Molecular Analysis of Rat Pituitary and Hypothalamic Growth Hormone Secretagogue Receptors
Karen Kulju McKee,
Oksana C. Palyha,
Scott D. Feighner,
Donna L. Hreniuk,
Carina P. Tan,
Michael S. Phillips,
Roy G. Smith,
Lex H. T. Van der Ploeg and
Andrew D. Howard
Department of Biochemistry and Physiology (K.K.M., O.C.P., S.D.F.,
D.L.H., C.P.T., R.G.S., L.H.T.VdP., A.D.H.) Merck Research
Laboratories Rahway, New Jersey 07065
Department of Human
Genetics (M.S.P.) Merck Research Laboratories West Point,
Pennsylvania 19486
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ABSTRACT
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GH release is thought to occur under the
reciprocal regulation of two hypothalamic peptides, GH releasing
hormone (GHRH) and somatostatin, via their engagement with specific
cell surface receptors on the anterior pituitary somatotroph. In
addition, GH-releasing peptides, such as GHRP-6 and the nonpeptide
mimetics, L-692,429 and MK-0677, stimulate GH release through their
activation of a distinct receptor, the GH secretagogue receptor
(GHS-R). The recent cloning of the GHS-R from human and swine pituitary
gland identifies yet a third G protein-coupled receptor (GPC-R)
involved in the control of GH release and further supports the
existence of an undiscovered hormone that may activate this receptor.
Using the human GHS-R as a probe, we report the isolation of a rat
pituitary GHS-R cDNA derived from an unspliced, precursor mRNA. The rat
cDNA encodes a protein of 364 amino acids containing seven
transmembrane domains (7-TM) with >90% sequence identity to both the
human and swine GHS-Rs. A single intron of
2 kb divides the open
reading frame into two exons encoding TM 15 and TM 67, thus placing
the GHS-R into the intron-containing class of GPC-Rs. The intron maps
to the site of sequence divergence between the human and swine type 1a
and 1b GHS-R mRNAs. In addition, determination of the nucleotide
sequence for the human GHS-R gene confirmed the position of an intron
in the human GHS-R gene at this position. A full-length contiguous cDNA
from rat hypothalamus was isolated and shown to be identical in its
nucleotide and deduced amino acid sequence to the rat pituitary GHS-R.
The cloned rat GHS-R binds [35S]MK-0677 with
high affinity [dissociation constant (KD) =
0.7 nM] and is functionally active when
expressed in HEK-293 cells. Expression of the rat GHS-R was observed
specifically in the pituitary and hypothalamus when compared with
control tissues.
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INTRODUCTION
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GH is an anabolic hormone responsible for postnatal growth,
weight gain, and whole body nitrogen retention (1, 2). The two
hypothalamic peptides GHRH and somatostatin coordinately regulate the
positive and negative control of GH release, respectively, from
somatotroph cells of the anterior pituitary (1, 3, 4, 5). In humans, GH
replacement therapies (i.e. injection of recombinant GH)
have afforded clinical benefit in GH-deficient children and in the aged
(6, 7). GH replacement therapy, however, suffers from three primary
drawbacks: 1) circumvention of the normal hypothalamic-pituitary axis
that governs pulsatile episodic release of GH; 2) low bioavailability;
and 3) side effects of administration (e.g. carpal tunnel
syndrome). After the synthesis of a class of enkephalin-like peptides,
the GH releasing peptides (GHRPs) were shown to stimulate GH release
in vitro and in vivo in all species tested via a
mechanism distinct from GHRH (8, 9, 10, 11). We sought to discover GHS
nonpeptide mimetics and identify and characterize their receptor and
mechanism of action. Two nonpeptide GHRP mimetics have been developed
and tested clinically in humans (12, 13). L-692,429 was shown to
elevate GH in young male volunteers but suffered from overall limited
oral bioavailability (14). Derived from a different structural class,
MK-0677 was active orally in humans and is currently undergoing further
clinical evaluation (15). Using high-specific activity
[35S]MK-0677, a low-abundance high-affinity G
protein-linked receptor of the anterior pituitary gland and
hypothalamus that presumably mediates the actions of the GHRPs was
identified (16, 17). GHRPs and nonpeptide mimetics induce a
transient increase in the concentration of intracellular calcium in
somatotrophs, an increase in the intracellular concentration of
inositol trisphosphate (IP3), and an associated increase in
the activity of protein kinase C (11, 18, 19). These data helped form
the rationale for an expression cloning strategy for the GHS-R.
Functional cloning of GHS-R cDNA from swine and human pituitary gland
revealed that the GHS-R is a member of the GPC-R heptahelical
superfamily with only limited sequence identity to previously cloned
GPC-Rs (20). Additional evidence that GHS signaling proceeds through
the activation of phospholipase C was given by the finding that
reproducible functional expression of the GHS-R in Xenopus
oocytes was strictly dependent on the coexpression of G
protein
11 (Ref. 20 and S. D. Feighner, D. L. Hreniuk, O.
C. Palyha, L. H. T. Van Der Ploeg, and A. D. Howard, unpublished
observations). From both human and swine pituitary, two types of cDNA
were isolated: type 1a, encoding a functional protein containing 7-TM
domains, and type 1b, encoding a protein containing TM-1 through 5 with
no measurable functional activity in cell-based assays. In
situ hybridization studies in rhesus brain and RNase protection
with RNA from human tissues demonstrated that GHS-R expression could be
detected in discrete nuclei of the hypothalamus. In this report, we
describe the cloning and characterization of the rat GHS-R type 1a gene
from the pituitary. An intron in the pre-mRNA locates to the same
predicted amino acid position at which the human and swine type Ia and
Ib GHS-R cDNAs diverged. Nucleotide sequence analysis of the human gene
shows that the human and swine Ib mRNA also contain an intron with a
short conserved intron-derived coding sequence. A full-length GHS-R
cDNA from the rat hypothalamus is identical to the rat type Ia GHS-R
from the pituitary gland. The KD for MK-0677 binding to the
GHS-R measured 0.7 nM, and the receptor is functionally
active when expressed in HEK-293 cells.
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RESULTS AND DISCUSSION
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Reduced stringency hybridization with a 32P-labeled
swine GHS-R cDNA probe was used to isolate a rat pituitary GHS-R cDNA
clone. Using a probe encompassing the complete open reading frame (ORF)
and 3'-untranslated region of the swine type Ia GHS-R, a single
hybridizing phage was identified out of
106 plaques. The
cDNA encoding the GHS-R was excised as a
6.5-kb BsiW1 fragment, and
its nucleotide sequence was determined. A noncontiguous ORF of
1.1
kb encoding seven transmembrane domains (TM) that exhibits a high
degree of sequence identity to both human and swine type 1a GHS-Rs was
identified (Fig. 1
; the location of the first ATG is
conserved compared with the human and swine GHS-Rs and is preceded by
several in-frame stop codons, assuring that the correct
translation initiation codon was identified). Approximately 2 kb of
noncoding intronic sequence at nt 790 (corresponds to a splice donor
site [(A/C)AG/GTAAGT)]) was found at the second amino acid of the
predicted TM-6. This intron divides the ORF into an amino-terminal
segment ending at leucine-263 (encompassing the extracellular domain,
TM-1 through TM-5, and the three intra- and first two extracellular
loops) and a carboxyl-terminal segment encoding TM-6, the third
extracellular loop, TM-7, and the C-terminal intracellular domain. The
position of the intron is highly conserved among rat, human, and swine
GHS-R genes (20), and, as previously observed for several other GPC-Rs,
the intron is located between the TM domains (21, 22). The cDNA clone
described here encodes a protein with significant homology to the type
1a GHS-R and presumably represents unspliced GHS-R pre-mRNA. Two types
of human and swine GHS-R cDNA (type 1a and 1b) that most likely arise
from a single gene by alternative mRNA processing were described
previously (20). This assertion was confirmed by determination of the
nucleotide sequence for the proposed human exon-intron boundaries and
the complete intron of the human gene. Type 1a cDNA encodes the
complete 7-TM GHS-R and results from a splicing event that removes the
intron. In type 1b cDNA, the intron is not removed, and an alternative
polyadenylation signal is presumably used in the intron. As a result,
the human and swine type 1b cDNA contain a short, 24-amino acid ORF
fused to leucine-263. Comparison of the rat sequence 3' to the proposed
splice acceptor site with the corresponding region of the human and
swine type 1b cDNA demonstrates significant sequence divergence,
suggesting that the intronic sequences differ among rat, human, and
swine. This genomic organization is also found in the murine GHS-R
gene, which shares
95% nucleotide sequence identity to the rat exon
and intron nucleotide sequence near the splice acceptor site (M.-H.
Jiang, A. D. Howard, L. H. T. Van Der Ploeg, and H. Zheng,
unpublished observations). Interestingly, the rat intron encodes an
80-amino acid extension to the ORF that bears no identity to any known
GHS-R protein sequence.

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Figure 1. Nucleic Acid and Deduced Amino Acid Sequence of Rat
Pituitary GHS-R Type 1a.
ORF DNA sequence is shown in capital letters, noncoding
sequence in lower-case type.
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We also isolated a full-length rat GHS-R type 1a cDNA (without the
proposed intron) encoding the complete 7-TM domain GHS-R from rat
hypothalamus. The deduced amino acid sequence for this cDNA is
identical to that of the proposed rat pituitary type 1a GHS-R cDNA ORF.
Comparison of the complete amino acid sequences of the rat, swine, and
human homologs revealed a high degree of sequence identity (rat
vs. human, 96.1%: rat vs. swine 94.5%; Fig. 2
). Compared with the human and swine GHS-R, the rat
GHS-R is two amino acids shorter, with a loss of one residue each in
the amino-terminal extracellular domain and the third intracellular
loop. Six amino acid substitutions were also noted: three being
localized to the N-terminal extracellular region, one in TM-1, and two
in the C-terminal intracellular domain. Several conserved features
ascribed to GPC-Rs were also identified in the rat GHS-R: the signature
aromatic triplet sequence (Glu-Arg-Tyr) adjacent to TM-3, Cys-115, and
Cys-197 in the first two extracellular loops capable of disulfide
bonding, putative amino-terminal N-glycosylation sites
(Asn-Xaa-Ser/Thr), phosphorylation sites in the carboxyl-terminus and
the third cytoplasmic loop, and conserved proline residues in TM-4, 5,
6, and 7 (23).

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Figure 2. Comparison of Protein Sequences for GHS-R Type 1a
from Rat, Human, and Swine
A contiguous ORF for the rat isoform was made by removing the
intronic sequence at the putative splice donor/acceptor site
(G/GT-AG/C; nt 791 to 2824). Both the human and swine isoforms are
given as described (20). Predicted transmembrane (TM) domains are
overlined. The alignment was constructed using the
Clustal method (Lasergene Software, DNASTAR, Madison, WI).
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Southern blot analysis of EcoRI-digested rat genomic DNA
utilizing a probe from amino acid 85 to 259 (TM-2 to third
intracellular loop) gave a single hybridizing restriction
fragment of approximately 6.6 kb using high-stringency
posthybridizational washing conditions consistent with the presence of
a single copy gene encoding the rat GHS-R (Fig. 3a
).
Related genes could not be identified under reduced stringency
conditions (lane 1). A similar hybridization profile indicative of a
single copy gene was also observed in other species such as rhesus
monkey, dog, mouse, and rabbit. Hybridizing sequences could not be
observed in yeast (A. D. Howard, C. P. Tan, and K. K. McKee,
unpublished observations). Interestingly, in humans the GHS-R gene is
found at a chromosomal location (3q26.2) in the vicinity of the
possible map position for the Brachmann-de Lange syndrome, a prenatal
and postnatal growth deficiency with developmental delay and dysmorphic
features (24). Determination of the putative involvement of the GHS-R
with the deficiency identified in the Brachman-de Lange syndrome is in
progress.

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Figure 3. Characterization of Rat GHS-R Gene
A, Southern blot analysis: 4 µg EcoRI restriction
enzyme-digested rat genomic DNA was size-separated on a 1% agarose
gel, transferred to nitrocellulose, and hybridized with a
32P-labeled GHS-R DNA fragment encompassing TM-2 through
the third intracellular loop (nt 255 to 799). The Southern blot was
washed under conditions of low (4 x SSC, 55 C; lane 1) or high
(0.1 x SSC, 65 C; lane 2) posthybridizational stringency. B,
RNase protection assay: Poly (A)+ mRNA (5 µg for GHS-R
identification, 0.1 µg for actin identification) from various rat
tissues (pituitary, lane 2; hypothalamus, lane 3; liver, lane 4;
kidney, lane 5; whole brain, lane 6) was hybridized with a GHS-R cRNA
antisense probe (top panel), or a human actin probe
(internal standard; bottom panel). Human pituitary is
given as a reference (lane 1). Precipitated fragments were subjected to
electrophoresis in Tris-borate-EDTA buffer containing 5% acrylamide
and 8 M urea. Exposure times on film at -80 C were 3 days
(GHS-R) or 18 h (actin). RNA size standards (Ambion Century
markers) in nt are 400, 300, 200, and 100.
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The rat GHS-R is expressed at a low level, as suggested by
radioligand binding (Bmax
6 fmol/mg pi-tuitary
membrane protein), PCR amplification of swine GHS-R sequences from
pools of an unamplified swine cDNA library (prevalence of 1:300,000),
Northern blot analysis (no signal detected with 10 µg poly
(A)+ mRNA), and in situ hybridization signals
(weak specific signals observed in the central nervous system when
compared with other control GPC-Rs; Refs. 20 and 25). Accordingly,
RNase protection methodology was used to perform an initial analysis of
the tissue-specific expression pattern of the rat GHS-R. The probe used
was designed against the human GHS-R type 1a and 1b sequences (89%
overall nucleic acid sequence identity between rat and human cDNAs) and
therefore contains mismatched residues when compared with the rat
nucleotide sequence. This probe revealed three protected fragments with
human pituitary poly A+ mRNA (Fig. 3b
, lane 1) which are
not found in a variety of other tissues, including liver, kidney, whole
brain, and testis (25). The size of the protected bands at 410, 304,
and 106 nucleotides corresponds to transcripts encoding: full-length
type 1a (410 nucleotides); unspliced pre-mRNA for type 1a (304
nucleotides); and type 1b GHS-R or unspliced pre-mRNA for type 1a (106
nucleotides). Using rat poly (A)+ mRNAs and allowing for
mismatches between the human and rat nucleotide sequences that generate
additional RNase digestion sites, a similar pattern of three bands
[
213 (nt 884-1097; spliced type 1a mRNA), 155 (nt 729884; spliced
type 1a mRNA) and 70 nucleotides (nt 730800; type 1b mRNA or
unspliced type 1a pre-mRNA)] was observed in rat pituitary and
hypothalamus (Fig. 3b
, lanes 2 and 3, respectively) but not in tissues
that contain no detectable GHS-R binding activity, such as liver,
kidney, or whole brain (lanes 46). These results were confirmed and
extended by mapping the expression of GHS-R mRNA in rat brain and
peripheral tissues by in situ hybridization, which reveals
that the strongest signals are found in hippocampal and multiple
hypothalamic nuclei and the pituitary gland (25).
A contiguous DNA fragment encoding the complete 7-TM rat ORF
(devoid of intervening sequence) was expressed in HEK-293 cells and
COS-7 cells. Radioligand binding studies were performed on membranes
prepared from transfected COS-7 cells. Binding was saturable
(Bmax of specific binding
30 pmol/mg), of high affinity
(KD = 0.7 nM) and specificity (> 90%) (Fig. 4A
). As shown in Fig. 4B
, both peptide and
nonpeptide GHSs compete with high affinity for the binding of
[35S]MK-0677, in a similar rank order of potency observed
for the native rat pituitary receptor and cloned human receptor
(Ki values for GHRP-2, GHRP-6, and MK-0677 are 0.5, 1.5,
and 0.7 nM, respectively). Functional activity of the
expressed receptor was determined by measuring MK-0677-dependent,
IP3-coupled mobilization of intracellular calcium and
concomitant calcium-induced aequorin bioluminescence. The rat GHS-R
expression construct was transiently transfected into an aequorin
reporter cell line [293-AEQ17; (25)]. After
40 h of expression,
the cells were charged with the essential chromophore coelenterazine
and harvested. A dose-response curve for the rat GHS-R is shown in Fig. 5
for MK-0677 concentrations ranging from
10-5 to 10-12 M giving an
EC50 value of approximately 4 nM. The rank
order potency of GHRP-2, GHRP-6, and MK-0677 in functional assays
(IP3 release) is also comparable to the EC50
values obtained in the GH release assay in primary rat pituitary cells.
One explanation for the
5-fold difference in IC50 for
MK-0677 in the radioligand binding assay and the EC50 in
the aequorin functional assay is that the high level of receptor
expression results in a large proportion of cell-surface receptors that
are not G protein-coupled and thus cannot tranduce the Ca2+
mobilization signal needed for aequorin bioluminescence. This
interpretation is corroborated by binding data showing a significant
proportion (
50%) of GTP
S-insensitive binding sites. As expected,
the dose-response curve in the functional assay can be shifted as a
function of the amount of cell-surface receptor expressed using
different amounts of DNA for transfection (our unpublished
observations).

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Figure 4. Binding of [35S]MK-0677 to Membranes
from Rat GHS-R Transfected COS-7 Cells
Two days after transfection with a rat GHS-R cDNA expression construct,
crude cell membranes were prepared and assayed for
[35S]MK-0677 binding (0.4 µg membrane protein). A,
Saturation isotherm: Total binding (filled squares),
nonspecific binding (+50 nM unlabeled MK-0677; open
circles), specific binding (filled triangles
facing up). B, Competition analysis using unlabeled MK-0677
(square), GHRP-2 (triangle), and GHRP-6
(inverted triangle). 100% of control
[35S]MK-0677 binding = 0.03 pmol bound (0.2
nM).
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Figure 5. Functional Activity of the Rat GHS-R Using the
Aequorin Bioluminescence Assay in 293-AEQ17 Cells
The HEK-293 cell line stably expressing aequorin [293-AEQ17; (26)]
was transfected with a rat GHS-R expression plasmid. Two days after
transfection, cells were charged with coelentrazine, scraped, and
transferred into ECB buffer, and centrifuged in plastic tubes for
luminometer measurements. Data for each point on the dose-response
curve represent the average of triplicate measurements for each sample
( 3 x 105 cells per tube).
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In summary, we have identified both pituitary and hypothalamic cDNAs
that are strikingly similar to the swine and human GHS-R type 1a. Use
of the receptor cDNA allows for the precise mapping of the GHS-R by
in situ hybridization in the central nervous system and
peripheral tissues of the rat, and it will facilitate the in-depth
analysis of its normal physiological role in rodent models of GH
release.
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MATERIALS AND METHODS
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Complementary DNA Cloning
Reduced stringency hybridization was performed to isolate
a rat GHS-R cDNA. Approximately 106 phage plaques of a
once-amplified rat pituitary cDNA library in
gt11 (RL1051b;
Clontech, Palo Alto, CA) were plated on Escherichia coli
strain Y1090r-. The plaques were transferred to
maximum-strength Nytran (Schleicher & Schuell, Keene, NH) denatured,
neutralized, and screened with a 1.6 kb
EcoRI/NotI fragment containing the entire coding
and untranslated regions of the swine GHS-R, clone 73 (20). The
membranes were incubated at 30 C in prehybridization solution [50%
formamide, 2 x Denhardts, 5 x SSPE (1 x is 0.18
M NaCl, 0.01 M NaH2PO4,
0.001 M Na2EDTA; pH 7.7), 0.1% SDS, 100
µg/ml salmon sperm DNA] for 3 h followed by overnight
incubation in hybridization solution (50% formamide, 2 x
Denhardts, 5 x SSPE, 0.1% SDS, 10% dextran sulfate, 100
µg/ml salmon sperm DNA) with 1 x 106 cpm/ml of
[32P]-labeled probe. The probe was labeled with
[32P]dCTP using a random priming kit (GIBCO BRL,
Gaithersburg, MD). After hybridization, the blots were washed two times
each with 2 x NaCl-sodium citrate (SSC), 0.1% SDS (at 24 C, then
37 C, and finally 55 C). A single positive clone was isolated after
three rounds of plaque purification. Phage containing the GHS-R was
eluted from plate plaques with 1 x
-buffer (0.1 M
NaCl, 0.01 M MgSO4, 35 mM Tris-HCl,
pH 7.5) after overnight growth of approximately 200 pfu/150 mm dish.
After a 10-min centrifugation at 10,000 x g to remove
debris, the phage solution was treated with 1 µg/ml RNAse A and DNAse
I for 30 min at 24 C, followed by precipitation with 20% polyethylene
glycol (8000)/2 M NaCl for 2 h on ice, and collection
by centrifugation at 10,000 x g for 20 min. Phage DNA
was isolated by incubation in 0.1% SDS, 30 mM EDTA, 50
µg/ml proteinase K for 1 h at 68 C, with subsequent phenol
(three times) and chloroform (twice) extraction before isopropanol
precipitation overnight. The GHS-R cDNA insert (
6.4 kb) was
subcloned from
gt11 into the plasmid vector Litmus 28 (New England
Biolabs, Beverly, MA). Two micrograms of phage DNA were heated to 65 C
for 10 min, then digested with 100 U of BsiWI (New England Biolabs) at
37 C (overnight). A 6.5-kb fragment was gel purified, electro eluted,
and phenol/chloroform extracted before ligation to BsiWI-digested
Litmus 28 vector. Double-stranded DNA was sequenced on both strands on
an ABI 373 automated sequencer using the ABI PRISM dye termination
cycle sequencing ready reaction kit (Perkin Elmer; Foster City, CA).
Isolation of full-length rat hypothalamus type 1a GHS-R cDNA was
performed by screening a plasmid-based cDNA library (
700,000 primary
clones) constructed in the vector pcDNA-3 (Invitrogen) using poly
(A)+ mRNA prepared from freshly dissected tissue. Two
independent positive clones were identified.
Complementary DNA Expression Studies
For sequence comparisons and functional expression studies, a
contiguous ORF for the rat GHS-R type Ia was generated by removal of
intervening sequences. PCR was used to synthesize an amino-terminal
fragment from Met-1 to Val-260 with EcoRI (5') and
HpaI (3') restriction enzyme sites, while a
carboxyl-terminal fragment was generated from Lys-261 to Thr-364 with
DraI (5') and NotI (3') restriction enzyme sites.
The construct was assembled into the mammalian expression vector pSV-7
(20) with EcoRI/NotI-digested pSV7,
EcoRI/HpaI-digested NH2-terminal
fragment, and DraI/NotI-digested C-terminal
fragment.
Transfection of COS-7 and 293-AEQ17 Cells
Transient transfections of the rat GHS-R pSV-7 construct were
conducted using the lipofectamine procedure (GIBCO BRL) according to
the manufacturers instructions. Transfections were performed in 60-mm
dishes (80% confluent cells) with 30 µg lipofectamine and 2.5 µg
GHS-R plasmid DNA. Receptor expression was allowed to proceed for
4872 h.
Radioligand Binding Assay
The binding of [35S]MK-0677 to crude
membranes prepared from COS-7 cell transfectants was performed
essentially as described (16, 20). Crude cell membranes were prepared
on ice 48 h after transfection. Each 60-mm dish was washed twice
with 3 ml PBS, once with 1 ml homogenization buffer [50 mM
Tris-HCl (pH 7.4), 5 mM MgCl2, 2.5
mM EDTA, 30 µg/ml bacitracin]. Homogenization buffer
(0.5 ml) was added to each dish, and cells were removed by scraping and
then homogenized using a Polytron device (Brinkmann, Syosset, NY; three
bursts of 10 sec at setting 4). The homogenate was centrifuged for 20
min at 11,000 x g at 0 C, and the resulting crude
membrane pellet (chiefly containing cell membranes and nuclei) was
resuspended in homogenization buffer supplemented with 0.06% BSA (0.5
ml/60-mm dish) and kept on ice. Binding reactions were performed at 20
C for 1 h in a total volume of 0.5 ml containing: 0.1 ml membrane
suspension (
25 µg protein), 10 µl [35S]MK-677
(0.05 to 1 nM; specific activity,
1200 Ci/mmol), 10 µl
competing drug, and 380390 µl homogenization buffer. Bound
radioligand was separated by rapid vacuum filtration (Brandel 48-well
cell harvester) through GF/C filters pretreated for 1 h with 0.5%
polyethylenimine. After application of the membrane suspension to the
filter, the filters were washed three times with 3 ml each of ice-cold
50 mM Tris-HCl (pH 7.4), 10 mM
MgCl2, 2.5 mM EDTA, and 0.015% Triton X-100,
and the bound radioactivity on the filters was quantitated by
scintillation counting. Specific binding (> 90% of total) is defined
as the difference between total binding and nonspecific binding
conducted in the presence of 50 nM unlabeled MK-0677.
Aequorin Bioluminescence Assay
The assay was carried out essentially as described with
modifications (26). Measurement of GHS-R expression in the
aequorin-expressing stable reporter cell line 293-AEQ17 was performed
using a Turner model 20E luminometer (Turner Designs, Sunnyvale, CA).
293-AEQ17 cells (60-mm dish, 8 x 105 cells plated
18 h before transfection) were transfected with 2.5 µg plasmid
DNA-30 µg lipofectamine. After approximately 40 h of expression,
the aequorin in the cells was charged for 2 h with coelenterazine
(10 µM) under reducing conditions (30 µM
reduced glutathione) in ECB buffer [140 mM NaCl, 20
mM KCl, 20 mM HEPES-NaOH (pH 7.4), 5
mM glucose, 1 mM MgCl2, 1
mM CaCl2, 0.1 mg/ml BSA], the cells were
harvested, washed, and pelleted by low speed centrifugation into
plastic luminometer tubes (75 x 12 mm, Sarstedt). ECB buffer (2.9
ml) was added without disturbing the cell pellet, and bioluminescence
measurements were triggered by the injection of 0.1 ml of 30 x
concentrated stocks of MK-0677. Recordings were followed for 2 min to
observe responses consistent with an IP3-mediated
kinetics.
Southern Blot Analysis and RNase Protection Assay (RPA)
A commercial blot (Clontech) containing
EcoRI-digested rat genomic DNA (4 µg) was hybridized
overnight with a 32P-labeled fragment of the rat GHS-R cDNA
(nt 253 to 775 of the ORF) in a solution containing 50%
formamide, 2 x Denhardts, 5 x SSPE, 0.1% SDS, 10% dextran
sulfate, 100 µg/ml salmon sperm DNA. The blot was subsequently washed
under low (4 x SSC, 55 C) and then high (0.1 x SSC, 65 C)
stringency conditions for three washes each (15 min/wash) and exposed
to X-omat film overnight at -70 C with two intensifying screens.
Synthesis of high-specific activity radiolabeled antisense probes and
the RPA was conducted using a kit (MAXIscript and HybSpeed RPA kits;
Ambion, Austin, TX) essentially as described by the manufacturer. The
antisense cRNA GHS-R probe was synthesized from a cDNA template
encompassing nt 681 to 1101 of the human GHS-R ORF inserted behind the
T7 promoter in pGEM-11Z(f)+ (Promega Biotech, Madison, WI).
Digestion of the construct with PvuI will generate a cRNA
transcript
550 nt in size with
140 nt of vector sequence. Input
poly A+ mRNA (Clontech) was 5 µg for the GHS-R probe and
0.1 µg for a control human actin probe. Precipitated fragments were
subjected to slab-gel electrophoresis (42 cm x 32 cm x 0.4
mm) in 5% acrylamide/Tris-borate-EDTA buffer containing 8
M urea. The gels were fixed, dried, and autoradiographed on
film (X-Omat; Kodak) for 3 days (GHS-R) or 18 h (actin).

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Figure 7. Dose Response of icv Administered Serotonin (Ser) on
Lordosis Response in PR Mutants in the Presence of Wild Type PR Male
Mice.
A, Ovariectomized wild type PR mice were subjected to weekly hormonal
priming and behavioral testing as described for Fig. 5A . Third cerebral
ventricle cannulations were performed on week 5, followed by EB priming
on week 6 and icv administration of serotonin (1200 ng) 48 h later.
The mice were tested for sexual responsiveness 30 min after icv
administration of serotonin. Controls were the same as described for
Fig. 6. A significant effect of serotonin was seen at a dose of 50 ng
as compared with the EB-treated controls (*, P < 0.05). B,
The effects of serotonin on lordosis response of homozygotes and wild
type PR mutants was examined using the paradigm described in Fig. 6B.
The EB-primed mice received serotonin (50 ng), 48 h after EB
administration. Controls were the same as described for Fig. 6. No
significant differences were seen in serotonin-facilitated responses
between the wild type and homozygous PR mutants. (P > 0.05)
(n = 6 animals in each group).
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ACKNOWLEDGMENTS
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We thank J. Shockey of MRL Visual Communications for expert
preparation of figures and D. Button and M. Brownstein for the gift
of aequorin plasmids and the 293-AEQ17 cell line. We are grateful to K.
Likowski for editorial assistance and D. Dean for radiolabeled
MK-0677.
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FOOTNOTES
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Address requests for reprints to: Dr. Lex H. T. Van der Ploeg, Department of Genetics and Molecular Biology, Merck Research Laboratories, Building RY80M-213, Rahway, New Jersey 07065.
Received for publication October 18, 1996.
Revision received January 16, 1997.
Accepted for publication January 16, 1997.
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