DuPont Merck Research Laboratories CNS Diseases Research Wilmington, Delaware 19880
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ABSTRACT |
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INTRODUCTION |
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Our interest in the function of the CRF system in humans led us to
clone the human CRF2 receptor cDNAs. We screened a human
amygdala cDNA library and confirmed, as reported by ourselves and
others (12, 13, 14), the existence of the human CRF2 and
CRF2ß receptor cDNAs and also provided evidence for a
third CRF2 receptor isoform, which we refer to as
CRF2
. This new isoform contains a predicted amino
terminus, which differs from the other reported isoforms.
CRF2
RNA is expressed at low levels in the human central
nervous system (CNS) in a pattern similar to that of the
CRF2ß receptor. Pharmacologically the CRF2
isoform is most like the CRF2
receptor. The discovery of
the CRF2
receptor adds additional diversity to the
CRF2 family of receptors, thereby providing an additional
receptor through which CRF, urocortin, or similar unknown peptides may
function.
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RESULTS |
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Expression Pattern of the CRF2
Isoform mRNAs in Human Brain
To examine the relative levels of CRF2 mRNA
expression in brain we performed PCR amplification on cDNAs from
various human brain regions using primers directed at the
CRF2
-specific exon and the first common exon. This
approach distinguishes potentially contaminating genomic DNA by
inclusion of an intron. The same approach was taken with
CRF2ß and CRF2
receptor primers to provide
comparative expression data for the other splice forms. PCR
amplification of CRF2
message from the human brain
cDNAs showed easily detectable expression in the septum and
hippocampus, and weaker expression in the amygdala. Very weak, but
still visually detectable, expression is found in nucleus accumbens,
midbrain, and frontal cortex (see Fig. 3A
). A Southern blot of the same gel
probed with a CRF2 oligo confirmed the weak expression in
the amygdala, nucleus accumbens, midbrain, and frontal cortex as well
as stronger expression in septum and hippocampus (see Fig. 3B
). PCR of
CRF2ß receptor RNA in these same tissues shows a pattern
similar to that observed for CRF2
receptor mRNA, with
expression in septum, hippocampus, and amygdala (see Fig. 3A
).
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Expression Pattern of CRF2
Isoform mRNAs in Selected Peripheral Tissues
To examine the expression of CRF2,
CRF2ß, and CRF2
message in human heart,
lung, and skeletal muscle, we again used PCR amplification of cDNA
(Fig. 3C
). As a positive control we used human hippocampus cDNA. A PCR
product for CRF2
was only very weakly detectable in lung
and undetectable in heart and skeletal muscle. CRF2ß RNA
was only detectable in skeletal muscle. CRF2
message was
detectable in all three of the tissues, most prominently in heart and
skeletal muscle.
Pharmacology of the Human CRF2
Receptor
The human CRF2 cDNA was subcloned into
the expression vector phchm3AR, a pHEBo derivative, and transfected
into 293-EBNA (Epstein-Barr nuclear antigen) cells (16). In parallel,
human CRF2
and CRF2ß receptor expressing
293-EBNA cells were prepared. Activation of these receptors with CRF
family agonists resulted in a dose-dependent elevation of cAMP, as
measured by whole cell adenylyl cyclase assays. The relative potencies
of the CRF-related agonists on all the CRF2 receptor cells
were similar, with EC50 values for
urocortin
sauvagine<urotensin<r/h CRF (Table 1
). (see Table
1A). The relative potencies we observed
for human CRF2
and CRF2ß
receptor-expressing cells were consistent with previous reports using
rodent and human receptors (6, 7, 8, 9, 12). The EC50 values
derived for CRF2
and CRF2
were very
similar, while those for CRF2ß were approximately 10-fold
lower for all peptides.
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DISCUSSION |
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We made numerous unsuccessful attempts to clone a rat version of the
CRF2 cDNA. In addition, a moderate stringency screen of
a Southern blot of rat genomic DNA reveals a lack of
CRF2
-like sequence in the rat genome (our
unpublished observation). While this does not rule out the possibility
of a rat homolog, it suggests that such a homolog would have relatively
weak sequence homology or a quite different expression pattern.
When heterologously expressed in 293-EBNA cells, the pharmacological
profile of the CRF2 receptor was quite similar to that
of the other CRF2 splice forms. The rank order of
EC50 values for CRF-related peptides was
urocortin
sauvagine<urotensin<rat/human (r/h) CRF. However,
the absolute EC50 values we report for CRF2
are closer to what we observe for CRF2
than for
CRF2ß. In adenylyl cyclase assays, CRF-related peptides
appeared approximately 10-fold more potent at the CRF2ß
receptor than the CRF2
and CRF2
receptors. These potency differences could be due to receptor reserve;
however, we did not observe any gross differences in Bmax
values or overall levels of adenylyl cyclase stimulation among the
three receptor- expressing cells that would suggest that receptor
reserve could account for a potency shift of this magnitude.
Thus, while the N-terminal amino acid differences do not allow
discrimination between the CRF2
and CRF2
receptors using these CRF-related agonists, our data do suggest that
the N terminus affects the affinity of peptide agonists for
CRF2ß.
Competition binding assays at the CRF2 receptor were
performed using [125I-Tyr0]-sauvagine and
other unlabeled CRF family agonists. The rank order of
IC50s we observed for the human CRF2
receptor was urocortin
urotensin<a-helical CRF<r/h CRF. These
data are in general agreement with the values previously reported for
the human CRF2
and CRF2ß receptors (13)
and the rank order of Ki values determined with the
orthologous rodent receptors (19, 20). The agonist IC50
values and the Kd values for the high- and low-affinity
binding sites for sauvagine were also similar between human
CRF2
and CRF2
(high- and low-affinity
sites for CRF2
: 60 pM and 5 nM,
and for CRF2
: 44 pM and 4 nM)
(18). Thus, the competition binding assays also show a very similar
pharmacological profile for the CRF2
and
CRF2
receptors.
In contrast, the expression pattern of human CRF2 RNA in
brain was most similar to our expression data for CRF2ß.
PCR amplification of human brain cDNAs using
CRF2ß-specific primers demonstrated expression of this
splice form in septum, hippocampus, and amygdala. These same tissues
were also found by PCR amplification to have the highest expression of
CRF2
message. CRF2
expression was
detected additionally in the nucleus accumbens, midbrain, and frontal
cortex by the more sensitive technique of Southern blotting the PCR
amplification products with a CRF2
-labeled
oligonucleotide probe. We did not examine CRF2ß
expression by Southern blot; therefore, it is unknown whether the
weakly expressing tissues determined for CRF2
also
weakly express CRF2ß. The regions with most abundant
CRF2
RNA expression, however, coincided with regions of
most abundant CRF2ß message expression. These regions are
limbic structures and suggest a possible role for both receptors in the
behavioral effects of CRF or the related peptide urocortin. In rat
central nervous system tissues the CRF2ß isoform appears
to be primarily associated with cerebral arterioles, perhaps playing a
role in stress-induced cerebrovascular dysfunction (11). Further
studies at the cellular level are necessary to determine whether
CRF2ß is also vascular in humans and whether the
CRF2
receptor shares cellular localization as well as
regional localization with the CRF2ß receptor.
The expression profile we determined for the CRF2
receptor message in human CNS tissues was widespread and generally in
agreement with findings in the rat (10). We observed strong PCR
products in limbic areas such as septum and hippocampus as was the case
with the other human splice forms. We also found significant
CRF2
expression in human hypothalamus. While this assay
was unable to discern regional localization within the human
hypothalamus, in rodents, strong CRF2
expression was
found in the supraoptic, paraventricular, and ventromedial hypothalamic
nuclei of the hypothalamus (10). The CRF2
receptors in the paraventricular nucleus colocalize with CRF-expressing
neurons and have been postulated to be presynaptic and function in a
short feedback loop. They may thereby regulate hypophysiotropic and/or
autonomic related CRF neurons. The rodent CRF2
receptors
in the VMH may play a role in autonomic outflow and gastrointestinal
function (10). As in rodents, we observed no detectable
CRF2
message expression in human cerebellum. In contrast
to rodent reports, however, we did detect CRF2
message
in frontal cortex. This result is supported by a recent report by Liaw
et al. (12) in which they obtained human CRF2
receptor cDNA clones from a human cortex library. It will be of
interest to determine the cell types expressing the CRF2
receptor and compare this distribution to that of the human
CRF1 receptor in cortex. While the human
CRF2
receptor may play a species-specific role in
cortex, the human CRF1 receptor is clearly more abundant in
this tissue (our unpublished observation).
In rodents, CRF2ß RNA is known to be expressed in a
number of peripheral tissues, namely, heart, lung, and skeletal muscle
(7, 8, 9, 10, 11). Given the parallels in expression we observed between the
CRF2ß and CRF2 messages in the CNS, we
looked for the presence of CRF2ß and CRF2
expression in human heart, lung, and skeletal muscle. Additionally, we
examined CRF2
message levels.
In contrast to data reported for rodents, we did not detect
CRF2ß message in human heart. However, as in rodents, we
did observe expression of CRF2ß message in skeletal
muscle (14). A recent report suggests that CRF2ß message
can be found in human heart when the PCR reaction is Southern blotted,
thereby enhancing the sensitivity of detection (13). However, given the
amplification power of PCR and the sensitivity of a Southern blot, it
is unclear whether such low expression levels have any biological
significance. Aside from a very weak amplification band in lung we were
unable to detect CRF2 message in any of these tissues.
Therefore the expression of human CRF2ß and
CRF2
RNAs are different in these peripheral tissues. Our
examination of CRF2
message levels suggest that it is
the predominant CRF2 receptor in these human peripheral
tissues. The cellular localization of the CRF2
receptor
in human heart is unknown; however, in rodents, CRF2
receptors have been localized by in situ hybridization to
heart perivascular cells and epicardium (8). Systemic administration of
CRF causes hypotension and tachycardia in a number of species including
humans (21, 22, 23, 24). Evidence for a direct effect on heart function has
come from work with isolated heart preparations. Direct injection of
CRF into the left atrium of an isolated rat heart causes a prolonged
dilatory effect on coronary arteries and a transient positive
ionotropic effect (25). Based on our expression data, if
CRF2 receptors mediate a direct cardiovascular effect in
human heart, this effect is most likely mediated by the
CRF2
receptor rather than the CRF2ß
receptor.
As a caveat to all studies examining gene expression, we cannot
conclusively demonstrate in situ expression of the protein
product of these gene transcripts without the benefit of specific
antibodies to each of the human CRF2 receptors. Nonetheless
we have shown that CRF2 RNA is expressed in human brain
in a regionally specific manner and that a functional receptor can be
heterologously expressed in tissue culture cells.
In conclusion, we have discovered a novel CRF2 receptor
cDNA in human brain, which we have termed CRF2. This
isoform is expressed in similar brain regions to the
CRF2ß isoform, primarily limbic brain structures that may
subserve some of the behavioral effects of CRF or other endogenous CRF-
related peptides, such as urocortin. The pharmacological profile of the
CRF2
receptor is typical for a CRF2
receptor, exhibiting closest similarity to the CRF2
splice form, with the highest apparent potency for the CRF-related
peptides urocortin and sauvagine. Given the similar peptide
pharmacology of CRF2
and CRF2
, at least
for the known CRF-related peptide agonists, and the similarity of
regional distribution of expression to CRF2ß, it is not
clear what specific role CRF2
might serve. Nonetheless,
the existence of a third splice isoform of the CRF2
receptor family provides another avenue to explore the complex
physiological and behavioral responses to CRF and other endogenous
related neuropeptides.
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MATERIALS AND METHODS |
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For the genomic library screen, a 467-bp CRF2 5'-RACE
clone containing both the CRF2
- and
CRF2ß-specific exons was labeled as described above and
used to screen approximately 4 x 105 plaques from an
EMBL-3 phage human genomic library (CLONTECH). Hybridizations were
performed at 65 C (hybridization solution as above) with the final wash
condition of 0.2xSSC and 0.1% SDS at 65 C. Hybridization-positive
clones were further characterized by restriction mapping and limited
DNA sequence analysis.
5'-RACE Reactions
5'-RACE was performed using the Marathon cDNA Amplification Kit
(CLONTECH) with human hippocampus cDNA (described below) and Marathon
Ready hippocampus cDNA (CLONTECH). The cDNA was initially amplified
using the kit- supplied anchor primer and the CRF2
receptor-specific primer 5'-GCAGAAGAGCTGAGGAAAGCCCAGGTC-3'. The
reaction products were then further amplified using a supplied nested
anchor primer and a nested CRF2
receptor-specific
primer, either 5'-AAAGCCCAGGTCCCTGTCTTCAGGC-3' or
5'-GCTGAGGAAAGCCCAGGTCCCTGTC-3'. DNA fragments of the proper size were
cloned using the TA Cloning kit (Invitrogen, San Diego, CA).
PCR of Human Genomic DNA
PCR amplification of the genomic region between the
CRF2ß and CRF2 exons was performed using
50 ng human genomic DNA (CLONTECH) and the Expand Long Template PCR
system (Boehringer Mannheim, Indianapolis, IN). The CRF2ß
sense and CRF2
antisense primers used were
5'-GATGCCCAAAGACCAGCCCCTGTG-3' and 5'-GCAGAAGAGCTGAGGAAAGCCCAGGTC-3',
respectively. The PCR reaction was carried out for 30 cycles with an
anneal temperature of 66 C. The reaction product was gel purified and
ligated into the pCR2.1 vector using the T/A cloning kit
(Invitrogen). Sequence analysis confirmed the identity of the
cloned genomic region.
PCR of Brain cDNAs
Human postmortem brain tissue was obtained from the St.
Elizabeths Hospital Brain Bank (courtesy of Dr. Thomas Hyde). All
tissue except for hypothalamus and frontal cortex came from the same
brain. Total RNA was extracted using RNAzol B (Tel-Test,
Friendswood, TX) and 10 µg of total RNA from each tissue was reverse
transcribed into first-strand cDNA using the Superscript
Preamplification System (GIBCO BRL). CRF2 receptor cDNAs
were amplified from 1 µl cDNA (from a total of 40 µl) using primers
to the CRF2 isoform of interest. For CRF2,
the exon-specific primer was 5'-TGGGCTTTCCTCAGCTCTTCTG-3', and the
antisense primer to the first common exon was
5'-CCATTCTCCAAGCATTCTCGATAG-3'. Amplitaq Gold (Perkin-Elmer,
Norwalk, CT) was used with an annealing temperature of 65 C for 36
cycles. The CRF2
and CRF2ß exon-specific
primers were 5'-GAGCTGCTCTTGGACGGCTGGGGGC-3' and
5'-GACCAGCCCCTGTGGGCACTTCTGG-3', respectively, and the antisense primer
to the common exon was 5'-GCAGACAGCGAATGCTCCGCAGGC-3'. Amplitaq Gold
(Perkin-Elmer) was also used for these primer pairs; however, the
reaction was a two-step PCR, alternating between 95 C and 68 C for 36
cycles. PCR reaction products were visualized by agarose gel
electrophoresis followed by staining with SYBR Green I (Molecular
Probes, Eugene, OR) (1:10,000). Gels were scanned into a STORM imaging
system (Molecular Dynamics, Sunnyvale, CA) using blue chemifluorescence
mode. The resulting image was analyzed using ImageQuant 4.1 software
(Molecular Dynamics). Gel photographs were produced by importing the
gel file into Adobe Photoshop 4.0 and printing the gel on a Fujix
Pictography 3000 printer.
The Southern blot of CRF2 PCR products was performed
using the Vistra fluorescent labeling system (Amersham, Arlington
Heights, IL) in conjunction with the STORM imager. Briefly, the PCR
products were blotted onto Hybond N+ membrane and hybridized with the
oligo 5'-CTACTCCTACTGCAACACGACCTTG-3', which had been previously
labeled at the 3'-end with fluorescein-11-dUTP. The hybridization was
carried out overnight at 42 C in a buffer consisting of 5xSSC, 0.1%
SDS, 5% dextran sulfate, and a 1:20 dilution of liquid block
(Amersham). The final wash condition was 1xSSC and 0.1% SDS twice at
50 C for 15 min each. The blot was then treated with the signal
amplification system (Amersham). Briefly, the blot was blocked again
with a 1:20 dilution of liquid block and incubated with an
antifluorescein alkaline phosphatase conjugate. The blot was
then washed and treated with Attophos reagent (Amersham).
Hybridization-positive PCR bands were detected using the STORM imager
(Molecular Dynamics) in blue chemifluorescence mode and ImageQuant 4.1
software (Molecular Dynamics).
Quantitative RT-PCR was performed to measure human CRH2
receptor RNA message levels. The procedure was carried out essentially
as described in the PCR Mimic kit (CLONTECH). This method relies on the
amplification of the target message in the presence of a known amount
of an artificial competitor. The competitor contains flanking sequences
identical to the primers used to amplify the desired target. The
primers used are described above. The CRH2
receptor
competitor was constructed by ligating the appropriate primer sequences
to the ends of a fragment of v-erbB. Initial quantitative RT-PCR
reactions were carried out using six reactions per tissue, each
containing competitor at a different 10-fold serial dilution, and a
constant amount of target cDNA. PCR amplification was performed as
described above using Amplitaq Gold (Perkin-Elmer), and products were
resolved by agarose gel electrophoresis and visualized by ethidium
bromide (EtBr) staining. RT-PCR was then repeated using 2-fold serial
dilutions of competitor, with the choice of dilutions based on the
first-round prediction of CRH2
message abundance. These
fine scale (2-fold) reactions were initially imaged by spiking with
[33P-
]-dCTP and resolving the products on a 5%
acrylamide gel, followed by quantification with a Molecular Dynamics
PhosphorImager. A nonradioactive method was also used in which reaction
products were resolved on an agarose gel, stained with EtBr,
photographed with Kodak 55 film (Eastman Kodak, Rochester, NY), which
produces transparent negatives, and quantitated with a Molecular
Dynamics densitometer. Comparable results were derived from both
techniques. Values for CRH2
receptor RNA message levels
were determined by plotting the log(CRH2 signal/competitor
signal) on the y-axis vs. the log(molecules of competitor
used) on the x-axis. Solving for x at y = 0 yields the amount of
CRH2 receptor RNA message present in the reaction.
Endogenous levels of the housekeeping gene, GAPDH, were also determined to normalize for differences in RNA quality between tissues. The GAPDH sense primer 5'-GGGAAGGTGAAGGTCGGAGTCAACG-3' and antisense primer 5'-CATCGCCCCACTTGATTTTGGAGGG-3' amplify a product spanning three introns (26). Quantitative PCR was performed as described above with the following modifications: cDNA was diluted 100-fold before use, and PCR was performed for 30 cycles with an annealing temperature of 64 C.
Cell Transfection, Receptor Binding, and Functional
Studies
The human CRF2 cDNA was subcloned into the
expression vector phchm3AR and transfected into 293-EBNA cells
(Invitrogen) using LipofectAMINE (GIBCO-BRL). This expression
vector, a modified pHEBo plasmid (27), places the cDNA sequence under
the control of the cytomegalovirus immediate early promoter and
contains the Epstein-Barr virus (EBV) oriP for maintenance of
the plasmid in an episomal state in EBNA-positive primate cells (16).
Human CRF2
and CRF2ß receptor expressing
293-EBNA cells were produced as described above from full-length cDNA
clones also obtained from the human amygdala library screen.
The whole cell adenylyl cyclase assays were performed by EIA (PerSeptive Diagnostics, Cambridge, MA). Briefly, cells were switched to low-serum growth media and incubated for 30 min with 0.1 mM 3-isobutyl-1-methyl-xanthine (IBMX). Test peptides were then added to the media, and the incubation was extended an additional 2530 min. After the incubation the media were removed and replaced with ice-cold sodium acetate buffer (with 1 mM IBMX) and the cells were disrupted by a freeze/thaw cycle followed by sonication. Supernatants were then assayed for cAMP levels. Cell supernatants were added together with rabbit anti-cAMP antibody to wells precoated with goat antirabbit antibody, followed by the addition of a cAMP-alkaline phosphatase conjugate to the wells. The mix was incubated for 1 h and then washed. p-Nitrophenyl phosphate substrate was added, and the enzyme product was allowed to accumulate for 3 h at 37 C. The absorbance was read at 405 nm and compared with values obtained from a standard curve.
The receptor binding studies were performed using buffered membrane
homogenates from CRF2 expressing 293-EBNA cells. Assays
were initiated by the addition of 1020 µg protein from cell
membrane homogenates to a final volume of 200 µl of assay buffer
containing 60 pM
[125I-Tyr0]-sauvagine and various
concentrations of inhibitors. Assay buffer consisted of 50
mM HEPES, 10 mM MgCl2, 15
mM EGTA, pH 7.0, containing 1 µg/ml each of aprotinin,
leupeptin, and pepstatin, as well as 0.1% ovalbumin and 0.15
mM bacitracin. After 4 h at room temperature, free
ligand was separated from bound by rapid vacuum filtration through
presoaked (0.3% polyethyleneimine for
2 h) GFF glass
fiber filters (Inotech, Dottikon, Switzerland) using an Inotech
96-well harvester. Filters were washed three times with ice-cold PBS,
pH 7.2 (with 0.01% Triton X-100), dried briefly, and analyzed with an
Isomedic 10/880
-counter (Titertek, Huntsville, AL).
Saturation isotherms were performed as described above, using 23
concentrations of sauvagine over a range of 4 pM to 100
nM. Sauvagine concentrations from 4 pM to 1
nM were achieved with
[125I-Tyr0]-sauvagine, while concentrations
of sauvagine higher than 1 nM consisted of 0.25
nM [125I-Tyr0]-sauvagine in the
presence of sufficient unlabeled sauvagine to reach the desired
concentration. Nonspecific binding was defined in the presence of 1
µM -helical CRF(941).
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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1 Authors contributed equally.
Received for publication January 26, 1998. Revision received March 26, 1998. Accepted for publication April 13, 1998.
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REFERENCES |
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