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


    ABSTRACT
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS AND DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
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 1–5 and TM 6–7, 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.


    INTRODUCTION
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS AND DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
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{alpha}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.


    RESULTS AND DISCUSSION
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS AND DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
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. 1Go; 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.

 
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. 2Go). 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).

 
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. 3aGo). 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.

 
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. 3bGo, 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 729–884; spliced type 1a mRNA) and 70 nucleotides (nt 730–800; type 1b mRNA or unspliced type 1a pre-mRNA)] was observed in rat pituitary and hypothalamus (Fig. 3bGo, 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 4–6). 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. 4AGo). As shown in Fig. 4BGo, 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. 5Go 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{gamma}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).

 
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.


    MATERIALS AND METHODS
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS AND DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
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 {lambda} 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 7–3 (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 Denhardt’s, 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 {lambda}-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 {lambda} 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 manufacturer’s 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 48–72 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 380–390 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. 5AGo. Third cerebral ventricle cannulations were performed on week 5, followed by EB priming on week 6 and icv administration of serotonin (1–200 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).

 

    ACKNOWLEDGMENTS
 
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.


    FOOTNOTES
 
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.


    REFERENCES
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS AND DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 

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