phenyImethylsulfonyl fluoride, 0.2 unitiml of aprotinin, 0.02 mM leupeptin, 0.5 mgiml pepstatin A, and centrifuged a t 15,000 x g for 15 min to remove particulate material. The supernatant served as the source of recombinant retinoid receptors used as standards for ligand binding assays and Western analysis, as described in the text. The amount of receptor in the Sf9 cell supernatant was quantified by ligand binding assay, as described below. Retinoid Receptor Ligand Binding Assay-Binding of I3HI9-cis-retinoic acid and C3H]CD367to recombinant retinoid receptors and nuclear extracts from human skin were performed by the dextran-coated charcoal adsorption method, as described previously (51,52). To determine retinoid receptor protein levels by ligand binding, [3H19-cis-retinoic acid . and [3H]CD367 wereused a t saturating concentrations of 10 n ~ Nonspecific binding, determined in the presence of 100-fold excess unlabeled ligand, was subtracted from total binding to give specificbinding. Competition experiments utilized 50 gm unlabeled competitor. Calculations of receptor levels werebased on a stoichiomet~of one forligand binding by receptors. Binding data were analyzed by the method of Scatchard (53). Immunoprecipitation and Western Analysis-Immunoprecipitations of recombinant retinoid receptors and retinoid receptors in nuclear extracts from human skin were performed as described previously (3941). For direct Western analysis, nuclear extracts (1ml containing 3 mg of protein) were concentrated to 0.1 ml using a Centricon 30 concentrator (Amicon, Inc.). Samples (105-210 pg of protein) were analyzed by SDS-polyacrylamide gel electrophoresis, using 4-20% acrylamide gels (Novex).The monoclonal antibodies utilized were: RAR-a, Ab9dF) (9a9A6) (39);RAR-p, Ab8p(F)2 (8P-lOE2)(40); RAR-7, Ab4$bF) (4y-7A11) (41); RXR-a!, 4RX-1F6; RXR+ (54), and RXR-y, 12RX-2D3. Immunoreactive proteins on Western blots were detected by ‘‘’I-goat anti-mouse IgG and radioactivity in bands quantified by a PhosphorImager (Molecular Dynamics).

UUMAN SKIN KERATOMEBIOPSY

1

NUCLEAR EXTRACTS {RETINOID RECEPTORS)

t

LIGAND BINDING

1

INDIVIDUA~RARs & RXRs SINGLE (L DOUBLE SUPERSHI~S

I

RAR~RXRDNA INTERACTIONS

J

FIG.1. Outline of methods usedto determine levels and interactions among retinoid receptor proteins in nuclear extracts from adult human skin. Electrophoretic MobilityShift Assay-Gel mobility shift assays were performed as described by Garner and Revzin (55) and Rochette-Eglyet al. (41). Double-stranded oligodeoxynucleotides containing the wildtype (shown in bold, with half-sites in capitals) (5”TCGACTAAGGaTTCAccpaaAG‘ITCACTCGCA-3‘)or mutated retinoic acid response element in the RAFt-pP promoter (56) were used as probes. T w o mutated probes were utilized, one with two mutations ( ~ d e r l i n e din ) one half- 3 ’ one ), site ( 5 ’ - T C G A C T ~ G ~ C A c c g a ~ G ~ C A C T C ~and with two mutations ~ u n d e r ~ n e din) both half-sites I5‘TCGACTAAGl’J”CAccgaaAGTT~TCGCA-3’). For antibody supershif’ts, monoclonal antibodies to RAR-y, AB4$hF) (4y-7All) (41);=-a, 4RX1F6; and general RxlE 4RX-1D12 (i.e. recognizesRXR-a, -p, and -7) and rabbit polyclonal antibodies to RAR-a, RPa!(F) (391,and RAR-0,RPp(F) (40) were utilized. Radioactive protein-DNA complexesin the gels were visualized and quantified by a PhosphorImager. RESULTS

Experimental Design-As outlined in Fig. 1, we employed ligand binding, Westernanalysis, andgel retardation assays to determine the levels and interactions among RARs and RXRs in nuclear extracts from human skin. Measurements of total RARs and RXRs were performed in two separate ways. The first method employed directbindingassaysusing labeled [3H]9-cis-retinoic acid, which binds with near equalaffinity to RARs and RXRs, to determine totalretinoid receptors (RARs+ RXRs), and [3H]CD367,a synthetic RAR-specific ligand, to delevel of total RXRs was termine totalRARs in human skin. The then calculated as thedifference between these two values. The second method utilized to determine totalRAR and RXR levels in human skinemployed competition of [3H]9-cis-retinoic acid binding by nonradiolabeled CD367 and SRI 11237, a synthetic RXR-specific ligand. The level of total RARs was calculated from the reduction of [’H]S-cis-retinoic acid binding by CD367, whereas totalRXRs was calculated from the reduction of I’H39cis-retinoic acid binding by SRI 11237. Levels of individual RARs in human skin were determined by measurement of residual f3H1CD367binding in supernatants of nuclear extracts,following mock-immunoprecipitation with isotype control IgG, or removal of RAR-a, RAR-p, or R.AR--y by immunoprecipitation with specific monoclonal antibodies. The levels of RAR-a, RAR-p, and RAR--y were calculated from decreased [3H]CD367binding, which resulted from specific immunoprecipitation of each receptor. Similarly, the level of =-a was calculated from decreased [3H]9-cis-retinoicacid binding, which resulted from RXR-a immunoprecipitation, taking into account the contribution of RARs to L3H19-cis retinoic acid binding. In addition, RAR-a, RAR-p, RAR-y, and RxR-cu were immu-

RAR and RXR Proteins in Human Skin

--

0.35-

-a

0.25-

A

120-

20631

B

0.30-

z 0* SIW‘ = z 80-

%n

a ; 60-

a

26

2,, 0

1

2

3

4

;:I\ 5

402008

6

7

0

a

1

0

RECEPTOR (FOLD CONCENTRATION)

C 0.08

2

Kd=0.56nM

0.04

0.

3

p

a

0.6

f

0.4

0

3

0

4

0

5

0

~

SRI 11237 ( p ”

D

12OOO

1.5

,

0.2

&;:I 2$

‘E

Q

2 8:0n-

Ih

a

Kdd2nM

e .O

0.

0.02

E

2

8oM) looo0

0.0

0.04

0.06

0.08

0.10 0.01.412

BOUND (pmol)

0.0

0.1

0.2

0.3 0.50.4

0.6

$1

6Ooo 4Ooo

0.3

2MH) 3 0

0.0

0



z

a 0.00 0.02

5;

0.6

RARs

RXRs

BOUND (pmol)

FIG.2. Measurement of total RAR and RXR protein levels in human skin determined by direct and competitive ligand binding assays.A, specific binding of [3H19-czs-retinoicacid (circles)and c3H1CD367(triangles) to recombinant human RAR-y1 (open symbols, dashed lines) and recombinant RXR-a (solid symbols, solid lines). Data are means 2 S.E. of duplicate determinations of two experiments.B , competition of [3H19-cis-retinoicacid binding to RAR-y (circles) and RXR-ar (triangles) by SRI 11237. Data are means of duplicate determinations of two experiments.C , Scatchard analysesof c3H1CD367binding to retinoic acid receptors innuclear extracts from humanskin. Binding was determined over the concentration range of 1 4 0 nM c3H1CD367,as described under ”Experimental Procedures.”Open and closed circles are data from two separate experiments.Kd value is mean of the two experiments.D , Scatchard analysesof [3H19-cis-retinoicacid binding to retinoid receptors in nuclear extracts from human skin. Binding was determined over the concentration range 0.2-20 n M [3H19-cis-retinoicacid, as described under “Experimental Procedures.” Open and closed circlesare data from two separate experiments.Kd value is mean of the two experiments.E , total RAR and RXR levels in nuclear extracts from human skin determined by direct (open bars) and competitive (hatched burs) ligand binding. TotalFtARs and RXRs were calculated from measurement of specific c3H1CD367 and [3H19-cis-retinoicacid binding, as described in the text. Right and left vertical axes display the same data in different units. Results are means * S.E. of 13 experiments for W s and 7 experiments for RXRs.

noprecipitated from human skin nuclear extracts and their levels in the immunoprecipitates quantitated by Western analysis by comparison with known amounts of immunoprecipitated recombinant receptors, used as standards. Each retinoid receptor antibody was titrated with recombinant receptors to ensurequantitativeimmunoprecipitation.Underthe conditions utilized immunoprecipitation of receptors was greater than 90% (data not shown). We also attempted to quantify RXR-p and RXR-y by direct Westernblot, using specific monoclonal antibodies (we could not perform immunoprecipitation with the RXR-p and RXR-y antibodies, because they were unsuitable for this purpose), by comparison with recombinant receptor standards. Finally, we utilized single anddouble antibody supershiftgel mobility retardationassaystodeterminerelative levels of RARs and RXRs and t o examine their interactions withDNA. Quantitation of Total RARs a n d RXRs in Human Skin by Direct a n d Competitive Ligand Binding-We exploited the ligand bindingspecificities of RARs and RXRs to determine their levels in nuclear extracts from human skin biopsies. Fig. 2A demonstrates the bindingspecificities of [3H]9-cis-retinoic acid and L3H1CD367to recombinant humanRAR-y and RXR-a. At a saturating concentrationof 10 nM, [3H19-cis-retinoicacid bound RAR-y and equallyto RAR-y and =-a. Bindingtoboth RXR-a was proportional to recombinant receptor concentration. In contrast, 10 nM [3HlCD367bound specifically to RAR-y, with no significant binding t o RXR-a. Binding of CD367 t o RAR-y was proportional to receptor concentration. The specificity of SRI 11237 binding toRXR is demonstrated in Fig. 2B. SRI 11237 effectively competed for [3H]9-cis-retinoic acid binding to recombinant RXR-a. Competition was doseSRI 11237 lowering binding of r3H]9-cisdependent, with 50 retinoic acid t o background levels. There wasno effect of 50 V M

SRI 11237 on [3H]9-cis-retinoic acid binding to recombinant “7.

Direct ligand binding assays performed with nuclear extracts from human skin revealed specific saturable binding of both [3H]9-cis-retinoic acid and L3H]CD367. Dissociation constants calculatedfrom Scatchard analysis for L3H1CD367 (Fig. 2C) and [3H]9-cis-retinoic acid (Fig. 2 0 ) binding t o retinoid receptors in nuclear extractsfrom human skin were 0.58 and 1.2 r m , respectively. Therefore, t o determine receptor protein levels by ligandbinding, both [3H19-cis-retinoic acid and L3H1CD367were utilizedat saturating concentrationsof 10 nM. Under these conditions, specific ligand binding accounted for 48 2 4% ( n = 7) and 80 2 3% ( n = 7) of total CD367 and 9-cis-retinoic acid binding, respectively. The results of these studiesrevealedthattotal RAR levels were 0.24 2 0.013 fmol/pg of protein ( n = 13), and total RXR levels were 1.26 2 0.08 fmol/pg of protein ( n = 7), in nuclear extractsfrom human skin (Fig. 273). Calculation of these values on an averdge per cell basis yielded 1790 2 100 RARs/cell and 9400 2 600 RXRs/ cell. Thus the level of RXRs wasapproximately five times greater than thelevel of RARs in human skin(Fig. 2C). To verify the above results, we performed competition binding assays as described above, utilizing CD367 and SRI 11237 t o specifically compete [3H]9-cis-retinoic acid binding to RARs and RXRs, respectively, in nuclear extracts from human skin. These studiesyielded levels of RARs and RXRs in human skin 2 nuclearextracts of 0.29 O.O2fmol/pg ( n = 6)and1.48 O.O2fmol/pg ( n = 6), respectively (Fig. 2 E ) . Calculation of these values on an average per cell basis yielded 2160 2 150 F U R S / cell and 11040 2 150 RXRs/cell (Fig. 2E). Based on these data RXRs comprise 84% and RARs comprise 16% of total retinoid receptors in human skin (Table I).

RAR and RXR Proteins in

20632

Human Skin

TABLEI Quantitation of total and individual RAR and RXR proteins in human skin by ligand binding and Western analysis

A

-r

Retinoid receptors fmoupg protein

Total RARs Direct binding Competitive binding Total m s Direct binding Competitive binding W - a 1mmpt.hinding Immpt./Western

R'4R-S

1mmpt.hinding Immpt./Western

& :& '

0.240.01 0.29 f 0.02

16

16

( n = 13) ( n = 7)

1.26 0.08 1.48* 0.02

84 84

( n = 7) ( n = 7)

0.03 * 0.004" 0.02 f 0.0026

2 (12)' 2 (1l)d

'

R A R a RAR-6

E

(n = 6) ( n = 6)

( n = 4) ( n = 4)

Not detected" Not detectedb

RAR-Y

1mmpt.hinding Immpt./Western

0.21f 0.05" 0.16 f 0.02b

15 (88)" 13 (89)d

( n = 6) ( n = 6)

1.12 f 0.09" 1.03 0.07*

76 (90)' 69 (82)e

( n = 6) ( n = 6)

RXR-a

1mmpt.hinding Immpt./Western

m - p

Direct Western

RXR-Y

Direct Western

Not detected

( n = 4)

Not detected

( n = 4)

a Calculated from reductions in ligand binding (binding) in supernatants of nuclear extracts following immunoprecipitation (immpt.) of the indicated receptor. Calculated from Western analysis (Western) on immunoprecipitates (immpt.) of the indicated receptor. e Percent of total F U R S calculated from the sum of FUR-a + FUR-., determined by immunoprecipitationfigandbinding analysis. Percent of total RARs calculated from sum of RAR-a + RAR-y determined by immunoprecipitation/Western analysis. e Percent of total RxRs calculated from the sum of total R A R s + total RxRs determined by direct ligand binding.

Measurement of RAR-a, RAR-p,RAR-y, and RXR-a Levels in HumanSkin by Immunoprecipitation followed by Ligand Binding-We next utilized specific monoclonal antibodies to immunoprecipitate RAR-a, RAR-p, RAR-7, and RXR-a in nuclear extracts from human skin andmeasured residual nonimmunoprecipitated receptors by ligand binding. Immunoprecipitation of RAR-a, -p and -y reduced L3H1CD367 binding by 11* 1.5%( n = 6), 0% ( n = 41, and 88.1 2 11%( n = 6), respectively (Fig. 3A). Immunoprecipitation of RXR-a in nuclear extracts from human skin reduced [3H]9-cis-retinoic acidbinding by 76 + 8% ( n = 6) (Fig. 3A).Based on these data, human skin nuclear extracts contain 0.026 0.004fmollpg RAR-a (190-c 30 RARdcell), no detectable RAR-p, 0.21+0.05 fmollpgRAR-y (1570 * 370 RAR-$cell), and 1.120.09fmollpg FER-a (8350 670 RXR-dcell) (Fig. 3B). Thus RAR-y comprises nearly 90% of total RARs, and RXR-a comprises nearly 90% of total RXRs in human skin nuclear extracts (Table I). Measurement of Individual RARs and RXRs by Immunoprecipitation and WesternAnalysis-To confirm the results obtained by ligand binding assays, RAR-a, -p, and -y and RXR-a in nuclear extracts from human skin and k n o w n amounts of recombinant receptors used as standardswere immunoprecipitated, as described above, with specific monoclonal antibodies and the immunoprecipitated receptors quantified by Western blot. Immunoprecipitation was necessary to reliably detect RARs; however, RXR-a could be detected in concentrated nuclear extracts by direct Western analysis, without immunoprecipitation. Immunoprecipitation of recombinant RARs and human skin nuclear extracts yielded bands of the expected size (-51 m a ) . Protein bands corresponding to RAR-a (Fig. 4A, upper panel ) and RAR-y (Fig. 4A, lower panel) were detected in human skin nuclear extracts. No immunoprecipitated protein band corresponding to RAR-p could be detected in nuclear ex-

*

*

FIG.3. RAR-a,RAR/3, R A R y , and RXR-a levels in human skin measured by immunoprecipitation and ligand binding. A, ligand binding following immunoprecipitation of FUR-a, RAR-p, RAR-y, and =-a from human skin nuclear extracts. Open bars demonstrate reductions in specific ['H]CD367 binding following immunoprecipitation of RARs in human skin nuclear extracts with the indicated mouse monoclonal antibodies. Hatched bar demonstrates reduction in specific ['Hl9-cis-retinoic acidbinding following immunoprecipitationof =-a. Reductions in specific ligand binding following immunoprecipitation of receptors are expressed as percent of control immunoprecipitations, which were performed with isotype control immunoglobulin. Data are means * S.E.of six experiments. B , RAR-a, RAR-p, FUR-y, and RXR-a levels in human skincalculated from data in A.

tracts (Fig. 4A, middle panel), although the background was rather high. Multiple species of both RAR-cy and RAR-y have been detected by immunoprecipitatioflestern analysis in cell lines and shown to result from phosphorylation (39, 41). The broad appearance of immunoprecipitated RAR-a and RAR-y bands in human skin nuclear extracts is compatible with the possibility of such post-translational phosphorylation. Fig. 4B demonstrates detection of RXR-a in nuclear extracts from human skin by direct Western analysis. Both recombinant RXR-a, used as a standard (Fig. 4B, lanes 1 and 21, and RXR-a in nuclear extracts from two samples (Fig. 4 B , lanes 4-6) appeared as a single major protein of 51 kDa. We also attempted to detect RXR-p and RXR-y by direct Western analysis of nuclear extracts. Their levels, however, were below the limit of detection, as determined with recombinant RXR-p and -y used as standards (i.e.