A Novel Spliced Variant of the Type 1 Corticotropin-Releasing Hormone Receptor with a Deletion in the Seventh Transmembrane Domain Present in the Human Pregnant Term Myometrium and Fetal Membranes

Dimitris K. Grammatopoulos1, Yalei Dai1, Harpal S. Randeva, Michael A. Levine, Emmanouil Karteris, Andrew J. Easton and Edward W. Hillhouse

Sir Quinton Hazell Molecular Medicine Research Centre (D.K.G., Y.D., H.S.R., E.K., A.J.E., E.W.H.) Department of Biological Sciences University of Warwick Coventry, CV4 7AL, United Kingdom The Johns Hopkins University School of Medicine (M.A.L.) Division of Pediatric Endocrinology Department of Pediatrics Baltimore, Maryland 21287


    ABSTRACT
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
CRH exerts its actions via activation of specific G protein-coupled receptors, which exist in two types, CRH-R1 and CRH-R2, and arise from different genes with multiple spliced variants. RT-PCR amplification of CRH receptor sequences from human myometrium and fetal membranes yielded cDNAs that encode a novel CRH-R type 1 spliced variant. This variant (CRH-R1d) is present in the human pregnant myometrium at term only, which suggests a physiologically important role at the end of human pregnancy and labor. The amino acid sequence of CRH-R1d is identical to the CRH-R1{alpha} receptor except that it contains an exon deletion resulting in the absence of 14 amino acids in the predicted seventh transmembrane domain. Binding studies in HEK-293 cells stably expressing the CRH-R1d or CRH-R1{alpha} receptors revealed that the deletion does not change the binding characteristics of the variant receptor. In contrast, studies on the G protein activation demonstrated that CRH-R1d is not well coupled to the four subtypes of G proteins (Gs, Gi, Go, Gq) that CRH-R1{alpha} can activate. These data suggest that although the deleted segment is not important for CRH binding, it plays a crucial role in CRH receptor signal transduction.

Second messenger studies of the variant receptor showed that CRH and CRH-like peptides can stimulate the adenylate cyclase system, with reduced sensitivity and potency by 10-fold compared with the CRH-R1{alpha}. Furthermore, CRH failed to stimulate inositol trisphosphate production. Coexpression studies between the CRH-R1d or CRH-R1{alpha} showed that this receptor does not play a role as a dominant negative receptor for CRH.


    INTRODUCTION
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
In mammals, hypothalamic CRH secretion is one of the most important regulatory mechanisms for coordinating the activity of the hypothalamic-pituitary-adrenal axis in response to stress (1, 2). In addition, CRH is involved in the control of the cardiovascular, gastrointestinal, immune, and reproductive systems (3, 4, 5). The actions of CRH are mediated via binding to, and activation of, specific seven-transmembrane domain (TMD) G protein-coupled receptors, which belong to the distinct family of R7G2 receptors [receptors for polypeptides of an intermediate length such as calcitonin/vasointestinal peptide/PTH/secretin/GHRH/glucagon] (6). To date, two distinct CRH receptor types (R1 and R2) have been found in the human, each being encoded by separate genes (7, 8). The CRH-R1 gene is located on the long arm of chromosome 17 at 17q12-q22 (9) while the CRH-R2 gene is located on the short arm of chromosome 7 at 7p21-p15 (10). Interestingly, the two receptors have distinct binding characteristics for CRH and CRH-like ligands. Pharmacological studies on the CRH-R1 receptor have shown that CRH and other ligands of the CRH-like peptide family (urocortin, sauvagine, and urotensin) have similar binding affinities and potency in generating cAMP. In contrast, the CRH-R2 family of receptors has distinct pharmacological characteristics and higher binding affinity for urocortin than CRH, suggesting that urocortin may be the native agonist for CRH-R2 receptors (11).

The two receptors share 70% homology at the amino acid level, and each of them exists as a family of related proteins produced from multiple alternatively spliced forms of mRNA. The cDNA of CRH-R1{alpha} (7) encodes a 415-amino acid protein, and two alternative forms have been identified so far; the first variant has 29 amino acids inserted into the first intracellular loop (CRH-R1ß), while the second (CRH-R1C) (12) has 40 amino acids deleted from the N-terminal domain. These structural differences reduce the receptor binding affinity for [125I]CRH, and the CRH-R1ß appears to have reduced coupling to adenylate cyclase, while the CRH-R1C appears to have no signal transduction properties (12, 13). The type 2 CRH receptors have distinct tissue distribution from the type 1 receptors and also exist in three splice variant forms, namely CRH-R2{alpha}, -R2ß, and -R2{gamma} (9, 14, 15). Structural comparison of these CRH-R2 subtypes showed that 377 amino acids at their C terminus are identical and they differ only in their N terminus; the 34 amino acids N terminal to CRH-R2{alpha} are replaced by a 61-amino acid sequence to form the CRH-R2ß or a 20-amino acid sequence to form the CRH-R2{gamma}. Moreover, in adenylate cyclase activation assays, CRH-related peptides appeared 10-fold more potent at the CRH-R2ß than CRHR-2{alpha} or CRH-R2{gamma}, which suggests that the N terminus of the receptor is involved in the ligand-receptor interaction (15). In these experiments, urocortin was found to be the most potent peptide for activation of the type 2 CRH receptors.

We have identified and characterized specific CRH-Rs in the human pregnant and nonpregnant myometrium (16, 17, 18, 19, 20) that mediate the actions of CRH on the myometrium during pregnancy and labor. Multiple subtypes are present as determined by isoelectric focussing (18) and RT-PCR (21), and our studies on the CRH-R subtype expression in the human myometrium demonstrated that during pregnancy there is differential CRH-R expression. These studies have led to the cloning and characterization of a cDNA from human pregnant, but not nonpregnant myometrial RNA that encodes a novel spliced variant of the human CRH-R1 receptor (termed CRHR-1d). This receptor isoform, which is also present in the human fetal membranes (amnion and chorion derived from spontaneous rupture) but not in placental biopsies at term, is generated by deletion of a sequence that corresponds to exon 12 of the human CRH-R1 gene (22) and encodes 14 amino acids in the putative seventh TMD. Characterization of the CRHR-1d isoform demonstrated that the protein has identical binding but different signal transduction characteristics to those of R1{alpha}.


    RESULTS
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
RT-PCR Cloning and Sequence Analysis-Fluorescent in Situ Hybridization (FISH)-Stable Transfection Studies
Using specific primers for a nested PCR, one DNA fragment of 1.2 kb was amplified from primary cultured pregnant myometrial cells and from pregnant (at term, 38–40 weeks of gestation) but not nonpregnant myometrial biopsies (Fig. 1Go). Other alternatively spliced CRH receptor mRNAs reported previously have been isolated by cDNA library screening. Interestingly, no DNA fragment was amplified from pregnant (preterm, 30–34 weeks of gestation) myometrial biopsies (data not shown). After RT-PCR and the identification of the CRH-R1d receptor variant mRNA transcripts, we used FISH to localize the receptor cellular distribution by using subtype-specific oligonucleotide probes. This identified the CRH-R1d receptor mRNA in pregnant (at term) but not nonpregnant or preterm myometrium (Fig. 1bGo). It is possible that the variant receptor is present in nonpregnant or preterm myometrial tissue but is below the level of detection by the RT-PCR technique, and therefore the physiological role in these tissues is unlikely to be significant. 293-R1d and 293-R1{alpha} cells were used as positive and negative controls, respectively (Fig. 1bGo). Furthermore, positive staining for the variant receptor was also observed in primary cultured human pregnant myometrial cells and human fetal membranes (amnion and chorion) derived from spontaneous rupture but not in placental biopsies at term. Using receptor subtype-specific oligonucleotide probes we showed that the CRH-R1d signal was localized primarily in the chorion, while that of the CRH-R1{alpha} was localized mainly in the amniotic epithelium (Fig. 1cGo).



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Figure 1. Identification of CRH-R1d in Pregnant Myometrium and Fetal Membranes

a, Nested PCR amplification of the human CRH-R1d receptor subtype from mRNA extracted from nonpregnant (lane 1), pregnant (lane 2) human myometrium, and primary cultured human myometrial cells taken at term (lane 3); lanes 2 and 3 show amplification of a 1.2-kb fragment, when compared with the DNA size marker (lane M). b, Distribution of the CRH-R1d receptor mRNA in human pregnant myometrium at term before the onset of labor (A), nonpregnant myometrium (B), and cultured human pregnant myometrial cells (C), by fluorescent in situ hybridization. D, 293-R1{alpha} cells were used as negative controls and these cells were stained using the DNA-specific dye 4',6-diamino-2-phenylindole (DAPI). Magnification, x400. c, Distribution of the CRH-R1d receptor mRNA in human fetal membranes (amnion and chorion) derived from spontaneous rupture at term, by fluorescent in situ hybridization. Magnification, x400.

 
After cloning into pBluescript II(+/-) and nucleotide sequencing, the nucleic acid sequence of the fragment was shown to be identical to the previously published CRH-R1{alpha} (7), except for an in-frame 42-bp deletion that encodes 14 amino acids (Gly356-Glu369) on the putative seventh TMD of the receptor protein. This 42-bp cassette corresponds exactly to exon 12 of the human CRH-R1 gene (22), suggesting that this novel isoform of CRH-R1 (CRH-R1d) is generated by alternative splicing of exon 12 (Fig. 2Go, a and b). Hydropathy analysis (23) of the amino acid sequences of the CRH-R1{alpha} and CRH-R1d receptors (Fig. 2cGo) demonstrated that the hydrophobic region corresponding to the seventh TMD was smaller, consistent with the predicted 14-amino acid deletion. To determine the functional significance of this deletion on receptor function, the variant CRH-R1d and the CRH-R1{alpha} receptors were subcloned into the expression vector pCI-neo (Promega Corp., Madison, WI), and three clones for each receptor were stably transfected into HEK 293 cells. After selection for transfected cells with the antibiotic G418, a total of 50 and 86 cell lines transfected with CRH-R1d and CRH-R1{alpha} receptor cDNAs (293-R1d and 293-R1{alpha}, respectively) survived, and three individual cell lines were used for each receptor binding and second messenger experiment.



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Figure 2. Schematic Representation of CRH-R1 Gene and Deduced Human CRH-R1 Peptide Sequences

a, CRH-R1 gene and its mRNA spliced variants and position of CRH-R1 exons in relation to signal peptide (SP), intracellular (IC), extracellular (EC), and transmembrane (TM) domains. b, Deduced human CRH-R1 subtype peptide sequences. Gray circles indicate the amino acid sequences that differ in the CRH-R1 splice variants, and black circles indicate the deleted amino acid sequence in the human CRH receptor novel variant R1d. c, comparison by Kyte-Doolittle hydrophobicity analysis of CRHR-1{alpha} and CRHR-1d amino acid sequence. The cDNA sequence has been submitted to the EMBL Data Library (accession no. AF180301).

 
Binding and Signal Transduction Characteristics of the CRH-R1d Receptor
The binding characteristics of the variant CRH-R1d receptor were determined in a stable transfection system by using a specific RRA in membranes prepared from 293-R1d cells and compared with those of the 293-R1{alpha}. Competitive displacement studies of 125I-[Tyr0]-oCRH by h/rCRH showed no differences in pharmacological characteristics (Fig. 3aGo); the EC50 was 10 ± 2.1 nM for both receptors, which is comparable to previously described data (7). The pharmacological specificity of both R1 receptors was assessed by use of AVP and oxytocin, and both were inactive in the RRA in displacing 125I-[Tyr0]-oCRH (data not shown). No specific binding was detected in cells transfected with the pCI-neo vector alone. Scatchard analysis of CRH binding for each of the CRH-R1d and R1{alpha} was consistent with the presence of a single population of high-affinity receptors, which displayed identical high-affinity binding for CRH with apparent dissociation constants (Kd) ranging between 0.90 ± 0.4–1.45 ± 0.4 nM and 1.60 ± 0.3–2.15 ± 0.6 nM for CRH-R1{alpha} and CRH-R1d, respectively (Fig. 3bGo). All three individual clones demonstrated comparable binding characteristics (Table 1Go). The maximum binding site concentrations (Bmax) were found to be similar for both receptors (3.4 ± 0.6 and 2.2 ± 0.7 nmol/mg protein for the R1{alpha} and R1d, respectively), confirming that transfection efficiencies were not different for the two receptors.



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Figure 3. Binding Characteristics of CRH-R1{alpha} and CRH-R1d

a, Competitive displacement curves of 125I-labeled [Tyr0]-oCRH in the presence of increasing concentrations of unlabeled h/rCRH in membranes prepared from HEK-293 cells stably transfected with CRH-R1{alpha} or CRH-R1d receptor subtypes. Results are mean ± SEM of three experiments. b, Scatchard analysis of 125I-labeled [Tyr0]-oCRH specific binding to membranes prepared from HEK-293 cells stably transfected with CRHR-1{alpha} or CRHR-1d receptor subtypes. Results indicate the presence of a single population of high-affinity receptors with comparable dissociation constants (Kd). Data are representative of three independent experiments for each receptor.

 

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Table 1. CRH-Binding and Signal Transduction Properties of HEK-293 Cells Stably Transfected with Different Clones of CRH-R1{alpha} or CRH-R1d Receptor Subtypes

 
Having established that the 14-amino acid deletion did not affect the binding characteristics or the cell-membrane receptor expression of the variant CRH-R1d receptor, the signal transduction characteristics of the R1{alpha} and R1d receptors were characterized. In 293-R1{alpha} cells, CRH or CRH-like peptides (urotensin or urocortin) elicited a dose-dependent increase in cAMP production with maximum effect observed at concentrations of 100 nM (Fig. 4aGo). For the individual 293-R1{alpha} cell lines tested, maximum cAMP response ranged between 80 ± 7 and 120 ± 15-fold above basal (Table 1Go). This effect is presumably mediated by coupling to Gs, which activates adenylate cyclase. In contrast, in 293-R1d cells a different pattern of response was observed; CRH, urocortin, or urotensin stimulated a weak cAMP response and both the receptor sensitivity and potency were reduced by 10-fold compared with that seen for 293-R1{alpha} cells. In all three individual 293-R1d cell lines tested, CRH could exert a significant cAMP production only at concentrations greater than 10 nM, and maximum cAMP responses ranged between 6 ± 2.1 and 19 ± 3.5-fold above basal (at a CRH concentration of 100 nM) (Fig. 4bGo). The possibility that CRH-R1d might act as a dominant negative receptor form was investigated by cotransfection of CRH-R1{alpha} and CRH-R1d; the cAMP response was identical to that of CRH-R1{alpha} (Fig. 4cGo). In all types of cells, the integrity of adenylate cyclase was tested by the use of forskolin (10-5 M) and no differences were found (data not shown). Furthermore, 293-R1d cells showed no inositol triphosphate (IP3) response to CRH challenge (with concentrations up to 1 µM), while significant dose-dependent inositol phosphate production was observed in 293-R1{alpha} cells with a CRH threshold of 75–100 nM and maximum response at 500 nM (maximum 90 ± 21% of basal (Table 1Go). Similarly, cotransfection of CRH-R1{alpha} and CRH-R1d receptors did not affect the IP3 response of the R1{alpha} (data not shown).



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Figure 4. Effect of CRH on cAMP Release from HEK-293 Cells Stably Transfected with CRH-R1{alpha}, CRH-R1d, or Both

cAMP release from HEK-293 cells stably transfected with CRH-R1{alpha} or CRH-R1d receptor subtypes (50 µg protein) in the presence of different concentrations of h/rCRH (a) or urocortin (b). c, cAMP release from HEK-293 cells (50 µg - amount of protein assayed in the cAMP assay) stably transfected with either CRH-R1{alpha}, CRH-R1d, or both in the presence of different concentrations of h/rCRH. Results are representative of one receptor clone and are expressed as the mean ± SEM of four estimations from three independent experiments. *, P < 0.05 compared with basal.

 
G Protein Activation Characteristics by the CRH-R1d and -R1{alpha} Receptors
These results suggested that the variant CRH-R1d receptor may not couple well to G proteins. In previous studies, the CRH-R1, like other members of this family, has been shown to couple to multiple G proteins and activate at least two signaling molecules, cAMP and IP3. Also, we recently showed that the rat cerebral cortex CRH receptor can activate multiple classes of G proteins (24).

Initially, the class of G proteins coupled to the CRH-R1{alpha} and -R1d receptors was investigated by using the nonhydrolyzable GTP analog 32P-GTP-AA to label the activated G proteins, and this was followed by immunoprecipitation with specific G protein antibodies (25). In preliminary experiments we found that for all classes of G proteins maximal GTP-AA incorporation was found at a concentration range of 100 nM CRH (data not shown). As shown in Fig. 5Go our results demonstrated that the stimulation of CRH-R1{alpha} by CRH resulted in activation of a 48-kDa G{alpha}s-protein, as well as three more types of G proteins: the G{alpha}q, G{alpha}i1/2, and G{alpha}o. G{alpha}s activation was found to be the most potent (4-fold increase over basal), followed by G{alpha}q and G{alpha}o activation (2.5- to 3-fold increase over basal) while G{alpha}i-protein activation was found to be the least potent (1.2- to 1.5-fold increase over basal). When urocortin was used to stimulate 293-R1{alpha} cells, a similar G protein activation pattern was seen with potency comparable to CRH (data not shown). Furthermore, in 293-R1d cells CRH and urocortin could only weakly activate G{alpha}s and G{alpha}o-proteins (0.5- to 0.9-fold increase over basal). No G{alpha}q or G{alpha}i activation was observed with either CRH or urocortin.



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Figure 5. Autoradiograghs (A) and Densitometry Scanning Quantitation (B) of Agonist-Induced Photolabeling with GTP-AA of Various G{alpha}-Proteins from HEK-293 Cells Stably Transfected with CRH-R1{alpha} or CRH-R1d Receptor Subtypes

Membranes were incubated with GTP-AA and 100 nM CRH, followed by UV cross-linking and immunoprecipitation of the G{alpha}-subunits using specific antibodies. Proteins were resolved on SDS-PAGE gels, followed by autoradiography. Identical results were obtained from four independent experiments. *, P < 0.05 compared with unstimulated controls.

 
To confirm the specificity of these data, a different method was employed to identify the specific G proteins coupled to the CRH-R1{alpha} and R1d receptors. Membranes from 293-R1{alpha} cells were incubated in the presence or absence of CRH (100 nM), solubilized, and immunoprecipitated with receptor antiserum. The G protein subunits co purified with the receptor were detected by immunoblotting with antibodies specific for different {alpha}-subunits (26). Nonimmunoprecipitated membranes from HEK293 cells were used as controls for the blotting antisera, and specific immunoreactivity was detected at 40–42 kDa with antibodies to G{alpha}i1/2 and G{alpha}q/11, 39 kDa with anti-G{alpha}o antibody, and 45–53 kDa with anti-G{alpha}s antibody. When the CRH-R1{alpha} was precipitated under basal conditions, small amounts of G proteins were found to be present. Our results confirmed that when the receptor was activated by preincubation with CRH (100 nM), the amount of G proteins, G{alpha}s, Gq, G{alpha}o, and G{alpha}i, that could be immunoprecipitated was significantly greater (Fig. 6Go). Thus, CRH binding determined the amount of G proteins coprecipitated with the receptor. Furthermore, when 1 µM blocking peptide was added during the immunoprecipitation step, no CRH-induced increase in the amount of G protein precipitated was observed. In our system, although the antibody used could detect three forms of G{alpha}s, the CRH-R1{alpha} could activate only the long form of G{alpha}s (48 kDa), but not the other two forms. Identical results were observed with the CRH-like peptide, urocortin (data not shown). In agreement with our previous observations in membranes from 293-R1d cells, CRH or urocortin had a reduced action on G protein activation, and only small amounts of Gs- and Go-protein were coprecipitated with the receptor (data not shown).



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Figure 6. Identification of G Proteins Coprecipitated with the CRH-R1{alpha} Receptor

293-R1{alpha} membranes were incubated in the presence or absence of 100 nM CRH. After solubilization, receptors were immunoprecipitated with anti-CRH-R1 antibody and protein A-Sepharose in the absence or presence of 1 µM blocking peptide. Precipitated proteins were solubilized and separated on 10% SDS-PAGE gels. Specific G protein {alpha}-subunits were identified by immunoblotting with G protein antibodies.

 

    DISCUSSION
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
The CRH-R1 receptor plays a key role in mediating the stress response and anxiety-related behavior as shown in CRH-R1-deficient mice (27). Three alternative spliced forms of the human CRH-R1 receptor family have been previously isolated and characterized (7, 12). Here we report the cloning of a cDNA encoding a distinct type 1 receptor splice variant isolated from human myometrium and fetal membranes, which we term CRH-R1d. The presence of the variant CRH receptor mRNA in the human pregnant myometrium and fetal membranes at term was demonstrated by FISH. Nucleotide sequencing revealed that alternative mRNA splicing results in the deletion of exon 12 and hence a 14-amino acid deletion in the putative seventh TMD, which results in a seventh TMD shorter by two thirds.

Receptor-spliced variants arising from similar exon deletions resulting in 14-amino acid deletions in the putative seventh TMD have been described for two other members of this receptor family, i.e. calcitonin (CTR{Delta}e13) (28) and PTH/PTHrP (29) receptors. The CRH-R1 exon/intron junctions are aligned to that of the PTH receptor after exons 3, 5, 7–10, and 12, and both receptors are similar in that amino acids 457–509 (of the CRH-R1) are divided into exons 11 and 12. The calcitonin receptor family is of particular interest since it appears to have a very similar isoform profile to the CRH-R1 receptor family; both receptors have splice variants that contain inserts in the first intracellular loop (16 and 29 amino acids in the calcitonin and CRH-R1 receptor, respectively) (7, 30) and exon deletions in the seventh TMD. Furthermore, analysis of the nucleotide sequence reveals that there are conserved splicing sites in the first intracellular loop (site of insertion) and the seventh TMD (site of exon deletion) (Table 2Go).


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Table 2. Alignment of Nucleotide and Deduced Amino Acid Sequences of CRH-R1 and CTR Splice Variants Arising from Conserved Splicing Sites in the First Intracellular Loop (Site of Insertion) or the Seventh TMD (Site of Exon Deletion)

 
Deletion of 14 amino acids from a TMD of the seventh TMD receptors might be expected to reduce or even abolish expression, binding, or signal transduction characteristics of the translated protein. Preliminary studies using immunocytochemistry (data not shown) showed that the novel CRH-R variant expression in the cell membrane was not affected. It is well established that in G protein-coupled receptors, residues in the transmembrane helical domains participate in the formation of ligand binding sites (31). However, our binding data on the CRH-R1d indicate that an intact seventh TMD is not required for efficient receptor-ligand interaction. Interestingly, in these binding experiments, both receptors (R1{alpha} and R1d) showed an affinity for CRH that was 15- to 20-fold less than that reported previously (16). Although the reason for this discrepancy is not known, it might be due to differences between the experimental systems used (myometrial membrane homogenates vs. transfected HEK-293 cells), although it is possible that as-yet-unidentified CRH receptor(s) with higher affinity for CRH than R1{alpha} are present in myometrium. Previous studies have identified regions important for high-affinity CRH binding to the second extracellular domain (ECD), the junctions of the third ECD/fifth TMD, and second ECD/third TMD (32). Also important sequences for high-affinity agonist binding to CRH-R1 are present in the N terminus (amino acids 42–50 and 76–84) and in the ECDs of the R1 receptor (33, 34). Interestingly, the CTR{Delta}e13 receptor showed reduced binding for calcitonin (28), a finding that demonstrates that the same receptor region might play distinct roles in different receptors.

The deletion of the amino acids encoded by exon 12 resulted in major differences in the signal transduction characteristics of R1d receptor. The CRH-R1{alpha} receptors can efficiently stimulate adenylate cyclase upon activation with subnanomolar concentrations of CRH and, like several receptors of the R7G2 family, can generate phospholipase C and IP3 production at high concentrations of CRH (13). In contrast, in 293-R1d cells CRH and CRH-like peptides were less potent in generating intracellular cAMP and were unable to stimulate IP3 production. Again the CTR{Delta}e13 splice variant had similar altered signal transduction characteristics (28). Previous studies on the CRH-R1 isoforms revealed that the insertion of 29 amino acids in the first intracellular loop of the CRH-R1ß had similar effects on the signal transduction characteristics (12). However, the CRH-R1ß variant has also reduced binding affinity for CRH.

Using techniques that demonstrate receptor-mediated G protein activation, we provide direct evidence for the first time that in HEK-293 cells upon stimulation by CRH or urocortin the CRH-R1{alpha} can activate multiple G proteins with order of potency G{alpha}s > G{alpha}q = G{alpha}o > G{alpha}i. This suggests that the CRH-R1{alpha} receptor can activate diverse intracellular signaling pathways. Alternative candidate signaling pathways include calcium channel modulation and the tyrosine kinase pathway, both of which have been shown to be modulated by CRH in various types of cells (35, 36). Interestingly, although the anti-Gs-antibody used could detect three types of G{alpha}s protein present in the HEK293 cells, our results showed that the CRH-R1{alpha} could only activate the long form of G{alpha}s (48 kDa). Also, consistent with the observations on cAMP and inositol phosphate production, the CRH-R1d variant had 75% reduced potency in stimulating Gs and Go proteins while its ability to activate G{alpha}q and G{alpha}i proteins was abolished. The observed reduction in cAMP release was much greater (10 fold) than the difference in G{alpha}s activation, which might be due to the differences in experimental conditions and/or the different sensitivities of each method. At present, the exact nature of signal transduction pathways that can be activated by these G proteins is unknown, and since it is possible that there is tissue-specific selectivity of G-protein activation, detailed analysis in various tissues is required.

Collectively, these results indicate that an intact seventh TMD is required for efficient coupling to G proteins, but not for high-affinity binding, and therefore it dissociates high-affinity binding of the ligand from its biological responsiveness and might provide a clue to the conformational switch that activates this receptor. It is possible that the deletion of the 14 amino acids in the seventh TMD could draw the proximal residues of the C-tail into the lipid bilayer and thus block their interaction with G proteins or that the CRH-R1d variant has a reduced hydrophobic character in its seventh TMD, which results in instability and failure to anchor the remaining sequence in the membrane. Also, our results suggest that sequences in the C terminus proximal to the membrane may be important for specific coupling to phospholipase C, while more distal residues in the C-tail are required for coupling to adenylate cyclase.

Of interest is the observation that during pregnancy there is an alteration in the pattern of myometrial CRH-R1d expression that is expressed only at term. This would suggest an important, yet unidentified, role of this receptor variant in the mechanism of labor. The cotransfection experiments in HEK 293 cells failed to demonstrate a dominant negative action for the CRH-R1d when overexpressed in conjunction with CRH-R1{alpha}. In the native tissue, however, the possibility exists that it could contribute to the decreased activation of adenylate cyclase by CRH at term (19). It is also important to note that in the fetal membranes, the CRH-R1d signal was localized mainly in the chorion, while that of the CRH-R1{alpha} was localized mainly in the amniotic epithelium. The functional significance of this differential receptor expression is unknown, but again it suggests a distinct functional role for the CRH-R1d. During pregnancy, CRH is produced by the placenta and feto-maternal and myometrial tissues but its biological role is unknown. Multiple CRH receptor mRNAs have been identified in the fetal membranes, placenta, and human myometrium with differential expression pattern during pregnancy (21), which argues for multiple roles for CRH and/or related peptides in myometrial function and suggests distinct functional roles for each receptor during pregnancy. Different mechanisms may be involved to increase myometrial sensitivity to different ligands dependent upon the presence of CRH receptor subtypes on the membrane of myometrial smooth muscle cells. The specific role of CRH-R1d during pregnancy is currently unknown, and cotransfection experiments between CRH-R1{alpha} and R1d receptors suggest that the R1d does not act as a dominant negative receptor by preventing activation of R1{alpha} and stimulation of adenylate cyclase or phospholipase C. However, the possibility remains that it might block other signaling cascades.

In conclusion, we have identified a novel CRH-R1 variant generated by alternative splicing of mRNA resulting in the absence of 14 amino acids from the seventh TMD. This variant receptor has impaired signal transduction properties and is present in the human pregnant, but not in the nonpregnant, myometrium as well as in fetal membranes, which argues for a physiological role during pregnancy and provides evidence that the generation of different CRH-R isoforms by alternative splicing of the CRH-R mRNA may define the balance of diverse biological responses induced by CRH.


    MATERIALS AND METHODS
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Chemicals
Radioiodinated ovine (o)-tyr-CRH and human/rat (h/r) CRH were obtained from Penninsula Laboratories (Merseyside, UK). The mammalian expression vector pCI-neo was obtained from Promega Corp. UK Ltd (Chilworth Research Centre, Southampton, UK), and the pREP4 was obtained from Invitrogen BV (Groningen, The Netherlands). Dithiothreitol, GTP and GppNHp, 2-[N-morpholino]ethane sulfonic acid, 1,4-dioxane, triethylamine, and all other chemicals were purchased from Sigma Ltd (Poole, Dorset, UK). Waters Sep-Pak C18 columns were obtained from Millipore Corp. Ltd (Watford, Herts, UK). The polyclonal G protein rabbit antibodies, RM/1 (anti-Gs{alpha}), AS/7 (anti-Gi1{alpha}, Gi2{alpha}), GC/2 (anti-Go{alpha}), QL (anti-Gq/11{alpha}), [3H]myo-inositol, and the cAMP assay kits were obtained from NEN Life Science Products (Hertfordshire, UK). Protein-A Sepharose beads (CL-4B) was purchased from Pharmacia Biotech (Uppsala, Sweden). [{alpha}-32P]-GTP, enhanced chemiluminescence, the DNA sequencing kit, and 35S-{alpha}-ATP were obtained from Amersham International (Little Chalfont, Buckinghamshire, UK). 4-Azidoanilide-HCl and 1-(3-dimethylamino propyl)-3-ethylenecarbodiimide hydrochloride were purchased from Aldrich (Gillingham, Dorset, UK). PCR and cloning reagents were purchased from Life Technologies, Inc. (Renfrewshire, UK). The DNA 3'-end labeling kit was purchased from Roche Molecular Biochemicals (Lewes, UK). Synthetic oligonucleotide probes and enzymes were purchased from Life Technologies, Inc. (Paisley, UK). The specific CRH receptor antibody was obtained from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA). This is a goat polyclonal antibody raised against a peptide corresponding to amino acids 425–444 mapping at the C terminus of the human CRH-R1 precursor. All other chemicals were purchased from BDH (Merck, Poole, UK).

Subjects and Sample Preparations
Pregnant myometrial biopsies (n = 13) were obtained from women undergoing elective cesarean section at term (n = 7) or preterm (n = 6) before the onset of labor for nonmaternal problems. The biopsy site was standardized to the upper margin of the lower segment of the uterus in the midline. This provides the closest approximation to the upper segment of the uterus. Nonpregnant myometrial tissues (n = 8) were obtained from premenopausal controls undergoing hysterectomy for nonmalignant conditions. The nonpregnant myometrial biopsies were obtained from the same location as the cesarean section myometrial biopsies to avoid possible differences in receptor expression patterns. The relative content of myometrial and fibrous tissue in these biopsies was identified by immunostaining using specific smooth muscle cell and fibroblast markers (actin and vimentin, respectively). The biopsies were immediately snap frozen in liquid nitrogen and subsequently stored at -70 C until use. Ethical approval was obtained from the local ethical committee and informed consent to the study was obtained from all patients.

Culture of Myocytes
The tissue was immediately placed in 20 ml of ice-cold DMEM culture medium containing 200 IU penicillin/ml, 200 mg streptomycin/ml. Myocytes were prepared by enzymatic dispersion as previously described (20). Briefly, pieces of myometrium were transferred into DMEM containing collagenase (300 U/ml), deoxyribonuclease (DNAase) (30 U/ml), penicillin (200 U/ml), and streptomycin (200 mg/ml) and incubated at 37 C for 30 min. After filtration and centrifugation, single myocytes were suspended in DMEM containing 10% FCS, penicillin (100 U/ml), streptomycin (100 mg/ml), and Fungizone (2.5 µg/ml). The purity of myometrial muscle cells was assessed by immunocytochemical staining. Mouse antihuman smooth muscle actin-specific monoclonal antibody (MAb) and peroxidase-conjugated rabbit antimouse antibody (Ab) were used. Human fibroblast cells and omission of the primary antibody were used as negative controls, while frozen myometrial tissue was used as a positive control. The cells were kept at 37 C in a humidified atmosphere of 95% air and 5% CO2 until confluent.

RT-PCR, Cloning, and Sequence Analysis
Polyadenylated RNA was isolated from pregnant and nonpregnant myometrium tissues by RNeasy Total RNA kit (Qiagen, Crawley, UK) and reverse transcribed to synthesise cDNA, by using RNase H Reverse Transcriptase (Life Technologies, Inc., Paisley, UK). This was used as a template for a first-round PCR, and the products of the first PCR reaction served as a template for the second round of amplification reaction. All PCR reactions were carried out using Elongase enzyme mix (Life Technologies, Inc.) with 200 ng cDNA for each amplification. Four primers (1S: 5'-AGCCGAGCGAGCCCGAGGATG-3'; 1A: 5'-GTCGACAAGCTT (T)18-3'; 2S: 5'-CGAGGAT GGGAGGGCACCCGC-3'; and 2A: 5'-TCAGACTGCTGTGGACTGCTT-3') were used for the nested PCR. Distilled water was used in place of the cDNA as a negative control for each reaction.

The products of the second PCR reaction were analyzed by using 1% agarose gel electrophoresis, purified by using QIAquick Gel Extraction Kit (Qiagen) and ligated using T4 DNA Ligase Kit (Life Technologies, Inc.) into plasmid pBluescript II SK (+/-)-derived T-vector for sequence analysis. Positive isolated clones were sequenced using internal primers for the whole gene, in an automated DNA sequencer, and the sequence data were analyzed using Blast Nucleic Acid Database Searches from the National Centre for Biotechnology Information (NCBI, Bethesda, MD).

FISH
Paraffin-embedded blocks of human myometrium previously fixed in 4% (wt/vol) paraformaldehyde in 10 mmol/liter PBS, were sectioned (7 µm) and mounted onto gelatin-coated slides. At the time of hybridization, sections were dewaxed using xylene, dehydrated by successive washes through ethanol, and air-dried. A synthetic oligonucleotide probe (Life Technologies, Inc.) with fluorescein conjugated at the 5'-end was used (F-CCGGATGGGAGAACGGACCTGGAAGGATTCCA GGAA-3').

After prehybridization, sections were covered with parafilm and incubated at 37 C for 5 h in a humidified chamber. Then hybridization solution (100 µl) containing 25% formamide, 4x SSC, 5% dextran sulfate, 0.2% dried milk powder, and 1 ng/µl of the probe was added, and the section was sealed with Sureseal (Hybaid Ltd, Teddington, Middlesex, UK) and allowed to hybridize at 37 C for 20 h. After hybridization, sections were washed twice with 2x SSC at 45 C and once with 0.1x SSC at 45 C. The sections were then rinsed in PBS, air dried, and visualized under a fluorescent microscope. Samples of pregnant and nonpregnant human myometrium were probed with both sense and antisense oligonucleotide probes of CRH receptors to ensure specificity.

Stable Transfection of HEK-293 Cells
Human CRH-R1{alpha} or the variant (R1d) CRH receptor subtypes were subcloned into the expression vector pCI-neo (Promega Corp.) under the control of the human cytomegalovirus immediate early promoter. Human embryonic kidney cells (HEK)-293 were transfected using Lipofectamine reagent (Life Technologies, Inc.). The cells were grown in DMEM in the presence of G418 (500 µg/ml) to select for transfected cells, and those surviving were subcultured. A number of these cell lines (293-R1{alpha} or 293-R1d) were selected for characterization of their binding and signaling properties.

For cotransfection studies CRH-R1{alpha} or the variant (R1d) CRH receptor subtypes were subcloned into the expression vectors pCI-neo (Promega Corp.) and pREP4 (Invitrogen), respectively, and transfected into HEK-293 cells. The cells were grown in DMEM in the presence of G418 (500 µg/ml) + Hugromycin (200 µg/ml) to select for transfected cells, and those surviving were subcultured and characterized for their signaling properties.

Membrane Preparation and CRH RRA
When confluent, 293 cells were washed with PBS and lysed with 0.2% NaCl. The cells were homogenized in extraction buffer A containing 10 mM Tris-HCl, 1 mM EDTA, 1 mM phenylmethylsulfonyl fluoride, 10 mM MgCl2, 0.1% BSA, and 0.1% bacitracin, pH 7.2. The homogenate was centrifuged at 600 x g for 30 min at 4 C to remove nuclei and unbroken cells. The supernatant was collected and centrifuged at 40,000 x g for 60 min at 4 C. The pellet was rinsed twice, resuspended in binding buffer B containing 10 mM Tris-HCl, 1 mM EDTA, 10 mM MgCl2, 0.1% BSA, and 0.1% bacitracin, pH 7.2, and aliquoted (50 µg in 50 µl aliquots) in microfuge tubes.

For competitive displacement studies, 293-R1{alpha} or 293-R1d cell membranes (50 µg) were incubated with 1 nM 125I-labeled oCRH in the presence or absence of unlabeled peptide (0.01–1000 nM). Nonspecific binding was measured in the presence of 1 µM unlabeled r/hCRH. For Scatchard analysis, membranes (50–100 µg of protein) were incubated with 125I-labeled oCRH (0.2–2 nM) and unlabeled r/h CRH (1,000 molar excess) in 50 µl of binding buffer B. The tubes were incubated at 22 C for 120 min. The reaction was terminated by adding 1 ml/tube of ice-cold 20% polyethylene glycol. After centrifugation at 10,000 x g for 15 min at 4 C, the pellets were washed once with 20% polyethylene glycol and counted in a {gamma}-counter (Packard Instrument Co., Meriden, CT) at 70% efficiency. Nonspecific binding was 18 ± 5% of the total added radioactivity.

The binding data were analyzed using the computer program EBDA (37), which provides initial estimates of equilibrium binding parameters by Scatchard and Hill analyses and then produces a file for the nonlinear curve-fitting program Ligand (38).

Second Messenger Studies
293-R1{alpha} or 293-R1d cells were plated in 96-well plates and cultured until 95% confluency. Before treatments, cells were washed once with 200 µl DMEM containing 0.1% BSA, followed by preincubation with DMEM containing 0.5 mM 3-isobutyl-1-methylxanthine for 30 min. Cells were then stimulated with hCRH (0.1–1000 nM) for 15 min at 37 C; reactions were terminated by addition of 0.1 M HCl. After an overnight freeze/thaw cycle, the cAMP levels were measured in the supernatants using RIA. The sensitivity of the assay was 0.025 pmol/liter with intraassay precision of CV 2.9% and interassay precision of CV 9.7%.

For the inositol phosphate stimulation assay, cells were plated in six-well plates and subcultured in DMEM until 95% confluency. After incubation with inositol-free DMEM with [3H]myo-inositol (10 µCi/well) for 24 h, cells were washed with inositol-free DMEM once and preincubated with inositol-free DMEM containing 0.1% BSA and 30 mM LiCl for 30 min at 37 C. Phosphoinositide turnover was stimulated with r/hCRH (10–500 nM) in the presence of 30 mM LiCl, and the reactions were stopped by addition of chloroform-methanol-hydrochloric acid (50:100:1) at specified time intervals. After transferring to borosilicate glass tubes and centrifugation, the upper phase was applied to Prefilled Poly-Prep columns (AG 1-X8 resin 100–200 mesh chloride form, Bio-Rad Laboratories, Inc., York, UK) and [3H]IPs were resolved and quantified as previously described (39, 40). The radioactivity was measured by a ß-counter.

Synthesis of GTP-AA and Photolabeling of G{alpha} Subunits
GTP-AA was synthesized after a method described previously (41). Briefly, [{alpha}-32P]GTP (1 mCi) was evaporated and resuspended in 60 µl of 0.1 M 2-[N-morpholino]ethane sulfonic acid containing 30 mg/ml 1-(3-dimethylamino propyl)-3-ethylenecarbodiimide hydrochloride, pH 5.6, plus 40 µl of a suspension of azidoanilido-HCl (40 mg/ml) in 1,4-dioxane. The mixture was incubated for 6 h at room temperature and the GTP-AA was purified using a C-18 Sep Pak cartridge. The cartidge was prewetted with methanol and equilibrated with 97.2% buffer A and 2.8% buffer B (buffer A was prepared by bubbling CO2 through 100 mM triethylamine until the pH reached 7; buffer B was prepared similarly with a 100 mM solution of triethylamine in ethanol). The sample was dissolved in 1 ml equilibrating buffer and applied to the cartridge. After a wash step (with 10 ml of equilibrating buffer), the GTP-AA was eluted with 5 ml of 10% buffer A and 90% buffer B and collected in 0.5-ml fractions. Aliquots of each fraction were added to scintillation fluid and 32P was measured by scintillation spectrophotometry. Fractions containing GTP-AA were combined, evaporated to dryness, and stored at -70 C for up to 1 month. The overall yield of GTP-AA varied from 30 to 50%. All procedures were carried out in a darkened room.

293-R1{alpha} or 293-R1d cell membranes (100 µg) were incubated with or without h/rCRH (1 pM to 100 nM) for 5 min at 30 C before the addition of 5 µCi of GTP-AA in 120 µl of 50 mM HEPES buffer, pH 7.4, containing 30 mM KCl, 10 mM MgCl2, 1 mM benzamidine, 5 µM GDP, 0.1 mM EDTA, in a darkened room. After incubation for 3 min at 30 C, membranes were collected by centrifugation and resuspended in 100 µl of the above buffer containing 2 mM glutathione, placed on ice, and exposed to UV light (254 nm) at a distance of 5 cm for 5 min.

G Protein Immunoprecipitation
GTP-AA-labeled 293-R1{alpha} or 293-R1d cell membranes were precipitated by centrifugation and solubilized in 120 µl of 2% SDS. Then 360 µl of 10 mM Tris-HCl buffer, pH 7.4, containing 1% (vol/vol) Triton X-100, 1% (vol/vol) deoxycholate, 0.5% (wt/vol) SDS, 150 mM NaCl, 1 mM dithiothreitol, 1 mM EDTA, 0.2 mM phenylmethylsulfonyl fluoride, 10 µg/ml aprotinin were added and insoluble material was removed by centrifugation. Solubilized membranes were divided into 100-µl aliquots, and each aliquot was incubated with 10 µl of undiluted G protein antiserum at 4 C for 2 h under constant rotation. Then 50 µl of protein A Sepharose beads [10% (wt/vol) in the above buffer] were added, and the incubation was continued at 4 C overnight under constant rotation. The beads were collected by centrifugation, washed twice with 1 ml of a 50 mM Tris-HCl buffer, pH 7.4, containing 10% NP-40, 0.5% SDS, 600 mM NaCl, and then were further washed twice with 1 ml of a 100 mM Tris-HCl buffer, pH 7.4, containing 300 mM NaCl, 10 mM EDTA and dried under vacuum in a Speed-Vac microconcentrator. The immune complexes were dissociated from protein A by reconstitution in Laemmli’s buffer (42) (100 µl) and boiling for 5 min. Samples were then subjected to gel electrophoresis using discontinuous SDS-PAGE slab gels (10% running; 5% stacking). Molecular weight markers dissolved in solubilization buffer were also electrophoresed. The gels were then stained with Coomassie blue, dried using a slab gel dryer, and exposed to x-ray film (Fuji Photo Film Co., Ltd., Dusseldorf, Germany) at -70 C for 2–5 days.

Activated Receptor-G Protein Complex Immunoprecipitation
After the CRH binding reaction, 293-R1{alpha} or 293-R1d membranes were centrifuged and solubilized as described above. Insoluble material was removed by centrifugation, and solubilized ligand-receptor complex was incubated with anti-CRH-R1 antiserum to a final concentration of 1:100 and incubated at 4 C for 3 h. Protein A-Sepharose 4B was then added (40 µl), and the incubation was continued at 4 C overnight. After centrifugation (15,000 rpm x 5 min), the pellet was washed twice with 1 ml of a 100 mM Tris-HCl buffer, pH 7.4, containing 300 mM NaCl, 10 mM EDTA and dried under vacuum in a Speed-Vac microconcentrator.

Immunoblotting
The pellet was resuspended in Laemmli’s buffer (100 µl) (42) and boiled for 5 min. Samples were then subjected to gel electrophoresis using discontinuous SDS-PAGE slab gels (10% running; 5% stacking). Membranes from HEK293 cells were used as positive controls for all G protein blotting experiments. Molecular weight markers dissolved in solubilization buffer were also electrophoresed. The resolved proteins were transferred to polyvinylidene difluoride membrane at 100 mA for 90 min. The membrane was then blocked with 5% nonfat dry milk at room temperature for 30 min and subsequently incubated at 4 C for 2 h with G protein subtype-specific antisera. Polyvinylidene difluoride membranes were washed twice with PBS-Tween 20 (0.05%) and incubated with goat antirabbit antibody conjugated with horseradish peroxidase for 1 h. After washing twice with PBS-Tween 20 (0.05%), immunoreactivity was detected by enhanced luminescence.

Statistical Analysis
Data are shown as the means ± SEM of each measurement. Comparison between group means was performed by ANOVA; P < 0.05 was considered significant. The relative density of the bands was measured by optical density scanning using the software Scion Image-Beta 3b for Windows (Scion Corp., Frederick, MD).


    FOOTNOTES
 
Address requests for reprints to: Dr. D. Grammatopoulos, Sir Quinton Hazell Molecular Medicine Research Centre, Department of Biological Sciences, The University of Warwick, Gibbet Hill Road, Coventry CV4 7AL, United Kingdom.

This work was supported by Wellcome Trust, Action Research, and Sports Action Research for Kids (SPARKS). D.G. is a Wellcome Trust Career Development Fellow. E.H. is the Warwickshire Private Hospitals (WPH) Charitable Trust Chair of Medicine.

1 D.G. and Y.D. should be considered equal first authors by virtue of their unique contributions to this work. Back

Received for publication March 8, 1999. Revision received July 7, 1999. Accepted for publication September 3, 1999.


    REFERENCES
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 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
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