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Molecular Endocrinology, doi:10.1210/me.2008-0177
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Molecular Endocrinology 22 (11): 2505-2519
Copyright © 2008 by The Endocrine Society

Structural Determinants Critical for Localization and Signaling within the Seventh Transmembrane Domain of the Type 1 Corticotropin Releasing Hormone Receptor: Lessons from the Receptor Variant R1d

Danijela Markovic, Hendrik Lehnert, Michael A. Levine and Dimitris K. Grammatopoulos

Endocrinology and Metabolism (D.M., H.L., D.K.G.), Clinical Sciences Research Institute, Warwick Medical School, University of Warwick, Coventry CV4 7AL, United Kingdom; and Division of Pediatrics and Diabetes (M.A.L.), The Children’s Hospital of Philadelphia and the University of Pennsylvania School of Medicine, Philadelphia, Pennsylvania 19104

Address all correspondence and requests for reprints to: Professor D.K Grammatopoulos, Sir Quinton Hazell Molecular Medicine Research Centre, Warwick Medical School, The University of Warwick, Gibbet Hill Road, Coventry CV4 7AL, United Kingdom. E-mail: d.grammatopoulos{at}warwick.ac.uk.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
The type 1 CRH receptor (CRH-R1) plays a fundamental role in homeostatic adaptation to stressful stimuli. CRH-R1 gene activity is regulated through alternative splicing and generation of various CRH-R1 mRNA variants. One such variant is the CRH-R1d, which has 14 amino acids missing from the putative seventh transmembrane domain due to exon 13 deletion, a splicing event common to other members of the B1 family of G protein-coupled receptors. In this study, using overexpression of recombinant receptors in human embryonic kidney 293 and myometrial cells, we showed by confocal microscopy that in contrast to CRH-R1{alpha}, the R1d variant is primarily retained in the cytoplasm, although some cell membrane expression is also evident. Use of antibodies against the CRH-R1 C terminus in nonpermeabilized cells showed that membrane-expressed CRH-R1d contains an extracellular C terminus. Interestingly, treatment of CRH-R1d-expressing cells with CRH (100 nM) for 45–60 min elicited functional responses associated with a significant reduction of plasma membrane receptor expression, redistribution of intracellular receptors, and increased receptor degradation. Site-directed mutagenesis studies identified the cassette G356-F358 within transmembrane domain 7 as crucial for CRH-R1{alpha} stability to the plasma membrane because deletion of this cassette caused substantial intracellular localization of CRH-R1 {alpha}. Most importantly, coexpression studies between CRH-R1d and CRH-R2β demonstrated that the CRH-R2β could partially rescue CRH-R1d membrane expression, and this was associated with a significant attenuation of urocotrin II-induced cAMP production and ERK1/2 and p38MAPK activation, suggesting that CRH-R1d might specifically induce heterologous impairment of CRH-R2 signaling responses. This mechanism appears to involve accelerated CRH-R2β endocytosis.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
IN MAMMALS, THE FUNDAMENTAL role of CRH in integrating central and peripheral responses to stressful stimuli is mediated through activation of two highly homologous heptahelical receptors (CRH-Rs) that transduce signals primarily through activation of G proteins. The type 1 CRH-R (CRH-R1) is crucial for activation of the hypothalamo-pituitary-adrenal axis (1), whereas the type 2 CRH-R (CRH-R2) appears to be important for homeostatic mechanisms controlling energy balance, cardioprotection, tissue angiogenesis, and gastrointestinal regulatory effects in the periphery (2, 3). The family of urocortins (UCNs), which are CRH-like stress peptides, appear to be the cognate agonists of the CRH-R2 subtype (4).

Both CRH-R1 and CRH-R2 belong to the class B1 subfamily of G-protein coupled receptors (GPCRs) (brain-gut neuropeptide receptors). In humans, the CRH-R1 gene, which spans over 50 kb, contains 14 exons, and the complete gene product is a 444-amino acid seven-transmembrane domain (TMD) protein receptor, termed CRH-R1β, that exhibits impaired agonist binding and signaling properties (4). The CRH-R1 gene is subject to significant alternative splicing, and a growing number of CRH-R1 mRNA splice variants have been described in humans and other species (4). Their distinct structural properties revealed important insights about the structural determinants of CRH-R1 functional characteristics. Although, their biological role(s) is only partially known, recent evidence suggests that these receptor variants might alter CRH cellular responsiveness, because coexpression of CRH-R1{alpha} and the truncated forms of CRH-R1, e and h, can either attenuate or amplify CRH-R1{alpha} signaling (5).

The CRH-R1 variants are generated by partial or complete deletions of various exons. Excision of exon 6, from the pre-mRNA results in a transcript encoding CRH-R1{alpha}, which appears to be the principal functional CRH-R1 receptor. CRH-R1{alpha} contains 415 amino acids and primarily mediates CRH (and UCN-I) actions. Another variant, termed CRH-R1d, has been identified in humans and hamsters (6, 7), and is missing amino acids encoded by exon 13 (exon 12 in the rodent R1 homolog) which leads to the loss of 14 amino acids from the C-terminal end of the putative TMD7 (Fig. 1Go). Specific molecular mechanisms appear to regulate CRH-R1d mRNA expression, and recent studies (8) demonstrated that the onset of human labor (either term or preterm) is associated with a significant up-regulation of myometrial CRH-R1d mRNA expression. Expression of this putative CRH-R1 variant might allow tissues to modulate their responsiveness to CRH because overexpression studies of recombinant CRH-R1d have previously demonstrated that the possible distortion of the C terminus tail does not affect its agonist binding characteristics, although it has profound effects on receptor-G protein coupling and signaling properties (6). This is not surprising given that many sites involved in GPCR signaling, through posttranslational modifications and docking of signaling proteins, are present in the C terminus. Similar splice variants arising from the deletion of the corresponding exon have been described for other members of the B1 subfamily of GPCRs, such as the calcitonin receptor (CTR{Delta}e13) (9), PTH receptor (10), and the type II receptor for vasoactive intestinal peptide and pituitary adenylate cyclase-activating polypeptide (11). Analysis of the nucleotide sequence reveals that there are conserved splicing sites within the TMD7 (site of exon deletion), shared among members of this GPCR family.


Figure 1
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  		Fig. 1. Exon Splicing Events Regulating CRH-R1 mRNA Variant Expression and Resulting Modifications in Receptor Protein Structural Characteristics (in brackets)

aa, Amino acids.

 
Recent studies (12, 13) investigating the structural/functional characteristics of CTR{Delta}e13 provided important information about the impact of the loss of 14 amino acids from TMD7. The CTR{Delta}e13 appears to be a six-TMD receptor with reduced cell surface expression that contains an extracellular C terminus. When coexpressed with the fully active CTR C1a isoform, the CTR{Delta}e13 can act as a dominant-negative regulator, by forming heterooligomers intracellularly. This process inhibited C1a receptor expression at the cell surface, leading to a reduction in the normal signaling response.

In this study we sought to investigate the structural and functional characteristics of the CRH-R1d receptor. Using coexpression studies between the CRH-R1d and CRH-R1{alpha} and/or CRH-R2β in human embryonic kidney (HEK)293 cells, we also investigated whether the R1d can modulate CRH-R1{alpha} or R2 signaling.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Distinct Subcellular Distribution of CRH-R1d
Previous studies demonstrated that deletion of 14 amino acids from TMD7 of CTR (CTR{Delta}e13 variant) reduced receptor cell surface expression (12). This finding, coupled with the strong intracellular immunostaining observed in native cells that endogenously express CRH-R1d mRNA (such as uterine smooth muscle cells) (8), led us to investigate the subcellular localization of CRH-R1d. Recombinant CRH-R1d was overexpressed in HEK293 cells, and indirect fluorescent confocal microscopy using a specific CRH-R1/2 antibody was performed. Results (Fig. 2AGo) showed significant intracellular accumulation of immunofluorescent signal in CRH-R1d-overexpressing cells with a weak plasma membrane signal; in contrast, CRH-R1{alpha}-overexpressing cells showed immunostaining that was exclusively localized around the plasma membrane in agreement with our previous data (14).


Figure 2
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  		Fig. 2. Visualization of Recombinant CRH-R1{alpha} and CRH-R1d Distribution Overexpressed in (A) HEK293 and (B) MSMCs by Indirect Immunofluorescent Confocal Microscopy

The cells were transiently transfected with recombinant CRH-R1{alpha} or CRH-R1d as described in Materials and Methods and grown on glass cover slips. The CRH-R were detected using specific primary antibody for CRH-R1/2 (1:100) and Alexa-Fluor 594 (1:400) (red) as described in Materials and Methods. The cell nuclei were stained with a DNA-specific dye-DAPI. Identical results were obtained from three independent experiments. Scale bar, 10 µm.

 
We also investigated whether this distinct pattern of receptor localization was evident when recombinant CRH-R1d was expressed in primary myometrial smooth muscle cells (MSMCs), a cell type that endogenously expresses CRH-R1d mRNA and also exhibits strong intracellular CRH-R staining (8). In native MSMCs CRH-R immunoreactivity was localized around the plasma membrane as well as intracellularly (Fig. 2BGo, left panel). Overexpression of CRH-R1{alpha} in MSMCs (Fig. 2BGo) led to a significant increase in CRH-R immunoreactivity in the plasma membrane that was uniformly distributed. However, CRH-R1d overexpression resulted in significant signal increase predominantly in the cytoplasm, and some in the plasma membrane, providing further evidence that the CRH-R1d is primarily expressed intracellularly.

To obtain more information about the structural differences between CRH-R1{alpha} and CRH-R1d, we analyzed the protein sequences of the two CRH-R1 variants using the TMHMM method (http://workbench.sdsc.edu), which allows prediction of topology of transmembrane helices and inverting loop regions based on a hidden Markov model (15). As expected, computational analysis predicted that CRH-R1{alpha} protein sequence forms a putative heptahelical protein (Fig. 3AGo). However, there was a 50% probability for the CRH-R1d TMD7 sequence to fold as a transmembrane helix. Potential failure of the truncated TMD7 to segregate into the lipid membrane phase would lead the plasma membrane CRH-R1d to fold as a hexahelical receptor and its EC3, the remainder of TDM7 and the C terminus, to be either outside the cell or within the lumen of intracellular space. To examine this, we tested the ability of a CRH-R1 C terminus-specific antibody for receptor binding in permeabilized or nonpermeabilized HEK293 cells stably expressing CRH-R1{alpha} or R1d. Indirect confocal microscopy experiments showed that when cells overexpressing CRH-R1{alpha} or CRH-R1d were permeabilized with Triton X-100, a strong immunofluorescent signal was detected for both receptor variants (Fig. 3BGo). By contrast, in nonpermeabilized cells, fluorescent signal was detected only in the surface of CRH-R1d-expressing cells, but not in nonpermeabilized CRH-R1{alpha}-expressing cells. The accessibility of CRH-R1d-C terminus in nonpermeabilized cells suggested that the plasma membrane CRH-R1d contains an extracellular C terminus.


Figure 3
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  		Fig. 3. Orientation of CRH-R1d Carboxy Terminus

A, Prediction of CRH-R1{alpha} and CRH-R1d topology of transmembrane helices and inverting loop regions. The protein sequences of CRH-R1{alpha} and CRH-R1d were analyzed by using the TMHMM program (available at http://workbench.sdsc.edu) to predict TMD and intracellular/extracellular loops. Red color represents TMD, the intracellular loops are presented in blue, and the extracellular loops are shown in pink. B, Orientation of the C terminus of CRH-R1{alpha} and CRH-R1d overexpressed in HEK293 cells: investigation by indirect fluorescent confocal microscopy. Cells transiently expressing recombinant CRH-R1{alpha} or CRH-R1d, as described in Materials and Methods, were either permeabilized with Triton X-100 (top panel) or not (bottom panel) before addition of specific antisera raised against the C terminus of CRH-R1/2. The distribution of immunoreactivity was monitored by indirect confocal microscopy. Identical results were obtained from three independent experiments. Scale bar, 10 µm.

 
Amino Acid Motifs within the TDM7 Important for Plasma Membrane Expression of CRH-R1
The 14 amino acids encoded by exon 13 of the CRH-R1 appeared to be crucial for receptor membrane expression and orientation of the C terminus, and their deletion might lead to misfolding of the TDM7 a-helix. Interestingly, TMD7 sequence alignment of the CRH-R1 with other members of the B1 family of GPCRs with similar short-TDM7 variants, identified a highly conserved amino acid sequence, Gly356-Phe357-Phe358, present at the start of the deleted sequence (Fig. 4Go, inset). To explore the role of this motif in CRH-R1 subcellular localization, a series of CRH-R1{alpha} and CRH-R1d mutant receptors were generated (Table 1Go). Mutant receptors were transiently expressed in HEK293 cells, and their subcellular localization was visualized using indirect confocal microscopy. Deletion of the Gly356-Phe357-Phe358 (G-F-F) cassette [mutant CRH-R1{alpha}-{Delta}(356–358)] from CRH-R1{alpha} resulted in a receptor with poor plasma membrane localization and strong intracellular expression (Fig. 4Go). This effect was directly relevant to the identity of amino acids deleted because deletion of the adjacent cassette Val359-Ser360-Val361 [mutant CRH-R1{alpha}-{Delta} (359–361)] did not alter receptor subcellular distribution. These results suggested that the G-F-F cassette plays a crucial role for CRH-R1 stability in the plasma membrane. This led us to investigate whether replacement of the missing G-F-F cassette in CRH-R1d would be sufficient to restore membrane expression. Results demonstrated that addition of residues Gly356-Phe358 or Gly356-Val361 into CRH-R1d sequence [mutants CRH-R1d(+356–358) and CRH-R1d(+356–361), respectively] did not result in altered subcellular localization. However, when a nine-amino acid cassette (Gly356-Cys364) was inserted [mutant CRH-R1d(+356–364)], the mutant CRH-R1d receptor was expressed exclusively in the plasma membrane (Fig. 4Go).


Figure 4
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  		Fig. 4. Amino Acid Motifs within the CRH-R1{alpha} and CRH-R1d TDM7 Important for Plasma Membrane Expression

To identify amino acid sequences present in TDM7 important for CRH-R1 subcellular localization, a series of CRH-R1{alpha} and CRH-R1d mutant receptors were generated (Table 1Go). The effect of these mutations on cellular expression of CRH-R1{alpha} and CRH-R1d was assessed by indirect confocal microscopy. HEK293 cells transiently expressing CRH-R1{alpha} and CRH-R1d (wild-type or mutant receptors) were grown on poly-D-lysine-coated glass cover slips. Receptor immunoreactivity was detected with CRH-R1/2-specific antibody and Alexa Fluor 594 secondary antibody. Identical results were obtained from three independent experiments. Inset, TMD7 sequence alignment of the CRH-R1 with other members of the B1 family of GPCRs with similar short-TDM7 variants. Scale bar, 10 µm. w.t, Wild type.

 

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  		Table 1. Amino Acid Modifications in CRH-R1{alpha} and CRH-R1d Mutant Receptors

 
Agonist-Induced Endocytosis of Membrane-Expressed CRH-R1d
Our previous studies showed that the CRH-R1β, another signaling-impaired CRH-R1 variant (14), exhibits normal internalization characteristics; therefore, we investigated the potential of plasma membrane-expressed CRH-R1d to internalize upon agonist activation in HEK293 cells transiently expressing CRH-R1d (293-R1d). Under basal conditions, CRH-R1d immunoreactivity was distributed primarily in the cytoplasm and some in the plasma membrane. Addition of 100 nM CRH for 5–60 min appeared to induce redistribution of intracellular receptor immunoreactivity from a diffuse to a more punctuated staining (Fig. 5AGo). The redistribution of intracellular receptor immunoreactivity, potentially indicative of receptor clustering in endocytotic vesicles, was more profound after 60 min treatment. At this time point, there was little or no immunoreactivity of the receptor on the plasma membrane. This was confirmed by quantification of subcellular (cell membrane and intracellular) fluorescence spectra using the Image J software of randomly selected 20 individual cells. After 30 min of CRH treatment, there was a 60% decrease of cell membrane receptor immunoreactivity, and after 60 min cell membrane fluorescent signal was reduced by 80–90% (Fig. 5AGo, inset). The specificity of staining was demonstrated by lack of staining when CRH-R1 antibody was preabsorbed with the synthetic blocking peptide (10-fold molar excess) before addition on the slides (data not shown).


Figure 5
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  		Fig. 5. Regulation of CRH-R1d Expression and Localization by CRH

A, CRH-induced internalization of plasma membrane expressed CRH-R1d: visualization by confocal microscopy. HEK293 cells transiently expressing CRH-R1d were grown on cover slips and after exposure to 100 nM CRH for various time points (0–60 min), CRH-R distribution was monitored by indirect immunofluorescence using CRH-R1/2-specific antibody and Alexa Fluor 594. Cell nuclei were stained with DAPI, a DNA-specific dye. Inset, Fluorescence intensity of intracellular or plasma membrane fluorescence CRH-R1d distribution after CRH treatment was measured by Image J software. Total cellular fluorescence measurements were carried out to normalize data. Results are expressed as percentage (%) of basal (time zero) fluorescence intensity and represent the mean ± SEM of three estimations from 20 individual cells. Scale bar, 10 µm. B, Time-dependent effects of CRH treatment on CRH-R1d expression. After treatment of 293-R1d cells with 100 nM CRH for different time periods (0–60 min), cell lysates were prepared and 50 µg of protein was fractionated on SDS-PAGE and subjected to immunoblotting with specific antibodies against CRH-R1/2 or ERK1/2 (total). Antibody complexes were detected by enhanced chemiluminescence. Results are expressed as percentage (%) of basal (time zero). *, P < 0.05 compared with basal (unstimulated). Identical results were obtained from four independent experiments. WB, Western blot.

 
Using a different approach, after CRH treatment for 5–60 min, cell membrane-rich fractions were also prepared as previously described (16), and the total amount of CRH-R1d available for CRH binding was determined by Scatchard analysis of 125I-[Tyr0]-oCRH binding. Results showed that CRH treatment for 30 or 60 min reduced the maximum CRH-R1d binding sites (Bmax) by 45% and 80%, respectively (35 ± 5 vs. 16 ± 8 and 7 ± 3 fmol/mg protein) without any significant changes in receptor binding affinity [dissociation constant (Kd)] (Table 2Go). The purity of the membrane-rich fractions and equal protein loading was confirmed by immunoblotting; results showed that all samples contained equal amounts of the plasma membrane protein cadherin, whereas glyceraldehyde-3-phosphate dehydrogenase, a cytosolic protein, was not detected in any of the samples (data not shown).


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  		Table 2. Binding Characteristics of CRH-R1d-Expressing Cells after CRH Pretreatment (5–60 min)

 
Interestingly, our confocal microscopy data suggested that CRH-R1 immunoreactive signal was also reduced in a time-dependent manner, suggesting potential receptor degradation. This was confirmed by Western blot analysis of 293-R1d total cell extracts; immunoblotting with a specific CRH-R1/2 antibody showed a 50% reduction in CRH-R1d immunoreactivity after 60 min treatment with CRH compared with basal (Fig. 5BGo). Equal protein loading was confirmed by immunoblotting with a specific ERK1/2 antibody.

Coexpression of CRH-R1d and CRH-R2β
Effects on Receptor Expression.
During the initial studies to characterize CRH-R1d structure and signaling, we observed an absence of CRH-R1{alpha}/CRH-R1d functional interactions (6). In this part of our study, we examined the potential of CRH-R1d to modify CRH-R2β localization by coexpressing V5 and myc-tagged (both at the C terminus) receptors in HEK293 cells, using the mammalian expression vector pBudCE4.1 that allows independent but simultaneous expression of two genes in cells. A CRH-R1{alpha}/CRH-R2β receptor combination was also coexpressed, to test for specificity of potential CRH-R1d effects. In preliminary studies, V5 or myc-tagged CRH-R1{alpha}, CRH-R1d, or CRH-R2β, individually subcloned in pBudCE4.1, was transiently expressed in HEK293 cells, and their subcellular localization was examined. Indirect confocal microscopy using anti-V5 or -myc antibodies confirmed plasma membrane expression of myc- and V5-tagged CRH-R1{alpha}, V5-CRH-R2β, and mixed intracellular/plasma membrane expression of myc-CRH-R1d (Fig. 6AGo). We next examined receptor localization of cells coexpressing either V5-CRH-R1{alpha}/myc-CRH-R1d, V5-CRH-R2β/myc-CRH-R1{alpha}, or V5-CRH-R2β/myc-CRH-R1d. Results (Fig. 6BGo) showed that the presence of myc-CRH-R1d did not alter V5-CRH-R2β and myc-CRH-R1{alpha} expression in the plasma membrane; furthermore, coexpression of V5-CRH-R2β and myc-CRH-R1{alpha} resulted in plasma membrane expression for both receptors. However, significant differences were observed in CRH-R1d subcellular localization that were dependent on the type of partner receptor coexpressed with CRH-R1d. Although, as expected, in V5-CRH-R1{alpha}/myc-CRH-R1d cells the CRH-R1d was primarily intracellular, a different pattern of CRH-R1d localization was observed in V5-CRH-R2β/myc-CRH-R1d-expressing cells, where substantial myc-CRH-R1d immunoreactivity was detected in the plasma membrane rather than the intracellular space, demonstrated by the significant increase in plasma membrane red signal (yellow in the overlap image) (Fig. 6BGo). These results suggested that in the presence of CRH-R2β, CRH-R1d was primarily localized in the plasma membrane and not intracellularly. Relative quantification of the plasma membrane myc-signal demonstrated a 3- to 5-fold increase in plasma membrane fluorescence in V5-CRH-R2β/myc-CRH-R1d expressing cells compared with myc-CRH-R1d-only or V5-CRH-R1{alpha} /myc-CRH-R1d cells (Fig. 6CGo). Moreover, Scatchard analysis of 125I-[Tyr0]-oCRH binding was also employed to determine the total amount of CRH-R1d available for CRH binding in each of the two cell populations expressing either V5-CRH-R2β/myc-CRH-R1d or myc-CRH-R1d alone. In some experiments, cells were also pretreated with antisauvagine 30 (1 µM) for 1 h, to eliminate potential 125I-[Tyr0]-oCRH binding to CRH-R2β. Results showed that the maximum myc-CRH-R1d binding sites (Bmax) were 20- to 30-fold higher in V5-CRH-R2β/myc-CRH-R1d expressing cells compared with myc-CRH-R1d alone (9 ± 3 vs. 180 ± 35 fmol/mg protein, respectively) (Table 3Go), without any significant changes in receptor binding affinity (Kd). Western blot analysis of total cell extracts using a specific myc antibody showed comparable levels of myc-CRH-R1d protein expression in both cell populations (Fig. 6CGo, inset).


Figure 6
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  		Fig. 6. Individual (A) or Combined (B) Expression of myc-CRH-R1{alpha}, V5-CRH-R1{alpha}, myc-CRH-R1d, and V5-CRH-R2β in HEK293 Cells

HEK293 cells were transiently transfected with a mammalian expression vector pBudCE4.1 containing either myc-CRH-R1{alpha} (A), V5-CRH-R1{alpha} (B), myc-CRH-R1d (C), or V5-CRH-R2β (D). In a different set of experiments (B), cells were transfected with a combination of V5-CRH-R1{alpha} and myc-CRH-R1d, myc-CRH-R1{alpha} and V5-CRH-R2β, or V5-CRH-R2β and myc-CRH-R1d as described in Materials and Methods. V5-tag was detected using mouse monoclonal antibody and secondary antimouse Alexa-Fluor 488 (green), whereas myc-tag was detected with mouse monoclonal myc-antibody conjugated with Texas Red (red) entity as described in Materials and Methods. The cell nuclei were stained with DAPI, a DNA-specific dye (blue). The cells were examined using indirect confocal microscopy. Scale bar, 10 µm. C, In some experiments, myc-fluorescence intensity in plasma membrane (reflecting CRH-R1d plasma membrane expression) was also determined by measuring fluorescence intensity of plasma membrane fluorescence by Image J software. Total cellular fluorescence measurements were carried out to normalize data. Results are expressed as the mean ± SEM of three estimations from 20 individual cells. *, P < 0.05 compared with myc-CRH-R1d only. Identical results were obtained from two independent transfections and four independent experiments. Inset, To determine myc-CRH-R1d protein expression, cell lysates were prepared and 25 µg of protein was fractionated on SDS-PAGE and subjected to immunoblotting with mouse monoclonal myc-antibody. Antibody complexes were detected by enhanced chemiluminescence. WB, Western blotting.

 

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  		Table 3. Binding Characteristics of myc-CRH-R1d in the Absence or Presence of CRH-R2b

 
Effects on UCN-II Signaling.
The effects of myc-CRH-R1{alpha} or myc-CRH-R1d coexpression with V5-CRH-R2β on receptor functional characteristics were also determined. In preliminary experiments we determined the signaling characteristics (cAMP and MAPK activation) of the tagged receptors in response to UCN-II (100 nM for 5 min). As expected, UCN-II treatment did not significantly alter cAMP levels or ERK1/2 and p38MAPK phosphorylation in cells expressing myc- or V5-CRH-R1{alpha}, or myc-CRH-R1d (Fig. 7AGo). In contrast, UCN-II treatment of V5-CRH-R2β-expressing cells significantly increased intracellular cAMP levels by 4.80 ± 0.15-fold. Coexpression of V5-CRH-R2β with myc-CRH-R1{alpha} did not significantly alter UCN-II-induced intracellular cAMP response (4.41 ± 0.25-fold increase above basal). However, in cells coexpressing V5-CRH-R2β and myc-CRH-R1d, UCN-II effect on cAMP production was reduced by 40–50%. Under the experimental conditions described above, UCN-II-induced ERK1/2 and p38MAPK phosphorylation was also investigated. Again, UCN-II treatment for 5 min did not stimulate ERK1/2 and p38MAPK phosphorylation in cells expressing myc- or V5-CRH-R1{alpha}, or myc-CRH-R1d, whereas in cells expressing V5-CRH-R2β, UCN-II increased ERK1/2 and p38MAPK phosphorylation above basal by 2.5- and 1.5-fold, respectively (Fig. 7BGo). Similar effects of UCN-II were found in cells coexpressing V5-CRH-R2β/myc-CRH-R1{alpha} cells. In contrast, in V5-CRH-R2β/myc-CRH-R1d-expressing cells, UCN-II-induced ERK1/2 phosphorylation was only 1.5-fold above basal whereas UCN-II had no effect on p38 MAPK phosphorylation (Fig. 7BGo).


Figure 7
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  		Fig. 7. Effect of CRH-R1{alpha} or R1d Coexpression on CRH-R2β Signaling Characteristics

HEK293 cells transiently expressing myc-CRH-R1{alpha}, myc-CRH-R1d, and V5-CRH-R2β, independent or in combination as described in Materials and Methods, were stimulated with 100 nM UCN-II for 5 min. cAMP production was measured by ELISA. For MAPK phosphorylation assays, after cell lysis and centrifugation, supernatants were subjected to SDS-PAGE and immunoblotted with antibodies for phospho- and total-ERK1/2 to determine the phosphorylated/activated ERK1/2 and secondary antibodies conjugated to IRDye 800 and Alexa Fluor 680 (near-infrared fluorophore dyes) as described in Material and Methods. Alternatively, samples were immunoblotted with antibody for phospho-p38 MAPK. Data are expressed as the ratio of agonist-stimulated over basal levels and represent the mean ± SEM of three estimations from four independent experiments. *, P < 0.05 compared with basal (unstimulated); +, P < 0.05 compared with V5-CRH-R2β-alone.

 
Effects on Agonist-Induced CRH-R2β Internalization.
The reduced functional activity of CRH-R2β in the presence of CRH-R1d could potentially be explained by altered rate of agonist-induced CRH-R2β endocytosis. The temporal characteristics of agonist-induced V5-CRH-R2β internalization were initially assessed by indirect confocal microscopy. Treatment of V5-CRH-R2β expressing cells with UCN-II (100 nM) for various time periods induced a time-dependent increase in intracellular V5-immunoreactivity, indicative of substantial receptor internalization (Fig. 8Go). Similar to our previous results (16), receptor internalization was evident only after 15min of agonist treatment and within 30 min of UCN-II treatment, intracellular CRH-R2β immunoreactivity was increased by 8-fold. Interestingly, coexpression of myc-CRH-R1d and V5-CRH-R2β accelerated agonist-induced CRH-R2β early (5 min) internalization (Fig. 9Go left panel); in V5-CRH-R2β/myc-CRH-R1d-expressing cells, UCN-II (100 nM) treatment for 5 min increased intracellular V5 cytoplasmic immunoreactivity. To assess the specificity of CRH-R1d effect on CRH-R2β internalization rate, V5-CRH-R2β was coexpressed with myc-CRH-R1{alpha}. Cells were stimulated with UCN-II (100 nM) for 5–30 min, and CRH-R2β internalization was monitored by indirect confocal microscopy. Results (Fig. 9Go, right panel) demonstrated that the presence of CRH-R1{alpha} did not significantly alter the rate of CRH-R2β internalization because UCN-II treatment for at least 15 min was required to induce substantial increase of intracellular V5 immunoreactivity. Furthermore, comparable levels of V5-CRH-R2β intracellular immunoreactivity, induced by 15 or 30 min of agonist treatment, was observed in the presence of either myc-CRH-R1d or myc-CRH-R1{alpha}.


Figure 8
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  		Fig. 8. UCN-II Induced Internalization of V5-Tagged CRH-R2β Transiently Expressed in HEK293 Cells

HEK293 cells transiently expressing V5-CRH-R2β were grown on cover slips and after treatment with 100 nM UCN-II for various time points (0–30 min), CRH-R2β distribution was monitored by indirect immunofluorescent confocal microscopy using V5-tag mouse monoclonal antibody and secondary antimouse Alexa Fluor 488 (green). The cell nuclei were stained with DAPI, a DNA-specific dye (blue). Scale bar, 10 µm. Intracellular fluorescence intensity was measured by Image J software. Data are expressed as the ratio of agonist-stimulated over basal (time zero) levels. Results are expressed as the mean ± SEM of three estimations from 20 individual cells. *, P < 0.05 compared with basal (unstimulated). Identical results were obtained from four independent experiments.

 

Figure 9
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  		Fig. 9. CRH-R2β Homologous Internalization in the Presence of CRH-R1d or CRH-R1{alpha} Coexpressed in HEK293 Cells

HEK293 cells transiently expressing V5-CRH-R2β with either myc-CRH-R1d or myc-CRH-R1{alpha} were treated with 100 nM UCN-II for various time points (0–30 min). A, CRH-R2β distribution was monitored by indirect immunofluorescent confocal microscopy using V5-tag mouse monoclonal antibody and secondary antimouse Alexa Fluor 488 (green). Intracellular fluorescence intensity was measured by Image J software. Data are expressed as the ratio of agonist-stimulated over basal (time zero) levels. Results are expressed as the mean ± SEM of three estimations from 20 individual cells. Alternatively (in panel B), cells were pretreated with 100 nM UCN-II for either 5 or 15 min and after preparation of cell membrane-rich fractions the total amount of plasma membrane V5-CRH-R2β (Bmax) available for agonist binding was determined by Scatchard analysis. Plasma membrane-rich fractions were prepared by using ProteoExtract Native Membrane Protein Extraction Kit (Calbiochem/ Merck Biosciences, Nottingham, UK) and radioiodinated antisauvagine 30 (10 pM to 10 nM) binding to membranes (100 µg) was carried out for 2 h at room temperature as described in Materials and Methods. Results are expressed as the mean ± SEM of four estimations from three independent experiments. *, P < 0.05 compared with basal (unstimulated); +, P < 0.05 compared with each other. Scale bar, 10 µm.

 
Scatchard analysis of 125I-[Tyr0]-antisauvagine30 binding was also employed to determine the total amount of V5-CRH-R2β binding sites available in plasma membrane-rich fractions, after pretreatment of cells expressing either V5-CRH-R2β/myc-CRH-R1d or V5-CRH-R2β/myc-CRH-R1{alpha} with UCN-II for 5 or 15 min. Results showed that UCN-II pretreatment for 5 min reduced by 28% plasma membrane V5-CRH-R2β Bmax in V5-CRH-R2β/myc-CRH-R1d but not in V5-CRH-R2β/myc-CRH-R1{alpha} cells. In contrast, pretreatment of cells for 15 min attenuated V5-CRH-R2β Bmax by 50% in both V5-CRH-R2β/myc-CRH-R1d or V5-CRH-R2β/myc-CRH-R1{alpha} (Fig. 9BGo).


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
MATERIALS AND METHODS
 REFERENCES
 
The class B1 GPCRs, which in humans contains 15 members, is characterized by an exon-intron organization that permits extensive alternate splicing. This offers a potential for versatile regulation of expression and tissue responsiveness to hormonal stimuli, because splicing of specific exons encoding critical domains can dramatically alter receptor function. The TMD7 of these receptors appears to be targeted by splicing mechanisms, resulting in deletion of specific amino acid sequences and putative receptor variants with substantially shortened TMD7 (9, 10, 11). One such receptor variant is the CRH-R1d (6), which contains a deletion of 14 amino acids from the distal part of the putative TMD7. In the present study we provide novel evidence demonstrating that this sequence deletion has a major impact on receptor subcellular localization because overexpression of recombinant CRH-R1d in HEK293 cells demonstrated that this receptor variant is predominantly expressed in the cytoplasm, and only a small fraction is localized to the plasma membrane. Increased intracellular CRH-R immunoreactivity was also observed when recombinant CRH-R1d was overexpressed in MSMC, a cell type that endogenously expresses CRH-R1d mRNA (6) and under native conditions shows intense intracellular CRH-R immunoreactivity (8). Although the lack of CRH-R1d-specific antibodies prevents us from conclusively demonstrating that intracellular CRH-R immunoreactivity represents CRH-R1d receptors, our observations raise the possibility that at least a fraction of intracellular staining observed in native MSMCs might be due to CRH-R1d immunoreactivity.

Furthermore, in receptors retained in the plasma membrane, this sequence modification appears to prevent proper anchoring of TMD7 in the lipid bilayer of the plasma membrane, resulting in an extracellular C terminus orientation, evident from the ability of specific antibodies against the receptor C terminus to recognize and bind the C terminus in nonpermeabilized cells. Interestingly, similar subcellular localization and structural characteristics have previously been reported for the calcitonin splice variant CTR{Delta}e13 (12), a finding that suggests presence of common structural determinants and characteristics in this class of GPCRs.

The C terminus of TMD7 appears to be highly conserved among members of the class B1 GPCRs. Sequence alignment indicates that the TMD7 of all 15 human class B1 GPCRs contains the motif -(H/S)-F-Q-G-(L/F)-X-V-S/A-X-X-F/Y-C-F/Y-X-N-X-E-V- (letters in bold depicting the 14-amino acid cassette deleted in short-TMD7 GPCR variants due to alternative splicing). Within the 14-amino acid sequence of the CRH-R1{alpha}, there are two amino acid cassettes, G356-F357-F358 and V359-S360-V361, that are conserved across most members of class B1 GPCRs. The sequence G-F-F or G-F-X is present in 10 of 15 class B1 GPCRs, whereas nine of 15 have either V-S-V, V-X-V or V-S-X. Our mutant receptor expression studies showed that the G356-F357-F358 motif appears to be necessary, but not sufficient, for successful CRH-R1 plasma membrane expression because deletion of this cassette from CRH-R1{alpha} results in intracellular receptor unable to stabilize in the plasma membrane and potentially exhibiting defective export trafficking properties. Moreover, this structural impact on receptor subcellular localization appears to be specific for the G-F-F cassette because deletion of the V359-S360-V361 cassette had no effect on CRH-R1{alpha} plasma membrane expression and subcellular localization. Interestingly, insertion of G-F-F into the R1d TMD7 sequence did not restore plasma membrane expression, suggesting that the missing G-F-F is not the sole cause of CRH-R1d-defective plasma membrane localization. In fact, the plasma membrane expression of R1d was restored only when the cassette G-F-F and six additional amino acids (-V-S-V-F-Y-C-) were inserted in the CRH-R1d TMD7, possibly indicating the minimum requirement for formation of a transmebrane {alpha}-helix that would prevent protein misfolding and allow the receptor to pass through the endoplasmic reticulum (ER) quality control mechanism and become available for export trafficking. At present, the molecular mechanisms and receptor structural determinants underlying export transport of CRH-R1 from the ER through the Golgi to the plasma membrane are unknown. Studies mainly on other membrane proteins, and non-class B1 GPCRs, identified a number of important ER-export signals (17), mostly present in the carboxyl-termini of GPCRs. Two of these motifs, double phenylalanine (FF) and F(x)3F(x)3F (F356-F357, F350-L-E-S-F-Q-G-F-F358 and F354-Q-G-F-F-V-S-V-F362) appear to be present within the CRH-R1 TMD7 sequence, raising the possibility that the TMD7 participate in CRH-R1 export trafficking.

Despite being signaling impaired (6), the CRH-R1d receptors that achieve anchoring to the plasma membrane appear to retain one of the fundamental properties of GPCRs: time-dependent endocytosis in response to agonist binding. Interestingly, plasma membrane CRH-R1d endocytosis was associated with redistribution, clustering, and subsequently rapid down-regulation of intracellular CRH-R1d levels, a pattern of endocytosis not previously observed with other CRH-R1 variants, R1{alpha} and R1β, during the time period (1 h) tested (14). Although, our study did not specifically investigate the specific subcellular compartments involved in cytoplasmic CRH-R1d distribution, it is possible that CRH induces CRH-R1d targeting to distinct compartments such as the lysosomal lumen involved in receptor degradation. It is also noteworthy that the levels of CRH-R1d down-regulation (~50% within 1 h of agonist stimulation) were comparable to CRH-R1{alpha} down-regulation induced after 12–14 h of CRH treatment (18). The characteristics of CRH-R1 endocytosis have not been fully elucidated, and previous studies demonstrated that receptor endocytosis can occur through different paths (18, 19); agonist-induced CRH-R1 endocytosis involves recruitment of β-arrestin and internalization of receptor-β-arrestin complexes (14, 18), whereas antagonists such as astressin induce receptor internalization in a phosphorylation and β-arrestin2-independent manner (18). At least two distinct CRH-R1 regions have been identified as important for receptor-β-arrestin interactions: a C-terminal motif in which specific Ser/Thr residues must be phosphorylated and an intracellular loop motif configured by agonist-induced changes in receptor conformation (20, 21).

Another important finding of our studies was the observation that CRH-R1d subcellular localization can be modified by coexpressing other CRH-Rs, and expression of CRH-R1d to the plasma membrane can be rescued when CRH-R2β was also present. This effect appears to require specific interactions between CRH-R2β and CRH-R1d because it was absent when CRH-R1{alpha} was coexpressed with CRH-R1d. It is possible that direct or indirect protein-protein interactions between CRH-R1d and CRH-R2β modulate cellular machinery responsible for CRH-R1d retention in the cytoplasm. Previous studies in other GPCR systems have demonstrated that GPCR dimerization plays an important role in receptor folding, and the transport of several GPCRs from the ER to the cell surface requires dimerization in the early secretory pathway (22).

Interestingly, enhancement of cell surface expression allows CRH-R1d to act as a dominant-negative regulator and heterologously attenuate CRH-R2β signaling activity in stimulating cAMP, ERK1/2, and p38MAPK cascades. Our results also suggest that this was achieved by accelerating kinetics of UCN-II-induced CRH-R2β early (5 min) endocytosis. This is the first evidence demonstrating negative regulation of CRH-R2β signaling activity, a receptor with crucial and diverse roles in mammalian pathophysiology. Moreover, under the experimental conditions used, this dominant-negative effect on CRH-R2β signaling appears to be specific for CRH-R1d, because coexpression of CRH-R1{alpha} had no effect on CRH-R2β signaling and endocytosis. Given that a similar dominant-negative role has been proposed for the CTR {Delta}e13 receptor (12), it is possible that short-TMD7 variants of GPCRs might act as important modulators of the biological activities of wild-type or fully active GPCRs.

In summary, deletion of 14 amino acids from the C-terminal end of the putative TMD7 of CRH-R1d has a major impact on receptor subcellular localization and C terminus orientation. Within the deleted sequence the amino acid cassette G356-F357-F358 appears to be a major structural determinant for efficient CRH-R1{alpha} anchoring to the plasma membrane. Specific interactions between CRH-R1d and CRH-R2β can rescue CRH-R1d plasma expression and allow CRH-R1d to exert dominant-negative effects on CRH-R2β signaling by accelerating agonist-induced CRH-R2β endocytosis. These results might suggest the presence of a new level of regulation of CRH and CRH-R biological activity.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
REFERENCES
 
Reagents
Radioiodinated ovine (o) CRH, human (h) CRH, and UCN-II were purchased from Bachem UK Ltd (Helens, Merseyside, UK). Radioiodinated antisauvagine 30 was purchased from Amersham [GE Healthcare UK Ltd. (Amersham Place, Buckinghamshire, UK)]. Forskolin was from Calbiochem/Merck Biosciences (Nottingham, UK). CRH-R1/2 and its blocking peptide, and c-myc-TRITC were from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA). Phospho-ERK1/2 (Thr202/Tyr204), total ERK1/2, phospho p38 MAPK (Thr180/Tyr182), and total p38 MAPK were from Cell Signaling (Chandlers Ford, Hampshire, UK). Secondary antibodies, Alexa Fluor 488, Alexa Fluor 594, Alexa Fluor 680, and Gold Slowfade mounting solution with 4',6-diamidino-2-phenylindole (DAPI) were purchased from Invitrogen/Molecular Probes (Paisley, UK); IRDye 800-conjugated goat antirabbit IgG was from Rockland Immunochemicals (Gilbertsville, PA). VECTASHIELD Mounting Medium without DAPI was from Vector Laboratories, Inc. (Orton Southgate, Peterborough, UK). Cell culture media, gentamycin (G-418), Lipofectamine 2000, pcDNA3.1(+), pBudCE4.1, restriction enzymes, and Pfu polymerase were from GIBCO/Invitrogen (Paisley, UK). Deoxynucleotide triphosphates and DNA ladder were purchased from Bioline Ltd. (London, UK). Primers were purchased from TAG Newcastle (VHBio, Gateshead, UK). All other chemicals were purchased from Sigma Aldrich Co. Ltd (Gillingham, UK).

Subjects and Myocytes Culture Preparation
Pregnant myometrial biopsies were obtained from women undergoing elective cesarean section at term (>37 wk of gestation; n = 4) 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. The biopsies were processed immediately for myocyte cell culture as previously described (8). Ethical approval was obtained from the local ethical committee, and informed consent to the study was obtained from all patients. The purity of MSMCs was assessed by immunocytochemical staining for {alpha}-actin with mouse monoclonal antibody specific for smooth muscle {alpha}-actin (Sigma Aldrich Company Ltd). Confluent cells up to third passage were used; no significant difference between results was obtained with cells from individual passages and cells obtained from different myometrial biopsies.

Transfection of CRH-R to HEK293 Cells and Myocytes
Lipofectamine 2000 reagent (Invitrogen) was used for transfection of HEK293 and MSMCs. Transient transfection were performed when cells seeded in 25-cm2 vented flasks reached 50–70% confluency. DNA [CRH-R1{alpha} (5 µg) or CRH-R1d in pcDNA3.1 (+)] and Lipofectamine 2000 reagent (5 µl) was mixed with 5 ml of OptiMEM+GlutaMax (GIBCO) and used for overnight transfection according to manufacturer’s instructions. Coexpression of CRH-R1{alpha} or R1d and CRH-R2 was achieved by subcloning both receptor cDNAs into pBudCE4.1, a mammalian expression vector that contains cytomegalovirus and elongation factor 1 (EF-1) promoter sites that allows independent expression of two genes from a single plasmid in mammalian cells. The morning after transfection the mixture was replaced with normal growth media (DMEM with 10% fetal bovine serum) and after 8 h, cells were transferred on poly-D-lysine (Sigma)-coated glass cover slips (for confocal microscopy studies) or to 12-well plates for signaling studies. All experiments were performed 48 h after transfection. Receptor expression was verified by confocal microscopy analysis.

CRH-R1 Mutagenesis
A series of CRH-R1{alpha} and CRH-R1d mutants were generated, by overlap extension PCR (23). The PCR mixture (20 µl) contained 5 ng of template cDNA, 5 ng/µl of each mutagenic primer, 1 U of Pfu polymerase, and 0.2 mM of each deoxynucleotide triphosphate. cDNAs were amplified at 48 C in a total of 35 cycles. The primers used for mutagenesis (Table 4Go) also introduced HindIII and XbaI restriction sites that allowed ligation to pcDNA3.1(+), which was performed with LigaFast Rapid DNA Ligation System (Promega). Moreover, when receptor cDNAs were subcloned into pBudCE4.1, stop codons were removed by PCR to allow tagging at the C terminus with either V5 or myc. The vector and the insert DNAs were mixed in a 3:1 insert to vector molar ratio in 10 µl containing 1 µl T4 ligase and 5 µl of accompanying buffer and incubated at room temperature for 5 min. After transformation into Escherichia coli DH-5{alpha} cells and purification, resulting plasmids were sequenced to ensure the fidelity of the mutant cDNAs and confirm presence of mutations (Molecular Biology Service, Department of Biological Sciences, University of Warwick).


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  		Table 4. Nucleotide Sequences of Primers Used to Create CRH-R1{alpha} and CRH-R1d Mutants

 
CRH-R1 and CRH-R2 Radioreceptor Assays
Plasma membrane-rich fractions were prepared from HEK 293 cells transiently expressing CRH-R1d, myc-CRH-R1d, V5-CRH-R2β/myc-CRH-R1d, or V5-CRH-R2β/myc-CRH-R1{alpha}, as previously described (16). Radioiodinated [Tyr0]-oCRH (50 pM to 5 nM) binding to membranes (100 µg) from CRH-R1d, myc-CRH-R1d, or V5-CRH-R2β/myc-CRH-R1d was carried out for 2 h at room temperature, as previously described (6, 14). Unlabeled oCRH (1000 molar excess) was used for displacement of radiolabeled [Tyr0]-oCRH. In some experiments, membranes were preincubated with 1 µM antisauvagine 30 (a CRH-R2-specific antagonist) for 60 min, to block CRH-R2β binding sites, before addition of 125I-[Tyr0]-oCRH. Nonspecific binding was 24 ± 5% of the total added radioactivity.

V5-CRH-R2β binding characteristics (Kd and Bmax) were determined by using radioiodinated antisauvagine 30 (10 pM to 10 nM) binding to membranes (100 µg) from V5-CRH-R2β/myc-CRH-R1d or V5-CRH-R2β/myc-CRH-R1{alpha} cells for 2 h at room temperature, as previously described (24). In some experiments, cells were pretreated with 100 nM UCN-II for either 5 or 15 min. Unlabeled antisauvagine 30 (1000 molar excess) was used for displacement of radiolabeled peptide. Nonspecific binding was 14 ± 8% of the total added radioactivity. The binding data were analyzed using the EBDA program (25) and LIGAND (26) (EBDA/LIGAND; Elsevier-Biosoft, Cambridge, UK).

Signaling Assays
MAPK (ERK1/2 and p38MAPK) activation was determined in transiently transfected HEK293 cells, seeded in 12-well plates to reach 60–70% confluency, as previously described using the Odyssey Infrared ImagingSystem (LI-COR Biosciences, Cambridge, UK) (16). Intracellular cAMP production was measured as previously described (16) by a commercially available ELISA Direct Cyclic AMP Enzyme Immunoassay Kit (Assay Designs, Inc., Ann Arbor, MI).

Internalization Studies and Immunofluorescent Confocal Microscopy
Transiently transfected cells were treated with 100 nM UCN-II or CRH for various time intervals and fixed with 4% paraformaldehyde. Nonspecific binding was blocked with 1% BSA in PBS-Triton X-100 (0.01%) for 1 h at room temperature. In some experiments the permeabilization step was omitted. After a 5-min wash with PBS, monoclonal mouse antibody against V5-tag (1:100 in PBS) was added for overnight incubation at 4 C. The next morning, slides were washed with PBS three times for 5 min, and donkey antimouse AlexaFluor 488 (1:400 in PBS) was added for 1 h at room temperature. After three PBS washes for 5 min each, c-myc-TRITC monoclonal antibody (1:100) was added for 2 h at room temperature. After three 5-min washes with PBS, slides were mounted with Gold Slowfade mounting solution with DAPI (Molecular Probes). In some experiments, CRH-R1/2 (1:100) primary antibodies were placed on slides for overnight incubation at 4 C. After three washes with PBS, slides were incubated with donkey antirabbit AlexaFluor 594 (1:400), before mounting with Gold Slowfade mounting solution with DAPI (Molecular Probes). The slides were examined under an oil immersion objective (x63) using a Leica model DMRE laser scanning confocal Microscope (Leica Microsystems, Buckinghamshire, Milton, Keynes, UK) with TCS SP2 scan head. Laser 543 nm at 50% of power and emission filter set at 555–620 nm were used to examine AlexaFluor 594 staining, and Laser 488 nm at 30% of power and emission filter set at 500–535 nm were used to examine AlexaFluor 488 staining. DAPI staining was examined with Laser 405 at 10% of power and emission filter set at 410–450 nm. The scan speed was set at 400 Hz, and the format was 1024 x 1024 pixels. Optical sections (0.5 µm) were taken, and representative sections corresponding to the middle of the cells are presented. After indirect immunofluorescent staining, no specific fluorescence was observed in cells treated with secondary antibody only or when blocking peptide was incubated with the primary antibody before incubation on cells. For each treatment, between 20 and 30 individual cells in five random fields of view were randomly selected and examined. Fluorescence intensity of specific regions of interest was quantified by using the Measure function of Image J software developed at the National Institutes of Health (http://rsb.info.nih.gov/ij/), as previously described (14).

CRH-R Western Blot
The amount of CRH-R1d expressed in 293-R1d cells was assessed by Western blot analysis using a specific antibody against the receptor C terminus as previously described (16). Myc-CRH-R1d expression was assessed by using mouse monoclonal myc-antibody (1:1000 dilution).

Statistics
The results obtained are presented as the mean ± SEM of each measurement. Data were tested for homogeneity, and comparison between group means was performed by one- or two-way ANOVA. Probability values of P < 0.05 were considered to be significant.


   FOOTNOTES
 
D.K.G. is supported by a Wellcome Trust University Award.

Author Disclosure: The authors have nothing to disclose.

First Published Online September 4, 2008

Abbreviations: CRH-R, CRH receptor; CTR, calcitonin receptor; DAPI, 4',6-diamidino-2-phenylindole; ER, endoplasmic reticulum; GPCR, G protein-coupled receptor; HEK, human embryonic kidney; MSMCs, myometrial smooth muscle cells; TMD, transmembrane domain; UCN, urocortin.

Received for publication June 9, 2008. Accepted for publication August 28, 2008.


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 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
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