| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
ARTICLES |
T4 Pituitary Cells Using Recombinant Adenovirus1
University Research Centre for Neuroendocrinology (J.N.H., M.T.M., H.M.E., T.H., J.B.U., C.A.M.), University of Bristol, Bristol, United Kingdom; Department of Cell Physiology and Pharmacology (G.B.W.), University of Leicester, Leicester LE1 9HN, United Kingdom; Medical Research Council Human Reproductive Science Unit (R.P.M.), Centre for Reproductive Biology, Edinburgh EH3 9ET, Scotland, United Kingdom; and Medical Research Council Research Unit for Molecular Reproductive Endocrinology (B.E.T., R.P.M., J.S.D.), Department of Medical Biochemistry, University of Cape Town, Observatory 7925, South Africa
Address all correspondence and requests for reprints to: Dr. Craig A. McArdle, University of Bristol, Department of Medicine, Bristol Royal Infirmary, Marlborough Street, Bristol BS2 8HW, United Kingdom. E-mail: craig.mcardle{at}bris.ac.uk
 |
Abstract |
|---|
 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
T4 cells (gonadotrope progenitors that lack
endogenous GnRH-R) transfected with a mammalian (human) or nonmammalian
(Xenopus laevis type I) GnRH-R. Because conventional
transfection strategies proved inefficient, recombinant adenovirus
expressing these receptors were constructed, enabling controlled and
efficient GnRH-R expression. When expressed in T4 cells at
physiological density, these GnRH-Rs retain the pharmacology of their
endogenous counterparts (as judged by ligand specificity in radioligand
binding and inositol phosphate accumulation assays) but do not activate
adenylyl cyclase and are not constitutively active. Moreover, the
Xenopus GnRH-R rapidly desensitizes and internalizes in
these cells, whereas the human GnRH-R does not, and the internalization
rates are not dependent upon receptor number. These data extend studies
in COS, HEK, and GH3 cells showing that other GnRH-R with
C-terminal tails desensitize and internalize rapidly, whereas tail-less
mammalian GnRH-R do not. Retention of these distinctions at
physiological receptor density in gonadotrope lineage cells, supports
the argument that the evolution of nondesensitizing mammalian GnRH-Rs
is functionally relevant and related to the development of mammalian
reproductive strategies. |
Introduction |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
GnRH is a hypothalamic decapeptide that acts via GPCRs on gonadotropes to stimulate the secretion of LH and FSH. Activation of GnRH-R causes a Gq/11 mediated stimulation of phospholipase C (PLC), which hydrolyzes membrane phosphoinositides yielding inositol phosphates (IPs), including Ins (1, 4, 5)P3, which mobilizes Ca2+ from intracellular stores (6, 7, 8, 9). GnRH also increases Ca2+ entry into gonadotropes, predominantly via voltage-operated Ca2+ channels, and the increase in cytosolic Ca2+ caused by GnRH is primarily responsible for the increase in exocytotic hormone release (6, 7, 8, 9, 10). Sustained stimulation of gonadotropes with GnRH and reduces GnRH-stimulated gonadotropin secretion, and this homologous desensitization underlies the suppression of the reproductive system, which is exploited in the major clinical applications of GnRH analogues (6, 11).
Most vertebrates investigated express at least two forms of GnRH.
Typically, the highly conserved chicken GnRH-II (cGnRH-II) is found
with one or more additional form of the peptide, and these distinct
forms may play different physiological roles. Amphibians, like
mammals, express mammalian GnRH, which is thought to regulate
reproductive function, and cGnRH-II, which may function as a
neuromodulator. The multiple forms of GnRH have apparently evolved in
parallel with distinct forms of the GnRH-R, which have different
pharmacological characteristics. Thus the cloned GnRH-R of nonmammalian
vertebrates (catfish, goldfish, chicken, and Xenopus
laevis) have high selectivity for cGnRH-II over GnRH,
whereas the opposite is true for the cloned mammalian GnRH-R (9, 12). Mammalian and nonmammalian GnRH show considerable
similarity in a number of key areas (12), including
conserved disulphide bridges (between extracellular loops I and II) as
well as conservation of residues involved in G protein coupling and of
residues in the TM domains implicated in ligand binding (but not
specificity). There are also, however, clear structural differences
between mammalian and nonmammalian GnRH-R (12). For
example, the interacting asparagine and aspartate acid of TM II and TM
VII (Asn87 and Asp318) in
hGnRH-R), which are necessary for mammalian GnRH-R function are not
conserved in nonmammalian GnRH-R (where aspartic acid is found in both
positions) and two cysteins in the amino terminus and extracellular
loop II (Cys14 and Cys184)
in the hGnRH-R), which are thought to form a disulphide bridge, are not
conserved in nonmammalian GnRH-R. However, the most striking structural
difference is that all cloned mammalian GnRH-Rs lack C-terminal tails,
whereas all cloned nonmammalian GnRH-Rs possess C-terminal tails with
multiple potential sites for phosphorylation (9, 12, 13, 14, 15, 16, 17).
The mouse GnRH-R of T3–1 cells (a gonadotrope-derived cell line)
does not show rapid homologous desensitization, and this has been
attributed to the lack of necessary C-terminal tail and its
phosphorylation sites (18, 19, 20, 21, 22, 23, 24, 25). In accord with this, it
has been shown that tailed GnRH-Rs (e.g. catfish and
chicken) desensitize and/or internalize more rapidly than nontailed
GnRH-Rs (e.g. rat and human) when expressed in COS or HEK
cells (15, 23, 25).
Although it was originally thought that the fundamental pharmacological
characteristics (e.g. ligand specificity, effector coupling,
and desensitization) would be constant for any given GPCR, it is now
clear that these features can vary according to cell type and receptor
density. Indeed, cell-to-cell differences in the stoichiometry of
receptors to effectors and accessory proteins can cause receptor
signaling and regulation to vary dramatically from cell to cell. For
example, in addition to activating PLC, 5-HT2C
receptors can stimulate adenylyl cyclase at low density, but inhibit it
at high density (26). Moreover, rapid desensitization of
thromboxane A2 receptors was pronounced at low receptor density and
absent at high density (27) and desensitization of TRH
receptors was more pronounced in two pituitary cell lines than in COS,
Hela, or HEK cells (28). GnRH-R signaling is also
dependent upon receptor density and cellular context. It appears, for
example, that GnRH-R expressed in Sf9 insect cells, COS cells, and
GH3 cells can activate Gs
(29, 30, 31, 32, 33), whereas there is no compelling evidence for such
activation in gonadotropes or in the gonadotrope-derived T3–1 cell
line (34, 35, 36). In addition, reducing GnRH-R number in
GH3 cells has been found to increase
GnRH-stimulated cAMP accumulation while reducing GnRH-stimulated IP
accumulation (37). Moreover, catfish GnRH-R have been
shown to desensitize more rapidly in COS cells than in HEK cells
(23). Because pituitary GnRH-R number can vary
dramatically (e.g. through the oestrous cycle) and GnRH-R
are also expressed in several extra-pituitary sites (e.g.
prostate, testes, ovary, and breast), such observations raise the
intriguing possibility that the pharmacology and/or signaling of GnRH-R
varies between different cell types and/or under different
physiological conditions. They also raise the question of whether
differences in receptor density, signaling, or cellular context have
contributed to the reported functional differences between mammalian
and nonmammalian GnRH-R. To address these issues, we have developed
recombinant adenovirus (Ad) expressing mammalian and nonmammalian
GnRH-R and have used these for a functional comparison of the human and
type I Xenopus laevis GnRH-Rs (hGnRH-R and XGnRH-R)
expressed in T4 cells, a gonadotrope/thyrotrope progenitor derived
cell line which expresses the gonadotropin -subunit, but not GnRH or
TRH receptors (38).
|
Materials and Methods |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
T3–1 and
T4 cells were cultured in serum-supplemented DMEM as described
(35, 38, 39). For experiments they were harvested by
trypsinization and then incubated for 1–3 days in flasks or culture
plates as described in the figure legends. For
Ca2+ imaging, cells were cultured
in 12-well plates (2 ml/well) containing untreated round glass cover
slips.
Generation of recombinant adenovirus
Recombinant, E1 deleted Ad, were produced according to standard
techniques (40, 41, 42, 43). Ad encoding the enhanced green
fluorescent protein (EGFP), were generated for assessment of
transfection efficiency. The reporter gene was excised from the plasmid
pEGFP-N1 (CLONTECH Laboratories, Inc., Basingstoke, UK)
and cloned into the Ad transfer vector pXCXCMV under control of the
human cytomegalovirus (CMV) 1E promoter enhancer fragment (663 bp of
pcDNA 1 from Invitrogen, Nu Leek, The Netherlands)
followed by a GH poly A tail. The recombinant virus (Ad-EGFP) was then
generated by homologous recombination with pJM17 (Microbix Systems
Inc., Toronto, Canada) in HEK-293 cells, grown to high titer and then
purified by CsCl density gradient centrifugation. The transfer vector
(pXCXCMV) and a control Ad construct which contains no insert in the E1
region (Ad0) was kindly provided by A. Byrnes (University of Oxford,
Oxford, UK). Ad-EGFP titer (determined using a plaque assay) was
0.75 x 1010 plaque forming U/ml. To
generate Ad encoding GnRH-R, DNA encoding human and type I
Xenopus GnRH-Rs was excised from pcDNA1/Amp plasmids
(12, 14) using XbaI and BamHI
(human) or XhoI and BamHI (Xenopus).
The inserts were purified and ligated into an identically digested
pXCXCMV which, after transformation and growth in Escherichia
coli was purified on a CsCl gradient. Homologous recombination was
then achieved by CaPO4 transfection of HEK-293
cells with 6 µg of pXCXCMV-GnRH-R construct and 6 µg pJM17 per 60
mm culture dish of 70% confluent cells. The cells were overlaid in
medium with 0.5% agarose and individual recombinant Ad plaques were
then picked and amplified. Restriction analysis (HindIII
digests) of the Ad genome was then used to confirm homologous
recombination. PCR was also performed amplifying viral DNA with primers
(Cruachem, Glasgow, UK) flanking the multiple cloning sites of pXCXCMV:
(forward, dACAACAGATGGCTGGCAAC; reverse, dAAATGGGCGGTAGGCGTG). The PCR
mixture was run on an agarose gel to verify the appropriate size of the
amplified bands and these were also excised and sequenced. After
sequence confirmation, Ad stocks were bulked up by infection of 8x
T175 flasks of HEK-293 cells followed by extraction and caesium
chloride gradient purification, filter sterilization and aliquoted
storage at -80 C. Viral titer was determined using a standard plaque
assay and is reported as multiplicity of infection (m.o.i.) where an
m.o.i. of 1 is defined as 1 plaque forming unit per plated T4
cell. Because T4 cells will have proliferated between plating and
infection, the values given over-estimate the actual m.o.i. at the time
of infection.
Flow cytometry
Flow cytometry was used to compare the efficiencies of different
transfection procedures by transfecting T4 cells with pEGFP-N1
plasmid (encoding EGFP) or with recombinant Ad encoding EGFP (Ad-EGFP).
Analyses were performed on a FACScalibur flow cytometer using CellQuest
software (both from Becton Dickinson and Co., Oxford, UK)
for data acquisition. Cells were seeded into 24 well plates (100,000
cells per well), transfected 24 h later and then collected for
flow cytometry after a further 18 h of culture. Standard
procedures were used for CaPO4 transfection and
manufacturers instruction were followed for transfections using 1 µg
of EGFP DNA and Fugene 6 (3 µl for 6 h), Lipofectin (2.5 µl
for 6 h) or Lipofectamine with the Plus Reagent (4 µl Plus
Reagent and 1 µl Lipofectamine for 3 h). For the Ad-mediated
transfections, cells were incubated in the presence of the Ad-EGFP at
an m.o.i. of 1–1000 as indicated. The cells were then trypsinized,
suspended in serum supplemented DMEM, and centrifuged (4 min, 300
x g, 4 C). The cells were washed in 4 ml FACS buffer (PBS,
5% FCS, 0.1% sodium azide), centrifuged and resuspended in FACS
buffer. The cells were then analyzed at 100–300 cells/sec and a
minimum of 1 x 104 gated single cell events
was acquired. The laser excitation wavelength was 488 nm and the
photo-multiplier band pass filter was Fl1, 505
nm. Plots of event number against fluorescence intensity were used for
calculation of mean fluorescence intensity, the proportion of cells
that were positive (e.g. fluorescence intensity above a
threshold set to exclude >90% of untransfected cells), and the
mean fluorescence intensity within the positive cell population.
Accumulation of [3H]inositol phosphates
([3H]IPx) and Ins
(1, 4, 5)P3 quantification
[3H]IPx
accumulation was used as a measure of PLC activity as described
(19) using cells labeled by preincubation with
[3H]inositol and stimulated in the presence of
LiCl. Cells were cultured in 24-well plates in 1 ml of media and 2
µCi [2-3H]inositol (14–16 Ci/mmol) was added
to each well for the final 16 h of incubation. After two washes in
physiological salt solution (PSS: 127 mM NaCl, 1.8
mM CaCl2, 5 mM KCl, 2
mM MgCl2, 0.5 mM
NaH2PO4, 5 mM
NaHCO3, 10 mM glucose, 0.1% BSA, and
10 mM HEPES, pH 7.4) each well was stimulated for the
period indicated in the figures with 200–250 µl of PSS containing 10
mM LiCl and the indicated concentration of GnRH, Buserelin,
or cGnRH-II. The stimulation was terminated by adding 1 ml of water at
95 C. The cells were lyzed by freezing and thawing, and
[3H]IPx was separated
from free [3H]inositol using anion exchange
chromatography in formate form Dowex-1 columns (19).
D-Ins (1, 4, 5)P3 mass was determined
using a RRA as described (19). Briefly, T4 cells
cultured and infected with Ad in 24 well plates were washed and
incubated for 30 min in 1 ml of Krebs/HEPES buffer at 37 C. The medium
was aspirated and replaced with 150 µl Krebs/HEPES with GnRH or
cGnRH-II (both at 10-7
M). Incubations were performed in duplicate and were
terminated at 5–300 sec by addition of 150 µl ice-cold 1
M TCA. The control (0 sec) time-point was obtained by
adding the stimulus after the TCA. The D-Ins
(1, 4, 5)P3 was then extracted as described
(19) using duplicate aliquots for each sample and
standards (0.1 nM–3 µM).
Dynamic video imaging of cytosolic Ca2+
Video imaging of fura 2 loaded T4 cells was performed as
described (39). Cells were loaded for 30 min in 1 ml PSS
containing 2 µM fura 2. They were then washed several
times in PSS and the cover slips were loaded into a holder which was
fitted into a heating chamber at 37 C. Image capture was typically
performed within 10–25 min of loading in approximately 500 µl of PSS
or in Ca2+-free PSS (containing 250
µM EGTA instead of CaCl2) using
MagiCal hardware, Tardis software and a Nikon Diaphot
microscope (39). The cells were excited alternately at 340
and 380 nm and emitted light was collected at 510 nm, averaging the
data from 8 or 16 video frames, and subtracting background values
before ratioing. The ratio of fluorescence at 340 and 380 nm, was
calculated on a pixel-by-pixel basis and used to determine the
Ca2+ concentration assuming a dissociation
constant of 225 nM for fura-2 and
Ca2+ at 37 C. Calibration was performed as
described (39), and where spike and plateau
Ca2+ values are reported these were defined as
the maximum response within 10 sec of stimulation and the response
after 1 min, respectively.
Radioligand binding
GnRH-R expression was assessed using whole cell binding assays
in which approximately 50,000 cells were incubated in suspension for 30
min at 21 C in 100 µl of PSS containing 1 mg/ml bacitracin with
approximately 10-10
M radiolabel and 0 or
10-11–10-5
M of the unlabeled competitor peptide (36).
Free and bound peptide were then separated by centrifugation through
oil (36). For human GnRH-R, the radiolabel was
[125I]Buserelin. For Xenopus GnRH-R
the radiolabel was [125I]cGnRH II. Receptor
internalization was quantified in a modified whole cell binding assay
in which approximately 50,000 cells were grown in 24-well plates, were
washed in PSS and then incubated at 37 C in 200 µl PSS containing
approximately 10-10
M radiolabel and 0 (total binding) or
10-6
M (nonspecific binding) of Buserelin or cGnRH-II.
After the required incubation period (2–60 min) the cells were rapidly
rinsed in ice-cold PSS and then incubated for 2 min either in PSS or in
150 mM NaCl with 50 mM
acetic acid (pH 3–4). The cells were then washed again in PSS and
solubilized in 0.5 ml of 0.2 M NaOH with 1% SDS.
Radiolabel in the solubilized cells was determined by 
-counting and
specific cell-associated radioactivity was determine by subtraction of
nonspecific from the total. Total specific binding is defined as the
specific binding in cells receiving no acid wash, whereas
acid-resistant (internalized) specific binding is defined as that seen
in the acid washed cells. For one series of experiments, an
internalization index was calculated by expressing acid-resistant
specific binding as a percentage of total cell-associated specific
binding.
Statistical analysis and data presentation
The figures show data from a single representative experiment or
the mean ± SEM of data pooled from "n"
independent experiments (raw data or data normalized as described in
the figure legends). Data are typically reported in the text as
mean ± SEM and statistical analysis was by Student’s
t test, accepting P < 0.05 as statistically
significant. EC50 values were estimated by
nonlinear regression using GraphPad Software, Inc. Prism
(GraphPad Software, Inc., San Diego, CA). For
Ca2+ measurements, image analysis was used to
quantify the mean ionized Ca2+ in all of the
cells in each field of view (which typically contained 10–50 cells) as
well as in individual cells. The figures show the mean (with or without
SEM) of data pooled from the indicated number of
fields of view or in individual cells, as indicated.
|
Results |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
T4 cells
with hGnRH-R by CaPO4 precipitation but
transfection efficiency was extremely poor. Because others have shown
efficient Ad-mediated transfection of pituitary cells
(28), we exploited EGFP expressing vectors to compare
transfection efficiencies using Ad and 4 other transfection strategies.
Flow cytometry (used to measure mean fluorescence intensity in the
entire population and reported in arbitrary units) revealed that mean
EGFP levels (expressed as arbitrary fluorescence units) were much
greater in cells transfected using the recombinant Ad (470,000) than in
control cells (100) or in cells transfected with pEGFP-N1 using
CaPO4 (150), Fugene 6 (1900), Lipofectin (30,000)
or Lipofectamine with the Plus Reagent (50,000). This distinction was
largely due to the fact that >95% of cells expressed EGFP after
infection with Ad-EGFP, whereas <15% expressed EGFP after
transfection using the other strategies (not shown). We therefore
developed recombinant Ad encoding human and Xenopus GnRH-R
(Ad hGnRH-R and Ad XGnRH-R). Insertion of receptor complementary DNA
(cDNA) into the adenoviral DNA was confirmed by HindIII
restriction digests, by PCR and by sequencing of the PCR products, as
described in Materials and Methods. In each case, inserts of
appropriate length and sequence were verified (not shown).
We next explored the relationship between viral titer and receptor
expression by infecting T4 cells with Ad hGnRH-R and constructing
competition binding curves using approximately 0.25 nM
[125I]Buserelin and varied amounts of unlabeled
Buserelin. No specific binding of
[125I]Buserelin was seen in the untransfected
T4 cells, but infection with Ad hGnRH-R at increasing titer (from
m.o.i. values of 3–300) increased
[125I]Buserelin binding and this was inhibited
in a concentration-dependent manner by Buserelin (Fig. 1A
, main panel). Fitting these
data to a single site, competition model revealed no dependence of
Kd values on viral titer, so
Bmax values were estimated by re-fitting the data
with the Kd fixed at the mean value of 2.0
nM (2.0 ± 1.2, n = 3). As shown (Fig. 1A, inset), increasing Ad titer from 3 to 100, increased
receptor number from approximately 300 to 30,000 sites/cell. Similar
studies performed with [125I]cGnRH-II and
cGnRH-II in T4 cells infected with Ad XGnRH-R (Fig. 1B, main
panel) revealed high affinity binding sites and again, no
relationship was observed between Ad titer and
Kd. The data were therefore fitted through the
mean Kd value of 3.0 nM
(3.0 ± 1.1, n = 4), revealing that increasing Ad titer from
3 to 100 increased receptor number from approximately 1,000 to 220,000
sites/cell (Fig. 1B, inset).
|
, upper
panel) GnRH did not stimulate
[3H]IPx accumulation in
untransfected T4 cells but clearly did so after infection, and this
effect was dependent upon both GnRH dose and viral titer. Similar
results were obtained in cells infected with Ad XGnRH-R and then
stimulated with cGnRH-II (Fig. 3, lower panel), although the higher levels of receptor
expression achieved with the Ad XGnRH-R (compare insets in
Fig. 1
) were not associated with greater
[3H]IPx responses
(compare upper and lower panels in Fig. 2). Indeed, a plot
of receptor number against maximal
[3H]IPx response (data
derived from those in Figs. 1 and 2) revealed half-maximal
[3H]IPx accumulation with
at least 30,000 XGnRH-R per cell, compared with only 6,000 hGnRH-R per
cell (Fig. 3). In parallel experiments, effects of
10-7
M GnRH and 10 µM
forskolin on cAMP accumulation were measured by stimulation of cells
infected with Ad hGnRH-R, for 30 min in medium with 1.0
mM IBMX. Infection with Ad hGnRH-R did not
measurably alter basal cAMP values. Moreover, although forskolin
clearly stimulated cAMP accumulation (11.1 ± 1.1 fold over basal,
n = 5), GnRH failed to do so in untransfected cells, or in cells
infected with Ad hGnRH-R at m.o.i. values of 10–300 (not shown).
Similarly, cGnRH-II (10-7
M) failed to increase cAMP accumulation in cells
infected with Ad XGnRH-R at m.o.i. values of 3–300 (not shown).
Because activation of PLC-coupled GnRH-R increases
[Ca2+]i in T3–1 cells
(39), we investigated this possibility in T4 cells.
After infection with Ad hGnRH-R, GnRH caused a robust increase in
[Ca2+]i that began to
decline after approximately 20 sec and returned rapidly to control
values on transfer to Ca2+-free medium (Fig. 4). A similar effect was seen when
XGnRH-R infected cells were stimulated with cGnRH-II
(10-7
M) although in this case a more pronounced
biphasic (spike-plateau) response was observed. Similar responses were
observed in parallel experiments using T3–1 cells (not shown, see
Ref. 39).
|
|
|
are from cells infected with Ad at an
m.o.i. of 100, but were taken from a series of experiments in which
m.o.i. values were varied between 0.3 and 1000. In these experiments,
GnRH failed to increase
[Ca2+]i in control cells
but caused a titer-dependent increase in
[Ca2+]i after infection
with Ad hGnRH-R (Fig. 5, upper
panel). Because these data were obtained by
[Ca2+]i imaging,
Ca2+ traces for individual cells were available,
so the proportion of cells responding to GnRH could be estimated. As
shown (Fig. 5, upper panel), The proportion of
GnRH-responsive cells also increased with increasing viral titer but,
interestingly, over 50% of the cells were judged as GnRH responsive
after infection with an m.o.i. of 3, whereas an m.o.i. of 30 was
required to achieve a 50% maximal increase in
[Ca2+]i. Assuming that
Ca2+ responses are only observed in GnRH-R
expressing cells, this implies that the vast majority of cells express
GnRH-R at low viral titer and that increasing titer then increases
responses by increasing the amount of receptor per cell, rather than
the proportion of cells expressing the receptor. To investigate the
relationship between viral titer and protein expression more directly,
flow cytometry was used to define both the proportion of cells
expressing EGFP and the intensity of EGFP fluorescence in the EGFP
positive cells, after infection with Ad-EGFP at varied titer (Fig. 5, lower panel). As shown, >95% of cells were positively
stained, and fluorescence intensity in these cells was increased to
>100 fold above basal, after infection with Ad-EGFP at an m.o.i. >10.
Importantly, increasing viral titer from m.o.i. values of 10 to 1000
increased mean fluorescence by 40-fold without appreciably altering the
proportion of cells fluorescing, demonstrating (as implied by the
Ca2+ imaging data) that increasing viral titer
above an m.o.i. of 10, causes an increase in protein expression per
cell rather than an increase in proportion of cell expressing the
protein. A further intriguing observation was that the
[Ca2+]i responses to GnRH
were qualitatively dependent upon viral titer. As shown, oscillations
in basal [Ca2+]i were
seen in many control cells (Fig. 6, left-hand traces), which were unaltered by GnRH but were
rapidly extinguished on transfer to Ca2+-free
medium. In cells infected with hGnRH-R at low m.o.i. (1 or 3) most
cells failed to respond to GnRH but where responses were observed, GnRH
increased the amplitude and/or frequency of the
[Ca2+]i oscillations and
these were, again, inhibited by transfer to
Ca2+-free medium (Fig. 6, central
traces). In contrast, addition of GnRH to cells infected with Ad
hGnRH-R at high titer (10–100), caused spike-plateau type responses
instead of oscillations (Fig. 6, right-hand traces).
|
|
T4 cells infected with Ad hGnRH-R (m.o.i.
100), all 3 peptides competed for
[125I]Buserelin with the rank order of potency
(Buserelin > GnRH>>cGnRH-II) expected for a mammalian GnRH-R
Fig. 7, upper left panel).
Similarly, all three peptides competed for binding of
[125I]cGnRH-II in cells infected with XGnRH-R
(m.o.i. 100, Fig. 7, upper right panel), but in this case
the rank order of potency was cGnRH-II>>Buserelin>>GnRH. Identical
rank-orders of potency were seen when T4 cell infected with Ad
hGnRH-R or Ad XGnRH-R were used for construction of dose-response
curves for [3H]IPx
accumulation (Fig. 7, lower left and right panels,
respectively).
|
T4 cells were infected
with Ad XGnRH-R or with Ad hGnRH-R (both at a m.o.i. values of
100–300) and then used in time-course experiments in which
accumulation of [3H]IPx
and Ins (1, 4, 5)P3 mass were measured. As shown
(Fig. 8, upper panel), GnRH
caused a rapid accumulation of
[3H]IPx in hGnRH-R
expressing cells, with no obvious reduction in accumulation rate over
time. In cells expressing XGnRH-R, cGnRH-II also caused a rapid
increase in [3H]IPx
accumulation, but in this case, the initial rate of accumulation was
not maintained beyond 2 min Thus, although the initial rates of
[3H]IPx accumulation
(0–2 min) were indistinguishable, the final rate of XGnRH-R mediated
accumulation (2–15 min) was only 30% of that with the hGnRH-R, and
only 40% of the initial XGnRH-R mediated rate (Fig. 8, upper
panel). The time-courses of receptor-mediated elevation of Ins
(1, 4, 5)P3 mass were also markedly different for
the two receptors (Fig. 8, lower panel). In Ad XGnRH-R
infected cells, cGnRH-II caused a rapid and pronounced increase in Ins
(1, 4, 5)P3, but this response was transient with a
maximum at 5 sec and a marked reduction thereafter. In contrast, the
response to GnRH in Ad hGnRH-R infected cells was less pronounced and
was not transient. GnRH increased Ins (1, 4, 5)P3
mass to a maximal level at 60 sec, and this was maintained for 5
min.
|
T4 cells were infected with
Ad hGnRH-R or with Ad XGnRH-R (both at a m.o.i. values of 100–300) and
then used in time-course experiments in which total specific binding
and acid-resistant specific binding were measured. The time-courses for
specific association of [125I]Buserelin with
hGnRH-R, and of [125I]cGnRH-II with XGnRH-R,
were indistinguishable (not shown). However, the acid-resistant
fraction of the specific binding was greater with the XGnRH-R at all
time-points (Fig. 9), such that after 60
min, 75% of specific binding to cells expressing XGnRH was
internalized (acid resistant), whereas only 25% of binding to hGnRH-R
was internalized. In the final experiments, the possible influence of
receptor number of GnRH-R internalization was assessed by calculation
of an internalization index (specific acid-resistant binding as a
percentage of total specific binding) for binding to T4 cells
infected with Ad hGnRH-R and Ad XGnRH-R at m.o.i. values of 12.5–200.
As shown (Fig. 10, upper
panel), the internalization index for XGnRH-R was greater than
that for the GnRH-R at all m.o.i. values tested.
|
|
|
Discussion |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
T4 cells.
In the first experiments, we compared the efficiency of transfection
using CaPO4 precipitation and liposome based
strategies to infection with Ad-EGFP and found Ad mediated transfection
to be far more effective (higher levels of EGFP expressed and larger
proportion of cells transfected). We therefore prepared recombinant Ad
expressing human GnRH-R and found that infection with these causes
expression of GnRH-R in T4 cells, which have the pharmacological
characteristics of their endogenous counterparts. Thus, the affinity of
[125I]Buserelin binding to Ad hGnRH-R infected
T4 cells (Kd approx. 2 nM), the
potency of GnRH-stimulated
[3H]IPx
accumulation (EC50 approx. 1 nM), the
rank orders of potency for the 3 peptides in
[3H]IPx accumulation
assays and competitive binding assays (Buserelin >
GnRH>>cGnRH-II) are all similar to data obtained with mammalian
GnRH-Rs in pituitary cells and with hGnRH-R expressed in other
heterologous systems (9, 30, 46, 47). Moreover,
Ca2+ imaging studies revealed that these
receptors, like endogenous mammalian GnRH-R in other systems
(6, 7, 8, 9, 19, 39) cause a biphasic (spike-plateau) increase
in [Ca2+]i. In each case
the spike response is mediated by Ca2+
mobilization from intracellular stores [presumably Ins
(1, 4, 5)P3 mediated], as judged by retention of a
spike response in Ca2+-free medium (not shown),
whereas the plateau phase is dependent upon Ca2+
entry across the plasma membrane, as demonstrated by loss of the
response on transfer to Ca2+-free medium.
Although XGnRH-R have recently been expressed in COS cells, receptor
levels were too low for radioligand binding studies (12).
This problem was obviated using Ad XGnRH-R infected T4 cells that
expressed receptors with high affinity for
[125I]cGnRH-II (Kd 
3
nM) and specificity for cGnRH-II (cGnRH-II>>Buserelin
> GnRH). These characteristics are similar to those reported for the
endogenous GnRH-R of amphibian pituitary extracts (48).
Moreover, the potency of cGnRH-II stimulated
[3H]IPx accumulation
(EC50 approx. 2 nM) and the rank
order of potency for the three peptides in
[3H]IPx accumulation
assays (cGnRH-II>>Buserelin > GnRH) parallel the binding and
functional data obtained with other nonmammalian GnRH-Rs and the
XGnRH-R (12, 48, 49). Similarly, the biphasic effect of
cGnRH-II on [Ca2+]i in
XGnRH-R infected T4 cells, parallels that seen on activation of
nonmammalian GnRH-R in gonadotropes (50).
Interestingly, when the relationship between viral titer and EGFP
expression (Ad-EGFP) was defined, increasing Ad titer from an m.o.i. of
3 to 1000 caused a major increase in EGFP fluorescence, without
appreciably altering the proportion of positively stained cells
(80–100%). This implies that increasing m.o.i. above 10 causes an
increase in protein per cell, rather than in the proportion of cells
expressing the protein. This apparently holds true for the GnRH-R
because in the Ca2+ imaging experiments,
increasing Ad hGnRH-R from an m.o.i. of 10 to 1000 did not increase the
proportion of cells responding to GnRH, but instead, increased the
amplitude of the response. Assuming that only GnRH-R expressing cells
show Ca2+ responses to GnRH, the vast majority of
cells must express GnRH-R after infection at an m.o.i. of 10, so that
the 20- fold increase in receptor number caused by increasing Ad
hGnRH-R from 10 to 300 (Fig. 1) reflects an increase in receptors per
cell rather than an increase in the proportion of cells expressing
GnRH-R. Thus, recombinant Ad provides a simple means of controlling the
number of GnRH-R in T4 cells, highlighting an important distinction
to conventional transient transfection strategies, where a relatively
low (and usually unknown) proportion of cells are transfected so that
it is unclear whether increasing DNA increases the proportion of cells
expressing the protein or the amount of protein per positive cell.
Using this strategy we are able to vary receptor number from
approximately 1,500 to over 30,000 sites per cell for the hGnRH-R. This
caused a corresponding increase in maximal GnRH-stimulated
[3H]IPx accumulation,
although receptor affinity (Kd for
[125I]Buserelin 2 nM) and
potency of GnRH (EC50 approx. 0.2 nM)
were not measurably influenced by viral titer. No evidence was obtained
for stimulation of cAMP accumulation or for constitutive receptor
signaling (e.g. elevation of
[3H]IPx accumulation or
cAMP levels in the absence of stimulus) at any Ad hGnRH-R titer. We
also found that stimulation of hGnRH-R in cells infected at low titer
tended to cause asynchronous oscillatory Ca2+
responses, whereas stimulation of cells infected at high titer caused
more sustained spike-plateau responses (Fig. 7). In rat gonadotropes,
low concentrations of GnRH cause oscillatory Ca2+
response and increasing GnRH concentration increases
spike-frequency until sustained responses are achieved (8, 51). Thus, it appears that this same relationship is seen when
the number of active receptors is controlled by altering ligand
concentration (in the face of a constant receptor number) or by
altering receptor number (in the face of constant ligand
concentration). These data were, however, unexpected because
oscillatory Ca2+ responses are not seen in
T3–1 cells (39, 51) implying that immortalization of
this cell line has somehow prevented them from displaying this
fundamental characteristic of gonadotropes. Clearly, this is not the
case for T4 cells, which may therefore prove to be valuable models
for exploration of the mechanisms and relevance of oscillatory
Ca2+ signals in gonadotropes.
GnRH-R expression in gonadotropes is hormonally regulated. The dynamic
range for rat GnRH-R is approximately 20,000–75,000 sites/gonadotrope
through the oestrous cycle (49, 52), and sheep GnRH-R can
be manipulated through a range of 500–20,000 sites/cell by in
vitro exposure to gonadal steroids (53). The levels
of hGnRH-R expression reported here 500–30(1,500–30,000 sites/cell)
therefore compare favorably to the physiological range, as well as to
the levels in stable GnRH-R expressing cells lines [approximately
10,000 sites/cell in GGH3–1' cells (32, 33) and 65,000
sites/cell in T3–1 cells (36)]. Higher expression
levels (up to 220,000 sites/cell) were obtained with Ad XGnRH-R,
although data are not available to relate this to physiological XGnRH-R
receptor density. The higher expression of XGnRH-R could reflect
differences in protein synthesis, elaboration to the cell surface or
stability. A similar distinction has been observed for rat and catfish
GnRH-R expressed transiently in GH3 cells where
levels were 5- to 8-fold greater with the tailed GnRH-R and expression
of the rat GnRH-R was increased by addition of the catfish GnRH-R tail
(54). The lack of a second glycosylation site toward the
amino terminus of the human GnRH-R may also contribute to the
relatively low expression level in T4 cells, as it does in COS cells
(55).
As with the hGnRH-R, no evidence was obtained for constitutive
signaling or for stimulation of cAMP accumulation with the XGnRH-R in
T4 cells. The latter observation was unexpected because the closely
related catfish GnRH-R was cloned by virtue of its ability to activate
a cAMP response element binding protein reporter in HEK-293 cells
(17) and because mammalian GnRH-R mediate activation of
Gs in GH3 and COS cells
(31, 32, 33). It therefore appears that the specific coupling
of GnRH-R to Gq seen in T3–1 cells, also
exists in T4 cells and is retained for both hGnRH-R and XGnRH-R.
When [3H]IPx responses
were compared in cells infected with Ad hGnRH-R and Ad XGnRH-R at
varied titer (Fig. 4), increasing receptor number increased
ligand-stimulated [3H]IPx
accumulation but [3H]IPx
responses were lower in XGnRH-R expressing cells, despite the fact that
XGnRH-R number was greater than that for hGnRH-R. Indeed, a plot of
maximal [3H]IPx response
against estimated receptor number revealed that a 50% maximal
[3H]IPx response required
activation of at least 30,000 XGnRH-R as opposed to only 6,000 hGnRH-R.
Because these data are obtained using a 30-min stimulation, this
difference could be due to more pronounced desensitization and/or
internalization of the XGnRH-R, occurring during the stimulation. The
hallmarks of rapid homologous desensitization of PLC-activating GPCRs
are a failure to maintain initial rates of ligand stimulated
[3H]IPx accumulation
(against a LiCl block of IP metabolism) and a transient elevation of
Ins (1, 4, 5)P3 mass caused by concomitant Ins
(1, 4, 5)P3 metabolism and a reduction in the rate
of Ins (1, 4, 5)P3 generation due to receptor
desensitization (20). These characteristics were observed
in XGnRH-R infected cells but not in hGnRH-R infected cells. In these
experiments, the initial rates of XGnRH-R and hGnRH-R mediated
[3H]IPx accumulation were
indistinguishable and this rate was sustained by the hGnRH-R, but not
by the XGnRH-R. Moreover, the XGnRH-R mediated a transient
(spike-plateau) increase in Ins (1, 4, 5)P3 mass,
whereas the hGnRH-R mediated only a monophasic increase to a maintained
plateau. A similar distinction was observed when receptor
internalization was assessed. Only 25% of specific binding to the
hGnRH-R was acid resistant (internalized) after incubation for 60 min
at 37 C, whereas over 75% of the binding to the XGnRH-R was acid
resistant under the same conditions. These receptor-specific
[3H]IPx and Ins
(1, 4, 5)P3 profiles are unlikely to reflect
differences in receptor number because we have shown (22)
that the monophasic Ins (1, 4, 5)P3 profile seen on
activation of mouse GnRH-R in T3–1 cells, is maintained at a wide
range of receptor densities (receptor density manipulated by partial
irreversible blockade). Moreover, the differences in receptor
internalization rates were not due to differences in receptor number
because when Ad titer was varied (m.o.i. 12.5–200) <10% of hGnRH-R
binding was internalized, whereas >50% of XGnRH-R binding was
internalized, irrespective of Ad titer. Using the data inset in Fig. 3, A and B, we estimate that hGnRH-R number was varied from 2,000 to
30,000 sites/cell in this experiment, whereas XGnRH-R was varied from
2,000 to 220,000 sites/cell. Thus, the greater rate of XGnRH-R
internalization was seen at a range of receptor densities encompassing
that for the hGnRH-R, and cannot therefore reflect a difference in
receptor number.
One of the most remarkable aspects of this receptor family is that all
cloned nonmammalian GnRH-Rs have C-terminal tails, whereas none of the
cloned mammalian GnRH-Rs have such tails (9, 12, 15, 16, 17).
Although GnRH-stimulated gonadotropin secretion clearly desensitizes in
mammals, the lack of C-terminal tails in mammalian GnRH-R raised the
question of whether receptor desensitization is involved. The
endogenous murine GnRH-R of T3–1 cells were found not to undergo
rapid homologous desensitization, and this was shown to be a
characteristic of the receptor rather than cell type or receptor
density (18, 19, 20, 21, 22). The implied causal relationship between
the unique structural and functional features of these receptors
(mammalian GnRH-R are the only GPCRs known to completely lack
C-terminal tails and the only PLC-activating GPCRs known not to rapidly
desensitize) is supported by the demonstration that activation of
heterologously expressed catfish GnRH-R (or rat GnRH-R with added TRH
receptor tails) causes ß-arrestin translocation along with receptor
phosphorylation, internalization, and desensitization, whereas all of
these processes were absent or slower with the rat GnRH-R (23, 25). Similarly, the chicken GnRH-R internalized about 4 times
more rapidly than the human GnRH-R, and this internalization rate is
reduced by successive C-terminal truncations (15). Thus,
although we have not tested this directly, the absence and presence of
C-terminal most likely underlies the differences in desensitization and
internalization rates of the hGnRH-R and XGnRH-R reported herein.
In summary, development of recombinant Ad encoding GnRH-R has enabled the first comparative studies of different GnRH-R in gonadotrope lineage cells. In these cells, variation of Ad titer (above an m.o.i. of 10) provides an efficient means of controlling receptor number (per cell) within the physiological range. Human and Xenopus GnRH-R expressed in this way had pharmacological characteristics comparable to their endogenous counterparts (in terms of ligand recognition profiles and activation of PLC/Ca2+ signaling). No evidence was obtained for coupling to adenylyl cyclase or for constitutive receptor signaling at any receptor density but Ad hGnRH-R titer (and hence hGnRH-R number) had both quantitative and qualitative effects on the [Ca2+]i responses to GnRH. Interestingly, measurement of [3H]IPx accumulation in 30-min incubations revealed that sustained activation of PLC by XGnRH-R is much less efficient than activation by hGnRH-R, possibly because of differences in desensitization and/or internalization occurring during the stimulation. Indeed, internalization of the XGnRH-R was found to be more rapid than for the hGnRH-R, and the XGnRH-R rapidly desensitized, whereas the hGnRH-R did not. The retention of such differences at physiological receptor density in gonadotrope lineage cells supports the argument that the evolution of nondesensitizing GnRH-R is related to the development of mammalian reproductive strategies.
|
Acknowledgments |
|---|
T3–1 and
T4 cells, to Prof. Sandow (Aventis Pharma GmbH, Frankfurt, Germany)
for providing the Buserelin and
[125I]Buserelin, and to Dr. A. Byrne
(University of Oxford, Oxford, UK) for providing vectors. |
Footnotes |
|---|
Received May 17, 2000.
|
References |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
1B-adrenergic receptor. J Biol Chem 269:22887–22893T3–1 cells due to uncoupling of inositol
1,4,5-trisphosphate generation and Ca2+
mobilization. J Biol Chem 271:23711–23717T3–1 cells: implications for
mechanisms of rapid desensitization. Biochem J 333:301–308
T3–1
cells is not due to GnRH receptor reserve or phosphatidylinositol 4,5
bis-phosphate pool size. Mol Cell Endocrinol 147:161–173[CrossRef]
T3–1 cell line.
Endocrinology 136:4864–4871[Abstract]
T3–1 cells. Mol Cell
Endocrinol 87:95–103[CrossRef][Medline]
T3–1,
gonadotrope-derived cell line. Mol Cell Neurosci 3:124–132[CrossRef]
This article has been cited by other articles:
 |
|
 |
|
  S. P. Armstrong, C. J. Caunt, and C. A. McArdle Gonadotropin-Releasing Hormone and Protein Kinase C Signaling to ERK: Spatiotemporal Regulation of ERK by Docking Domains and Dual-Specificity Phosphatases Mol. Endocrinol., April 1, 2009; 23(4): 510 - 519. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 A. R. Finch, C. J. Caunt, S. P. Armstrong, and C. A. McArdle Agonist-induced internalization and downregulation of gonadotropin-releasing hormone receptors Am J Physiol Cell Physiol, January 1, 2009; 297(3): C591 - C600. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
J. Xie, K. H. Allen, A. Marguet, K. A. Berghorn, S. P. Bliss, A. M. Navratil, J. L. Guan, and M. S. Roberson Analysis of the Calcium-Dependent Regulation of Proline-Rich Tyrosine Kinase 2 by Gonadotropin-Releasing Hormone Mol. Endocrinol., October 1, 2008; 22(10): 2322 - 2335. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 C. J. Caunt, C. A. Rivers, B. L. Conway-Campbell, M. R. Norman, and C. A. McArdle Epidermal Growth Factor Receptor and Protein Kinase C Signaling to ERK2: SPATIOTEMPORAL REGULATION OF ERK2 BY DUAL SPECIFICITY PHOSPHATASES J. Biol. Chem., March 7, 2008; 283(10): 6241 - 6252. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 A. J. Pawson, E. Faccenda, S. Maudsley, Z.-L. Lu, Z. Naor, and R. P. Millar Mammalian Type I Gonadotropin-Releasing Hormone Receptors Undergo Slow, Constitutive, Agonist-Independent Internalization Endocrinology, March 1, 2008; 149(3): 1415 - 1422. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 K. R Sedgley, A. R Finch, C. J Caunt, and C. A McArdle Intracellular gonadotropin-releasing hormone receptors in breast cancer and gonadotrope lineage cells J. Endocrinol., December 1, 2006; 191(3): 625 - 636. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
C. J. Caunt, A. R. Finch, K. R. Sedgley, L. Oakley, L. M. Luttrell, and C. A. McArdle Arrestin-mediated ERK Activation by Gonadotropin-releasing Hormone Receptors: RECEPTOR-SPECIFIC ACTIVATION MECHANISMS AND COMPARTMENTALIZATION J. Biol. Chem., February 3, 2006; 281(5): 2701 - 2710. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 J. N Hislop, C. J Caunt, K. R Sedgley, E. Kelly, S. Mundell, L. D Green, and C. A McArdle Internalization of gonadotropin-releasing hormone receptors (GnRHRs): does arrestin binding to the C-terminal tail target GnRHRs for dynamin-dependent internalization? J. Mol. Endocrinol., August 1, 2005; 35(1): 177 - 189. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 C. K. Cheng and P. C. K. Leung Molecular Biology of Gonadotropin-Releasing Hormone (GnRH)-I, GnRH-II, and Their Receptors in Humans Endocr. Rev., April 1, 2005; 26(2): 283 - 306. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
C. J. Caunt, J. N. Hislop, E. Kelly, A.-L. Matharu, L. D. Green, K. R. Sedgley, A. R. Finch, and C. A. McArdle Regulation of Gonadotropin-Releasing Hormone Receptors by Protein Kinase C: Inside Out Signalling and Evidence for Multiple Active Conformations Endocrinology, August 1, 2004; 145(8): 3594 - 3602. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 A. R. Finch, L. Green, J. N. Hislop, E. Kelly, and C. A. McArdle Signaling and Antiproliferative Effects of Type I and II Gonadotropin-Releasing Hormone Receptors in Breast Cancer Cells J. Clin. Endocrinol. Metab., April 1, 2004; 89(4): 1823 - 1832. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
F. Liu, D. A. Austin, and N. J. G. Webster Gonadotropin-Releasing Hormone-Desensitized L{beta}T2 Gonadotrope Cells Are Refractory to Acute Protein Kinase C, Cyclic AMP, and Calcium-Dependent Signaling Endocrinology, October 1, 2003; 144(10): 4354 - 4365. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
A. J. Pawson, S. R. Maudsley, J. Lopes, A. A. Katz, Y.-M. Sun, J. S. Davidson, and R. P. Millar Multiple Determinants for Rapid Agonist-Induced Internalization of a Nonmammalian Gonadotropin-Releasing Hormone Receptor: A Putative Palmitoylation Site and Threonine Doublet within the Carboxyl-Terminal Tail Are Critical Endocrinology, September 1, 2003; 144(9): 3860 - 3871. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
H. M. Everest, J. N. Hislop, T. Harding, J. B. Uney, A. Flynn, R. P. Millar, and C. A. McArdle Signaling and Antiproliferative Effects Mediated by GnRH Receptors After Expression in Breast Cancer Cells Using Recombinant Adenovirus Endocrinology, November 1, 2001; 142(11): 4663 - 4672. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
J. N. Hislop, H. M. Everest, A. Flynn, T. Harding, J. B. Uney, B. E. Troskie, R. P. Millar, and C. A. McArdle Differential Internalization of Mammalian and Non-mammalian Gonadotropin-releasing Hormone Receptors. UNCOUPLING OF DYNAMIN-DEPENDENT INTERNALIZATION FROM MITOGEN-ACTIVATED PROTEIN KINASE SIGNALING J. Biol. Chem., October 19, 2001; 276(43): 39685 - 39694. [Abstract] [Full Text] [PDF]
|
|
| |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
| Endocrinology | Endocrine Reviews | J. Clin. End. & Metab. |
| Molecular Endocrinology | Recent Prog. Horm. Res. | All Endocrine Journals |
