. 266, N O . 33, Issue of November 25, PP. 22645”22652,1991
THEJOURNAL
OF BIOLOGICALCHEMISTRY
Printed in U . S . A .
k! 1991 by The American Society for Biochemistry and Molecular Biology, Inc.
The Nuclear Growth Hormone ReceptorBinding Protein
ANTIGENIC AND PHYSICOCHEMICALCHARACTERIZATION*
(Received for publication, April 23, 1991)
Peter E. LobieS, Ross Barnard, and Michael J. Waters
From the Department of Physiology and Pharmacology and the Centre for Molecular Biology,The Universityof Queensland,
Queensland 4072, Australia
The mechanisms involved in transcriptional regulation by growth hormone (GH) remain unknown. We
report here that GH receptor immunoreactivity can be
demonstrated in the nuclei of GH-responsive rat and
rabbit tissues at both the light and electron micrograph
level using monoclonal antibodies to the receptor extracellular domain. Nuclear staining is heterogeneous
and associated with both chromatin and the nuclear
membrane.
To confirm these observations, nuclei were isolated
from rabbit liver by two methods, one involving extensive nonionic detergent washes. Scatchard analysis of
nuclear fractions revealed high affinity somatogenic
receptor in nuclear membranes, nucleoplasm, and
chromatin fractions. A panel of GH receptor monoclonal antibodies was used to further define these nuclear binding sites as being antigenically identical to
microsomal receptor in all but one case. In addition,
affinity cross-linking experiments showed the somatogenic binding subunit to have a reduced M , of
67,000, similar to the M , of the GH binding protein.
We propose that the association of a GH binding
protein with the nucleus may provide a means whereby
GH can regulate the transcription of specific genes
either directly or indirectly through nuclear kinase C
activation. This speculation is congruent with the recent demonstration of a GH response element by Yoon
et al. (Yoon, J. B., Berry, S. A., Seelig, S., and Towle,
H. C. (1990)J. Biol. Chern. 265, 19947-19954).
unknown. It was hoped that elucidation of the GH receptor
sequence (5) would provide insight into this problem. However, while both the GH (5) and prolactin receptor (6, 7) are
homologous with membersof the lymphokinefamily (8), there
is no homology in the cytoplasmic region with any receptor
other than the prolactin
receptor (9) and no indication of
consensus sequence for tyrosine kinase, serine/threonine kinase, or other known signal transduction elements.
Steroid hormones mediate their
biological actions via intracellular receptors. These receptors bind withhigh affinity to
specific hormone-responsiveelementsonthe
genome, and
induced conformational changes lead to transcriptional activation of appropriate hormone-responsivegenes (10). Several
polypeptide hormones andgrowth factors have also shown an
association with thenucleus. Nuclear binding sitesfor insulin
(11) are associated predominantly with the
nuclear membrane
whereas gonadotropin (12) binds to bothnuclear membranes
and chromatin. Chromatin binding siteshave been reported
for angiotensin I1 (13), epidermal growth factor, nerve growth
factor,platelet-derived growth factor (14), and fibroblast
growth factor (15).
This evidence has largely been ignored as itdoes not fit the
current conceptual framework that mandates the lysosomal
destruction of endocytosed polypeptides. However, the failure
t o elucidate the intracellular mechanisms by which GH induces transcription has prompted us explore
to
the possibility
that GH action is mediated at the nuclear
level.
EXPERIMENTALPROCEDURES
Materials
In addition to its short-term
metaboliceffects,GH’
has
oGH (S-14),
oPrl (S-16),
rPrl (B-3),pPrl, and hPLwere gifts from
been reported to induce a number of RNA species in mamtheNationalHormoneandPituitaryProgram(Baltimore,MD).
maliantissues (1-4). Thesignaltransductionmechanisms
hGH was a generous gift from GenentechInc.The
responsible for increased levels of mRNA and rRNA remain Recombinant
monoclonal antibodies used in this study were prepared with the
*This work was supported by aNationalHealthand
Medical
Research Council (Australia) grant (to M. J . W.). Portions of this
work were presented to the United States Endocrine
Society Meeting
at Atlanta, GA, June 1990. The costs of publication of this article
were defrayed in part by the payment of page charges. This article
must therefore be hereby marked “aduertisement” in accordance with
18 U.S.C. Section 1734 solely to indicate this fact.
$ Supported by theAustralianKidneyFoundation,Australian
Medical Association, J. G. Hunter research fellowship, and the William Nanthaniel Robertson and Douglas H. K. Lee research scholarships. To whom reprint requests should he addressed.
’ The abbreviations used are: GH, growth hormone; mAb, monoclonal antibody; PBS, phosphate-buffered saline;
BSA, bovine serum
albumin;EGTA,
[ethylenebis(oxyethylenenitrilo)]tetraaceticacid;
RRA, radioreceptor assay buffer; Hepes, 4-(2-hydroxyethyl)-l-piperazineethanesulfonic acid; hGH, human growth hormone; PEG,polyethylene glycol; BP, binding protein; oGH, ovine growth hormone;
rPr1, rat prolactin; oPrl,
ovine prolactin; pPrl, porcine prolactin; hPL,
human placental lactogen; bGH, bovine growth hormone; EGF, epidermal growth factor.
assistance of AGEN Ltd.,Acacia Ridge, Queensland. Goat anti-mouse
IgG andstreptavidinbiotinhorseradish
peroxidasecomplex were
purchased from Amersham (Australia). Normalgoat serum and rabbit
anti-mouse immunoglobulins were purchased from DAKO Immunoglobulins (Glostrup, Denmark). Triton X-100 (membrane research
grade) was from Boehringer Mannheim. Hoechst 33528, aminoacetonitrile, benzamidine, and aprotinin
were purchased from Sigma and
disuccinimidyl suberate from Pierce Chemical Co. Protein molecular
weight standards and sodium dodecyl sulfate gel reagents were obtained from Bio-Rad.
Production and Characterizationof GH Receptor Monoclonal
Antibodies
Monoclonal antibodies (mAbs 1, 5 , 7, and 43)to the GH receptor
were produced by application of hybridoma technology to splenic
lymphocytesfrom mice immunizedwithahuman(GH)affinitypurified preparationof rabbit liver GH receptor (16).These antibodies
recognize independent epitopes on the extracellular portion of the
receptor, do not cross-react with insulin or prolactinreceptors in the
appropriate receptor assays, and react
specifically with the GH recep-
22645
22646
Nuclear GH Receptor
tor in immunoblots (5,17). mAb 7 recognizes a species-specificepitope g) were homogenized in 2 volumes of homogenization buffer (25 mM
on the rabbit GHreceptor. mAb 263 recognizes a cross-species deter- Tris-HC1, with 0.1% Triton X-100, 3 mM MgCI2, 0.32 M sucrose, 2
minant with high affinity (17) and was obtained from miceimmunized mM EGTA, 0.1 mM spermine, 2 mM phenylmethylsulfonyl fluoride,
with purified rat GH receptor. mAb 263 is reactive against the GH
24 trypsin inhibitory units aprotinin/100 ml, and 25 pg/ml leupeptin
receptor in a number of species but does not inhibit I""Iinsu1in or at pH 7.4, 4 "C). After the low speed spin in 0.2 M sucrose, the crude
"'I-prolactin binding to rabbit or rat
liver microsomes. Under certain nuclear pellet was resuspended in a final sucrose concentration. of
conditions mAb 263 precipitates rat and rabbit GH receptor, although
55% and spun at 50,000 X g for 60 min at 4 "C. The 55% sucrose
it can also compete for hormone binding to subtypes
of the GH
pellet was then washed in 50 ml of homogenization buffer containing
receptor, as does mAb 7 (17).
0.4% Nonidet P-40 but no Triton X-100 and collected by centrifugation (2000 rpm X 15 min) to give a white pellet of intact nuclei as
Light Microscopy
visualizedmicroscopicallybyGiemsa
staining.Thisfraction
was
Tissue Preparationfor
Immunohistochemistry-Neonatal
and suspended either in4 ml of homogenization buffer minus 0.1% Triton
X-100 or in100 volumes of homogenization buffer with 2% v/v Triton
adult rats and adult rabbits were anesthetized by an intraperitoneal
injection of pentobarbitone.Animals were perfused intracardially X-100 for preparation of chromatin. After stirring for 15 min, chromatin was harvested with a spin of 90 min a t 140,000 X g. Fractions
with phosphate-buffered saline, p H 7.4, until blanching, then with
Bouin's solution (0.9% picric acid, 9% (v/v) formaldehyde, 5% acetic were kept on ice and assayed within 24 h. Where soluble nuclear
acid). Tissueswere dissected and post-fixed inBouin's solution for 4 membrane was being assayed, nuclear pellets were suspended in 2
h at 4 "C. The tissues were then embedded in paraffin by standard volumes of 2% Triton X-100, tumbled for 10 min a t 4 "C, and then
histological procedures. Semi-serial 5-pm sections were collected onto centrifuged for 90 min a t 140,000 x g.
The method of Touster etal. (24) does not require the inclusion of
gelatin/chromic potassium sulfate-coated slides.
nonionic detergents in preparation buffers so that nuclei have intact
Immunohistochemistry-Sectionsweredeparaffinized
andsubjected to immunohistochemical staining according to the following outer nuclear membranes by electron microscopy, but there is substantial contamination with endoplasmic reticulum. In the experischedule: (a)elimination of endogenous peroxidase activity with0.5%
HjO, in phosphate-buffered saline (PBS) for 15 min a t 20 "C; (b) ments characterizingsoluble nucleoplasm describedhere, this method
elimination of nonspecific protein binding by incubation at 20 "C was used to obtainednucleoplasm, since removal of the outer nuclear
with 10% normal goat serum for 1 h; (c) incubation overnight at4 "C membrane by the morerigorous method was associated with considwith mouse anti-GH receptor monoclonal IgG (100 pg/ml (mAbs 1, erable leakage of soluble '*'I-GH binding from the nuclei. Solubilized
inner and outer nuclear membranes were obtained by treating these
5, 7,43, and Brucella) or 25 pg/ml (mAb 263) in PBS, 1%bovine
serum albumin (BSA)); (d) incubation with goat anti-mouse biotin- nuclei with 2 v/v 1%Triton X-I00 and 2% Triton X-100, respectively.
ylated IgG (diluted 1:150 in PBS, 1% BSA) for 2 h a t 25 "C, ( e )
Binding Assays
incubation with avidin (streptavidin)-biotin horseradish peroxidase
complex (diluted 1350 in PBS, 1% BSA) for 1 h a t 25 "C; and (f)
Assays were performed in tripIicate in 12 X 75-mm borosilicate
treatment with 0.05 mg/ml diaminobenzidine in PBS containing 1 % tubes according to Ref. 25. Displacing ligands were diluted in radiHjOj for 5 min a t 25 "C. Between each step sections were washed 3 oreceptor assay buffer (RRA; 50 mM Tris-HC1, 10 mM MgCI2, 0.1%
timesinPBSand
once inPES, 1% BSA. All incubations were bovine serum albumin, pH 7.4). Total incubation volume was 0.5 ml,
performed in a humidified chamber. Sections were counterstained in consisting of 100 p1 of RRA, 100 11 of appropriate nuclear fraction,
Mayer's hematoxylin, dehydrated, and mounted. Controls included
100 p1 of displacing ligand, or 100 p l of RRA and 100 pI of "'I-hGH
were: (a) omission of the primary antibody; and (b) replacement of
(approximately 100,000 cpm/tube). The assays were incubated on an
the anti-GH receptormouse IgG by unrelated primary antibodies
agitating platform for 12-16 h a t 18 "C. For all nucleoplasm and
(Brucella abortus and mAb 7 (for rat tissues)) of the same isotype chromatin or intactnuclei prepared by the Buckley et al. (22) method,
(IgGK1) andat the same concentration.
mAbs 43 or 1 were added to the incubation mixturea t a final dilution
of 10 pg/ml to make thelabeled complex precipitable by polyethylene
glycol (PEG). These assays were terminated by the addition of 1 ml
Electron Microscopy
of bovine y-globulin (0.1%) and 1ml of PEG 6000 (25% w/v), followed
For nuclear purity assessments, rabbit liver nuclei and chromatin by vortex mixing and centrifugation for 30 min at 1600 X g (4 "C).
Other chromatin assays were terminated by the addition of 3 ml of
(2% Triton X-I00 insoluble) were fixed in 3.0% glutaraldehyde in0.1
RRA and centrifugationfor 30 min at 1600 X g (4 "C).Supernatants
M cacodylate buffer, pH 7.4, for 4 h a t 4 " C ,postfixed in 1%osmium
tetroxide in cacodylate buffer, dehydrated in ethanol, andembedded were removed and pellets counted on an LKB 1274 y-spectrometer.
Scatchard analysiswere then undertaken with the LIGAND
program
in Spurr's resin. 60-nm sections were cut and stained with uranyl
(26).
acetate and Reynolds
lead citrate prior to examination
wit.h a Hitachi
H800 electron microscope a t 100 kV.
Precipitation Assays
For immunogold histochemistry, tissues were fixed and embedded
in LRwhite, and receptor immunoreactivity was detected with protein
Titration of precipitating mAbs against I2'II-hGH.GH receptor.BP
A-conjugated 12-nm gold particles ongold grids according to Slot and complexes in nucleoplasm, 2% Triton X-100 solubilized inner nuclear
Geuze (18). mAb 263 or a control mAb (B. abortus) at 25 pg of membrane receptor, or solubilized microsomes was performed by the
protein/ml was used for these studies.
standard double antibody method as described previously for serum
and liver cytosol (27). Titration of precipitating mAbs against nuDNA and Protein Estimations
cleoplasmwasalso
performed by using PEG to separate ternary
complexes.
Toquantitatethenumber
of nuclei assayedandthereforeto
determine receptor number/nucleus, the DNA content of the chroInhibition Assays
matin (2% Triton X-100 insoluble) fraction was determined. DNA
concentration was obtained by the Labarca and Paigen method (19)
A 100-pl portion of eitherthe nucleoplasm orchromatin was
using Hoechst 33528 (bisbenzamide). Protein concentrations were incubated for 12 h a t 4 "C with serial100-pl dilutions of the testmAb
estimated by the method of Lowry et al. (20) after alkaline solubiliza- (7 or 263). 100p1 of 1pg/ml mAb 43 was alsoadded to the
nucleoplasm
tion of particulate fractions.
to make the nucleoplasm complex PEG-precipitable. Parallel serial
dilutions of an unrelatedmAb of the sameisotype (B. abortus IgGIK)
Iodination
were used as binding controls.A 10O-pl aliquot of "'I-hGH was then
hGH was iodinated by the lactoperoxidase method of Thorell and added together with 200 p1 of RRA or 100 111 of RRA and 100 pl of
Johansson (21) and fractionated on a Sephadex G-100 column. Spe- excess unlabeled oGH (10 pg/ml) in the binding control tubes and
incubated a t 20 'C for 8 h. The respective assays were terminated as
cific activity was 120-150 pCi/pg.
described above.
Nuclear Fraction Preparation
Affinity Cross-linking
Adult female rabbit liver nuclei were prepared either by a modifiChromatin and liver microsomes were washed in cold cross-link
cation (22) of the method of Wang (23) using 0.1% Triton X-100 in
all buffers and ending witha thorough wash in 0.4% Nonidet P-40 or buffer (50 mM Hepes buffer, pH 7.4, containing 0.07% Triton X-100,
by the method of Touster et al. (24). In the first protocol, livers (45 10 mM MgCI,, leupeptin,andpepstatin
(each 10 pg/ml), 10 mM
Nuclear GH Receptor
22647
henzamidine, and1 mM phenylmethylsulfonyl fluoride). Washed
chromatin (approximately 15 pg of DNA) and microsomes (approximately 25 p g of protein) were then incubated for 16 h at 16 " C with
""I-hGH (200,000 cpm/min) in 1 ml of cross-link huffer & 2 pg/ml
oCH or rPrl. For coupling, disuccinimidvl suherate was added as a
20-fold concentrate in dimethyl sulfoxide with vortex mixing to give
a final concentration of 0.5 mM. Chromatin and microsomes were left
shaking for 15 min at 4 "C to cross-link before reaction termination
with100 pl of 1 M Tris, pH 7.4. Chromatin and microsomes were
harvested by centrifugation (1600 X g, 30 min) before being washed
in cross-link huffer, thoroughly drained, andresuspended in Laemmli
sample huffer containing 0.1 M dithiothreitol. Samples were boiled
for 3 min, microcentrifuged, and subjected to slab gel electrophoresis
by the method of Laemmli using 7.5% gels (28). Gels were dried and
autoradiographed using Kodak AR5 film with intensifying screensa t
-80 "C.
RESULTS
B
n
FIG. 1. Detection of the NHAerminal domain of the GH
receptor.BP in nuclei of rat andrabbit tissues by immunohistochemistry. Magnification bar represents 50 pm in A and C-F and
55 pm in H . A. mAh 263 immunoreactivity in hepatocytes of adult rat
Immunohistochemistry-In a variety of rat and rabbit tissues strong immunoreactivity was observed with mAbs 263
and 43, and weaker immunoreactivity was seen with mAbs 1
and 5. No immunoreaction was seen with control mAb 50.8
in both species and with mAb 7 in the rat. Immunoreactivity
was seen at identical locations with different antibodies and
appeared mainly in the cytoplasm and a variable proportion
of nuclei (Fig. 1,A - D ) . Four distinctnuclear staining patterns
were observed on light microscopy: A, no nuclear immunoreaction; R, nuclear immunoreaction associated with the nuclear envelope; C, nuclear immunoreaction associatedwith
both the nuclearenvelope and chromatin(mostly heterochromatin); and D, dense nuclear immunoreaction with no euchromatin/heterochromatin distinction.
Immunogold electron microscopy for the GH receptor. BP
showed strong nuclear localization in tissues immunoreactive
at thelight microscope level (Fig. 2, A and R ) . In the nucleus
gold particles were preferentially localized to heterochromatin
and the nucleolus. Gold particles were also located on both
the inner and outer nuclear
membranes. In thecytoplasm the
GHreceptor.BP was localized tothe rough endoplasmic
reticulum or the cytosol. Gold particles were not localized to
cellular organelles such asmitochondria or zymogen granules.
Purity of Isolated Nuclei-Nuclei were prepared in good
yield by the method of Buckley et al. (22) and judged to be
clean both by visual inspection after Giemsa stain and by
electron microscopy (Fig. 3). Assay of NADPH-cytochrome c
reductase (30) in freshly isolated nuclei showed no detectable
activity in 10-15 x 10" nuclei (threepreparations) while
microsomes showed 54 k 5 nmol/min/mg of protein ( n = 3).
After fractions hadbeen assayed to determine"'I-hGH binding, nuclear fractions were again assayed for NADPH-cytochrome c reductase a t a dilution giving equivalent hormone
binding as themicrosome fraction, and no detectable
enzyme
activity was found, despite the fact that the
microsomes gave
0.04-0.08 absorbanceunits/min.
Nuclei preparedwithout
nonionic detergent (24) did have some contamination with
endoplasmic reticulum, although NADPH-cytochromereducliver (see "Experimental Procedures"). Note that only a proportion
of nucleiwas immunoreactive whereas cytosolic immunoreactivity
was evident in all cells. R, adjacent control section to that of A , mAb
7; hematoxylin counterstain. C. adjacent control section, mAh 263
preadsorhed with purified rabbit
GH receptor (31) at 10 pg/ml. 11,
mAh 43 immunoreactivity in pancreatic acinar cells of the adult rat.
Note bothcytoplasmic and nuclear immunoreactivity and theabsence
of immunoreactivity in some nuclei. E, mAh 263 immunoreactivity
in pyramidal neuronesof the internal pyramidal layer of the cerehral
cortex of a neonatal rabbit. Note the intense
nuclear immunoreaction.
I.: adjacent section to that of 6 where the primary antibody was an
unrelated IgG of the same isotype ( R . nbortus). Structures are still
evidentbecause of the hematoxylin counterstain, hut noimmunoreaction is apparent.
Nuclear GH Receptor
22648
. .,,
. j ..>
0
,
.
.
.
*
.
.
.
1
2
3
4
5
6
7
0
I
22
HOURS
FIG. 4. Time course of'""IhGH binding to rabbit liver chromatin (0)and microsomes (A) and nonspecific binding of 1251hGH binding to chromatin ( 0 ) and microsomes (A). Points
represent means of triplicate determinationswith standard deviation
indicated. Somatogenic sites were assayed with oGH (final concentration, 2 pg/ml) as displacing ligand. Chromatin was a t a final DNA
concentration of 45 pg/ml. Incubation was performed a t 20 "C.
by 8 h at 20 "C,although a small increase in specific binding
was observableafter this time. GH
B P present in nucleoplasm
was PEG 6000-soluble and required a precipitating antibody
FIG. 2. Subcellular localization of the GH receptor.BP by tomakethe'"1-hGH.BP
complex PEG-precipitable. Apimmunogold electron microscopy. A, rat pancreatic acinar cell
immunostained with mAb 263. Note the prominent association of proximately 95% of specific ""I-hGH binding in 1% Triton
X-100-solubilized outer nuclear membranes was precipitable
gold particles with the nucleus (especially heterochromatin) andwith
endoplasmicreticulum. R, nucleus of rat gastric parietal cell with by PEG alone (as compared with PEG-precipitable specific
prominent localization of the receptor to the nucleolus and absence binding with the addition of a precipitating antibody mAb
of gold particles over mitochondria.
263). In 2% Triton X-100-solubilized inner nuclearmembranes approximately45% of specific hGH bindingwas PEGprecipitable without the addition
of antibody, presumably due
to release of nuclear PEG-soluble GH BP. Scatchard analysis
(withoGHas
displacingligand, II. = 5) revealed affinity
constants in the rangeof 2-3 X lo9 M" for all fractions (Fig.
5, B-D). This is identical with affinity constant values obtained with livermicrosomes and cytosol under the same
conditions (see also Ref. 27). To test thespecificity of somatogenic sites, oPrl, rPrl, pPrl, and
hPL were alsoused as
displacing ligands (Fig. 5 A ) . oPrl (known to bind to the GH
receptor withlower affinity (25))displaced '"'I-hGH, but only
a t 10-fold oGH concentrations. rPrl and pPrl did not affect
'"I-hGH binding to chromatin unless present
a t high concentration (>1 pg/1 ml). hPL (92% homologous to hGH) displaced with 1% of GH potency. The relative subcellular distribution of GH BPin rabbit liver as determinedby Scatchard
analysis of nuclei (Touster et al. (24)) showed the majority of
GH receptor. B P was confined to the cytosolic fraction (48 f
3%) followed by the nucleus (31 f 3%) and the microsomal
fraction (21 f 1%). Within thenucleus differential partitioning of the GH receptor.BP was observed (Fig. 6). Based on
the percentage of total nuclear receptor content the outer
nuclear membrane is the
major site of GH binding(60 f 10%)
followed
by
the
nucleoplasm
(25 f 6%), chromatin(10 f 2%),
FIG. 3. Electron micrograph of rabbit liver nuclei purified
k 2%). Assuming 5 pg of
by sucrose gradient centrifugation accordingto Buckley et al. and the inner nuclear membrane (5
( 2 2 ) .A, field, X 4000; R, showing lack of outer nuclear membrane, X
DNA/nucleus and based on DNA estimation and Scatchard
:10,000.
analysis of receptor number, it is calculated that the Touster
et al. (24)method gives a preparation withapproximately
tase activity was still lO-fold less than the activity in the
5400 GH receptors/rabbit liver nucleus. Of these 538 f 90
microsomal fraction (5.4 f 0.4 nmol/min/mg of protein).
(S.E.) are intimatelyassociated with chromatin as determined
Hormone Binding to Nuclear Fractions-Previous studies by resistance to solubilization by 2% Triton X-100.
havedemonstrateddifferential solubilization of outer and
Since the Touster et al. (24) preparation is contaminated
inner nuclear membranes with 1 and 2% Triton X-100, re- with endoplasmic reticulum (-10% by NADPH-cytochrome
spectively (29). The residual 2% Triton X-100-insoluble ma- c reductase assay),nuclei were prepared innonionic detergent
terial constitutes chromatin. A fourth assayable fraction was (30). This method gave 3220 f 540 ( n = 3) receptors/nucleus
formed upon homogenization of intact nuclei and constituted and 1750 f 430 receptors/cellular chromatin (based on DNA
nucleoplasm and soluble material released from the nuclear estimation). The affinity constant for GH binding to chrosac. High affinityoGH-displaceable "'I-hGH binding was matin prepared by this procedure was not significantly differseen in all fourfractions. Binding was both saturable and
ent from the soluble GH-binding protein (1 x lo9 M"). No
time-dependent (Fig. 4). Equilibriumwas achieved essentially detectable binding was found in 2% Triton X-100 extracts of
B
Nuclear GH Receptor
I
I
22649
80
I
r
0 NUCLEOPLASM
ONM
m
FIG.6. Relativesubnuclear GH receptor .BP contentin
adult female rabbit liver.Relative subnuclear distribution of the
GH receptor.BP wasdoneinnuclei
prepared by the method of
Touster et al. (24). Receptor number was estimated from Scatchard
analysis with appropriate corrections for receptor dilution. GH BPS
of nucleoplasm were made PEG-precipitableby the addition of mAb
43 at a final concentrationof 1 pg/ml. Error bars represent standard
deviations; meansof three preparations.
BOUND (U) x 10"l
FIG.7. Release of GH BP on rupture of nuclei by sonication
and homogenization. Scatchard analysis of nuclei prepared by the
method of Buckley et al. (22) is shown. 0, intact nuclei; 0, ruptured
nuclei from the same preparation, assayed at 13.6 pg of DNA/ml. 12'1hGH was used as ligand displaced with2 pg/ml oGH.
A
0.05
D
n.on
0
20
40
60
80
1no
120
140
IGO
1 0 " ' ~BOI!Nt (M)
FIG.5. Hormone competition and Scatchard analysis
of 12'1hGH binding to rabbit liver nuclear fractions.
A, hormone
competition curve of '"I-hGH binding to rabbit liver chromatin (2%
Triton X-100 insoluble). Displacing ligands were:
0 ,pPrl; A, rPrl; A,
hPL; 0, oPrl; and 0, oGH. Specific and nonspecific binding of I2'IhGH with oGH as displacing ligand was 30 and lo%, respectively.
Chromatin was at a final DNA concentration of 45pg/ml. Points
represent means of triplicate determinations. B, Scatchard analysis
of"'SI-hGH binding to nucleoplasm with oGH as displacing ligand.
Nucleoplasmwas at an added 1:2 (v/v) dilution of fraction 1 (see
"Experimental Procedures"). Specific and nonspecific binding
was 56
and 14%,respectively. The specific radioactivity of "'SI-hGH was 65
pCi/pg. C, Scatchard analysisof '"SI-hGH binding t o the outer nuclear
membrane (2% Triton X-100 solubilized) of rabbit liver. oGHwas
the displacing ligand. Inner nuclear membranes were used undiluted
(fraction 5). Specific and nonspecific binding was 17and 9%, respectively. The specific radioactivity of '"I-hGHwas65pCi/pg.
D,
Scatchard analysisof "'SI-hGH binding to chromatin (2% Triton X100 insoluble) of rabbit liver. oGH was displacing ligand. Chromatin
wasused at a final DNA concentration of 40 pg/ml. Specific and
nonspecific bindingwas 24 and 12%, respectively. The specific radioactivity of '"I-hGH was 65 pCi/pg.
nuclearcontent,fractions
were assayedimmediately after
preparation. Breakage of nuclei by sonication and glass homogenization resulted in the appearance of additional hormone binding sites, provideda precipitating mAb was included in the assay(Fig. 7).
Immunoprecipitation of Nuclear Associated GH ReceptorFour mAbs (mAbs 1, 2, 5, and 43) were tested in parallel for
GH bindingproteins from the
theirabilitytoprecipitate
nucleoplasm, outernuclearmembranes
(1%Triton X-100
solubilized), and inner nuclear membranes(2% Triton X-100
solubilized), respectively (Fig. 8, A-C). The orderof titers for
all three fractions was mAb 43 > mAb 1 > mAb 5 > mAb 2.
mAb 263 was also able to precipitate GH BPS
from these
fractions, but the order
of its titer depended on whether bound
andfreehormone
were separated by PEG or the double
antibody method. These results show that soluble GH BPS
from thenucleoplasm and solubilized GH BPS from the outer
and inner nuclear membranes are antigenically identical to
hepatic cytosolic forms of G H BPS at four independent epitopes.
mAb Inhibitionof '"I-hGH Binding to Nuclear Fractions-
mAbs 7 and 263 inhibit components of GH binding to rabbit
liver microsomes(16), cytosol, serum BP, and affinity-purified
nucleiprepared by thisprocedure,andtheouternuclear
GH receptor (17, 27). In the present study mAbs 7 and 263
membrane was absent in electron micrographs (Fig. 2). The were tested against both
nucleoplasm and chromatin (2%
outer membrane is presumed to have been removed by the Triton X-100-insoluble)(Fig. 9). mAb 7 totally inhibited I2'INonidet P-40 wash,
which contained 18pmol of GH receptor. hGH binding to the GH BPS of the nucleoplasm. The same
BP per nucleus, and inner nuclear binding islow even in the result was obtained from mAb 7 inhibition of binding to GH
Touster method. Incubationof isolated nuclei for 18 h at 0 "C BPS of livercytosol.However,for
chromatinthe mAb 7
resulted in therelease of 20% of nuclear GH binding into the inhibitory titer was approximately 100-fold lower relative to
medium, so thatinordertoobtain
reliable estimates of liver microsomes (Fig. 9). The increase in binding observed
Nuclear GH Receptor
22650
Y
I
I
I
IO"IO"
I
2
Y
lo-z
IO"
IO"
16'
ADDEODILUTION OF MAb
10-l
o
AOOED DILUTION OF MAb
ADDED DILUTION OF MAb
- ".
10"
o
FIG.8. Titration of precipitatingmAbs against nucleoplasm
( A ) , 1% Triton X-100-solubilized outer nuclear membranes
( B ) , and 2% Triton X-100-solubilized inner
nuclear
membranes (C).The ordinatesshow the antibody-precipitable binding as a percentage of mAb 43-precipitable specific binding. Points
represent means of triplicate determinations. 0, mAb 1; 0, mAb 2;
A, mAb 5; A, mAb 43; 0, mAb 263. For A, bound and free hormones
were separated by PEG precipitation, whereasfor B and C bound and
freehormones were separated by the double antibodymethod as
described for liver cytosol (19). For A, B, and C, respectively, dilutions
were 1:2 (fraction I), 1:3 (fraction4),andundiluted(fraction5).
oGH-displaceable specific binding of lYsI-hGHwas 36, 39, and lo%,
and nonspecific binding was 8, 4, and 2%, respectively.
FIG.9. mAb inhibition of '2511-hGH
binding to the nuclear
GH receptor. A, inhibition of '"I-hGH binding to chromatin by
mAb 263 ( 0 )and mAb 7 (0).Inhibition of "'I-hGH binding to liver
microsomes by mAb 7 (A). oGH-displaceable specific binding was
20% (chromatin) and 14% (microsomes). Nonspecific binding was
5% (chromatinand microsomes). Chromatin was at afinalDNA
concentration of 45 pg/ml. Error bars represent standard deviations.
B, mAb 263 (0)and mAb 7 (0)inhibition assay of "'I-hGH binding
to nucleoplasm.Nucleoplasmwas
used at a 1:2 (v/v) dilution of
fraction 1. oGH-displaceable specific binding was 23%, and nonspecific binding was 7% of radioactivity added. Points represent mean of
triplicate determinationswith error bars representing standarddeviations. mAb 43 a t a final concentration of 1 pg/ml was also used to
make the complex PEG-precipitable.
I 2
3
u.5"
116.
97-
4s31.
with mAb 263 and nucleoplasm has been observed with liver
cytosolic GH BPS andpresumably results from the formation
of macromolecular aggregates which rendersthe complex
more easily PEG-precipitable. mAb 263 displayed only 15%
inhibition of I2"I-hGH binding to chromatin, paralleling the
result observed for liver microsomes under similar conditions
(16).
Affinity Cross-linking of the ChromatinGH Receptor- BPThe GH receptor B P exists asat least two different molecular
weight species (55,000-60,000 and 130,000) (34, 35). The
smaller form has anidentical amino-terminal sequence to the
130,000 receptor and is released by cleavage of the full-length
receptor to yield the GH BP
of rabbit serum(31) or in rodents
is derived from alternate mRNA splicing (32, 33). To determine which species of receptor was associated with the nucleus, l2'1-hGH was cross-linked to chromatin, and thecomplex was reduced and sodium dodecyl sulfate-solubilized, then
subjected to polyacrylamide gel electrophoresis (Fig. 10). A
single diffuse hormone binding subunit of M , 67,000 was
observed, after subtracting the hormone component. Microsomes from the same preparation
gave three subunitM , values
(>140,000, 93,000, and 60,000 after subtraction of the hormone component). No displacementof '"I-hGH binding was
S
FIG.10. Affinity cross-link gel for chromatin and microsomal GH receptor, run according to the methods described
under "Experimental Procedures.". Binding assay without(lanes
1 and 4 ) or with 2 pg/ml bGH (lanes 2 and 5 ) or 2 pg/ml rat prolactin
(lanes 3 and 6 ) . Lanes 1-3, chromatin a t 15 pg of DNA/ml; lanes 46, microsomes a t 50 pg/ml protein. Position of standards is shown on
the left.
observed with microsomes in the presence of excess unlabeled
rat prolactin (2 pg/ml), and minimal displacement was seen
with chromatin. Total displacement was seen with the same
concentration of recombinant bovine GH for both microsomes
and chromatin.
DISCUSSION
We report that a GH receptor.BP, similar to the cytosolic
GH BP, is intimately associated with rabbit liver nuclei. The
basis for this claim has been established by several criteria:
( a ) immunohistochemicaldetection of theamino-terminal
Nuclear GH Receptor
22651
domain of the GH receptor in the nucleus of known GH- actionthrough nuclear receptors. The localization of GH
responsive tissues with a panelof mAbs at light and EMlevel; receptor-BP immunoreactivity in both the cytoplasm and
( b ) demonstration of specific high affinity binding of hGH to nucleus of cells and in different nuclear fractions suggests
somatogenic sites in both soluble and insoluble fractions of that receptor-mediated intracellular GH transportis possible.
morphologically and enzymatically characterized nuclei and Nuclear accumulation of polypeptide hormones by target cells
chromatin, somatogenic specificity of this interaction being has been observed for insulin (38,39),prolactin (40,621, nerve
determined by failure of related lactogenic ligands to displace growth factor (41,42), epidermal growth factor (43), plateletl2'1-hGH binding; (c) antigenic identity (in all but one case) derived growth factor (14), fibroblast growth factor (151, and
between soluble and insoluble nuclear receptor .BP and p - and &interferons (44). In support of this contention Bonplasma membrane-derived GH receptor; ( d ) demonstration ifacino et al. (45) have reported an in vivo accumulation of
in crude nuclear fractions of rat liver
of a somatogenic binding subunit in chromatin similar in size lZ5I-hGH and lZ5I-bGH
to the soluble GH BP, but not to the
membrane receptor. and kidney. Some abrogation of this accumulation was
Confirmation of the existence of a nuclear GH receptor in achieved in the presence of excess unlabeled ligand with male
incubated with human liver
human liver has come recently from Hoquette et al. (59). We rats.Furthermore,[3H]hGH
have also found nuclear immunostaining in the rat with a slices was found to be preferentially incorporated intoa
monoclonal antibody specific for the alternatively spliced nuclear fraction (46).
Recent studies directed toward the elucidation of GH sighydrophilic carboxyl terminus of the GH BP (32).'
While a high affinity GH receptor was associated with the naling mechanisms have implicated protein kinase C in rapid
nucleoplasm and chromatin, lZ5I-GHbinding was not detect- responses to GH (47-49). Smal et al. (48) have demonstrated
able in solubilized inner nuclear membrane (2% Triton X- that hGH-dependent lipogenesis in rat adipocytes is abolished
100) of clean nuclei prepared by the method of Buckley et al. by acridine orange, an inhibitor of protein kinase C, and
(22), where outer nuclear membrane was absent in electron down-regulation of protein kinase Cresults in a marked
micrographs. It is likely that receptor is present in the outer decrease of the maximal hGH effect (47). Similarly, an apnuclear membrane in a similar fashion to theinsulin receptor parent hGH-induced protein kinase C activation of c-fos but
(38, 39), and indeed with more gentle procedures for nuclear not of insulin-like growth factor gene expression in preadipose
preparation (24) considerable binding was found in the 1% Ob1771 cells has been reported (49). Since protein kinase C
Triton X-100 extract of such nuclei. What proportion of this is also nuclear (50) and prolactin and erythropoietin (both in
the GH lymphokine family (60)) have been reported to stimbinding is outer nuclear membrane and what contribution
derives from the contiguous endoplasmic reticulum is difficult ulate hepatic kinase C activity (51,52), it is possible that GH
to determine. The chromatin bound GH receptor. BP remains acts on protein kinase C at the nuclear level to regulate
insoluble in 2% Triton X-100 and has alower reactivity with transcriptionthrough specific trans acting elements (53).
mAb 7 than either cytosolic or membrane-bound GH receptor. Equally, it is possible that the GH BP.GH complex acts
The lower inhibitory titer of mAb 7 for lZ5I-hGHbinding to either directly or indirectly as a transcriptional control elechromatin as compared with microsomes suggests the pres- ment, and this would be supported by the apparently tight
ence of a chromatin/histone-associated proteinwithin 3.5 nm binding of the GH BP to chromatin and the evidence of
(36) of the mAb 7 epitope or a different receptor type. A association with heterochromatin in immunogold electron
similar phenomenon was noticed for the rabbit adipose mem- microscopy. In preliminary experiments4 we have been unable
brane receptor although this inhibition of mAb 7 accessibility to show aconsistent
increase in oligo(dT)-hybridizable
was lost upon Triton X-100 solubilization (37), suggesting mRNA or in total RNA synthesis as a result of adding bGH
steric hindrance of access rather thana receptor subtype. The at physiological levels to isolated rat liver nuclei. This would
antigenic and physicochemical similarity between the GH BP imply that any regulation of transcription is gene-specific,
of serum and liver cytosol and the GH BP of the nucleus and indeed Yoon et al. (61) have now demonstrated a GH
suggests that these are derived from the same gene. However, response element 5' of the Spi 2.1 gene in rat liver.
the difference in subunit size between chromatin and memA variety of polypeptide hormones has been reported to
brane proteins (67,000 versus 60,000) may be indicative of display specific nuclear effects. InsulinandEGF
regulate
alternate cleavage or mRNA splicing of the full-length GH poly(A) mRNA efflux from intact nuclei by influencing the
receptor, as is seen in the mouse and rat (32, 33). We have activity of nuclear envelope nucleoside triphosphatase (54,
observed a 1.8-kilobase mRNA species in the rabbit at low 55), which provides energy for mRNA transport andregulates
levels which may correspond to such an alternatively spliced the phosphorylation state of the nuclear envelope mRNA
form."
transporter (55). Direct effects by polypeptide hormones on
It is not possible to be categoric about the absence of full- chromatin have also been reported. Thus, binding of angiolength receptor in the nucleus on the basics of cross-linking tensin I1 enhances the susceptibility of chromatin to nuclease
experiments, since this is rapidly cleaved in the rabbit liver digestion (13), consistent with induction of transcriptional
to the binding protein (31). However, since full-length and activity. In contrast, binding of nerve growth factor to its
partially processed receptors ( M , > 140,000 and 93,000) were receptor in chromatin (14) results in increased resistance to
seen in membranes prepared at the same time and protease nuclease digestion. Bouche et al. (15) found that transport of
inhibitors were always present,it seems likely that only fibroblast GF to thenucleus and its nucleolar localization are
binding protein is present in the nucleus. This wouldbe
correlated with stimulation of transcription of ribosomal
predicted on thermodynamic grounds and is supported by the genes, associated with induction of nucleoli. Fibroblast GF
finding that monoclonal antibody to thehydrophilic carboxyl was also claimed to have a direct stimulatory effect on RNA
terminus of the rat binding protein can be localized to the polymerase I in isolated nuclei. Likewise, Miller (56) reported
nucleus of rat cells by immunohistochemistry.'
that insulin applied directly to isolated frogoocyte nuclei
GH must be transported to the nucleus if it is to exert an markedly stimulated RNA synthesis.
We have no information at present relating to the mode of
P. E. Lobie, J. Garcia-Aragon,B. S. Wang, W. R. Baumbach, and uptake of GH into thenucleus, i.e. whether it arrives through
M. J. Waters, submitted for publication.
:' P. E. Lobie, R. Barnard, and M. J. Waters, unpublished data.
G . Norstedt and P. E. Lobie, unpublished data.
22652
Nuclear GH Receptor
the endocytotic lysosomal route (57) in association with the
receptor or the BP, or whether it arrivesmore directly complexed to the cytoso~ic BP. It has been reported (58) that
plasma membrane-bound EGF receptor'EGF
find
their way to the nucleus and stimulate both nucleocytoplasmic
transportand DNA synthesis. It would appearthatthere is a
number of intriguing possibilities relating to the role of the
GH BP in GH action
at the nuclear level.
REFERENCES
1. MacGeoch, C., Morgan, E. T., Cordell, B., and Gustaffsson, J . - k
(1987) Biochem. Biophys. Res. Commun. 1 4 3 , 782-788
2. Le Cam, A., Pages, G., Auberger, P., Le Cam, G., Leopold, P.,
Benaro's, R.9 and G1aichenhaus, N. (1987) EMBO J. 6, 12251232
3. Hynes, M. A.,Van Wyk, J. J., Brooks, p. J., D'Ercole, A. J.,
Janson, M., and Lund, P. K. (1987) Mol. Endocrinol. 1, 233239
4. Yoon, J. B., Towle,H.C., and Seelig, S. (1987) J. Biol. Chem.
262,4284-4289
5 . Leung, D. W., Spencer, S. A. Cachianes, G., Hammonds, R. G.,
Collins, C., Henzel, W. J., Barnard, R., Waters, M. J., and
Wood, W. I. (1987) Nature 330,537-543
6. Boutin, J. M., Jolicouer, C., Okamura, H., Gagnon, J., Edery, M.,
Shirota, M., Banville, D., Dusanter-Fourt, I., Djiane, J., and
Kelly, P. A. (1988) Cell 53, 69-77
7. Waters, M. J., Barnard, R., Hamlin, G., Spencer, S. A., Hammonds, R. G., Leung, D. W., Henzel, W. J., Cachianes, G., and
Wood, W. I. (1988) Prog. Endocrinol. 1, 601-607
8. Bazan, J. F. (1989) Biochem. Biophys. Res. Commun. 1 6 4 , 788795
9. Edery, M., Jolicoeur, C., Levi-Mayrueis, C., Dusanter-Fourt, I.,
Petridau, B., Boutin, J., Lesueur, L., Kelly, P. A., and Djiane,
J. (1989) Proc. Natl. Acad. Sci. U. S. A . 86, 2112-2116
10. Beato, M. (1989) Cell 56, 335-344
11, vigneri, R., Goldfine, 1. D., Wong, K, y.,smith, G, J., and
Pezzino, V. (1978) J. Biol. Chem. 253, 2098-2103
12. Rajendran, K.G., and Menon, K. M. J , (1983) Biochem, Biophys.
Res. Cornmun. 111, 127-134
13, R ~ R.
, N,,vizard, D. L,, B
~ J., and
~ B ~ ~ s,~ E,
~ (1984)
, ~
Biochem. Biophys. Res. Commun. 1 1 9 , 220-227
14. Rakowicz-Szulczynska,E. M., Rodeck, U., Herlyn, M., and Koprowski' H' (1986)
sei' " " A' 83' 37283732
15. Bouche, G., Gas, N., Prats, H., Baldin, V., Tauber, J. P., Teissie,
J., and Almaric, F. (1987) PrOC. NQtl. ACad. SCi. u. s.A. 84,
6770-6774
16. Barnard, R., Bundesen, P. G., Rylatt, D.B., and Waters, M. J.
(1984) Endocrinology 1 1 5 , 1805-1813
17. Barnard, R., Bundesen, P. G., Rylatt, D. B., and Waters, M. J.
(1985) Biochern. J. 2 3 1 , 459-468
18. Slot, J. w., and Geuze, H. J. (1985) Eur. J . Cell Biol. 38, 87-93
19. Labarca, C., and Paigen, K. (1980) Anal. Biochem. 102, 344-352
20. Lowry, 0. H., Rosebrough, N. J., Farr, A. L., and Randall, R. J.
(1951) J. Biol. Chem. 193,265-275
21. Thorell, J. I., and Johansson, B.G. (1971) Bioehim. Biophys. Acta
25 1,363-369
22. Buckley, A. R.,Crowe, P. D., and Russell, D. H. (1988) Proc.
Natl. Acad. Sci. U. S. A. 85, 8649-8653
23. Wang, T.Y. (1967) Methods Enzymol. 1 2 , 417-421
24. Touster, O., Aronson, N. N., Dulaney, J. T., and Hendrickson,
H. (1970) J . Cell Biol. 47,604-618
25. Waters, M. J., and Friesen, H. G. (1979) J. Biol. Chem. 254,
6815-6825
26. Munson, P. J., andRodbard, D. (1980) Anal. Biochem. 107,220239
27. Barnard, R., and Waters, M. J. (1986) Biochem. J . 237,885-892
28. Laemmli, U. K. (1970) Nature 2 2 7 , 680-685
29. Bumen, S. J., and Jones, A. L. (1989) Trends Biochem. Sci. 12,
159-162
30. de Dwe, C., Pressman, B. C., Gianetto, R., Wattiaux, R., and
Appelmans, F. (1955) Biochem. J . 60, 604-617
31. Spencer, S. A., Hammonds, R. G., Henzel, W. J., Rodriguez,
H.,
Waters, M. J., and Wood, W. I. (1988) J. Biol. Chem. 2 6 3 ,
7862-7867
32. Baumbach, W.R., Horner, D. R., and Logan, J. S. (1989) Genes
& Deu. 3, 1199-1206
33. Smith, W. C., Kuniyoshi, J., and Talamantes, F. (1989) Mol.
Endocrinol. 3 , 984-990
34. Ymer, S. I., and Herington, A. C. (1987) Biochem. J. 242, 713720
35. Hughes, J. P., Simpson, J. S. A., and Friesen, H. G. (1983)
Endocrinology 112, 1980-1985
36. Tzartos, S. J., Rand, D. E., Enarson, B. L., and Lindstrom, J. M.
(1981) J. Biol. Chem. 256,8635-8645
37. Barnard, R., Rowlinson, S. W., and Waters, M. J. (1990) Biochem.
J. 267,471-478
38. Goldfine, I. D., Jones, A.L., Hradek, G. T., Wong, K. Y., and
Mooney, J. S. (1978) Science 2 0 2 , 760-763
39. Podlecki,
A., D.
Smith, R. M., Kao, M., Tsai, P., Huecksteadt,
T., Brandenburg, D., Lasher, R. S., Jarrett, L., and Olefsky, J.
M. (1987) J. Biol. Chem. 262,3362-3368
40. Giss, B. J., and Walker, A. M. (1985) Mol. Cell. Endocrinol. 42,
259-267
41. Yanker, B.A., and Shooter, E. M. (1979) Proc. N d . Acad. Sci.
U. S. A. 7 6 , 1269-1273
42. Marchisio, P. c., Naldin, L., and Calissano, P. (1980) Proc. Natl.
Acad. Sci. U. S. A. 7 7 , 1656-1660
Vlodavsky, I., and Gospodarowicz, D. (1981) J. Biol.
43. Savion, N.,
Chem. 256,1149-1154
44. Macdonald, H. S., Kushnaryov,
M.,
V.
Sedmak, J. J., and Grossberg, S. E. (1986) Biochem. Biophys. Res. Commun. 138, 254260
45. Bonifacino, J. S., Roguin, L. P., and Paladini, A. C. (1983)
Biochem. J. 2 1 4 , 121-134
46. Rezvani, I., Maddaiah, v. T.,ColliPP, p. J., Thomas, J., and
Chen, S. Y. (1973) Biochem. Med. 7, 432-440
47. Smal,
and De Mefls, p. (l987) Biochem. Biophys. Res.
mun. 147,1232-1240
~48. Smal,
,
J., Kathuria, S., and De Mefls, P. (1989) FEBSLett. 2 4 4 ,
465-468
49. Doglio, A., Dani, C., Grimaldi, P., and Ailhaud, G. (1989) Proc.
Natl. Acad. Sci. U. S . A . 86, 1148-1152
50. Masmoudi, A., Labourdette, G., Mersel, M., Huang, F.
L., Huang,
K.-P., Vincendon, G., and Malviya, A. N. (1989) J. Biol. Chem.
264,1172-1179
51. ~ ~ ~D. ~
H, (1989)
~ 1 Trends
1 , Pharmaco~,
sei, 10, 40-44
52. Mason Garcia, M., Weill, C.L., and Beckman, B. S. (1990)
Biochem. Biophys. Res. Commun. 168,490-497
53. Elsholtz, H. P., Mangalam, H. J., Potter, E., Albert, V. R.,
Supowit, S., Evans, R. M., and Rosenfeld, M. G. (1986) Science
234, 1552-1557
54. purello, F., Burnham, D. B., and Goldfine, 1. D. (1983) Proc, Natl.
A c Q ~Sci.
. U. S. A. 80, 1189-1193
55. Schroder, H. c., Wenger, R., Ugarkovic, D., Friese, K., Bachmann, M., and Muller, W. E. G. (1990) Biochemistry 29,23682378
56. Miller, D. S. (1988) Science 2 4 0 , 506-508
57. Roupas, P., and Herington, A.C. (1987) Endocrinology 1 2 1 ,
1521-1530
58, Jiang, L-W., and Schindler, M. (1990) J. Cell Biol. 1 1 0 , 559-568
59. Hoquette, J. F.,Postel Vinay, M. C., Kayser, C., de Hemptinne,
B., and Amar-Costesec, A. (1989) Endocrinology 125, 21672174
60. Bazan, J. F. (1990) Zmmunol. Today 1 1 , 350-354
61. Yoon, J. B., Berry, S. A., Seelig, S., and Towle, H. C. (1990) J.
B i d . Chern. 265, 19947-19954
62. Clevinger, C. V., Sillman, A. L., and Prystowsky, M. B. (1990)
Endocrinology 1 2 7 , 3151-3159
J.7