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
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ABSTRACT
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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.
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INTRODUCTION
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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 (0.40.5 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 (60120 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.
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RESULTS
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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. 1
). 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. 1
). 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.
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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. 2
. 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. 3
, 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. 3
(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 TM1TM7, 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.
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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
(7.497.54) 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 (5.465.52). The closely
related (56% identical) motilin receptor (GPR38, Fig. 2
) has similarly
high aromatic character, YFFLPF (5.465.51). 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. 4
). 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.
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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. 5
). 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. 5
). 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.
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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. 5
). Close inspection of Fig. 5
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. 5
). 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.
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DISCUSSION
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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. 1
). 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. 1
). 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. 2
and 3
) 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. 2
and 3
). 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. 3
).
These residues are hypothesized to form part of the binding pocket and
to make contact with the ligand and are conserved in 78B7 (Figs. 2
and 3
), 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. 4
).
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.
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MATERIALS AND METHODS
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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 TM34(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. 
Received for publication August 12, 1999.
Revision received October 6, 1999.
Accepted for publication October 12, 1999.
 |
REFERENCES
|
---|
-
Jansson JO, Eden S, Isaksson O 1985 Sexual dimorphism in
the control of growth hormone secretion. Endocr Rev 6:128150[Abstract]
-
Steiner RA, Stewart JK, Barber J, Koerker D, Gooner CJ, Brown
A, Illner P, Gale CC 1978 Somatostatin: a physiological role in the
regulation of growth hormone secretion in the adolescent male baboon.
Endocrinology 102:15871594[Medline]
-
Miller JD, Tannenbaum GS, Cole E, Guyda HJ 1982 Daytime
pulsatile growth hormone secretion during childhood and adolescence.
J Clin Endocrinol Metab 55:989994[Abstract]
-
Mayo KE, Godfrey PA, DeAlmeida V, Miller T 1996 Structure,
function, and regulation of the pituitary receptor for growth hormone
releasing hormone. In: Bercu BB, Walker RF (eds) Growth
Hormone Secretagogues. Springer-Verlag, New York, pp 5371
-
Mayo KE, Godfrey PA, Suhr ST, Kulik DJ, Rahal JO 1995 Growth
hormone-releasing hormone: synthesis and signaling. Recent Prog Horm
Res 50:3573[Medline]
-
Raynor K, Murphy WA, Coy DH, Taylor JE, Moreau JP, Yasuda K,
Bell GI, Reisine T 1993 Cloned somatostatin receptors: identification
of subtype-selective peptides and demonstration of high affinity
binding of linear peptides. Mol Pharmacol 43:838844[Abstract]
-
Huhn WC, Hartman ML, Pezzoli SS, Thorner MO 1993 24-hour
growth hormone (GH)-releasing peptide (GHRP) infusion enhances
pulsatile GH secretion and specifically attenuates the response to a
subsequent GHRP-6 bolus. J Clin Endocrinol Metab 76:12021208[Abstract]
-
Chapman IM, Hartman ML, Pezzoli SS, Thorner MO 1996 Enhancement of pulsatile growth hormone secretion by continuous
infusion of a growth hormone-releasing peptide mimetic, L-692,429, in
older adults a clinical research center study. J Clin
Endocrinol Metab 81:28742880[Abstract]
-
Chapman IM, Bach MA, Van C, E, Farmer M, Krupa DA, Taylor AM,
Schilling LM, Cole KY, Skiles EH, Pezzoli SS, Hartman ML, Velduis JD,
Gormley GJ, Thorner MO 1996 Stimulation of the growth
hormone (GH)-insulin-like growth factor-I axis by daily oral
administration of a GH secretagogue (MK-0677) in healthy elderly
subjects. J Clin Endocrinol Metab 81:42494257[Abstract]
-
Howard AD, Feighner SD, Cully DF, Arena JP, Liberator PA,
Rosenblum CI, Hamelin M, Hreniuk DL, Palyha OC, Anderson J, Paress PS,
Diaz C, Chou M, Liu KK, McKee KK, Pong S-S, Chaung L-YP, Elbrecht A,
Dashkevicz M, Heavens R, Rigby M, Sirinathsinghji DJS, Dean DC, Melillo
DG, Patchett AA, Nargund RP, Griffin PR, DeMartino JA, Gupta SK,
Schaeffer JM, Smith RG, Van der Ploeg LHT 1996 A receptor
in pituitary and hypothalamus that functions in growth hormone release.
Science 273:974977[Abstract]
-
Smith RG, Feighner S, Prendergast K, Guan X, Howard A 1999 A
new orphan receptor involved in pulsatile growth hormone release.
Trends Endocrinol Metab 10:128135[CrossRef][Medline]
-
Momany FA, Bowers CY, Reynolds GA, Chang D, Hong A, Newlander
K 1981 Design, synthesis, and biological activity of peptides which
release growth hormone, in vitro. Endocrinology 108:3139[Abstract]
-
Bowers CY, Momany FA, Reynolds GA, Hong A 1984 On the in
vitro and in vivo activity of a new synthetic
hexapeptide that acts on the pituitary to specifically release growth
hormone. Endocrinology 114:15371545[Abstract]
-
Bowers CY, Reynolds GA, Durham D, Barrera CM, Pezzoli SS,
Thorner MO 1990 Growth hormone (GH)-releasing peptide stimulates GH
release in normal man and acts synergistically with GH-releasing
hormone. J Clin Endocrinol Metab 70:975982[Abstract]
-
Smith RG, Cheng K, Schoen WR, Pong S-S, Hickey GJ, Jacks TM,
Butler BS, Chan WW-S, Chaung L-YP, Judith F, Taylor AM, Wyvratt Jr MJ,
Fisher MH 1993 A nonpeptidyl growth hormone secretagogue.
Science 260:16401643[Medline]
-
Patchett AA, Nargund RP, Tata JR, Chen M-H, Barakat KJ,
Johnston DBR, Cheng K, Chan WW-S, Butler BS, Hickey GJ, Jacks TM,
Scleim K, Pong S-S, Chaung L-YP, Chen HY, Frazier E, Leung KH, Chui
S-HL, Smith RG 1995 The design and biological activities of
L-163,191 (MK-0677): a potent orally active growth hormone
secretagogue. Proc Natl Acad Sci USA 92:70017005[Abstract]
-
Smith RG, Van der Ploeg LHT, Cheng K, Hickey GJ, Wyvratt Jr
MJ, Fisher MH, Nargund RP, Patchett AA 1997 Peptidomimetic regulation
of growth hormone (GH) secretion. Endocr Rev 18:621645[Abstract/Free Full Text]
-
McKee KK, Palyha OC, Feighner SD, Hreniuk DL, Tan C, Smith RG,
Van der Ploeg LHT, Howard AD 1997 Molecular analysis of growth hormone
secretagogue receptors (GHS-Rs): cloning of rat pituitary and
hypothalamic GHS-R type 1a cDNAs. Mol Endocrinol 11:415423[Abstract/Free Full Text]
-
Guillemin R, Brazeau P, Bohlen P, Esch F, Ling N,
Wehrenberg W 1982 Growth hormone-releasing factor from a human
pancreatic tumor that caused acromegaly. Science 218:585587[Medline]
-
Rivier J, Spiess J, Thorner MO, Vale W 1982 Characterization
of a growth hormone-releasing factor from a human pancreatic islet
tumour. Nature 300:276278[Medline]
-
Smith RG, Pong S-S, Hickey GJ, Jacks TM, Cheng K, Leonard RJ,
Cohen CJ, Arena JP, Chang CH, Drisko JE, Wyvratt Jr MJ, Fisher MH,
Nargund RP, Patchett AA 1996 Modulation of pulsatile GH
release through a novel receptor in hypothalamus and pituitary gland.
Recent Prog Horm Res 51:261286[Medline]
-
Guan X-M, Yu H, Palyha OC, McKee KK, Feighner SD,
Sirinathsinghji DJS, Smith RG, Van der Ploeg LHT, Howard AD 1997 Distribution of mRNA encoding the growth hormone secretagogue receptor
in brain and peripheral tissues. Mol Brain Res 48:2329[CrossRef][Medline]
-
Feighner SD, Tan CP, McKee KK, Palyha OC, Hreniuk DL, Pong
S-S, Austin CP, Figureoa D, MacNeil D, Cascieri MA, Nargund R,
Bakshi R, Abramovitz M, Stocco R, Kargman S, ONeill G, Van Der Ploeg
LHT, Evans J, Patchett AA, Smith RG, Howard AD 1999 Receptor for motilin identified in the human gastrointestinal
system. Science 284:21842188[Abstract/Free Full Text]
-
McKee KK, Tan CP, Palyha OC, Liu J, Feighner SD, Hreniuk DL,
Smith RG, Howard AD, Van der Ploeg LHT 1997 Cloning and
characterization of two Human G protein-coupled receptor genes related
to the GH secretagogue and neurotensin receptors. Genomics 46:426434[CrossRef][Medline]
-
Tan CP, McKee KK, Liu Q, Palyha OC, Feighner SD, Hreniuk DL,
Smith RG, Howard AD 1998 Cloning and characterization of a human and
murine T-cell orphan G protein-coupled receptor similar to the GH
secretagogue and neurotensin receptors. Genomics 52:223229[CrossRef][Medline]
-
Hinegardner R 1968 Evolution of cellular DNA content in
teleost fishes. Am Naturalist 102:517523[CrossRef]
-
Brenner S, Elgar G, Sandford R, Macrae A, Venkatesh B,
Aparicio S 1993 Characterization of the pufferfish
(Fugu)genome as a compact vertebrate genome. Nature 366:265268[CrossRef][Medline]
-
Baxendale S, Abdulla S, Elgar G, Buck D, Berks M, Micklem G,
Durbin R, Bates G, Brenner S, Beck S 1995 Comparative sequence analysis
of the human and pufferfish Huntingtons disease genes. Nat Genet 10:6776[Medline]
-
Elgar G, Rattray F, Greystrong J, Brenner S 1995 Genomic
structure and nucleotide sequence of the p55 gene of the puffer fish
Fugu rubripes. Genomics 27:442446[CrossRef][Medline]
-
Mason PJ, Stevens DJ, Luzzatto L, Brenner S, Aparicio S 1995 Genomic structure and sequence of the Fugu rubripes
glucose-6-phosphate dehydrogenase gene (G6PD). Genomics 26:587591[CrossRef][Medline]
-
Macrae AD, Brenner S 1995 Analysis of the dopamine receptor
family in the compact genome of the puffer fish Fugu
rubripes. Genomics 25:436446[CrossRef][Medline]
-
Macrae AD, Brenner S 1994 One armed PCR (OA-PCR):
amplification of genomic DNA from a single primer domain. Genomics 24:176178[CrossRef][Medline]
-
Feighner SD, Howard AD, Prendergast K, Palyha OC, Hreniuk DL,
Nargund RP, Underwood D, Tata JR, Dean DC, Tan C, McKee KK, Woods JW,
Patchett AA, Smith RG 1998 Structural requirements for the
activation of the human GH secretagogue receptor by peptide and
non-peptide secretagogues. Mol Endocrinol 12:137145[Abstract/Free Full Text]
-
van Rhee AM, Jacobson KA 1996 Molecular architecture of G
protein-coupled receptors. Drug Dev Res 37:138[CrossRef]
-
Ballesteros JA, Weinstein H 1995 integrated methods for the
construction of three-dimensional models and computational probing of
structure-function relations in G protein-coupled receptors. In:
Sealfon SC (ed) Methods in Neurosciences. Academic Press,
San Diego, CA, pp 366428
-
Yang L, Morriello G, Patchett AA, Leung K, Jacks T, Cheng K,
Schleim DD, Fenney W, Chan W, Chiu S-HL, Smith RG 1998 1-[2(R)-(2-Amino-2-methylpropionyl-amino)-3-(1H-indol-3-yl)propionyl]-3-benzylpiperidine-3-(S)-carboxylic
acid ethyl ester (L-163, 540): a potent, orally bioavailable, and
short-duration GH secretagogue. J Med Chem 41:24392441[CrossRef][Medline]
-
Nargund RP, Patchett AA, Bach MA, Murphy MG, Smith RG 1998 Peptidomimetic GH secretagogues - design considerations and therapeutic
potential. J Med Chem 41:31033127[CrossRef][Medline]
-
Murphy D, Si-Hoe S-L, Brenner S, Venkatesh B 1998 Something
fishy in the rat brain: molecular genetics of the
hypothalamo-neurohypophysial system. Bioessays 20:741749[CrossRef][Medline]
-
Carmignac DF, Bennett PA, Robinson IC 1998 Effects of GH
secretagogues on PRL release in anesthetized dwarf (dw/dw) rats.
Endocrinology 139:35903596[Abstract/Free Full Text]
-
Bennett PA, Thomas GB, Howard AD, Feighner SD, Van der
Ploeg LHT, Smith RG, Robinson ICAF 1997 Hypothalamic GH
secretagogue-receptor (GHS-R) expression is regulated by GH in the rat.
Endocrinology 138:45524557[Abstract/Free Full Text]