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Department of Biology, University of Victoria (D.B.P., M.E.P., N.M.S.), Victoria, British Columbia, Canada V8W 2Y2; The Clayton Foundation Laboratories for Peptide Biology, The Salk Institute (J.R.), La Jolla, California 92037; National Marine Fisheries Service (P.S.), Seattle, Washington 98112; Joslin Diabetes Center (D.B.P.), Boston, Massachusetts 02215; and the Swiss Federal Institute for Environmental Science and Technology (M.E.P.), Duebendorf, Switzerland
Address all correspondence and requests for reprints to: Dr. Nancy Sherwood, Department of Biology, University of Victoria, Victoria, British Columbia, Canada V8W 2Y2. E-mail: nsherwoo{at}uvic.ca
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Abstract |
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Introduction |
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More recently, a neuropeptide named pituitary adenylate cyclase-activating polypeptide (PACAP) was reported to release GH in mammals (17, 18). This peptide is encoded on a separate gene from GRF in mammals (19). In addition, there is another peptide that has structural similarity to GRF and is encoded in the PACAP gene. This molecule, named PACAP-associated peptide (PRP), has no known function to date (20). PACAP and PRP are structurally related to GRF, and all three peptides are members of a superfamily of peptides that includes glucagon, vasoactive intestinal peptide (VIP), peptide histidine-methionine, glucose-dependent insulin-inducing peptide (GIP), and secretin.
In 1992 a GRF-like peptide isolated from carp brains was found to have structural similarity with mammalian GRF peptides and to stimulate GH release (21). We reported that a cDNA isolated from salmon brain encoded two peptides in the same precursor (22). One of these peptides is related to the carp GRF, mammalian PRP, and mammalian GRF. The other peptide is PACAP, which has a sequence identity of 92% to amphibian (23) and mammalian PACAP (17).
It is clear from these studies that structural analysis of the fish gene and physiological studies of the encoded peptides are needed to determine whether the regulation of growth in fish is distinct from that in mammals. We identify the salmon (s) GRF-like/PACAP gene and its tissue distribution and examine whether the two peptides encoded in this gene have a role in the central control of growth in fish. Exon skipping in the fish gene is considered a mechanism to express the more potent of the GH-releasing peptides. We also consider whether the encoding of GRF and PACAP in a single gene in fish implies a coordinated function that should be considered in mammals, although the two neuropeptides are coded in separate genes.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Fix II (Stratagene, La
Jolla, CA), provided by Dr. R. Devlin, was screened with an 135-bp
sockeye salmon cDNA probe labeled with [32P]deoxy-CTP
(DuPont, Wilmington, DE). This probe, amplified by PCR with primers
CSC33 (5'-CA[CT]-GCIGA[CT]GGIATGTT[CT]AA-3') and NMS 1
(5'-TGACAGAGGCTCTGTGTC-3'), contained the entire coding sequence of the
GRF-like region from positions 243–378 as described by Parker et
al. (22). Filters were washed four times for 5 min each time at
room temperature in 2 x SSC (standard saline citrate)-0.1% SDS,
twice at 55 C for 20 min each time in 1 x SSC-0.1% SDS, and then
for 20 min at 55 C in 0.2 x SSC-0.1% SDS. Before rescreening the
first round of positive clones, 10 µl DNA lysate from each clone were
amplified using PCR with 40 pmol of the primers CSC33 and NMS1. PCR was
performed with 50 mM KCl, 10 mM Tris-HCl, 0.1%
Triton X-100, 1.5 mM MgCl2, 200 µm
deoxy-NTPs, and 2.5 U Taq DNA polymerase (Promega, Madison,
WI). DNA was amplified using a step program of 1 cycle at 94 C for 3
min, 50 C for 2 min, and 72 C for 5 min, followed by 36 cycles of 94 C
for 1 min, 50 C for 1 min, and 72 C for 1 min 30 sec
(Perkin-Elmer/Cetus, Norwalk, CT). The last cycle had a final extension
of 5 min at 72 C. PCR-positive lysates were rescreened to obtain single
isolated plaques. Hybridization and washing stringencies were increased
in subsequent screenings, with a final wash at 60 C in 0.1 x
SSC-0.1% SDS.
Subcloning and sequencing
GRF/PACAP clones were digested with the restriction enzymes
HaeIII, SacI, AccI, and
XbaI, and the fragments were subcloned into pBluescript
KS+ II by blunt end ligation as previously described (22).
Plasmid DNA for exonuclease III (ExoIII) digestion was
prepared using Qiagen Plasmid Preps (Qiagen Corp., Chatsworth, CA).
ExoIII deletions and S1 nuclease digestions were performed
in accordance with the Promega Erase-a-Base System (Protocols and
Applications Guide, Promega Corp.). Double stranded DNA sequencing was
carried out using the dideoxy chain termination method with Sequenase
version 2.0 (U.S. Biochemical Corp., Cleveland, OH) or by thermocycle
sequencing with vent (exo-) DNA polymerase (Circumvent, New England
Biolabs, Beverly, MA) according to the manufacturer’s instructions.
Sequencing products were run on 5% polyacrylamide in 7 M
urea wedge and normal gels (Bio-Rad Laboratories, Richmond, CA). Gels
were dried under vacuum at 80 C and then exposed to XAR-5 film (Eastman
Kodak, Rochester, NY) for 16–24 h. Sequence-specific primers were
synthesized at the Regional DNA Synthesis Laboratory, University of
Calgary (Calgary, Canada), or at the HSC/Biotechnology Center (Toronto,
Canada). M13 universal and reverse primers were obtained from
Stratagene.
Primer extension
Primer extension analysis was performed as previously described
(24) with modifications. A 30-bp primer
(5'-TCGCCCCCATCCTCTCGGAATGTAACACTG-3') was end labeled with 200 µCi
[32P]ATP in 1x One-Phor-All Buffer PLUS (Pharmacia,
Piscataway, NJ) with 9.2 U T4 PNK. The reaction mixture was
incubated at 37 C for 40 min, followed by heat denaturation of the
enzyme at 65 C for 5 min. Labeled primer (2.5 ng, 106 cpm)
was ethanol precipitated with 20 µg sockeye salmon messenger RNA
(mRNA) overnight at -20 C. The mRNA-primer mix was dissolved in 10
µl diethylpyrocarbonate-treated water plus 4 µl 5 x BRL first
strand buffer [250 mM Tris-HCl (pH 8.3), 375
mM KCl, and 15 mM MgCl2] and
heated for 5 min at 85 C, followed by a 10-min incubation at 50 C. RNA
guard (Pharmacia) was added, and the mixture was incubated for 4.5
h at 50 C. Reverse transcription (RT) was performed at 42 C for 1
h with the addition of dithiothreitol to 10 mM, dNTPs to
0.5 mM, and 200 U SuperScript ribonuclease H-
reverse transcriptase (Life Technologies, Rockville, MD). The sample
was phenol/chloroform extracted and precipitated with 2.5
mM ammonium acetate and 2.5 vol ethanol. DNA was dissolved
in TE buffer (10 mM Tris·Cl, 1 mM EDTA), pH
8.0, and running buffer (New England Biolabs, Beverly, MA), and a
sample was run on 5% polyacrylamide-urea gel.
RT-PCR isolation of GRF/PACAP from salmon species and tissues
Sockeye salmon [Oncorhynchus nerka (O.
nerka)], coho salmon (O. kisutch), chinook salmon
(O. tshawytscha), rainbow trout (O. mykiss), and
Atlantic salmon (Salmo salar) were killed by decapitation,
and the brains were immediately excised and frozen in liquid nitrogen.
Heart, intestine, liver, and pyloric cecum/pancreas from sockeye salmon
were also recovered and rapidly frozen. RNA was isolated by the
guanidium isothiocyanate method of Chomczynski and Sacchi (25).
Polyadenylated [poly(A)+] mRNA was isolated by means of
oligo(deoxythymidine)-cellulose using the FastTrack mRNA kit
(Invitrogen, San Diego, CA). Single stranded cDNA was synthesized from
1 µg mRNA by random priming with
oligo(deoxythymidine)18–20 (Pharmacia). The cDNA was
diluted 1:10, and 1 µl was used as the template in PCRs. The primers
NMS10 (5'-AATGCAGATCTCTCCACACGGTAATAGCAGG-3') and NMS5
(5'-AGTCACCTAGCGAGAACACAAG-3') were synthesized to correspond to the
5'- and 3'-untranslated regions of the sockeye sGRF/PACAP cDNA
sequence, respectively (22). DNA was amplified in 1 x Promega
buffer as described above, using a program of 5 cycles of 94 C for 2
min, 58 C for 2 min, and 72 C for 2 min, followed by 30 cycles of 94 C
for 1 min, 57 C for 1.5 min, and 72 C for 1.5 min.
PCR products were separated on 1.5% agarose gels, the bands were excised, and the DNA was recovered by centrifugation through ProSpin columns (Millipore Corp., Bedford, MA). The eluted DNA was phenol/chloroform extracted, then ethanol precipitated and ligated into pGEM Vector-T plasmid (Promega). Plasmids were electroporated into Escherichia coli JM109 cells using a Bio-Rad Gene Pulser following the manufacturer’s instructions. Colonies were picked by blue and white selection, and the bacteria were grown overnight at 37 C. Plasmid DNA was isolated using a Wizard Miniprep kit (Promega), and the inserts were sequenced.
RT-PCR products of the various sockeye tissues were recovered as described above and sequenced directly using the Circumvent Thermocyle Sequencing kit (New England Biolabs) with primers NMS5 and NMS10.
RT-PCR isolation of PRP/PACAP from mouse brain
The brains were removed from two mice that had been killed with
CO2. The brains were frozen in liquid N2.
Poly(A)+ and cDNA were prepared from the brain as described
for the salmon tissues. Primers PA1
(5'-ATGACCATGTGTAGCGGAGCAAGGTTGGC-3') and PA2
(5'-CGCTACAAGTACGCTATTCGGCGTCC-3') were synthesized corresponding to
the 5'-end of the signal peptide and the 3'-end of PACAP of the rat
PACAP gene (26). PCR was performed as described above, except that a
program of 35 cycles of 94 C for 1.5 min, 56 C for 1.5 min, and 72 C
for 1.5 min was used. The PCR products were vector T cloned and
partially sequenced to confirm the identity of the band.
Peptide synthesis
Sockeye sGRF
(His-Ala-Asp-Gly-Met-Phe-Asn-Lys-Ala-Tyr-Arg-Lys-Ala-Leu-Gly-Gln-Leu-Ser-Ala-Arg-Lys-Tyr-Leu-His-Ser-Leu-Met-Ala-Lys-Arg-Val-Gly-Gly-Gly-Ser-Thr-Met-Glu-Asp-Asp-Thr-Glu-Pro-Leu-Ser-OH)
and sockeye sPACAP
(His-Ser-Asp-Gly-Ile-Phe-Thr-Asp-Ser-Tyr-Ser-Arg-Tyr-Arg-Lys-Gln-Met-Ala-Val-Lys-Lys-Tyr-Leu-Ala-Ala-Val-Leu-Gly-Lys-Arg-Tyr-Arg-Gln-Arg-Tyr-Arg-Asn-Lys-NH2)
were synthesized using standard solid phase methodologies on a
chloromethyl (2 g) and a paramethylbenzhydrylamine (2 g) resin to yield
the peptide-free acid (GRF) and the amide (PACAP), respectively. The
tertiobutyloxycarbonyl strategy with trifluoroacetic acid deblocking
and final hydrofluoric acid cleavage and deprotection was used (27, 28). Purification using preparative reverse phase HPLC and several
eluting solvent systems yielded highly purified peptides (29) (yield
for GRF, 62 mg; yield for PACAP, 176 mg). Purified peptides were
characterized by amino acid analysis giving the expected ratios,
capillary zone electrophoresis (GRF >95% pure, PACAP >70% pure),
and liquid secondary ion mass spectrometry (monoisotopic mass for GRF:
calculated, 4937.46; found, 4937.5; average mass for PACAP: calculated,
4655.41; found, 4654.9).
In vitro pituitary cell assays
For each experiment, 8–12 coho salmon (200–300 g) were
anesthetized in MS 222 and decapitated, and the pituitaries were
removed and placed in modified Hanks’ Balanced Salt Solution (HBSS;
136.9 mM NaCl, 5.4 mM KCl, 0.8 mM
MgSO4, 0.3 mM Na2HPO, 8.0
mM NaHCO3, 1.26 mM
CaCl2, 2.0 mM glucose, 10 mM HEPES,
and phenol red) on ice. Pituitaries were washed once in HBSS containing
5-fold concentrated antibiotics, then three times in HBSS containing
1-fold concentrated antibiotics. After removal of the HBSS wash,
several drops of 0.1% trypsin (1:250; Life Technologies, Grand Island,
NY) in HBSS containing 0.3% BSA were added. The pituitaries were
minced to approximately 0.1 mM using a sterile razor blade
and subsequently transferred to a flask for trypsinization. Pituitary
tissue was stirred gently for 30 min at 15 C in air, and centrifuged at
200 x g to pellet the cells. The cells were suspended
in 10 ml trypsin inhibitor (25 mg/ml HBSS and 0.3% BSA) and incubated
for 5 min at 15 C with gentle shaking. The cells were centrifuged and
suspended in 10 ml deoxyribonuclease II (100 µg/10 ml HBSS and 0.3%
BSA), incubated, and centrifuged as previously described. Pituitary
cells were washed once in 10 ml Ca2+-free HBSS and 2
mM EGTA, once in Ca2+ free HBSS and 1
mM EGTA, and once in Ca2+-free HBSS, then
suspended in 10 ml Ca2+-free HBSS and filtered through a
100-mesh stainless steel screen. Cell density was determined using a
hemocytometer, whereas cell viability was determined by trypan blue
staining. Cells were centrifuged and suspended in Waymouth’s medium
(10 mM HEPES, 8 mM NaHCO3, 2
mM glucose, 100 U/ml penicillin G, 100 µg/ml streptomycin
sulfate, and 10 µg/ml gentamycin, pH 7.63) containing 2% UltroSer Sf
(Life Technologies). Dispersed pituitary cells were incubated in
96-well culture plates [Falcon 3872 (Becton Dickinson, Oxnard, CA);
250 µl/well at 2.5 x 105 cells/ml] at 15 C in air
for 72 h, then overnight with HBSS.
Peptides were dissolved in HBSS and 0.1% BSA immediately before addition to the cell cultures. The pituitary cells were then incubated with peptide in HBSS and 0.3% BSA for 4 or 24 h. In the assays, ascorbic acid was added to the peptide solution and media to a final concentration of 0.5 mM. Six replicates were used for each peptide concentration. Culture medium was either analyzed immediately or frozen at -80 C. Medium was analyzed for GH by RIA (30, 31). The recombinant chum sGH was provided by Kyowa Hakko Kogyo Co. (Tokyo, Japan), whereas the primary antiserum (HU-85) was raised in a rabbit against the recombinant chum sGH and provided by Dr. Akihiko Hara, Hokkaido University (Hakodate, Japan). Data analyses were performed by one-factor ANOVA followed by Fischer’s protected least significant difference test for significance at P < 0.05.
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Results |
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shows that exon I encodes the 5'-untranslated
region. Exon 2 encodes 10 bp of the 5'-untranslated region, the signal
peptide, and part of the 5'-cryptic peptide. The majority of the
5'-cryptic peptide is encoded on exon 3. Exon 4 encodes the last 3
amino acids of the cryptic peptide and the first 32 amino acids of the
GRF-like peptide. Exon 5 encodes the remaining 13 amino acids of the
GRF-like peptide, a dibasic amino acid processing site, PACAP, and the
3'-untranslated region.
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Transcription initiation site
Analysis of the sGRF-like/PACAP gene by primer extension was used
to determine the potential transcription start site(s). Three major
extension products from brain were observed corresponding to
nucleotides that are 465, 472, and 473 bp upstream of the translation
start site (Fig. 3). Two minor bands were also evident
between the major bands. The negative control, which was mRNA prepared
from heart tissue, did not result in an extension product (Fig. 3).
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and 5A. Sequence analysis shows that
the two bands represent long and short precursors encoded in the
sGRF-like/PACAP gene (Fig. 6). The region deleted in the
short cDNA precursor corresponds to the excision of exon 4 containing
most of the GRF domain of the gene (Figs. 1
and 6). This exon codes for
the last 3 amino acids of the cryptic peptide and the first 32 amino
acids of the GRF-like molecule. The gene structure (Fig. 1) shows
exon/intron splice sites at nucleotide positions corresponding to the
exon splice sites indicated in Fig. 6.
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).
Alternative splicing occurs not only in the brain, but also in other
specific tissues (Fig. 4A). Two bands of the same size as the
full-length and short forms of the cDNA precursor were expressed in the
pyloric cecum/pancreas and the large and small intestine in 1.5-yr-old
fish (Fig. 4A). We did not find any GRF-like/PACAP mRNA expression in
liver, heart (Fig. 4A), or gonads (not shown) in fish of the same age.
Tubulin was consistently amplified in all tissues as a control of mRNA
quality (Fig. 4B).
Tissue expression of mouse brain PACAP cDNA
Only one band resulted from the RT-PCR of mouse brain cDNA with
primers that matched the sequence of the rat PACAP cDNA. The sequence
of the mouse PCR product was that of the long precursor containing
full-length PRP and PACAP (Fig. 5B). In contrast, analysis of five
salmonid species (sockeye salmon, coho salmon, chinook salmon, Atlantic
salmon, and rainbow trout) showed that both the long and short cDNA
precursors were expressed in the brains of all of these species (Fig. 5A). Both the long and short bands were sequenced in their entirety for
each species. The excision of exon 4 occurred in each species.
In vitro effects of GRF and PACAP on GH release
In all bioassays PACAP stimulated GH release in a
concentration-dependent manner (Fig. 7). PACAP
stimulated GH release above control levels at both 4 and 24 h of
incubation. Although the levels of GH in the incubation medium were
higher at 24 h than at 4 h, the fold stimulations at these
two time points were similar (Fig. 7A). The release of GH by PACAP was
significant at a dose as low as 0.1 nM compared to the
control value. Ascorbic acid was added in the experiments to prevent
oxidation of the peptides because sPACAP has one methionine and
sGRF-like peptide has three methionines.
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). sGRF-like peptide was effective at
0.001 nM at 4 h, but did not consistently result in a
significant release of GH at the higher doses. There was not a
significant release of GH at 24 h in response to GRF-like
peptide. It should be noted that synthetic sockeye sGRF and sockeye PACAP are each one amino acid different from the peptides deduced from the gene reported here. Each peptide, however, is identical to those deduced from one of the two cDNAs that we isolated earlier from a sockeye brain library (22); the other cDNA and the gene encode identical peptides.
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Discussion |
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Structural organization of the GRF-like/PACAP gene was determined by comparison to the two sockeye cDNA sequences previously obtained by Parker et al. (22). The gene contains five exons; the GRF code spans exons 4 and 5, whereas PACAP is exclusively encoded on exon 5. Structural organization of the salmon gene also shows that it is more closely related to the hPRP/PACAP gene than to the hGRF gene. PACAP and the region reported to be the biologically active core of GRF-like peptide (amino acids 1–32) (32) are encoded on distinct exons of the salmon gene. The presence of active peptides encoded on separate exons is characteristic of members of the glucagon superfamily (33). The size and sequence identity of the exons encoding peptide domains are well conserved between the salmon and human PACAP genes.
The introns of the sGRF-like/PACAP gene contain the consensus GT/AG splice donor/acceptor sites (34), a pyrimidine-rich region just upstream of the 3'-splice site and a branch point recognition sequence, all associated with intron splice recognition. The main difference in the structural organization of the sockeye salmon gene compared to that of the human, is the smaller introns in the former. The sGnRH genes also have smaller introns than their mammalian counterparts (35, 36). Whether regulatory differences exist in the shorter introns of fish compared to those of mammals is not yet known.
sPACAP is more tightly conserved than sGRF-like peptide
A comparison of the biologically active core of sGRF-like
peptide-(1–29) to hPRP-(1–29) shows 59% and that to hGRF-(1–29)
shows 55% sequence identity. This suggests that the sGRF-like peptide
has structural similarity to both human peptides. However, sGRF-like
peptide is closer to hPRP (54%) than GRF (40%) if the full-length
(44–48 amino acids) peptide is considered. A more striking sequence
identity was observed for the PACAP molecules. sPACAP has 90% sequence
identity with hPACAP-(1–38) and 100% identity with hPACAP-(1–27).
This implies that a greater functional constraint exists on PACAP than
on the GRF-like peptide and may explain the past difficulty in
identifying GRF for vertebrates other than mammals.
The potential cleavage sites in the salmon precursor are also conserved. There are dibasic residues that are potential processing sites, the cleavage of which would result in PACAP-38, and GRF-like peptide-45 or their truncated versions of PACAP-27 or GRF-29.
The gene has multiple start sites and polyadenylation sites
To define sequences that may be important for expression of the
GRF-like/PACAP gene, we sequenced the 5'-flanking region of the gene.
The length of the 5'-untranslated region is not known for the PACAP
cDNA species reported to date, although it is at least 0.5–0.6
kilobases in rat and sheep. In our study of the transcriptional
initiation site, we found three major and two minor extension products
upstream of the translation start site. The two major bands at
nucleotides 472 and 473 upstream from the start site may represent a
single initiation site at the adenine; the band at the thymidine may be
part of the "cap effect," which is caused by stopping the
polymerase 1–2 nucleotides before the capping site. However, the third
band, which corresponds to a guanidine, is seven nucleotides downstream
from the A and may be a distinct initiation site.
Possible regulatory elements were identified in the 5'-flanking region, including a TATA motif that begins 32 nucleotides upstream from the most 5'-start site. Consensus sites for activating protein-2 (AP-2), CAAT, and a half-cAMP response element (CRE) are also present. The initiation site was not determined for the hPACAP gene (19), but the 5'-flanking region of the sGRF-like/PACAP gene resembles the human and rat genes for VIP (37, 38), glucagon (39), GRF (12, 16), GIP (40), and secretin (41) in that they all have a TATA sequence, most have a CAAT sequence, and a few have a full or half-CRE and/or an AP-2 consensus site.
Sequence analysis of the 3' region of the gene revealed three polyadenylation signals. We had previously only identified a cDNA terminating with a poly(A) tail 16 nucleotides after the first polyadenylation signal. Downstream of the first polyadenylation signal were several (U)TG-rich elements (TTTTTCT, TGTTTT, and TTTGTA), which were reported to be involved in polyadenylation signal recognition and 3'-cleavage (42). These elements overlap and are located between 6–20 bp downstream from the end of the mRNA cleavage site.
The mRNA is alternatively spliced in several tissues, resulting in
greater expression of PACAP
Sequence analysis of the two RT-PCR products from brain cDNA shows
that the two bands represent long and short precursors encoded in the
sGRF-like/PACAP gene, confirming our earlier suggestion that the short
precursor might be obtained by exon skipping during processing of the
RNA transcript (22). The region deleted in the short cDNA precursor
corresponds to the excision of exon 4 containing most of the GRF domain
of the gene.
Alternative splicing occurs not only in the salmon brain, but also in the pyloric cecum/pancreas and the large and small intestine in 1.5-yr-old fish. In mammals, GRF has been isolated as a peptide or mRNA from brain, testis, ovary, placenta, and pancreatic tumors (43). PACAP peptide and mRNA have been identified to date from mammalian brain and testis (43). sGRF-like/PACAP expression is unlike that of mammalian GRF or PACAP, because the fish mRNA is found in normal pyloric cecum/pancreas and intestine. Although we did not find GRF/PACAP mRNA in salmon gonads, the explanation may be that young fish were used. In adult catfish, we found that GRF-like/PACAP was expressed in both ovary and testis as well as in stomach (44).
Mouse PACAP mRNA is not alternatively spliced in contrast to salmon
mRNA in five species
It is interesting that salmon have a short precursor that has not
been identified in mammals. To confirm this, we used mouse brain to
show that only the long precursor containing full-length PRP and PACAP
is expressed. Both the long and short cDNA precursors were expressed in
the brain of each of the five salmonid species; in each case, exon 4
was excised in the short precursor.
Alternate splicing of the RNA means that GRF-like peptide and PACAP
need not be released in equimolar quantities as expected from cleavage
of the long precursor, but that additional quantities of PACAP could be
released from the short precursor. The ratio of expression of the long
and short precursors can vary in the brains of different salmonid
species, as shown in Fig. 5. However, the physiological relevance of
altering the ratio of the two peptides that both release GH may stem
from other actions not yet identified for the salmon GRF-like and PACAP
peptides.
PACAP is a potent releaser of GH in vitro
Our data suggest that sPACAP, compared with sGRF-like peptide, is
the more potent releaser of GH, in that PACAP resulted in a good
concentration-release curve and in GH release that was prolonged, as
measured at both 4 and 24 h. GRF-like peptide had a lower
threshold concentration than PACAP at 4 h, but the GH level was
variable and was not significantly elevated at the highest
concentration of sGRF-like peptide. Our results agree with studies on
carp GRF-like peptide showing that GH is released in vitro
from fish pituitary cells. However, in contrast to our studies, carp
GRF-like peptide released GH in a concentration-dependent manner from
rainbow trout pituitary cells (but only with ascorbic acid) (32) and
from goldfish pituitary cells (even without ascorbic acid) (21). The
reason that we did not obtain a concentration-dependent response to
sGRF is not thought to be due to a difference between salmon and carp
GRF structures, because the peptides are 91% identical. A more likely
explanation is that our experiments used a homologous assay in which
the peptides and pituitary cells were from the same species. Hence,
both studies suggest that at least one physiological function of salmon
and carp GRF is to release GH from the pituitary. The possibility
cannot be discounted that a more effective sGRF has yet to be found.
Meanwhile, the reason for the release of both PACAP and GRF together is
not yet clear.
Gene duplication may explain separate PACAP and GRF genes in
mammals
At present, only one GRF-like/PACAP gene has been identified in
salmon. Our Southern blot analysis suggested that more than one gene or
different allelic polymorphisms are present (22). Because salmon are
tetraploid, they contain duplicates of their genes, but not all
duplicates have been retained during evolution (45). Two genes for
melanin-concentrating hormone and two precursors for vasotocin and CRF
(46) have been identified in salmon.
The identification of the GRF-like/PACAP gene in a fish provides insight into the evolution of this superfamily of hormones. In mammals, the related structures and similar localization in the brain and gut of GRF, PACAP, VIP, peptide histidine-methionine, glucagon, secretin, and GIP suggest that the peptides evolved due to repeated gene duplications. The tandem arrangement of some of the peptides in precursors implies that exon duplication has occurred. We hypothesize that fish contain a gene encoding both GRF and PACAP, although it is possible that a gene encoding GRF-like peptide separately has yet to be found. Gene duplication in evolution after the fish-tetrapod split may have led to two separate genes in mammals: GRF and PRP/PACAP. Indeed, a function has been identified for neither PRP in the mammalian PACAP genes nor C peptide (or cryptic peptide) encoded in the GRF gene. Hence, the tandem coding in the mammalian genes does not appear to produce a functional peptide after the proposed gene duplication. Regardless of the origin, separate regulation of the GRF and PACAP genes in mammals may provide finer control of the two neuropeptides compared to that in fish.
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Acknowledgments |
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Footnotes |
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Received July 24, 1996.
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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 Q. Jiang, M. He, X. Wang, and A. O. L. Wong Grass carp somatolactin: II. Pharmacological study on postreceptor signaling mechanisms for PACAP-induced somatolactin-{alpha} and -{beta} gene expression Am J Physiol Endocrinol Metab, August 1, 2008; 295(2): E477 - E490. [Abstract] [Full Text] [PDF]
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 J. M. Lugo, A. Rodriguez, Y. Helguera, R. Morales, O. Gonzalez, J. Acosta, V. Besada, A. Sanchez, and M. P. Estrada Recombinant novel pituitary adenylate cyclase-activating polypeptide from African catfish (Clarias gariepinus) authenticates its biological function as a growth-promoting factor in low vertebrates J. Endocrinol., June 1, 2008; 197(3): 583 - 597. [Abstract] [Full Text] [PDF]
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 K. H. Sze, H. Zhou, Y. Yang, M. He, Y. Jiang, and A. O. L. Wong Pituitary Adenylate Cyclase-Activating Polypeptide (PACAP) as a Growth Hormone (GH)-Releasing Factor in Grass Carp: II. Solution Structure of a Brain-Specific PACAP by Nuclear Magnetic Resonance Spectroscopy and Functional Studies on GH Release and Gene Expression Endocrinology, October 1, 2007; 148(10): 5042 - 5059. [Abstract] [Full Text] [PDF]
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Y. Wang, J. Li, C. Y. Wang, A. H. Y. Kwok, and F. C. Leung Identification of the Endogenous Ligands for Chicken Growth Hormone-Releasing Hormone (GHRH) Receptor: Evidence for a Separate Gene Encoding GHRH in Submammalian Vertebrates Endocrinology, May 1, 2007; 148(5): 2405 - 2416. [Abstract] [Full Text] [PDF]
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 L. T. O. Lee, F. K. Y. Siu, J. K. V. Tam, I. T. Y. Lau, A. O. L. Wong, M. C. M. Lin, H. Vaudry, and B. K. C. Chow Discovery of growth hormone-releasing hormones and receptors in nonmammalian vertebrates PNAS, February 13, 2007; 104(7): 2133 - 2138. [Abstract] [Full Text] [PDF]
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 L. Chi-Wei, S.-L. Chang, and C.-F. Weng Pituitary Adenylate Cyclase-Activating Polypeptide (PACAP) Regulates the Expression of PACAP in Cultured Tilapia Astrocytes Experimental Biology and Medicine, February 1, 2007; 232(2): 262 - 276. [Abstract] [Full Text] [PDF]
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M. Amano, S. Moriyama, M. Iigo, S. Kitamura, N. Amiya, K. Yamamori, K. Ukena, and K. Tsutsui Novel fish hypothalamic neuropeptides stimulate the release of gonadotrophins and growth hormone from the pituitary of sockeye salmon. J. Endocrinol., March 1, 2006; 188(3): 417 - 423. [Abstract] [Full Text] [PDF]
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Y. Wang, A. O. L. Wong, and W. Ge Cloning, Regulation of Messenger Ribonucleic Acid Expression, and Function of a New Isoform of Pituitary Adenylate Cyclase-Activating Polypeptide in the Zebrafish Ovary Endocrinology, November 1, 2003; 144(11): 4799 - 4810. [Abstract] [Full Text] [PDF]
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 S. L. Gray, K. J. Cummings, F. R. Jirik, and N. M. Sherwood Targeted Disruption of the Pituitary Adenylate Cyclase-Activating Polypeptide Gene Results in Early Postnatal Death Associated with Dysfunction of Lipid and Carbohydrate Metabolism Mol. Endocrinol., October 1, 2001; 15(10): 1739 - 1747. [Abstract] [Full Text] [PDF]
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 N. M. Sherwood, S. L. Krueckl, and J. E. McRory The Origin and Function of the Pituitary Adenylate Cyclase-Activating Polypeptide (PACAP)/Glucagon Superfamily Endocr. Rev., December 1, 2000; 21(6): 619 - 670. [Abstract] [Full Text]
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Z. Hu, V. Lelievre, J. Tam, J. W. Cheng, G. Fuenzalida, X. Zhou, and J. A. Waschek Molecular Cloning of Growth Hormone-Releasing Hormone/Pituitary Adenylyl Cyclase-Activating Polypeptide in the Frog Xenopus laevis: Brain Distribution and Regulation after Castration Endocrinology, September 1, 2000; 141(9): 3366 - 3376. [Abstract] [Full Text] [PDF]
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 D. Vaudry, B. J. Gonzalez, M. Basille, L. Yon, A. Fournier, and H. Vaudry Pituitary Adenylate Cyclase-Activating Polypeptide and Its Receptors: From Structure to Functions Pharmacol. Rev., June 1, 2000; 52(2): 269 - 324. [Abstract] [Full Text] [PDF]
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D. Alexandre, Y. Anouar, S. Jegou, A. Fournier, and H. Vaudry A Cloned Frog Vasoactive Intestinal Polypeptide/ Pituitary Adenylate Cyclase-Activating Polypeptide Receptor Exhibits Pharmacological and Tissue Distribution Characteristics of Both VPAC1 and VPAC2 Receptors in Mammals Endocrinology, March 1, 1999; 140(3): 1285 - 1293. [Abstract] [Full Text]
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A. O. L. Wong, M. Y. Leung, W. L. C. Shea, L. Y. Tse, J. P. Chang, and B. K. C. Chow Hypophysiotropic Action of Pituitary Adenylate Cyclase-Activating Polypeptide (PACAP) in the Goldfish: Immunohistochemical Demonstration of PACAP in the Pituitary, PACAP Stimulation of Growth Hormone Release from Pituitary Cells, and Molecular Cloning of Pituitary Type I PACAP Receptor Endocrinology, August 1, 1998; 139(8): 3465 - 3479. [Abstract] [Full Text] [PDF]
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) incubation. B, The effect of
adding synthetic sGRF-like-45 after 4-h (
) or 24-h (
) incubation.
Ascorbic acid was added to the peptide solution and medium. Significant
differences (P < 0.05) compared with the control
values are indicated by a star. Pretreatment values are
isolated points on the left side of each panel.