Ligand Activation Domain of Human Orphan Growth Hormone (GH) Secretagogue Receptor (GHS-R) Conserved from Pufferfish to Humans

Oksana C. Palyha, Scott D. Feighner, Carina P. Tan, Karen Kulju McKee, Donna L. Hreniuk, Ying-Duo Gao, Klaus D. Schleim, Lihu Yang, Gregori J. Morriello, Ravi Nargund, Arthur A. Patchett, Andrew D. Howard and Roy G. Smith

Department of Biochemistry and Physiology (O.C.P., S.D.F., C.P.T., K.K.M., D.L.H., K.D.S., A.D.H.) Department of Medicinal Chemistry (L.Y., G.J.M., R.N., A.A.P.) and Department of Molecular Design (Y.-D.G.) Merck Research Laboratories Rahway, New Jersey 07065
Huffington Center on Aging and Department of Molecular and Cellular Biology (R.G.S.) Baylor College of Medicine Houston Texas 77030


    ABSTRACT
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Synthetic ligands have been identified that reset and amplify the cycle of pulsatile GH secretion by interacting with the orphan GH-secretagogue receptor (GHS-R). The GHS-R is rhodopsin like, but does not obviously belong to any of the established G protein-coupled receptor (GPCR) subfamilies. We recently characterized the closely related orphan family member, GPR38, as the motilin receptor. A common property of both receptors is that they amplify and sustain pulsatile biological responses in the continued presence of their respective ligands. To efficiently identify additional members of this new GPCR family, we explored a vertebrate species having a compact genome, that was evolutionary distant from human, but where functionally important genes were likely to be conserved. Accordingly, three distinct full-length clones, encoding proteins of significant identity to the human GHS-R, were isolated from the Pufferfish (Spheroides nephelus). Southern analyses showed that the three cloned Pufferfish genes are highly conserved across species. The gene with closest identity (58%) was activated by three synthetic ligands that were chosen for their very high selectivity on the GHS-R as illustrated by their specificity in activating the wild-type human GHS-R but not the E124Q mutant. These results indicate that the ligand activation domain of the GHS-R has been evolutionary conserved from Pufferfish to human (400 million years), supporting the notion that the GHS-R and its natural ligand play a fundamentally important role in biology. Furthermore, they illustrate the power of exploiting the compact Pufferfish genome for simplifying the isolation of endocrinologically important receptor families.


    INTRODUCTION
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
In all species studied, GH is released episodically (1 2 3 ). This process was thought to be regulated by two hypothalamic hormones, GH releasing hormone (GHRH) and somatostatin, through their respective receptors (4 5 6 ). However, these two hormones and their receptors are insufficient to explain the physiological and pharmacological control of pulsatile GH release mediated by the synthetic GH secretagogues GHRP-6, L-692.429, and MK-0677 (7 8 9 ).

In 1996, we reported the cloning of an orphan G protein coupled receptor (GPCR) that is highly conserved across species (10 11 ) and is the mediator of the action of the synthetic GH secretagogues (12 13 14 15 16 ) but is not activated by GHRH (19 20 21 ). Based on its deduced peptide sequence, the GH secretagogue receptor (GHS-R) is related to the rhodopsin superfamily but is not obviously related to known subfamilies of GPCRs (10 17 18 ). The closest neighbor (34% identity) is the neurotensin receptor (10 ).

The GHS-R plays an important regulatory function in endocrinology. Administration of low doses of specific ligands stimulates GH release across a wide variety of species, from chickens to humans, where they reset and amplify the ultradian cycle of pulsatile GH secretion (11 ). The mechanism involved is remarkable, because amplification occurs at three different levels. First, ligand activation of the GHS-R results in functional antagonism of the negative regulator of GH release, somatostatin. Second, GHS-R ligands activate arcuate neurons to increase GHRH secretion. Third, these ligands act directly on somatotrophs to amplify the action of GHRH on GH release (21 ). In humans, daily treatment of 70- to 90-yr-old subjects with the synthetic GHS-R ligand MK-0677 restores the amplitude of pulsatile GH and the levels of insulin-like growth factor-1 (IGF-I) to those of subjects in their late twenties (9 ). These data suggest that production of an unidentified endogenous GHS-R ligand decreases during aging (17 ).

GHS-R is expressed exclusively in the anterior pituitary gland and central nervous system (11 ). In addition to expression in centers of the brain controlling GH release, the GHS-R (22 ) is expressed in brain regions involved with biological rhythms, cognition, learning, memory, mood, and behavior. Hence, the importance of the GHS-R and its ligand is likely to be even more profound than the rejuvenating effects on the GH/IGF-I axis. We recently showed that a close GHS-R homolog, GPR38, is the motilin receptor (23 ). Motilin administration induces rhythmic contraction of the gut. Indeed, both GHS-R and GPR38 in the continued presence of their specific ligands stimulate pulsatile biological activity in vivo; this invites speculation that the GHS-R family is involved in establishing biological rhythmicity and reinforces the importance of isolating additional GHS-R family members to test this hypothesis.

We reported the isolation of three GHS-R homologs (GPR38, GPR39, and FM3) from a human genomic PAC library (24 25 ). However, screening this library produced positive hybridization signals of varying intensity for 325 PAC clones (24 ). The relatively low percentage of coding sequences in the human genome makes characterization of potential GHS-R family members from this library a major undertaking. To seek a more efficient alternative approach, we investigated the potential of exploiting a vertebrate organism with a more compact genome. A species was selected that was evolutionary distant from human, but where homologous genes were likely conserved because of their functional importance. Teleost fish have the specialized functions of higher vertebrates; therefore, genes necessary for complex functions are present in the fish genome.

In 1968, Hinegardner (26 ) determined the DNA content of more than 200 teleost species. Among teleosts, the Tetraodontidae family has the lowest DNA content (0.4–0.5 pg DNA/haploid genome). Pufferfish are members of this family and considered to be evolutionarily advanced. The loss of DNA during their evolution is presumably explained by specialization (26 ). The Japanese Pufferfish (Fugu rubripes) genome was proposed as a compact model genome for vertebrates by Brenner et al. (27 ). The Pufferfish genome (400 Mb) contains approximately the same number of genes as the mammalian genome, has very little repetitive DNA and about 90% of the DNA is unique, and lacks pseudogenes (27 ). A comparison of the genomic structure of Pufferfish genes with their mammalian homologs indicates that the intron-exon organization has been conserved; the intron size is generally smaller (60–120 bp) and intergenic sequences are greatly reduced (28 29 30 ). Similarities between Pufferfish and mammalian genes have already been established. Four Fugu dopamine receptors and three neurokinin receptor analogs have been identified that closely resemble their mammalian counterparts (31 32 ).

Based on the studies described above, we wished to evaluate the Pufferfish for its potential use in cloning GHS-R family members. The potential advantage is that about 20% of the Pufferfish genome contain coding sequences compared with less than 3% in the human genome. This compactness allows facile gene isolation, characterization, and expression. Our objectives were to investigate whether this vertebrate species provided a shortcut for identifying new GHS-R family members and investigate further the relevance of this species to mammalian endocrinology by testing whether the orphan GHS-R and its functional domain had been evolutionarily conserved.


    RESULTS
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Isolation of Pufferfish Clones with Homology to Human GHS-R
A genomic DNA library from Spheroides nephelus was screened using two radiolabeled probes. One, an approximately 0.28-kb DNA fragment representing the putative transmembrane domains (TM) 3 and 4 of the human GPR38 orphan receptor (24 ) (the family member with 52% identity to GHS-R); and the other representing the complete coding sequence of the GHS-R (~1.1 kb). After relatively high-stringency washing, three dominant signals were observed on the filters. We identified, cloned, and sequenced three human GHS-R homologs, 78B7, 75E7, and 1H9.1 The gene structures, with the exception of gene 1H9, are identical to the structure of the GHS-R. The intron in the mammalian GHS-R between TM5 and TM6 is conserved in 78B7, 75E7, and 1H9 (Fig. 1Go). The genes are predicted to encode similar size proteins but the introns in the Pufferfish, as predicted, are smaller. In the human GHS-R gene, the intron is 2200 bp, whereas the corresponding intron is 330 bp in 78B7, 585 bp in 75E7, and 150 bp in 1H9, reflecting the more compact genome of the Pufferfish. Gene 1H9 contains two additional introns. Intron 1 (140 bp) is positioned at the beginning of TM1, and intron 2 (1234 bp) is located between TM2 and TM3 (Fig. 1Go). The predicted amino acids present at the exon-intron boundary are well conserved, and the spacing of introns between TM helices is typical of GPCRs.



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Figure 1. Gene Structure of S. nephelus Clones

Schematic representation of Pufferfish GHS-R-related genes in comparison with GHS-R and GPR38 (FM-1) with several notable features highlighted. E/DRY motif responsible for G protein binding is shown. Putative TM regions numbered from 1 to 7 are represented by open boxes. Intron location and amino acids present at exon/intron boundary after TM-5 are noted. For 1H9, predicted extended loop domains are shown by open ovals.

 
Comparison of S. nephelus Protein Sequences
The protein sequences predicted from the nucleotide sequences were aligned using a Pileup program and are shown in Fig. 2Go. Accordingly, 78B7, 75E7, and 1H9 are 58, 47, and 41% identical to the human GHS-R, respectively. An even higher conservation is revealed if one compares the human and Pufferfish sequences predicted to compose the seven TM helices. Conservation between human GHS-R and 78B7 is readily appreciated from the illustration in Fig. 3Go, where differences are highlighted. Experiments with peptide antibodies specific for extracellular domain 3 and the intracellular C-terminal domain confirm the GHS-R extracellular/intracellular orientation shown in Fig. 3Go (33 ). Strikingly, although there is only 58% identity between the predicted protein sequences of the human and Pufferfish receptors, TM2, TM3, TM6, and TM7 are more highly conserved. The homologous domains in clone 78B7 for TM1–TM7, respectively, are 52%, 80%, 92%, 76%, 52%, 87%, and 88% identical to the human GHS-R. Site-directed mutagenesis of the human GHS-R shows that the important residues required for ligand activation are conserved in 78B7. For example, a key amino acid residue in TM3, E124, is conserved. Site-directed mutagenesis suggests that this residue forms a salt bridge with the basic amine in the aminoisobutyric acid side chain of MK-0677. Mutation of E124 to Q results in more than 100-fold rightward shift in the MK-0677 activation dose-response curve, whereas, the conservative E124D substitution has the same characteristics as the wild-type receptor (33 ). Further support for interaction with E124 is provided by comparing the activity of L-163,740, where the basic amine in MK-0677 is replaced by a hydroxyl moiety. L-163,740 is inactive on wild-type GHS-R but activates the E124Q mutant, presumably because of hydrogen bonding interactions (33 ). The overall retention of critical amino acid residues in the TM domains of the Puffer fish and human receptors is remarkable given the 400 million years of evolution that separates these two species.



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Figure 2. Comparison of S. nephelus Protein Sequences

Protein sequences were aligned using the Pileup program [Wisconsin Package Version 9.1, Genetics Computer Group (GCG), Madison, WI.; gap extension 4, gap creation 12]. Identical residues are boxed. The top panel is a graphic representation of sequence identities. The GAP alignment program (GCG) was used to determine amino acid identity for the complete protein or corresponding transmembrane helices (TM). Sequences used with GenBank, EMBL, or SWISSPROT database assession numbers were: huGHS-R1a, human type 1a GH secretagogue receptor (U60179); human GPR38 (||AF034632); puffer fish clones 78B7 (AF082209), 75E7 (AF082210), and 1H9 (AF082211).

 


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Figure 3. Homology between Human GHS-R and Pufferfish 78B7

Conserved (white circles) and altered (red circles) amino acid residues in the deduced protein sequences are illustrated by a schematic representation of receptor transmembrane topology (N terminus: extracellular; C terminus: intracellular). To equalize spacing in the N-terminal domain, open circles denote residues present in the human GHS-R and absent in the Pufferfish 78B7 protein.

 
The deduced protein sequence of 78B7 is typical of rhodopsin-like GPCRs. 78B7 has the ERY/DRY signature motif at the C terminus of TM3. Either E, or the conservative substitution D, of this motif occurs in 97% of GPCRs; R is fully conserved and in 84% of GPCRs the Y residue is present (34 ). Based on identifiers assigned to amino acids in the TMs of GPCRs (35 ), it is clear that other important residues are conserved in 78B7. In TM2, D is conserved at identifier 2.50 (2.50). For TM4, W (4.50) and P (4.59), found in 96% and 66% of GPCRs, respectively, are also conserved. In TM5, the P (5.50) residue typical of 86% of GPCRs is present, and the highly conserved residues in TM6, F (6.44), C (6.47), and W (6.48), are all found in 78B7. Toward the C terminus, TM7 contains the NPXXY (7.49–7.54) motif typical of 82% of GPCRs (34 ). A distinct feature of 78B7, which is shared by the human GHS-R, is the highly aromatic character of TM5, which contains FFFXPVF (5.46–5.52). The closely related (56% identical) motilin receptor (GPR38, Fig. 2Go) has similarly high aromatic character, YFFLPF (5.46–5.51). This feature is not conserved in the next most closely related receptors such as the neurotensin receptor (36% identical) and the TRH receptor (29% identical).

Activation of Pufferfish GHS-R Homologs by GHS-R Ligands
Having isolated Pufferfish homologs of the human GHS-R, it was important to test whether the Pufferfish clones could be activated by GHS-R ligands. Receptor function was assessed using an assay that is dependent upon ligand activation of the phospholipase C signal transduction pathway resulting in inositol triphosphate (IP3) -coupled mobilization of intracellular Ca2+. To measure ligand-induced changes in intracellular free Ca2+, we used the aequorin bioluminescence assay that had previously been exploited to expression clone the GHS-R (10 17 ).

The presumed open reading frames (ORFs) from 78B7, 75E7, and 1H9 were inserted into the expression plasmid pcDNA-3.1. The ORFs were FLAG-tagged so that expression of the respective sized proteins from each clone could be confirmed by Western blot analysis. The expression vectors containing 78B7, 75E7, and 1H9 ORFs were transfected into HEK-293-AEQ17 cells by the lipofectamine procedure. Forty hours later, the cells were charged with coelenterazine and treated with a broad spectrum of more than 500 potential ligands that included serotonin, somatostatin, corticostatin, dynorphin A, Substance P, oxytocin, vasoactive intestinal peptide, bombesin, ß-endorphin, endothelin, Neuropeptide Y, neurotensin, neurokinin, melanocyte stimulating hormone, GHRP-6, MK-0677, and L-163,540. A dose-dependent increase in bioluminescence was observed after treatment of HEK293-AEQ17 cells expressing 78B7 with the structurally distinct GH secretagogues (Fig. 4Go). The synthetic hexapeptide, GHRP-6, increased bioluminescence with an EC50 of 200 nM. The spiroindoline, MK-0677 (16 ), and benzylpiperidine, L-163,540 (36 ), GH secretagogues were also active (EC50 of 1 µM and 50 nM respectively). All other ligands tested were inactive. GHRP-6, MK-0677, and L-163,540 activate the human GHS-R at low nanomolar concentrations. Although the ligand concentrations necessary to activate 78B7 are approximately 10- to 100-fold higher, it is remarkable that of more than 500 potential ligands, activation by the same three ligands occurred at all considering that GHS-R and 78B7 are orphan receptors from species separated by 400 million years of evolution. By contrast, closely related ligands that were shown to activate both the wild-type GHS-R and its E124Q mutant failed to activate 78B7 (data not shown). HEK293-AEQ17 cells expressing the homologs, 75E7 and 1H9, were not activated by any ligand tested.



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Figure 4. Functional Activation of S. nephelus GHS-R Clone 78B7 by Various GH-Secretagogues.

Shown is the structure of GHS-R ligands and dose-response curves for the activation of 78B7 and the human GHS-R. HEK-293-AEQ17 cells (8 x105 cells) were transfected by the lipofectamine procedure with a contiguous ORF encoding 78B7 in the expression plasmid pcDNA-3. Approximately 40 h after transfection, the apo-aequorin in the cells was charged for 4 h with coelenterazine. In a 96-well plate assay format, 5 x104 cells were then injected into the test plate containing MK-0677, L-163,540, and GHRP-6 (13 16 36 ), and the integrated light emission was recorded over 30 sec followed by an injection of lysis buffer (0.1% final Triton X-100 concentration) recorded over 10 sec. The "fractional response" values for each well were calculated by taking the ratio of the integrated response to the initial challenge to the total integrated luminescence including the Triton X-100 lysis response.

 
Southern Analysis of S. nephelus GHS-R-Related Sequences
Southern analysis of S. nephelus GHS-R related sequences was performed across species. Genomic DNA from Drosophila, Caenorhabditis elegans, Pufferfish, chicken, mouse, rat, guinea pig, hamster, rabbit, cat, dog, cow, pig, monkey, and human was digested with EcoRI and BamHI. The digested DNA was subjected to Southern analysis by hybridizing with radiolabeled probes specific for human GHS-R, 78B7, 75E7, and 1H9 (Fig. 5Go). The results of hybridization with the human GHS-R probe are consistent with the presence of GHS-R homologs in the fish, chicken, and mammalian species (Fig. 5Go). With the exception of the cat and hamster, which to our knowledge have not been tested, all of these animals have receptors that are known to respond to GHRP-6 and MK-0677.



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Figure 5. Southern Blotting Analysis of S. nephelus GHS-R-Related Sequences

A genomic Southern blot (EcoRI and BamHI-digested DNA, 10 µg/lane) was hybridized with radiolabeled complete ORF DNA probes specific for (A) the human GHS-R (panel A); clone 78B7 (panel B); clone 75E7 (panel C); and clone 1H9 (panel D). Posthybridizational washing stringencies were: 4x SSC/0.1% SDS at 25 C, 2x SSC/0.1%SDS at 42 C, 1x SSC/0.1%SDS at 55 C, and 0.1x SSC/0.1% SDS at 65 C, after which the filters were exposed to x-ray film for 3 days at -80 C. Lambda HindIII DNA markers (in kilobases) are 23.1, 9.4, 6.6, 4.4, 2.3, and 2.1.

 
Southern analysis using the Pufferfish clones as hybridization probes was used to investigate conservation across species. When 78B7, the fish gene with 58% identity to the human GHS-R, was used, hybridization to mammalian and chicken DNA was somewhat reduced and not as discrete in comparison to the GHS-R probe. However, hybridization to both Drosophila and C. elegans DNA was evident (Fig. 5Go). Close inspection of Fig. 5Go is consistent with the presence of a mammalian homolog of 78B7. Hybridization results with the 75E7 probe were unremarkable. By contrast, Southern analyses using 1H9 as probe suggest that a homolog of 1H9, distinct from the GHS-R gene, is present in humans as well as other species (Fig. 5Go). The sizes of the DNA restriction fragments are clearly different from those obtained with the GHS-R probe. Based on this apparent conservation, the 1H9 gene is a potentially important new family member. We have been unable to find any closely related homologs of 1H9 in the human EST database other than GHS-R and GPR38. When the TM domains of 1H9 are aligned with either GHS-R or 78B7, the predicted structure is unusual because it contains a very large E2 loop. This characteristic, to our knowledge, makes it unlike other GPCRs.


    DISCUSSION
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
The genome of the Pufferfish, Fugu rubripes, was selected as a model of the mammalian genome because it contains approximately 90% of all mammalian genes in an 8-fold compacted form (27 ). Because of import restrictions, we were forced to select an alternative Pufferfish species, Spheroides nephelus. We identified and cloned three human GHS-R homologs, 78B7, 75E7, and 1H9, from the genome of this Tetraodontidae. The gene structures, with the exception of gene 1H9, were identical to the structure of the GHS-R. The intron in the mammalian GHS-R between TM5 and TM6 is conserved in 78B7, 75E7, and 1H9 (Fig. 1Go). The genes are predicted to encode similar size proteins and, as anticipated, the introns in the fish are smaller. Gene 1H9 contains two additional introns: intron 1 is positioned at the beginning of TM1 and intron 2 is between TM2 and TM3 (Fig. 1Go). The spacing of introns between the TM helices is typical of GPCRs.

Functional studies had suggested that GHS-R is well conserved across mammalian species and chickens. The high conservation in TM2 and TM3 of the Pufferfish 78B7 and human GHS-R (Figs. 2Go and 3Go) is notable because site-directed mutagenesis of the human GHS-R had defined key amino acid residues in TM2 and TM3 that were essential for binding and activation by ligands. All of the homologous residues selected for mutagenesis in the human GHS-R are conserved in 78B7 (33 ). Interestingly, the important residues in TM2 and TM3 of the human GHS-R that are absolutely required for activation by the GHS-R ligands, such as E124, D99, and R102, are conserved in 78B7 and 75E7 (Figs. 2Go and 3Go). Based on molecular models, residues F119 and Q120 in TM3 of the human GHS-R are positioned about one and one-half helical turns above E124 (Fig. 3Go). These residues are hypothesized to form part of the binding pocket and to make contact with the ligand and are conserved in 78B7 (Figs. 2Go and 3Go), but not in 75E7 or in 1H9. Hence, these differences may explain why GHRP-6, MK-0677 and L-163,540 fail to activate 75E7 and 1H9.

We have shown that gene 78B7 is activated by three structurally distinct synthetic ligands, GHRP-6, MK-0677, and L-163,540 (Fig. 4Go). The concentrations necessary to activate 78B7 were higher than those needed to activate the GHS-R. However, it is remarkable, that from a collection of more than 500 potential ligands the same three ligands activate orphan receptors from species separated by 400 million years of evolution. Most importantly, although GHRP-6, MK-0677, and L-163,540 belong to different structural classes, based on GHS-R/ligand computer-docking models and GHS-R mutagenesis studies, all three occupy the same binding pocket (11 33 ). To critically evaluate the ligand specificity of the activation of 78B7, we selected structurally related ligands that occupy an overlapping pocket in the GHS-R. These ligands activate the wild- type GHS-R and the E124Q mutant GHS-R, whereas the more selective agonists, GHRP-6, MK-0677, and L-163,540, are inactive on the E124Q mutant (11 33 ). The closely related more promiscuous compounds were completely inactive on 78B7, illustrating conservation of a highly selective binding pocket from Pufferfish to human. Collectively, these data suggest that GHRP-6, MK-0677, and L-163,540 mimic a highly conserved endogenous ligand that occupies a common pocket in GHS-R and 78B7.

The evidence for the existence of a conserved natural ligand for the GHS-R is circumstantial but nevertheless compelling. First, it has been shown that ligand binding to the GHS-R activates an endocrinologically important pathway by an elegant mechanism (17 ). Second, a rational structure-activity relationship has emerged from the development of several series of GHS-R agonists having subnanomolar and nanomolar potency (37 ). Third, the elusive nature of the natural ligand is not unusual; historically, the discovery of synthetic ligands for orphan receptors has preceded identification of their natural ligands. Fourth, we recently identified a natural ligand for the very closely related (52% identical) GHS-R family member, GPR38, (23 24 ). Finally, in all species tested, from chickens to humans, and now in Pufferfish, the GHS-R pathway has been functionally conserved. In spite of the lack of direct proof, the collective evidence that supports conservation of a physiologically important natural ligand for the GHS-R is overwhelming.

Southern analysis using GHS-R, 78B7, 75E7, and 1H9 as hybridization probes reveals that homologs of Pufferfish 78B7 and 1H9 might be present in C. elegans and Drosophila. Pufferfish GHS-R family member 1H9 is of interest because this homolog appears to be highly conserved across species including human. When GPR38, the closest related human family member is used as a probe, a simple hybridization pattern consistent with a single conserved gene encoding GPR38 is observed across species. Clone 75E7 contains an ORF of 363 amino acids that is approximately 54% identical at the deduced protein level to GPR38. We recently showed that the natural endogenous ligand for GPR38 is motilin (23 ). Motilin, by activating GPR38, regulates episodic gut motility. Thus, a common feature of the GHS-R and its closest family member, GPR38, is that both control pulsatile biological events in the continued presence of their ligands. Because 75E7 contains a similar exon-intron structure to the human GPR38, 75E7 was thought to represent the ortholog of the human GPR38; however, motilin failed to activate 75E7.

The identification of three homologs of the human GHS-R in the Pufferfish provides a new example of the value of the Tetraodontidae genome for the facile cloning of gene families of interest to endocrinologists. This report, and other examples cited above, illustrate that the Pufferfish can be exploited to efficiently isolate and express gene family members directly from genomic DNA. A fascinating aspect of the Pufferfish model is the example of conservation of gene-regulatory elements between Fugu and rat (38 ). This was demonstrated in transgenic rats, where 40 kb of genomic DNA from the Fugu isotocin locus (isotocin is the fish homolog of the mammalian oxytocin gene) had been introduced into the rat genome. In the transgenic rats, the isotocin gene was expressed selectively in oxytocin neurons and was regulated by physiological perturbations that normally regulate oxytocin (38 ). Hence, despite 400 million years of separation between these two animal lineages, the regulatory regions in the isotocin and oxytocin genes are conserved. This work establishes a precedent for extending the application of the Pufferfish model for the GHS-R family. In rats, GHS-R expression is positively regulated by estrogen and negatively regulated by GH (39 40 ). By introducing the 78B7 genomic locus into rats we can test whether regulatory elements have been conserved in the 78B7 gene by investigating its hormonal regulation.

In summary, our experiments have shown that the functional structure of the ligand-binding domain of the GHS-R has been conserved for at least 400 million years, implying that an unknown natural ligand for the GHS-R has also been conserved. These observations strongly suggest that the GHS-R ligand plays a fundamentally important biological role and provide a strong rationale for investigating, in detail, the endocrine and physiological function of the GHS-R and its family members across species. Our experiments also extend and compliment previous studies showing the relevance of the Pufferfish genome as a model for higher vertebrates and illustrate the utility of this model for the facile isolation of receptor family members.


    MATERIALS AND METHODS
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Genomic Library Screening
A Pufferfish (Spheroides nephelus) PAC genomic library was obtained from Genome Systems (St. Louis, MO) consisting of two filters with individual clones double spotted in an array. It represented 3x genome coverage with an average insert size of 80 kb. The filters were prehybridized in 50% formamide/5xSSPE/2x Denhardts/0.1% SDS/100 µg/ml single-stranded DNA (ssDNA) at 30 C for 2 h followed by hybridization in 50% formamide/5x SSPE/2x Denhards/10% dextran sulfate/0.1% SDS/100 mg/ml ssDNA at 30 C overnight. To identify clones of interest, two distinct DNA fragments were selected and random-prime radiolabeled using the Stratagene Prime-It II kit (La Jolla, CA). Probe 3f/4r was PCR generated employing sense 5'-ATGCGGACCACCACCAACTTG-3' and antisense 5'-AGAGCACCGCGATGAGCGCGCGGA-3' primers encoding TM3–4(3f/4r) of the human orphan GPCR GPR38 (24 ) and used at 1 x 106 cpm/ml of hybridization solution. A 1.1-kb DNA fragment that spans the ORF of the human GHS-R type 1a (10 17 ) was also PCR derived using sense 5'-ATGTGGAACGCGACGCCCAGCGAA-3' and antisense 5'-TAGTTTAGCGGCCGCTCATGTATTAATACTAGA-3' primers and purified by electrophoresis. PCR conditions included denaturation at 94 C for 1 min, annealing at 50 C for 1 min, and extending at 72 C for 3 min for a total of 30 cycles in a 10 µl reaction volume. After hybridization, the filters were washed at moderate stringency at 1x SSC/0.1% SDS at 55 C. The filters were exposed to XA-R film (Kodak, Rochester, NY) at -80 C for 3 days. After hybridization with probe 3f/4r, the filters were stripped in 0.4 N NaOH at 42 C for 30 min, then incubated in 0.2 M Tris HCl pH 7.4/0.1x SSC/0.1% SDS at 42 C for 30 min, and rehybridized with the complete ORF probe. Sixteen clones were chosen for characterization based on their hybridization signal (moderate to strong). Nine of these clones cohybridized to both probes; three clones were specific to the GPR38 orphan receptor, and four clones were specific for the human GHS-R type 1a. The clones were characterized by digestion with KpnI, BamHI, EcoRI, PstI, XbaI, XhoI, NdeI, Bgl2, SmaI, PvuII, SphI, SalI, NotI, or HindIII at 37 C for approximately 3 h and resolved on 0.8% Seakem GTG agarose gel (FMC, Rockland, ME) in 0.5x Tris-borate-EDTA + EtBr at 24 V overnight. The gel was then transferred onto NYTRAN PLUS membranes (Schleicher & Schuell, Inc., Keene, NH) in 20x SSC overnight by capillary action. The membranes were hybridized with complete ORF probes. All clones were grouped based on their hybridization pattern, resulting in analysis of three clones: 78B7, 75E7, and 1H9. PAC plasmid DNA was isolated from 30 ml overnight cultures grown in LB/25 mg/ml kanamycin, by an alkaline lysis protocol (Genome Systems, St. Louis, MO) and dissolved in TE buffer (10 mM Tris-HCl, pH 8.3, 1 mM EDTA). The gene for each of the three clones was isolated and transferred into pUC18 plasmid, and their nucleotide sequences were determined by sequencing both strands utilizing dye terminator cycle sequencing Ready Reactions (Perkin-Elmer Corp., Foster City, CA). ORF constructs were built by overlapping PCR.

Transfection and Functional Expression in HEK-293/aeq17 Cells
Ligand activation of GHS-R expressed in the aequorin-expressing stable reporter cell line 293-AEQ17 was performed using a Luminoskan RT luminometer (Labsystems Inc., Gaithersburg, MD) controlled by custom software written for a Macintosh PowerPC 6100. 293-AEQ17 cells (8 x 105 cells plated 18 h before transfection in a T75 flask) were transfected with 22 mg of plasmid DNA containing 78B7, 75E7, and 1H9 ORFs in the presence of 264 mg lipofectamine. The cells were incubated for approximately 40 h, after which coelenterazine (10 mM) was added under reducing conditions (300 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) to charge the apo-aequorin. Four hours later the test compounds at various concentrations were added to the culture medium and changes in bioluminescence were monitored.

Southern Blotting Analysis
Genomic DNA (10 µg) was digested with EcoRI or BamHI and size separated in a 0.7% agarose gel followed by capillary transfer to maximum strength Nytran (Schleicher & Schuell, Inc.) nylon membrane. The membranes were incubated at 30 C in prehybridization solution (50% formamide, 2x Denhardts, 5x SSPE, 0.1% SDS, 100 mg/ml salmon sperm DNA) for 2 h. They were then incubated overnight in hybridization solution (50% formamide, 2x Denhardts, 5x SSPE, 0.1% SDS, 10% dextran sulfate, 100 mg/ml salmon sperm DNA) containing complete ORFs of GHS-R, 78B7, 75E7, and 1H9 as hybridization probes. Three identical Southern blots were washed at posthybridization stringencies of 4x SSC, 55 C; 1x SSC, 55 C, and 0.1x SSC, 65 C.


    FOOTNOTES
 
Address requests for reprints to: Roy G. Smith, Department of Molecular Cellular Biology and Huffington Center on Aging, Baylor College of Medicine, One Baylor Plaza, M320, Houston, Texas 77030.

1 GenBank accession numbers: AF082209, AF082210, AF082211. Back

Received for publication August 12, 1999. Revision received October 6, 1999. Accepted for publication October 12, 1999.


    REFERENCES
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 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
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