Apr 6, 1994 - Koenig, R. J., Warne, R. L., Brent, G. A,, Harney, J. W., Larsen, P. R., and. 11. Strait, K. A,, Schwartz, H. L., Perez-Castillo,A., and Oppenheimer,.
Vol. 269, No. 40, Issue of October 7,pp. 24777-24782, 1994 Printed in U S . b
THEJOURNAL OF BioL0Cr~c.aC H E M I ~ Y
0 1994 by The American Society for Biochemistry and Molecular Biology, Inc.
Widespread Distributionof Immunoreactive Thyroid HormonePZ Receptor ~ T R ~ in Z ) the Nucleiof ~ x t r a p i t u i t a Rat ~ “issues* (Received for publication, April 6, 1994, and in revised form, July 15, 1994)
Harold L. SchwartzS, MitchellA. Lazars, and JackH.OppenheimerSV From the $Thyroid Research Unit, Section of Diabetes, Endocrinology and Metabolism, Department of Medicine, University of Minnesota, Minneapolis, Minnesota 55455 and the 5Department of Medicine, University of Pennsylvania, P h ~ Z * ~ l p h i ~ , P e n n s 1y 9 ~1 v ~~ ~4 i-a~ 0 6 ~
The precipitated protein may Messenger RNA for thyroid hormone receptor (TR) mRNA by Northern blot analysis. isoforms a1 and P l are widely distributed in rat tissues.be a novel T,-binding receptorpossessing a cross-reacting ~ t e ~ a t i v e l y , tunusually his high p r o t e i ~ m R N A r a t i o Until recently,TRPZ mRNA was believed to be limited epitope. to the pituitary and the assumption was made that TRPZ could result from high translational effkiency of the TRPZ protein was similarly restricted. We determined the dis- mRNA, rapid fractional degradation of the mRNA, slow turntribution of TRpZ protein in selected adult and fetal ratover of the protein product, ora combination of these factors. tissues using three anti-TRpZ antiseradirected to differ- We initiated the current studies for three reasons: 1)to deA 5 domain terminewhetheranti-TRP2precipitableT,-bindingactivity ent amino acid sequences of the distinctive of TRp2. The proportionof total nuclear binding capac-can be demonstrated in other nonpituitary tissues despitethe ity cleared by each antiserum was determinedby satu- reported absence of the corresponding mRNA from such tis10-20%of total binding capacity in adult sues; 2) availability of an antibody to a distinctive TRP2 seration analysis. brain, liver, kidney, and heart was ~ m ~ o p r e c i p i t a t e dquence that did not overlapthe sequences previously used alby each antiserum. Use of specific antibodies to TRpl lowed us to test the hypothesis that the anti-TRP2 antiserum and TRal showed these isofoms accounted for the re- was interacting with a homologousepitope in an unknown mainder of total Tsbinding. Fetal liver and brain, how-T,-binding protein, and 3) use of a specific antiserum t o T R a l ever, contained onlyTRa1. Immunohistochemical anall TRP2 allowedus to directly withantisera t o T R pand ysis of the adult tissues showed TRPZ present in nuclei. together assess the contribution of the i n ~ v i d receptor ~ l isofoms to Reverse ans script ion polymerase chain reaction dethe total T3 nuclear binding capacity in selected tissues. Previtected low levels of TRp2 mRNA in the adult tissues.We the assumption that infer thatTRP2 accounts for a significant fraction TRof ous studies from our laboratory made T R p l a n d T R a l were the only T,-binding isof‘orms in these in adult rat tissues despite the low levels of its mRNA tissues and that the contribution of TRal could be inferred
In humans and rats, thyroidhormonenuclearreceptors (TR)l are the products of two genes, designated a and P. Two mRNAs arise from each of these genes, TRal and TRa2, T R p l and TRP2 (1). TRa2, however, lacking the terminal 40 amino acids, does not bind T3 a n d its function is unknown. Initial studies using Northern blot analyses indicated the widespread tissue distribution of‘ the mRNAs for TRal, TRa2, and TRp1, whereas TRP2=RNA appeared to be confhed tothe pituitary (2, 3). However, Bradley et ab. (4) detected TRp2 mRNAin the developing rat hippocampus and striatum byin situ hybridization histochemistry. Cook et al. (5) using PCR techniques, identified the mRNA for TRP2 in selected areas of the adult rat brain. Lechan et al. (6),in a collaborative study with our laboratory, have shown that TR+2 protein is widely distributed among cells of the rat brain. Furthermore, two independently prepared but overlapping antisera against sequences in the uniqueAIB domain of‘TRP2 cleared about20% ofthe T3binding capacity from brain nuclear extracts. Two potential explanations could account for the detection of substantial levels of TRPZ in brain despite an inability t o detect the corresponding
from the difference between total nuclear binding capacity and that immunoprecipitated with anti-TRpl (7).
~ T E R I AND ~ S METHODS Adult male Sprague-Dawley rats, weighing 200-225 g, and timed pregnant females were purchased from Harlan (Madison, WI) and bad ad dibitum access to food (Purina Chow) and tap water. Preparation ofAntisera-Anti-TR antisera were prepared in rabbits (8). We earlier reported development and characterization of specific antisera againstTRpl(7) and TRp2 (6) as well as one capable of interacting with a sequence common to the c~boxyl-termina~ region of the known T,-binding T R TRrul, -@land -p2 (71. This last antiserum was designated TRrrllp. Current studies were carried out with three separate antisera to peptide sequences in the unique Ana domain of TRP2. Two of these were prepared at the University of Pennsylvania, IgGp2 (PA1) (amino acids 131-145), IgGp2 (PA2) (amino acids 92-114), and one at the University of Minnesota, IgGp2 (MN)(amino acids 111-142). The studies earlier reported by Lechan et ad. (6) employed IgGp2(PA11 and IgGp2 (MN).A specific anti-TRd antiserum prepared against the last 8 amino acids of the carboxyl terminus of the TRLvl (amino acids 403-410) was a gift from Dr. L. J. DeGroot (University of Chicago).The specificity of these antiserawas demonstrated by immunoprecipitation of the appropriate in vitro translated proteins which could be blocked by excess peptide. IgG fractions were prepared from preimmune serum and each of the antisera using protein G-Sepharose(Pharmacia Biotech * This work was supported by National Institutes of Health Grants Inc.) as described by the ofmanufacturer. I m ~ u n o ~ r e ~ ~ p ~ ~Binding a t w n Proteins-Nuclear extracts were AM19-812 (to J. H. 0.)and DK43806 (toM. A. L.).The costs of publication of this article were defrayed in part by the payment of page prepared as described (7). One-hundred fifty pg of the various IgG charges. This article must therefore be hereby marked “advertisement” preparations were added to identical aliquots of receptor extract and incubated overnight at 4 “C. Total volumewas equalized by addition of in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. ll To whom all correspondence should be addressed: University of phosphate-buffered saline (0.15 M NaC1, 0.015 M sodium phosphate, pH Minnesota, Dept. of Medicine, Box 91, UMHC,Minneapolis, MN 55455. 7.4).Control incubations contained an appropriate volume o f phosphate-buffered saline alone. The immune complexes werecleared with Tel.: 612-624-5150;Fax: 612-626-3840. protein A and the residual binding capacity measured as described I The abbreviations used are: TR, thyroid hormone receptor; PCR, polymerase chain reaction; RT, reverse transcription; T3, triiodothyro- previously (7) with the modification that bound and free hormone were nine. separated by filtration 19).
24777
p2 in Rat Tissues
Thyroid Hormone Receptor
24778 LIVER
0.50 o Control + NIS
0.40
IgGBl lgGR2 (MN)
0
a
?!
T
s
IgGal/O
0.30
c
0'
0.20
m 0.10
l
0.00
0.10
0.20
0.30
0.40
0.70
0.50 0.60
Bound (Mxl0"O) 0.35
-,
0.00 0.00
0.10
0.20
0.40
0.30
0.50
Bound (MxlO-lO) 0.50
BRAIN
Heart
0.40 -
I 0.00
0.05 0.150.10
0.20
Bound (Mxl O-10)
0.25
0.30
0.00
0.10
0.20
0.30
0.40
0.50
0.60
0.70
Bound (MxlO-10)
FIG.1. Immunoprecipitation of TR isoforms from nuclear extracts of adult rat tissues. Equal aliquots of nuclear extracts were incubated withIgG fractions from the indicated antiseraas detailed under "Materials and Methods." After clearing immune complexes with protein A, residual binding activity was assayed by saturation analysis (7). The anti-02 antiserum used in these studies was (MN) IgGp2 prepared against the amino acid sequence 111-142. Polymerase Chain Reaction-Oligonucleotide primers were syntherose gel (NuSieve GTG-agarose, Rockland, ME) together with a DNA sized with a DNA synthesizer, model 391, Applied Biosystems (Foster ladder. Ethidium bromide staining was t used o determine the size of the City, CAI. Primer positions were chosen to distinguish between ampli- resultant product. Following alkaline denaturation the products were fication of genomic DNA and cDNA. The upstream primer (5'-GAA- transferred to a nylon membrane (Zetabind, Cuno, Inc., Meridan, CT) GAAAAGCCTTTTCCTCA-3') is a 20-mer identical to nucleotides 447and hybridized using the 35S-labeledXbaIIPstI TRp2 cDNA fragment 466 coding for aminoacids 131-137 intheuniqueamino-terminal specific for TR-02. domain of TR-p2 (3). The downstream primer (5'-TATCAGCTTCCTCTImmunohistochemistry-Tissue sections were prepared as described TGGCTAG-3') is a sequence coding for amino acids 234-240 (nucleo- by Strait et al. (11). Five-pm sections were cuton a cryostatfrom tissue tides 756-776)of the 02 hinge region corresponding to amino acids blocks frozen inO.C.T. mounting solution (Miles,Inc., Elkhart, IN) and 181-187 of the TR-01 (10). mounted on slidescoatedwithgelatin.Slideswere fixed with 4% Total RNA was extracted from tissues as described previously (11). paraformaldehyde in phosphate-buffered saline and dehydrated Total cDNA was synthesized using random hexanucleotide primersa as throughaseries of ethanolwashes.Sectionswerestainedwitha template for the reverse transcription reaction. Four pg of total RNA 1 5 0 dilution of antireceptor antiserum followed by a 1:32 dilution of from each tissue or 0.5 pg of total RNA from pituitary was combined fluorescein-conjugated goat anti-rabbitIgG (Kallestad Laboratories). with 2 pl of 10 x PCR buffer (500 mM Tris, pH 8.3,750 mM KCI, 100 mM dithiothreitol, 30 mM MgCI,), 1 mM dNTPs, 2.5 p~ random hexamers RESULTS (Boehringer Mannheim),1 unitipl RNasin (Promega, Madison, WI), and 2.5 units/pl of MMLV reverse transcriptase (Life Technologies, Inc.) in For each tissue two independent pools of nuclear extract a total volume of 20 p1. The mixture was incubated in aDNA thermal were analyzed. Fig. 1 illustrates the results of one study of cycler (Tempcycler 11, model llOS, Coy Corp., Grass Lake, MI) at 37 "C for 60 min, then94 "C for 10 min, and finally soakedat 5 "C for 5 min. nuclear extracts from adult liver, kidney, brain, and heart. ImTo this mixture was added sufficient MgC1, and KC1 to bring the final munoglobulin p2 (MN) was used for this series. Addition of an concentrations to2.6 and 55 mM, respectively, oligonucleotide primers to IgG fraction from preimmune serum hadno effect on the meas0.25 pmol/pl, and distilled H,O t o bring the finalvolume t o 100 pl. The ured binding capacity. Treatment of extracts with anti-TRallp, mixture was heated to 94"C for 10 min to denature the primers, then directed to the common ligand-binding domain, cleared an av2.5 units of Taq polymerase (Boehringer Mannheim) were added. The erage of 96 2 2%(mean 2 S.D.) ofthe totalbinding capacity. The incubation profile used was 1) denaturation for 60 s a t 94 "C, 2) primer annealing for 60 s a t 50 "C, and 3) extension for 60 s a t 72 "C. This percentage of binding activity clearedby the various IgG preparations is summarized inTable I. The fraction of total binding sequence was repeated for 35 cycles. approximates those Twenty pl of each reaction mixture was electrophoresed on 4% aga- accounted for by TRpl in these studies
.8-,
24779
Thyroid Hormone Receptor p2 in Rat Tissues TABLEI Contribution of the T3nuclear receptor isoforms to the total binding activity in adult rat tissues The fraction of the total binding activity was estimated from the reduction in binding activity following incubation of nuclear extracts with each of the specific anti-TR IgG.
0.30 0.25
Q)
TR-P1
Tissue
TR-62
TR-a1
