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Endocrine Unit, Massachusetts General Hospital and Harvard Medical School, Boston, Massachusetts 02114
Address all correspondence and requests for reprints to: Dr. Abdul Abou-Samra, Endocrine Unit, Massachusetts General Hospital and Harvard Medical School, Boston, Massachusetts 02114. E-mail: samra{at}helix.mgh.harvard.edu
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Two CRF receptor subtypes, CRF-R1 and CRF-R2, have been identified in human (14, 15), rat (16, 17, 18), mouse (19, 20, 21), and Xenopus (22). Mammalian CRF-R1, which is the major CRF subtype expressed in the pituitary, has similar binding affinities and activation potencies for CRF, SVG, UI, and UC (19). In contrast, the mammalian CRF-R2 binds sauvagine and UI/UC with higher affinity than CRF (15, 18, 20). Interestingly, unlike other species, Xenopus (x) CRF-R1, which is also expressed in pituitary and brain, binds UI/UC and CRF with higher affinities than SVG (22).
The stress response in teleost fish is similar to that observed in the mammalian system despite being a phylogenetically older species. Handling and confinement stress increase the plasma concentrations of cortisol and POMC peptides within minutes (23). The presence of an additional neuroendocrine organ, the urophysis, which is not present in higher vertebrates, suggests that the fish species have a unique neuroendocrine control system that may be more complex than that of mammals. Here we report the molecular cloning of three distinct complementary DNAs (cDNAs) encoding different CRF receptors from a diploid species of catfish, Ameiurus nebulosus (24). In addition to the two subtypes, CRF-R1 and CRF-R2, known in other species, we identified a third catfish CRF receptor (cfCRF-R3) that has distinct structural and functional properties and tissue expression patterns.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Cell transfection
Full-coding cDNAs encoding cfCRF-R1, cfCRF-R2, cfCRF-R3,
mCRF-R1, or mCRF-R2ß were inserted into pcDNA1 and transfected (10
µg) into 75–95% confluent COS-7 cells in a 15-cm culture dish using
diethylaminoethyl-dextran transfection (25). Twenty-four
hours after transfection, cells were seeded in 24-well plates at a
density of 105 cells/well. Binding and cAMP
accumulation assays were performed 72 h after transfection.
RRA of cloned receptors
Transiently transfected COS-7 cells were incubated with a
modified SVG analog,
125I-[Tyr0,
Gln1, Leu17]sauvagine
(YQLS) (100,000 cpm/well) developed in our laboratory (26)
or commercially available
125I-[Tyr0]sauvagine
(Amersham Pharmacia Biotech, Piscataway, NJ; 100,000
cpm/well) in the presence of increasing concentrations (0–1000
nM) of unlabeled human/rat CRF, UI, SVG, or YQLS at
room temperature for 2 h in a Tris-based binding buffer [50
mM Tris-HCl (pH 7.6), 100 mM NaCl, 5
mM KCl, 2 mM CaCl2, 5%
heat-inactivated horse serum, and 0.05% heat-inactivated FBS]. The
cells were rinsed three times with binding buffer and lysed with 1
M NaOH, and the radioactivity of the cell lysates was
determined by a 
-counter (ICN Micromedic Systems, Horsham, PA). The
apparent dissociation constant, Kd, was derived
from competition studies and is defined as the concentration that
competes off 50% of the bound radioligand, which is referred to as
Ki.
cAMP accumulation assay
Three days after transfection, cells were stimulated with
increasing concentrations of human/rat CRF, UI, or SVG in
HEPES-buffered DMEM containing 20 mM HEPES (pH.7.4), 0.1%
BSA, 100 mg/ml aprotinin, and 2 mM isobutylmethylxanthine
for 15 min at 37 C. After incubation, the supernatant was removed, and
the cells were frozen immediately on dry ice and kept at -80 C until
RIA was performed. Intracellular cAMP was extracted by thawing the
cells in 50 mM HCl and was measured by a specific cAMP RIA
(25).
Northern blot analysis
Total RNA (20 µg) from brain, heart, liver, spleen, and gut
were run on a denaturing formaldehyde/agarose gel and transferred to
GeneScreen Plus nylon membrane (NEN Life Science Products,
Boston, MA). The blot was prehybridized (2–4 h) and hybridized
(overnight) at 42 C in 50% formamide buffer (for cfCRF-R1 and cfCRF-R2
hybridization). Full-length cfCRF-R1, cfCRF-R2, and cfCRF-R3 cDNAs
(
1.2 kb) were labeled by random priming using
[32P]deoxy-CTP (Roche,
Indianapolis, IN). The blot was washed twice at 42 C in 2 x
SSC/0.1% SDS (15 min each), then in 0.2 x SSC/0.1% SDS once at
42 C and once at 68 C. Hybridization of the cfCRF-R3 probe was
performed in the commercially available hybridization buffer (QuickHyb,
Stratagene, La Jolla, CA) following the manufacturer’s
protocol.
In situ hybridization
Catfish brain, pituitary, heart, liver, and gut were removed
immediately after decapitation and frozen on dry ice in OCT compound
(Miles, Elkhart, IN). Frozen sections (12 µm) were prepared and
stored at -80 C. In situ hybridizations were performed as
previously described (27), using
[35S]UTP-labeled probes [500 bp clones
initially cloned; MA3-MA5 (encoding the last four transmembranes and
carboxyl- terminal regions of the receptor) for cfCRF-R1 and
cfCRF-R3, 2d1f-2xr (encoding amino-terminus and first transmembrane
region) for cfCRF-R2]. Sense probes were used as negative controls.
Slides were exposed (Kodak NTB-2 emulsion, Eastman Kodak Co., Rochester, NY) for 2–4 weeks and stained with
hematoxylin and eosin.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The nucleotide sequences of the three full-length cfCRF receptors are
73–78% identical, and their amino acid sequences show 80–85%
sequence conservation (Fig. 1
and Table 1). The cfCRF-R1 (446 amino acids) is
structurally closer to the mammalian CRF-R1 (93% identical), whereas
the cfCRF-R2 (406 amino acids) has the highest homology to the
mammalian CRF-R2 (88% identical). The third receptor subtype, cfCRF-R3
(428 amino acids), has a higher homology to the mammalian CRF-R1 than
CRF-R2 (89% vs. 80% identical, respectively; Table 1). The
amino-termini (NT) of all three catfish CRF receptors vary
significantly from each other and from other known CRF receptors (Fig. 1). The NT of cfCRF-R1 is longer than the corresponding region of the
mammalian receptors and has nine potential N-linked
glycosylation sites, whereas cfCRF-R2 and cfCRF-R3 contain only five
and six of these sites, respectively. The NT of one of the four cloned
cDNAs encoding the cfCRF-R3 was 16 amino acids shorter than the others;
this suggests alternative splicing of the NT of cfCRF-R3.
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Phylogenetic analysis of CRF receptors
An evolutionary tree based on amino acid sequence alignments of
known CRF was constructed (28). The alignment showed that
the cfCRF-R1 and cfCRF-R2 are more closely related to the mammalian
CRF-R1 and CRF-R2, respectively. The cfCRF-R3 is phylogenetically
closer to CRF-R1 than to CRF-R2; however, it diverges earlier from all
known CRF-R1 sequences (Fig. 2).
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).
Dose-dependent increases in cAMP accumulation, which correlated well
with binding profiles, were seen in COS-7 cells expressing cfCRF-R1.
Half-maximal stimulation (EC50) by CRF, UI, and
SVG were observed at 0.4 ± 0.2, 0.6 ± 0.2, and 1.6 ±
0.4 nM, respectively, and the values were comparable to
those obtained with cells expressing the mCRF-R1 (0.5 ± 0.2,
0.8 ± 0.5, and 1.5 ± 0.6 nM, respectively;
Table 2).
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). Furthermore, both CRF-R2 species bound YQLS
with a higher apparent affinity than sauvagine. Thus, competition of
[125I]YQLS requires a higher concentration of
CRF-related peptides than those previously reported using radiolabeled
sauvagine (Fig. 3). Five- to 10-fold
lower Ki values were obtained for all CRF
receptor agonists tested in COS-7 cells expressing mCRF-R2 and cfCRF-R2
when [125I]sauvagine was used instead of
[125I]YQLS in this system (our
unpublished observation). Competition binding studies in COS-7 cells
expressing mCRF-R2 and cfCRF-R2 yielded about 4- and 10-fold lower
Ki values for SVG than for CRF (Table 2 and Fig. 3). The cells expressing cfCRF-R2 demonstrated similar affinities for
UI and CRF, whereas COS-7 cells expressing the mCRF-R2 bound UI with
approximately 3-fold lower Ki than when binding
to CRF. CRF, UI, and SVG increased cAMP accumulation in COS-7 cells
transfected with mCRF-R2 with EC50 values of
1.2 ± 0.9, 0.5 ± 0.2, and 0.7 ± 0.4 nM,
respectively; the corresponding values for cells expressing the
cfCRF-R2 were 2.4 ± 0.3, 2.5 ± 0.2, and 0.7 ± 0.2
nM, respectively (Table 2). These data indicate that unlike
in the mouse, UI binds and activates cfCRF-R2 less efficiently than
SVG, but similarly to CRF.
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). Although
both cfCRF-R1 and mCRF-R1 had minimal ligand selectivity with regard to
this second messenger, significant differences were observed in
competition binding assays using cells expressing the cfCRF-R3. The
cfCRF-R3 had an approximately 4- to 5-fold higher affinity for CRF than
for UI or SVG, i.e. Ki values were
1.4 ± 0.1, 6.5 ± 0.3, and 7.1 ± 1.3
nM, respectively (Table 2). The shorter form of
cfCRF-R3, although missing 16 amino acids at the amino-terminus,
increased cAMP accumulation to a level similar to that produced by the
cfCRF-R3 with a longer amino-terminus (not shown).
Distinct tissue distribution patterns of the three catfish CRF
receptors
Northern blot analysis of total RNA revealed the presence of high
levels of cfCRF-R1 messenger RNA (mRNA) in brain (Fig. 4A), whereas cfCRF-R2 expression was seen
predominantly in heart (Fig. 4B). Using the same hybridization
condition as that described above, no cfCRF-R3 transcript was detected
in the tissues tested. However, the use of a more sensitive
hybridization system produced a distinct hybridization signal in brain
and heart (Fig. 4C). The size of cfCRF-R3 mRNA was significantly
smaller (3 kb) than those of cfCRF-R1 and cfCRF-R2 (>7 kb).
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) and in distinct hypothalamic
circumventricular structures such as paraventricular organ (Fig. 6A) and posterior recess organ (Fig. 6B).
In addition, expression of cfCRF-R1 was seen throughout other
circumventricular structures such as nucleus recess posteriosus and
succus vasculosus (SV; not shown). Moderate expression was seen in the
intermediate lobe nuclei (Fig. 6A) and facial and vagal lobes (not
shown), which are involved in gustatory functions (29).
Low levels of cfCRF-R1 expression were also detected in telencephalic
structures (not shown). Sense probe hybridization was included in each
experiment and produced no signals in adjacent sections of the areas
mentioned above.
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), and its mRNA
distribution in brain correlated well with that of the mammalian
CRF-R2. The transcript was detected over the myocytes, but not over the
epicardium or the vessel walls. In brain, cfCRF-R2 exhibited high
expression in two hypothalamic nuclei that are known to extend their
axonal termini near the pituitary stalk (30, 31, 32), namely
the nucleus preopticus and the nucleus lateral tubularis (Fig. 6, A and
B). In the cerebellum, cfCRF-R2 was expressed in the distinct
superficial molecular layer that surrounds the inner layer, where high
levels of cfCRF-R1 and low levels cfCRF-R3 mRNA signals were detected
(Fig. 5).
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). This novel receptor
was also expressed in cell bodies of distinct neurosecretory cells
terminating in the urophysis (Fig. 7B). Expression of cfCRF-R3 was
generally low in brain; however, unique distribution patterns for
cfCRF-R3 mRNA were observed in areas such as the intermediate lobe
(Fig. 6A), which is thought to mediate gustatory function, and the
posterior hypothalamic nucleus and nucleus anterior tubularis (Fig. 6B). These areas do not express either cfCRF-R1 or cfCRF-R2. |
Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The stress response system in teleost fish is similar to that in mammals in that it consists of two components: the brain-sympathetic-chromaffin cell axis, which is equivalent to the brain-sympathetic adrenomedullary system, and the brain-pituitary-interrenal axis, which is equivalent to the brain-pituitary-adrenal axis (33). In fish, pituitary ACTH and other POMC peptides are regulated by factors released in the hypothalamus as in other vertebrates. CRF and UI exert ACTH-stimulating action on the pituitary (34, 35, 36), yet until now no receptors have been identified for either of these ligands in fish. Both CRF and UI have been reported to coexist in the hypothalamic region of the fish brain (37). A recent study on rainbow trout demonstrated an increase in CRF mRNA- positive perikarya in the preoptic nucleus when the fish were stressed by confinement (8). However, the physiological significance of CRF and UI has yet to be determined.
In teleost fish, the hypothalamic control of hypophyseal cells is known to be mediated by the hypophysiotropic neurons, which directly innervate the cells of the pituitary gland (38). In addition to the hypothalamic-hypophyseal system, fish possess an additional neurosecretory organ, the urophysis (39), which has been implicated in osmoregulation, in the maintenance of ionic balance, and in reproduction (39). UI was isolated from the urophysis of several fish species, such as sucker fish (40) and carp (9). Its roles in vasodilation (39) and osmoregulation (41) have been previously demonstrated both in vitro and in vivo; however, the role of UI in pituitary ACTH release has only been shown in vitro (34, 35), and the precise role of this peptide in fish physiology remains to be established.
To investigate the potential sites of CRF and UI action in catfish, we conducted a Northern blot and in situ hybridization analysis to identify the tissues that express cfCRF receptors.
CRF-R1 was the predominant subtype expressed in catfish brain, and its expression was widely distributed. The relatively large size and the well described anatomical structure of the catfish brain (29, 31, 42) facilitated detailed anatomical analysis of the distribution of this and other receptor subtypes. The highest cfCRF-R1 mRNA was detected in the cerebellum, where mammalian CRF-R1 is also abundantly expressed (16, 43). Relatively high cfCRF-R1 expression was also detected throughout highly vascularized circumventricular organs, such as the paraventricular organ, the posterior recess organ, the nucleus recess posteriosus, and succus vasculosus. No expression of CRF-R1 has been reported in the similar circumventricular structures in mammalian brain. In contrast, CRF-R2 is highly expressed in the choroid plexus of mammalian brain (43, 44) which could be considered equivalent to the circumventricular structures in catfish. In the catfish brain, expression of cfCRF-R2 was limited to the molecular layer of the cerebellum and the major hypothalamic nuclei in nucleus lateral tubularis and nucleus preopticus, where high levels of CRF mRNA and immunoreactivity (30, 45) and moderate levels of UI mRNA were reported (6). The most abundant transcript of cfCRF-R2 was detected in the atrium of the heart, which correlates well with that reported for mammalian and Xenopus CRF-R2 (17, 20, 21, 22). The cfCRF-R3 was minimally expressed in the catfish brain; however, distinct mRNA signals for this receptor subtype were identified in the hypothalamic regions, where neither cfCRF-R1 nor cfCRF-R2 mRNA was detected except in the SV. The SV located adjacent to the pituitary gland expresses high level of all three cfCRF receptors. The population of nuclei in the intermediate lobe that express cfCRF-R3 was also distinct from the nuclei that express cfCRF-R1. These results suggest an independent gene expression system for this novel third CRF receptor.
CRF-R1 is the major form of CRF receptor expressed in the pituitary gland of both rodent and Xenopus species. Physiological, molecular, and pharmacological manipulation of the receptor suggest that hypothalamic CRF exerts its action through the pituitary CRF-R1 to stimulate ACTH release and, consequently, glucocorticoid production from the adrenal cortex (46, 47, 48). Recently, however, CRF-R2 was also found to be expressed in the primate pituitary (43). This implies a role for this receptor subtype in controlling pituitary ACTH release and suggests that the control of ACTH release may be more complicated than previously believed. Interestingly, in catfish, cfCRF-R3 was found to be the dominant subtype expressed in the pituitary. This suggests that the third CRF receptor subtype, cfCRF-R3, may be important for the secretion of POMC peptides in fish. The importance of this novel CRF receptor subtype in catfish neuroendocrine physiology is also supported by its expression in urophyseal neurosecretory cells.
In contrast to their distinct structure and anatomical distribution pattern, cfCRF-R1 and cfCRF-R3 demonstrated only small differences in ligand binding properties. No significant differences were observed in the stimulation of cAMP accumulation by CRF, UI, and SVG. The cfCRF-R3 bound CRF with approximately 5-fold higher affinity than UI and SVG. The cfCRF-R1, however, bound these three ligands with similar affinity, which is consistent with the binding properties reported for the mammalian CRF-R1 (14, 16, 19). The functional characterization of cfCRF-R2 combined with its structural homology indicate that this receptor subtype is clearly a catfish homologue of the mammalian CRF-R2. However, SVG is more potent in cfCRF-R2 than mCRF-R2, whereas UI is more potent in mCRF-R2 than cfCRF-R2. The fact that SVG is the most potent ligand for cfCRF-R2 raises the possibility that the catfish may express an as yet unidentified SVG-like peptide. It is interesting to note that the Ki values observed in this study for CRF-R2 are higher than those reported in the literature. The high Ki values could result from the high binding affinity of the radioligand used; this increases the concentration of other peptides required for half-maximal competition. Additionally, apparent binding Kd values in intact COS-7 cells were higher than those measured in stably transfected cell lines (25, 49). The decreased binding affinity in transiently transfected COS-7 cells may reflect binding to receptors that are not coupled to the G protein-coupled receptor(s).
All three catfish CRF receptors, despite their sequence variation in the amino-terminal domains, possess 5 or more potential N-linked glycosylation sites and 6 conserved cystein residues. One of the splice variants for cfCRF-R3 that lacks 16 amino acids but retains all of the glycosylation sites and cystein residues in its amino-terminus, was well expressed and functioned as well as the longer cfCRF-R3 splice variant. These results suggest that sequence variations in the amino-terminal domain are relatively well tolerated. Interestingly, however, a human CRF-R1 splice variant with a 40-amino acid deletion in the amino-terminal domain, which lacked several cystein residues, failed to bind [125I]ovine CRF with high affinity (50). This indicates that the number of cysteine residues in the NT may be critical for proper folding and positioning of the molecule within the cell membrane.
In summary, we have cloned two CRF receptors from the catfish that are highly homologous to their mammalian counterparts. In addition, we have isolated a third novel CRF receptor that is distinct from the other two receptor subtypes. The cfCRF-R3, which bound CRF with higher affinity than UT and SVG, was highly expressed in the catfish pituitary and moderately in the urophysis. The presence of a third CRF receptor subtype in a diploid catfish species has an important evolutional significance, and it appears possible that the genomes of other species, such as mammals, contain additional, as yet uncharacterized genes that encode other CRF receptor subtypes. Alternatively, catfish may represent a unique evolutionary species in which the biological effects of CRF and UI are mediated by three distinct CRF/UI receptors.
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Acknowledgments |
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Footnotes |
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2 This work was supported by NIDDK Grant 45020–04A1 from the
NIH.
Received May 10, 2000.
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),
sauvagine (
), or YQLS (
) were performed for 2 h at room
temperature. Specific binding was computed and expressed as a
percentage of maximal specific binding (%B/B0). Data represent the
mean ± SD of three independent experiments. Each
experiment was performed in triplicate for each peptide concentration
tested.
