Molecular Identification and Analysis of a Novel Human Corticotropin-Releasing Factor (CRF) Receptor: The CRF2{gamma} Receptor

Walter A. Kostich1, Airu Chen1, Karen Sperle and Brian L. Largent

DuPont Merck Research Laboratories CNS Diseases Research Wilmington, Delaware 19880


    ABSTRACT
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
We report the discovery of a new CRF2 receptor splice isoform found in human brain, which we have termed the CRF2{gamma} receptor. The CRF2{gamma} cDNA encodes for a 397-amino acid receptor that has an amino terminus with no significant homology to the already reported {alpha}- and ß-termini. When expressed in 293-EBNA (Epstein-Barr nuclear antigen) cells, the CRF2{gamma} receptor responds in a dose-dependent manner to CRF and related peptides with a rank order of potency of urocortin>=sauvagine>urotensin>r/h CRF, with EC50 values more similar to CRF2{alpha} than CRF. Equilibrium saturation isotherm analysis with radiolabeled sauvagine reveals a two site/state model for binding to CRF2{gamma} with a 60 pM Kd high-affinity site and a 5 nM Kd low-affinity site. Analysis of CRF2{gamma} RNA expression in human brain demonstrates expression in septum and hippocampus, with weaker but detectable expression in amygdala, nucleus accumbens, midbrain, and frontal cortex.


    INTRODUCTION
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Corticotropin releasing factor (CRF) is a neuropeptide that plays a role in the integration of autonomic, neuroendocrine, and behavioral responses to stress (for recent review see Ref. 1). These effects are mediated through two recently cloned receptor families, CRF1 (2, 3, 4, 5) and CRF2 (6, 7, 8, 9). These G protein- coupled receptors have relatively nonoverlapping expression patterns in rodents and exhibit distinct pharmacological profiles (10). The CRF2 receptor family has additional diversity in that two isoforms, which utilize alternative amino termini, have been previously described (6). In situ hybridization studies in rodents suggest that the CRF2{alpha} receptor is expressed primarily in subcortical neuronal populations (10, 11). The CRF isoform message is expressed in nonneuronal cells in the central nervous system (e.g. cerebral arterioles and choroid plexus) (11). Peripherally, CRF message is found in heart, lung, and skeletal muscle (7, 8, 9, 11).

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{alpha} and CRF receptor cDNAs and also provided evidence for a third CRF2 receptor isoform, which we refer to as CRF2{gamma}. This new isoform contains a predicted amino terminus, which differs from the other reported isoforms. CRF2{gamma} RNA is expressed at low levels in the human central nervous system (CNS) in a pattern similar to that of the CRF receptor. Pharmacologically the CRF2{gamma} isoform is most like the CRF2{alpha} receptor. The discovery of the CRF2{gamma} 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.


    RESULTS
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Cloning of the Human CRF2{gamma} Receptor cDNA
Approximately 8.5 x 105 phage plaques from a human amygdala cDNA library were screened with a rat CRF2{alpha} receptor probe. Seven hybridization positive plaques were isolated and characterized. Four of the phage clones encoded known CRF receptors, namely human CRF1, CRF2{alpha}, and CRF. The three remaining positive plaques contained cDNAs for a novel splice form of the human CRF2 receptor, which we are referring to as the {gamma}-isoform. This isoform differed at the amino terminus from the CRF2{alpha} and CRF receptors. One clone was completely sequenced and was found to contain a 1558-bp insert with a 1190-bp open reading frame encoding a predicted 397-amino acid protein (see Fig. 1Go). The putative in-frame start codon was in an adequate translational context, containing a G at +4 and a pyrimidine at -3, and was preceded by an upstream in-frame stop codon (15). The start codon and 5'-untranslated region sequence were verified by performing 5'-RACE (rapid amplification of cDNA ends) using human hippocampus cDNA with CRF2{gamma}-specific primers to obtain multiple independent clones that were characterized by sequencing.



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Figure 1. Nucleotide and Amino Acid Sequence of the CRF2{gamma} cDNA

Potential N-linked glycosylation sites are boxed. Cysteines available for disulfide bonding are circled. Predicted transmembrane domains are underlined. This sequence has been deposited in Genbank under accession no. AF019381. Arrow refers to splice junction between CRF2{gamma}-specific exon and common region.

 
Cloning of the CRF2{gamma} Genomic Locus
Examination of the genomic structure of the CRF2 gene served to further verify the authenticity of the CRF2{gamma} cDNA clone by demonstrating the hallmarks of an exon in the CRF2{gamma} splice form specific genomic DNA. While the genomic structure of a significant portion of the human CRF2 gene was recently described (12), this description did not extend 5' of the CRF2{alpha} alternative exon. Previous PCR-based expression studies by our laboratory suggested that both the CRF and CRF2{gamma} exons lie in this undescribed 5'-region. We had examined the expression of human CRF receptor RNAs in human brain using RT-PCR and had obtained products containing the CRF2{gamma} or CRF2{alpha} exons interposed between the CRF exon and the first common exon (our unpublished observation). These alternatively spliced products indicated that the CRF exon lies upstream of the CRF2{gamma} and CRF2{alpha} exons. In addition, the size of the CRF2{gamma} exon precluded it from being located in the region between the CRF2{alpha} exon and the first common exon based on a previous human CRF2 genomic study (12). We therefore hypothesized the genomic order of the alternative exons to be ß-{gamma}-{alpha}. As a direct test of the genomic position of the CRF exon relative to the CRF2{gamma} exon, we performed PCR amplification on genomic DNA to amplify the region between the CRF and CRF2{gamma} exons. We used a primer to the CRF exon 3'-end and a primer to the 5'-end of the CRF2{gamma} specific exon and generated a 3.2-kb fragment. Sequence analysis of this fragment confirmed the presence of CRF and CRF2{gamma} exon sequence at the 5'- and 3'-ends, respectively. The CRF and CRF2{gamma} exons possess good consensus splice junctions flanking an apparent 2.9-kb intron (see Fig. 2Go). Similar attempts to amplify the genomic region between the CRF2{gamma} and CRF2{alpha} specific exons were unsuccessful. Based on the results of the genomic screen described below, this is likely due to the large intervening distance between the exons.



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Figure 2. Schematic of Alternative Exon Region of CRF2 Genomic Locus

The CRF2 alpha, beta, and gamma alternative exons are represented as solid boxes (not drawn to scale). The distance between the {gamma}-exon and the {alpha}-exon is not known; however, it is at least 5.8 kb and may be significantly more. The intron/exon boundary sequences flanking the CRF2{gamma} exon and its preceding intron are shown, with the consensus sequences for intron/exon boundary sequences depicted below them. Underlined sequences are invariant. Y, Pyrimidine; R, purine; N, any base.

 
To confirm and extend our results with genomic DNA amplification by PCR, we screened a human genomic library to identify clones corresponding to the CRF and CRF2{gamma} exons. Approximately 4 x 105 phage plaques were screened with a probe containing both the CRF and CRF2{gamma} alternative exons. Eight positive plaques were isolated. An approximately 7-kb positive fragment contained all of the CRF2{gamma} exon with 1 kb of 5'- and approximately 5.8 kb of 3'-flanking genomic sequence. None of the genomic fragments from the positive plaques had a hybridization signal when probed using an oligo directed to the CRF2{alpha} exon, suggesting that the intervening sequence between the CRF2{gamma} exon and the CRF2{alpha} exon(s) exceeds 5.8 kb (see Fig. 2Go).

Expression Pattern of the CRF2 Isoform mRNAs in Human Brain
To examine the relative levels of CRF2{gamma} mRNA expression in brain we performed PCR amplification on cDNAs from various human brain regions using primers directed at the CRF2{gamma}-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 CRF and CRF2{alpha} receptor primers to provide comparative expression data for the other splice forms. PCR amplification of CRF2{gamma} 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. 3AGo). 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. 3BGo). PCR of CRF receptor RNA in these same tissues shows a pattern similar to that observed for CRF2{gamma} receptor mRNA, with expression in septum, hippocampus, and amygdala (see Fig. 3AGo).



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Figure 3. Expression of CRF2 Isoform RNAs in Selected CNS Tissues

A, Composite figure showing results from CRF2{gamma}, CRF, and CRF2{alpha} PCRs using human brain cDNAs. Samples were electrophoresed through 1.5% agarose gels and were stained after electrophoresis with SYBR Green I (Molecular Probes) and scanned into a STORM imaging system (Molecular Dynamics). B, Southern blot of CRF2{gamma} PCRs using human brain cDNAs probed with a fluorescently labeled CRF2 oligo. The oligo is a 25 mer that is homologous to DNA sequence internal to the original PCR primers. Closed arrow indicates PCR product of expected size for CRF2{gamma}. C, Expression of CRF2 isoform RNAs in selected peripheral tissues. Composite figure showing results from CRF2{gamma}, CRF, and CRF2{alpha} PCR reactions using human peripheral tissue cDNAs. Hippocampus cDNA was included as a positive control. Samples were electrophoresed through 1.5% agarose gels and were stained after electrophoresis with SYBR Green I (Molecular Probes) and scanned into a STORM imaging system (Molecular Dynamics).

 
Human CRF2{alpha}, in contrast, was expressed at visually detectable levels in most of the tissues we examined (see Fig. 3AGo). Expression was consistently strongest in hypothalamus, hippocampus, septum, and cortex. Weaker, but detectable, expression was found in thalamus, nucleus accumbens, pons, amygdala, medulla, and midbrain. Expression was absent from cerebellum. The expression pattern of human CRF2{alpha} was confirmed by quantitative PCR using known quantities of a competitor that would also be amplified by the CRF2{alpha} primers. The expression profile mirrors the data shown from the noncompetitive PCR (data not shown). In an attempt to control for potential differences in RNA quality we also performed the same experiment with glyceraldehyde-3-phosphate dehydrogenase (GAPDH). For the sake of comparison the values obtained for human CRF2{alpha} message were then normalized to a uniform GAPDH level. The only significant result of this normalization was a decrease in relative hypothalamic expression, which results in the hypothalamus being a site of more moderate expression, with levels closer to those observed in septum (data not shown). This result is not surprising in that the hypothalamic tissue came from a different brain source than the others and may have provided RNA of a higher quality.

Expression Pattern of CRF2 Isoform mRNAs in Selected Peripheral Tissues
To examine the expression of CRF2{gamma}, CRF, and CRF2{alpha} message in human heart, lung, and skeletal muscle, we again used PCR amplification of cDNA (Fig. 3CGo). As a positive control we used human hippocampus cDNA. A PCR product for CRF2{gamma} was only very weakly detectable in lung and undetectable in heart and skeletal muscle. CRF RNA was only detectable in skeletal muscle. CRF2{alpha} message was detectable in all three of the tissues, most prominently in heart and skeletal muscle.

Pharmacology of the Human CRF2{gamma} Receptor
The human CRF2{gamma} 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{alpha} and CRF 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 1Go). (see Table 1A). The relative potencies we observed for human CRF2{alpha} and CRF receptor-expressing cells were consistent with previous reports using rodent and human receptors (6, 7, 8, 9, 12). The EC50 values derived for CRF2{alpha} and CRF2{gamma} were very similar, while those for CRF were approximately 10-fold lower for all peptides.


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Table 1. Pharmacological Profile of Human CRF2{gamma} Receptor

 
Previous work has shown that one obtains specific, saturable binding with [125I-Tyr0]-sauvagine on CRF2{alpha} receptor-expressing cells (17, 18). We used this ligand with the human CRF2{gamma} receptor cells in competition binding assays with other CRF family agonists. The following rank order of IC50 values was observed for the displacement of [125I-Tyr0]-sauvagine from these membrane preparations: urocortin<=urotensin<=a-helical CRF<r/h CRF (see Table 1B) Saturation isotherm analysis with [125I-Tyr0]-sauvagine revealed saturable binding with the data best fitting a two site/state model (a 60 pM Kd high-affinity site and a 5 nM Kd low-affinity site) as would be expected with a G protein-coupled receptor (see Fig. 4Go).



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Figure 4. Equillibrium Saturation Isotherm with [125I-Tyr0]-Sauvagine Using Membrane Homogenates from CRF2{gamma} Receptor Expressing 293-EBNA Cells

Inset, Scatchard transformation of specific binding, indicating a two-site fit with high and low affinity Kd values of 60 pM and 5 nM, respectively. Apparent equilibrium Kd calculated using the nonlinear iterative curve-fitting computer program (LIGAND) of Munson and Rodbard (28 ).

 

    DISCUSSION
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
We report the cloning of a cDNA encoding a third CRF2 receptor splice isoform from human brain, which we term the CRF2{gamma} receptor. The human forms of the CRF2{alpha} and CRF receptors have been recently described (12, 13, 14). While no group has previously found evidence for more than two CRF2 splice forms in rodents or humans, there have been reports that this gene is frequently misspliced. Given this precedence, our initial suspicion was that the CRF2{gamma} sequence was an unspliced intron or misspliced product. Analysis of the genomic locus, however, showed this sequence to have the hallmarks of a genuine exon. In addition, the start codon contained a reasonable Kozak consensus sequence, and the transcript encoded a functional protein in vitro. While preparing this manuscript we found the CRF2{gamma} exon sequence in the Genbank database, where it had been submitted as an aberrant splicing event that disrupts the reading frame of CRF (13).

We made numerous unsuccessful attempts to clone a rat version of the CRF2{gamma} cDNA. In addition, a moderate stringency screen of a Southern blot of rat genomic DNA reveals a lack of CRF2{gamma}-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{gamma} 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{gamma} are closer to what we observe for CRF2{alpha} than for CRF. In adenylyl cyclase assays, CRF-related peptides appeared approximately 10-fold more potent at the CRF receptor than the CRF2{alpha} and CRF2{gamma} 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{alpha} and CRF2{gamma} receptors using these CRF-related agonists, our data do suggest that the N terminus affects the affinity of peptide agonists for CRF.

Competition binding assays at the CRF2{gamma} receptor were performed using [125I-Tyr0]-sauvagine and other unlabeled CRF family agonists. The rank order of IC50s we observed for the human CRF2{gamma} 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{alpha} and CRF 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{gamma} and CRF2{alpha}(high- and low-affinity sites for CRF2{gamma}: 60 pM and 5 nM, and for CRF2{alpha}: 44 pM and 4 nM) (18). Thus, the competition binding assays also show a very similar pharmacological profile for the CRF2{gamma} and CRF2{alpha} receptors.

In contrast, the expression pattern of human CRF2{gamma} RNA in brain was most similar to our expression data for CRF. PCR amplification of human brain cDNAs using CRF-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{gamma} message. CRF2{gamma} 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{gamma}-labeled oligonucleotide probe. We did not examine CRF expression by Southern blot; therefore, it is unknown whether the weakly expressing tissues determined for CRF2{gamma} also weakly express CRF. The regions with most abundant CRF2{gamma} RNA expression, however, coincided with regions of most abundant CRF 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 CRF 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 CRF is also vascular in humans and whether the CRF2{gamma} receptor shares cellular localization as well as regional localization with the CRF receptor.

The expression profile we determined for the CRF2{alpha} 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{alpha} expression in human hypothalamus. While this assay was unable to discern regional localization within the human hypothalamus, in rodents, strong CRF2{alpha} expression was found in the supraoptic, paraventricular, and ventromedial hypothalamic nuclei of the hypothalamus (10). The CRF2{alpha} 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{alpha} receptors in the VMH may play a role in autonomic outflow and gastrointestinal function (10). As in rodents, we observed no detectable CRF2{alpha} message expression in human cerebellum. In contrast to rodent reports, however, we did detect CRF2{alpha} message in frontal cortex. This result is supported by a recent report by Liaw et al. (12) in which they obtained human CRF2{alpha} receptor cDNA clones from a human cortex library. It will be of interest to determine the cell types expressing the CRF2{alpha} receptor and compare this distribution to that of the human CRF1 receptor in cortex. While the human CRF2{alpha} 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, CRF 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 CRF and CRF2{gamma} messages in the CNS, we looked for the presence of CRF and CRF2{gamma} expression in human heart, lung, and skeletal muscle. Additionally, we examined CRF2{alpha} message levels.

In contrast to data reported for rodents, we did not detect CRF message in human heart. However, as in rodents, we did observe expression of CRF message in skeletal muscle (14). A recent report suggests that CRF 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{gamma} message in any of these tissues. Therefore the expression of human CRF and CRF2{gamma} RNAs are different in these peripheral tissues. Our examination of CRF2{alpha} message levels suggest that it is the predominant CRF2 receptor in these human peripheral tissues. The cellular localization of the CRF2{alpha} 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{alpha} receptor rather than the CRF 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{gamma} 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{gamma}. This isoform is expressed in similar brain regions to the CRF 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{gamma} receptor is typical for a CRF2 receptor, exhibiting closest similarity to the CRF2{alpha} splice form, with the highest apparent potency for the CRF-related peptides urocortin and sauvagine. Given the similar peptide pharmacology of CRF2{gamma} and CRF2{alpha}, at least for the known CRF-related peptide agonists, and the similarity of regional distribution of expression to CRF, it is not clear what specific role CRF2{gamma} 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.


    MATERIALS AND METHODS
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Library Screens
Briefly, a PCR fragment (coding nucleotides 1–535) from the rat CRF2{alpha} receptor was labeled with [32P-{alpha}]-dCTP using a random primed labeling kit (GIBCO BRL, Gaithersburg, MD) and was used to screen approximately 8.5 x 105 plaques from a {lambda}gt11 human amygdala cDNA library (CLONTECH, Palo Alto, CA). Hybridizations were performed at 60 C [6x saline sodium citrate (SSC), 0.25% nonfat dry milk, 100 µg/ml herring DNA], and the final wash condition was 0.5xSSC and 0.1% SDS at 60 C.

For the genomic library screen, a 467-bp CRF2{gamma} 5'-RACE clone containing both the CRF2{gamma}- and CRF-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{gamma} receptor-specific primer 5'-GCAGAAGAGCTGAGGAAAGCCCAGGTC-3'. The reaction products were then further amplified using a supplied nested anchor primer and a nested CRF2{gamma} 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 CRF and CRF2{gamma} exons was performed using 50 ng human genomic DNA (CLONTECH) and the Expand Long Template PCR system (Boehringer Mannheim, Indianapolis, IN). The CRF sense and CRF2{gamma} 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. Elizabeth’s 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{gamma}, 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{alpha} and CRF 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{gamma} 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{alpha} 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{alpha} 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{alpha} message abundance. These fine scale (2-fold) reactions were initially imaged by spiking with [33P-{alpha}]-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{alpha} 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{gamma} 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{alpha} and CRF 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 25–30 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{gamma} expressing 293-EBNA cells. Assays were initiated by the addition of 10–20 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 {gamma}-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 {alpha}-helical CRF(9–41).


    ACKNOWLEDGMENTS
 
The authors thank Thomas M. Hyde of the NIMH Neuroscience Center for his gift of human brain tissue and Julie C. Bunville and Karen L. Krakowski in the DuPont Merck Sequencing Facilities for their excellent technical support.


    FOOTNOTES
 
Address requests for reprints to: Brian Largent, DuPont Merck Research Laboratories, CNS Diseases Research, P.O. Box 80400, Wilmington, Delaware 19880-0400. E-mail: Brian.L.Largent{at}DuPontMerck.com

1 Authors contributed equally. Back

Received for publication January 26, 1998. Revision received March 26, 1998. Accepted for publication April 13, 1998.


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