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Characterization of GPRA, a Novel G Protein–Coupled Receptor Related to Asthma

Department of Medical Genetics, and Department of Anatomy, Biomedicum Helsinki, Department of Pathology, Haartman Institute, University of Helsinki; GeneOS Ltd.; and Department of Medicine, Helsinki University Central Hospital, Helsinki, Finland; and Department of Biosciences at Novum and Clinical Research Centre, Karolinska Institutet, Huddinge, Sweden

Correspondence and requests for reprints should be addressed to Tarja Laitinen, GeneOS Ltd., Tukholmankatu 2, 00251 Helsinki, Finland. E-mail: tarja.laitinen{at}geneos.fi

	Abstract

Top Abstract Introduction MATERIALS AND METHODS RESULTS DISCUSSION References

 
We recently identified a novel positional asthma susceptibility gene, GPRA, which belongs to the G protein-coupled receptor family. In the present studies, we show that isoform specific activation of GPRA-A with its agonist, Neuropeptide S (NPS) resulted in significant inhibition of cell growth. GPRA has several variants due to extensive alternative splicing. We observed that only the full-length variants, GPRA-A and GPRA-B, with 7 transmembrane topology are transported into the plasma membrane, while the truncated proteins retain intracellular compartments. To clarify disease mechanism, we studied co-expression of the variants without finding any indication that truncated variants would inhibit the receptor transport into the plasma membrane. By using in situ hybridization and immunohistochemistry, we detected ubiquitous expression of GPRA-B, and frequent expression of GPRA-A in the epithelia of several organs including bronchi and gastrointestinal tract. Furthermore, we observed aberrant mRNA and protein expression levels of GPRA in the asthmatic bronchi. Finally, we demonstrate that GPRA and NPS are co-expressed in bronchial epithelium. In summary, this study provides evidence that GPRA might have functional relevance in modulating asthma by increased expression levels in the relevant tissues under diseased state and by potential inhibitory effect of GPRA-A activation on cell growth.

Key Words: asthma • susceptibility • GPRA • G protein–coupled receptor • Neuropeptide S

	Introduction

Top Abstract Introduction MATERIALS AND METHODS RESULTS DISCUSSION References

 
We have recently implicated by genetic linkage and association analyses a locus for asthma-related traits on chromosome 7p15-p14 (1, 2). The significant linkage mapping result was confirmed in two independent cohorts: families with asthma from Quebec and families with allergy from Finland. We could identify seven common haplotypes spanning across a 133-kb region in all three cohorts. The susceptibility haplotypes were closely related and distinct from the nonrisk haplotypes, and associated significantly with high total serum IgE values or asthma (2).

Within the locus, there are two genes of which GPRA, a novel member of the G protein–coupled receptor superfamily (GPCRs), showed to be of particular interest. GPRA has two main transcripts, GPRA-A (also known as GPR154, PGR14, VRR1) and GPRA-B with alternative 3' exons, encoding proteins of 371 and 377 amino acid residues in length, respectively. Both isoforms have 7 transmembrane (TM) domains and the 14 conserved amino acid residues characteristic to the G protein–coupled receptor class A (2, 3). Structurally the closest homolog to GPRA-A is the vasopressin receptor V1a (27% amino acid similarity) (4). When bronchial biopsies from patients with asthma were compared with biopsies from control subjects by using immunohistochemistry, we found altered expression of GPRA-B in the smooth muscle cell layer among all the patients (n = 8) but not among the control subjects (n = 10) (2).

The strongest disease associations are to noncoding SNPs (single nucleotide polymorphisms) within GPRA introns, and all but one SNP (Asn107Ile in exon 3 encoding the first exoloop of the receptor) in the susceptibility haplotypes are noncoding. These genetic associations have recently been confirmed in two independent large cohorts of childhood asthma (5, 6). We hypothesized that intronic SNPs may affect alternative splicing of mRNAs. A large diversity of 7 TM receptor variants with different TM topology (less than 7 TM domains) due to alternative splicing events have been shown to exist, but biological functions of these GPCR isoforms are elusive (7–9). However, studies with gonadotropin-releasing hormone receptor have suggested that truncated receptor proteins may inhibit the transport or signaling of the wild-type receptor (10, 11).

A recent proteomic screening for endogenous ligands for GPRA identified a linear 20-residue peptide, named Neuropeptide S (NPS), that activated the receptor by increasing both intracellular cAMP and Ca²⁺ levels (12, 13). In the present study, we confirmed this interaction by using stable GPRA-A–expressing cell clones and show that this activation has an effect on cell growth via isoform-specific signaling pathways. We analyzed the cellular location of transiently expressed recombinant GPRA splice variants and the effect of co-transfections with truncated variants on the transport of the full-length receptors into the plasma membrane. Finally, we report the expression pattern of GPRA in several cell and tissue types, and show endogenous expression of NPS in some of these. Our results suggest that the GPRA signaling pathway may be active in an autocrine or paracrine fashion in several tissues of relevance in allergies as well as asthma, and that GPRA-A–specific signaling can mediate effects on cell growth.

	MATERIALS AND METHODS

Top Abstract Introduction MATERIALS AND METHODS RESULTS DISCUSSION References

 
Antibodies
Anti–GPRA-A and -B antibodies have been described previously (2). In addition, two nonisoform specific polyclonal antibodies were produced. Antibodies against the amino terminus were raised by immunizing a goat with the peptide TEGSFDSSGTGQTLDSSPVAKKG (corresponding to the residues 6–25 of GPRA) (University of Oulu, Oulu, Finland). Rabbit antibodies were produced against the third cytoloop SSYNRGLISK (corresponding to the residues 258–267 of GPRA; Sigma Genosys Ltd, the Woodlands, UK). Antisera were purified by affinity chromatography with Sulfolink (N-terminus) and Ultralink Immobilization (cytoloop-3) kits according to the manufacturer's (Pierce, Rockford, IL) instructions. Blocking experiments using molar excess of free peptide as a competitor were also performed to demonstrate antibody specificities. Monoclonal anti-myc and anti-HA antibodies were purchased from Berkeley antibody company (Richmond, CA). Horseradish peroxidase (HRP)-conjugated goat anti-mouse or goat anti-rabbit secondary antibodies were from Jackson ImmunoResearch Laboratories Inc. (West Grove, PA).

Construction of Expression Vectors
The cDNAs encoding different GPRA isoforms were subcloned from the pCR 2.1 TOPO -vector into the pCVM-Script expression vector (Stratagene, La Jolla, CA) by restriction enzyme digestion. N-terminally Myc-tagged and C-terminally HA-tagged expression constructs were generated by PCR using the corresponding non-tagged cDNA in the pCMV-Script vector as a template. The resulted PCR products were subcloned into the pCMV-Tag3A vector (Stratagene). All expression constructs were verified by sequencing.

Cell Culture
COS-1 cells (ATCC, Teddington, UK) were cultured in Dulbecco's modified Eagle's medium (Gibco BRL/Invitrogen, Carlsbad, CA) supplemented with 10% fetal bovine serum (Perbio, Northumberland, UK), 1% penicillin/streptomycin (Gibco BRL) and 1x nonessential amino acids (Gibco BRL). Human lung epithelial carcinoma cell line NCI-H358 (ATCC) was cultured in RPMI 1640 medium (Gibco BRL) supplemented with 1 mM sodium puryvate (Gibco BRL), 10% fetal calf serum (Biological Industries, Kibbutz Beit Haemek, Israel), and 1% penicillin/streptomycin. BEAS-2B cell line, which originates from normal human bronchial epithelium, was cultured in Basal Medium (Cambrex, East Rutherford, NJ) supplemented with Bullet Kit (Cambrex). Myoblast cells were isolated from normal human skeletal muscle (kindly provided by Dr. P. Salmikangas, University of Helsinki, Finland) and cultured in Dulbecco's modified Eagle's medium (Gibco BRL) supplemented with 15% fetal bovine serum (Perbio), 4% Ultroseer G (BioSepra, Fremont, CA) and 1% penicillin/streptomycin. All cell lines were cultured at 37°C in a CO₂-conditioned, humidified incubator.

Western Blot Analysis
Human cell lysates were obtained by homogenizing the cell samples in RIPA-buffer (1x phosphate-buffered saline [PBS], 1% Nonidet P-40, 0.5% sodium deoxycholate, 0.1% SDS) with Mini Complete protease inhibitors (Roche, Basel, Switzerland). Human tissue lysates from spleen, skeletal muscle, uterine muscle, colon muscle, colon epithelium, testes, and prostate were obtained by mechanically homogenizing frozen tissue samples in 10 mM Tris HCl, 100mM NaCl, 2% Triton X-100 buffer with Complete Mini protease inhibitors (Roche). The amount of protein was measured with the BCA Protein Assay Reagent kit (Pierce).

For crude membrane preparations, transfected COS-1 cells were harvested in TE buffer (10 mM Tris, 0.1 mM EDTA, pH 7.5). The membrane fractions were separated by suspending cell pellets in TE buffer/0.32 mM sucrose, homogenizing mechanically, and centrifuging for 15 min at 380 x g at 4°C. Supernatant was further centrifuged for 30 min at 40,600 x g at 4°C. The pellet was suspended into sucrose-free TE buffer and centrifuged as above.

Lysates were run on 12.5% SDS-PAGE gels and electroblotted to the PVDF membranes according to standard procedures. Nonspecific protein binding was prevented by incubating the membrane with 5% milk/ 0.1% Tween 20/TBS (TBST) for 1 h at room temperature. Thereafter, membranes were incubated with anti–GPRA-A, anti–GPRA-B, or anti–GPRA-cytoloop-3 antibodies for 1 h at 37°C, washed with TBST, and then incubated with a dilution of 1:2,000 of HRP-conjugated anti-rabbit IgG antibody in 5% milk/ 0.1% TBST for 30 min at room temperature. The protein bands were visualized by using an ECL detection kit (Amersham Biosciences, Buckinghamshire, UK).

Stable GPRA-A– and GPRA-B–Positive 293H Cell Clones
To get GPRA-A and GPRA-B stable cells, GPRA-A and -B cDNA were cloned into a pQM vector under CMV or SR promoters (produced by Quattromed AS, Tarto, Estonia). 293H cells were transfected with Lipofectamine 2000 (Gibco BRL) and clones were cultured under puromycin selection. GPRA-A– and -B–positive clones were characterized by RT-PCR or real-time PCR, and by Western blotting (data not shown). Minimum of three different GPRA-A– and -B–positive, GPRA-A–negative clones and parental 293H cell line were used for different studies in which the clones were cultured in 293 SFM II medium (Gibco BRL) with or without 0.8 µg/ml puromycin (Sigma-Aldrich, St. Louis, MO).

Cell Growth Assay
GPRA-A–positive cells were seeded at 0.3 x 10⁶ cells per ml and cultured in 293 SFM II medium (Gibco BRL) containing 0.8 µg/ml puromycin for up to 4 d. Cell numbers were counted in a hemocytometer.

GPRA-A–positive and –negative clones, and GPRA-B–positive clones and 293H parental cells, were split into 96-well plates, 2 x 10⁴ cells per well. NPS (1 µM) was added and the relative amount of viable cells was determined after 3 d by using CellTiter 96 One solution Cell Proliferation Assay (Promega, Southampton, UK) according the instructions of the manufacturer.

GPRA Activation Assay
GPRA activation in parental 293H cells, in three GPRA-A– and in five GPRA-B–positive clones and in two GPRA-A–negative clones was determined by using nonradioactive GTP-Eu binding assay (Perkin Elmer, Wellesley, MA) according to the instructions of the manufacturer. GPRA activation was measured by comparing the GTP binding in the absence and presence (1 µM) of the NPS (SFRNGVGTGMKKTSFQRAKS), which was synthesized by MedProbe (Oslo, Norway).

Characterization of the Alternatively Spliced GPRA Transcripts
Poly A⁺ RNAs from human lung epithelial carcinoma cell line NCI-H358 (ATCC) were isolated by Dynabeads mRNA DIRECT Kit (Dynal, Oslo, Norway) according to the manufacturer's instructions and subsequently reverse transcribed to cDNA by using SMART RACE cDNA amplification Kit (BD Biosciences, Franklin Lakes, NJ). PCR was performed in 20 µl volume using 2.0 µl NCI-H358 cDNA as template, 1x PCR Gold buffer, 1.5 mM MgCl₂, 0.2 mM dNTPs (Finnzymes, Espoo, Finland); 1 µM of 3'-tgagcaattgataactctgtgggtcctc-5' and 3'-gaatggtggggaaggaaggcgttt-5' or 3'-tgagcaattgataactctgtgggtcctc-5' and 3'-ggccatcctgctgtgacccatttt-5'; and 0.5 U AmpliTaqGold (Applied Biosystems, Foster City, CA) under the following conditions: 94°C for 10 min; 40 cycles of 94°C for 30 s, 66°C for 30 s, 72°C for 1 min followed by final extension of 72°C for 10 min. PCR products were cloned into the pCR 2.1 TOPO vector using TOPO TA cloning kit (Invitrogen) according to manufacturer's instructions and plasmid DNAs were purified using QIAprep Spin Miniprep Kit (Qiagen, Hilden, Germany). The cloned RT-PCR products were verified by automated sequencing with dye-terminator chemistry (MegaBACE 1000; Amersham Biosciences).

Transient Transfections
COS-1 cells were transiently transfected using Fugene6 transfection reagent (Roche) according to the manufacturer's protocol. The empty pCMV vector, ß-gal-pCMV, and myc-tagged luciferace-pCMV vectors (Stratagene) were used as controls. In transfections, 12 µl of Fugene6 and 2.5 µg of DNA (GPRA-C, -D, -E, -F, and -B_short vectors) or 8 µl Fugene6 and 2.0 µg of DNA (GPRA-A and -B vectors) were used for 5 x 10⁶ cells in 35-mm cell culture dishes.

To study the effects of different splice variants on the translocation of the full-length GPRA-A or -B receptors to the plasma membrane, COS-1 cells were cotransfected with myc-tagged GPRA-A or -B with 0.3-, 1-, 3-, and 10-fold amount of pCMV-GPRA-A, -B, -Bshort, -C, -D, -E, or -F. The empty pCMV-vector and ß-gal vector were used as controls. In all transfections, 2 µg of DNA and 8 µl of Fugene6 per 5 x 10⁶ cells were used. The cells from one well of 6-well plate were divided into 16-wells of 96-well plate 24 h after transfection. The cells were analyzed with cell-based enzyme-linked immunosorbent assay (ELISA) 48 h after transfection.

Cell-Based ELISA
Transfected cells were fixed with 3.5% paraformaldehyde in PBS for 15–20 min at room temperature. Cells were blocked with TBS (25mM Tris-150 mM NaCl, pH 8.0) containing 2% milk powder and 1% goat normal serum at 37°C for 30 min. Cells were then incubated with 1:1,000 dilution of anti-myc antibodies for 1 h at 37°C, washed three times with TBS and thereafter incubated with a dilution of 1:2,000 of HRP-conjugated anti-mouse IgG antibodies for 30 min at room temperature. TMB-substrate (Sigma Genosys) was added to cells for 3–6 min. The reaction was stopped by adding an equal amount of 1.5 M HCl and absorbance was measured at 450 nm. Half of the cells were permeabilized to detect the total expression level of the corresponding construct by adding 0.5% TX-100 in PBS for 10–15 min after fixation. The results were normalized using the absorbance values obtained from pCMV and ß-gal control experiments.

Immunofluorescence Microscopy
Transfected cells grown on coverslips were fixed in 3.5% paraformaldehyde in PBS, permeabilized with 0.1% Triton X-100, blocked with PBST (PBS/0.01%/Tween 20) containing 0.5% BSA at room temperature for 30 min, and then incubated in PBST/0.1% BSA with anti–GPRA-A, anti–GPRA-B, or anti-HA antibodies for 1 h at room temperature and then washed three times with PBST. Thereafter, the cells were incubated in PBST with (10 µg/ml) Alexa Fluor 488 goat anti-rabbit IgG or Alexa Fluor 488 goat anti-mouse IgG antibodies (Molecular Probes, Eugene, OR) for 30 min at room temperature and washed three times with PBST. Samples were visualized under fluorescence microscopy.

In Situ Hybridization
Antisense and sense probes of GPRA (full-length GPRA-A cDNA in pCMV-Script vector) and NPS in pCMV-Script vector were transcripted by T3 or T7 RNA polymerases in the presence of digoxigenin-11-uridine-5'-triphosphate (Dig-11-UTP; Roche) by MAXIscript in vitro transcription kit (Ambion, Austin, TX) according to the manufacturer's instructions. The agonist cDNA sequences were amplified by PCR from a human pancreas cDNA sample (Human Multiple Tissue cDNA Panel; BD Biosciences)

Nonradioactive in situ hybridization on tissue sections was performed with Ventana Discovery device (Ventana Medical Systems, Tucson, Arizona). In brief, the samples were frozen sections or deparaffinized with heat treatment followed by post-fixation and RiboClear pretreatment. Samples were protease treated for 18 min and hybridized for 6 h at 65°C with both antisense and sense probes. Slides were then washed three times with 0.1x SSC (15 mM NaCl, 150 nM Sodium citrate, pH 7.0) at 75°C followed by the detection step, which includes 20 min incubation with biotinylated anti-DIG antibody (Jackson ImmunoResearch Laboratories) and 2 h incubation with the BCIP/NBT substrate. After color reaction the slides were washed, dehydrated and mounted with Mountex (HistoLab, Gothenburg, Sweden). All reagents for Discovery were provided by Ventana Medical Systems except for protease K (Roche), which was used at a concentration of 350 ng/µl.

Immunohistochemistry
GPRA expression was studied in formalin-fixed, paraffin-embedded specimens of normal human bronchus, skin, and colon, and MaxArray microarray slide (Zymed Laboratories Inc., San Francisco, CA) containing 30 normal human tissues. The slides were heated in a microwave oven in 10 mM citrate buffer, pH 6.0 for 5 min. Immunohistochemical analyses were performed using the ABC method (Vectastain Elite ABC kit; Vector Laboratories, Burlingame, CA). Omission of primary antibodies and staining with preimmune sera were used as negative controls for parallel sections.

Statistical Analysis
Student's t test was used for analysis of differences between groups.

	RESULTS

Top Abstract Introduction MATERIALS AND METHODS RESULTS DISCUSSION References

 
Specificity of GPRA Antibodies
To study expression of GPRA, we raised four different polyclonal antibodies by immunization in rabbit or goat. We characterized the antibodies by peptide competition assays, by immunohistochemistry, and by Western blotting of cell lysates and tissues. Anti–GPRA-A and -B antibodies were specific for the two alternative carboxy termini. Anti–GPRA-N and anti–cytoloop-3 recognized the shared segments of the GPRA-A and -B isoforms.

Myc-tagged GPRA-A and -B constructs were transiently expressed in COS-1 cells. Cells were harvested 48 h after transfection and the crude membrane preparations were isolated and subsequently analyzed using anti-myc, anti–GPRA-A, and anti–GPRA-B antibodies. Calculated molecular weights of GPRA-A and -B are 42.7 and 43.1 kD, respectively. As shown in Figure 1A, anti–GPRA-A and anti–GPRA-B antibodies recognized recombinant proteins at 35–50 kD, indicating that there are no extensive post-translational modifications in produced recombinant GPRA proteins. In addition, GPRA was not recognized as an one protein band but instead, as a ladder in Western blots, which is typical for hydrophobic GPCR proteins.

View larger version (60K): [in this window] [in a new window]   Figure 1. (A) Specificity of the GPRA antibodies demonstrated by recombinant GPRA expression. The crude membranes of transiently transfected cells were isolated and analyzed by Western blotting using either anti-myc, anti–GPRA-A, or anti–GPRA-B antibodies. (B) Detection of endogenous GPRA-A and -B of BEAS-2B and myoblast cells by Western blotting with indicated antibodies. (C) Western blot analysis of human tissues. The antibodies raised against the third cytoloop of GPRA (detecting both GPRA-A and -B); GPRA-A and -B specific antibodies were used.

When expression was further studied in human tissues by Western blot analyses, GPRA-B had broader expression pattern than the A isoform (Figure 1C). Analyses with anti–GPRA-A antibodies reveled one intensive polypeptide band corresponding to molecular weight of 50 kD in smooth muscle containing tissues: uterine muscle, colon muscle, and prostate, but not in spleen and testis rich in epithelial tissue. However, the band in colon epithelium demonstrates that also epithelium-derived GPRA-A exists. With GPRA-B antibodies, a 50-kD polypeptide band was detected in all studied tissues, except skeletal muscle. An additional 39-kD band was detectable in testis. The results were verified by anti–cytoloop-3 antibodies, which recognize both isoforms A and B in overlapping locations. As expected, all the studied tissues except skeletal muscle were positive for GPRA. Tissue extracts were homogenized into a different buffer than cell culture samples, giving rise to different mobility rates in Western blots. In addition, endogenous GPRA might have more post-translational modifications (e.g., glycosylation) than the recombinantly produced proteins.

Cell Growth of Stably GPRA-A–Overexpressing Cell Lines
To study biological functions of GPRA, we engineered 293H cell lines stably overexpressing GPRA-A and -B. This human kidney epithelial cell line 293H does not express endogenous GPRA (determined by real-time PCR; data not shown). After transfection, the GPRA-A– and -B–positive clones were identified by RT-PCR or real-time PCR and by Western blot analyses. Three GPRA-A– and -B–positive clones and two GPRA-A–negative clones and the parental 293H cell line were selected for further experiments. First of all, cell counting at different time points showed that stably GPRA-A–overexpressing cells grow slower than the GPRA-A–negative cells in serum-free medium. After 2 and 4 d of culture, GPRA-A–positive cells had grown 18% (p < 0.01) and 14% (p < 0.05) slower than the GPRA-A–negative cells, respectively (Table 1). The results were similar with or without puromycin in the culture medium. Cell growth of GPRA-B–overexpressing positive clones was determined by Cell Titer 96 one solution proliferation assay after 3 d of culture (with or without 1 µM NPS). The cell growth of GPRA-B–overexpressing cell clones did not show any change when compared with parental 293H cells (Figure 2D).

View larger version (20K): [in this window] [in a new window]   Figure 2. Specific GPRA-A and GPRA-B receptor activation and its effect on cell growth. Activation of GPRA-A (A) and GPRA-B (B) by 1 µM NPS was measured by comparing the GTP-binding in different GPRA-A– and GPRA-B–positive and –negative cells. One GPRA-A–positive clone was used as control in GTP-binding experiments of GPRA-B. GPRA-A–positive clones and -negative clones (C) or GPRA-B–positive clones and 293H cell line as negative control (D) were cultured for 3 d in the presence (black columns) or absence (gray columns) of NPS (1 µM) before determining relative cell numbers. The cell numbers were compared with the relative cell number of untreated negative clones (1.0). The receptor activation and the cell growth assays were done in triplicates and the results represent means ± SEM (*P < 0.05).

Characterization of the Splice Variants of GPRA
To analyze the alternative GPRA transcripts, we performed RT-PCR analyses using mRNA isolated from the human bronchoalveolar carcinoma cell line (NCI-H358). Five alternatively spliced mRNAs were identified in addition to GPRA-A and -B (Figure 3). The shortest transcript, GPRA-C (encoding a 94 aa peptide), had only three exons. Variants GPRA-D (encoding a 158 aa peptide) and -E (encoding a 136 aa peptide) had the deletion of either exon 3 or 4, respectively, resulting in an early stop codon. GPRA-F (encoding a 305 aa peptide) lacks both the exons 3 and 4, but preserves the rest of the reading frame of GPRA-A. GPRA-B_short (encoding a 366 aa peptide) has an in-frame deletion of 33 bp (11 aa) at the beginning of exon 3, whereas the rest of the downstream exons are the same as in GPRA-B (Figure 3).

View larger version (21K): [in this window] [in a new window]   Figure 3. Schematic representation of the GPRA-A, GPRA-B, GPRA-Bshort, GPRA-C, GPRA-D, GPRA-E, and GPRA-F transcripts. GPRA-A and -B encode the full-length G protein–coupled receptor proteins with 7 TM domains. The other variants encode for truncated proteins. Gray shading indicates the alternate open reading frames. Exon lengths are shown below exons and GenBank accession numbers are shown under the transcript names.

View larger version (81K): [in this window] [in a new window]   Figure 4. Cellular location of the GPRA isoforms. COS-1 cells were transfected with HA-tagged pCMV plasmid encoding one of the variants. Cells grown on glass slides were fixed 48 h after transfection and stained with GPRA-A, GPRA-B, or HA-specific rabbit IgG antibodies and thereafter with Alexa Fluor 488–conjugated goat anti-rabbit IgG secondary antibodies. Cells were visualized by fluorescence microscopy. GPRA-A and GPRA-B are located in the plasma membrane, whereas all the other variants stay in the intracellular compartments.

Tissue Expression Pattern of GPRA by In Situ Hybridization
In situ hybridization (ISH) of paraffine sections of 30 human tissues with a GPRA specific antisense probe resulted in positive staining of epithelial cells in all tissues relevant to asthma and allergy including bronchus, the gastrointestinal tract (esophagus, stomach, small intestine, colon), and skin. In addition, strong staining was observed in the submucosal epithelial cells of spleen, kidney, pancreas, prostate, uterus, breast, and in some glandular epithelia (e.g., that of salivary gland). In heart muscle, weak staining was observed, whereas skeletal muscle and smooth muscle were negative. Peripheral nerves, cerebral cortex, and cerebellum were negative. In tonsils, a few positive inflammatory cells were detected. When the corresponding sections were stained with the sense probe, no specific signal was seen (Figure 5).

View larger version (118K): [in this window] [in a new window]   Figure 5. Expression pattern of GPRA by using ISH (left panel) and immunohistochemistry (right panel). Three different antibodies were used in immunostainings as indicated. Original magnification: x200.

View larger version (151K): [in this window] [in a new window]   Figure 6. ISH of bronchial section from a patient with asthma shows large amount of smooth muscle (SM) staining positively with GPRA-specific antisense probe (left panel). Arrows show the basement membrane.

The specificity of immunostaining was further verified with the GPRA-N antibodies (raised against the amino terminus of GPRA). Staining with the GPRA-N antibodies was overall weaker than with GPRA-A and -B antibodies, which detected intracellular epitopes of the receptor, but consistently, the GPRA-N antibodies recognized GPRA-A and -B in overlapping positions, that is, in different epithelial cells and in smooth muscle cells (Figure 5).

Expression Pattern of Endogenous NPS
The 20-residue NPS (12, 13) is a C-terminal proteolytic fragment of a precursor polypeptide. Corresponding cDNA sequences were amplified from a human pancreas cDNA sample, subcloned into a transcription vector allowing synthesis of antisense and sense probes for ISH. The NPS mRNA was detected in the epithelium of human colon and bronchi in overlapping locations as compared with GPRA. Hybridization of the corresponding sections with the sense probe did not show any specific signal (Figure 7).

View larger version (132K): [in this window] [in a new window]   Figure 7. Expression of NPS in human bronchial and colon epithelia by using ISH.

	DISCUSSION

Top Abstract Introduction MATERIALS AND METHODS RESULTS DISCUSSION References

In parallel with our positional cloning of GPRA as an asthma susceptibility gene, Vassilatis and coworkers reported GPRA-A (called PGR14) as an orphan receptor most homologous to the vasopressin receptor V1a (4). Gupte and colleagues designed a chimeric GPRA-A (called VRR1) to study GPRA signaling induced by vasopressin (13). That study suggested that upon activation, GPRA-A couples to both Gq and Gs signaling pathways. We have expanded on this finding and showed here that the activation of GPRA-A by the endogenous agonist NPS results in growth inhibition of stable GPRA-A–overexpressing cell lines. We also demonstrated the endogenous expression of the agonist by ISH in epithelia of bronchi and colon, coinciding with GPRA-A expression. Based on these results, we also hypothesize that GPRA-A is activated by a paracrine or autocrine mechanism. GPRA (named NPS receptor) and NPS expression pattern in rat tissues have also been studied by others. Notable coexpression of GPRA and NPS was seen especially in different brain sections, for example, amygdala, thalamus, and hypothalamus (14). Interestingly, these results suggested that those brain regions coincide with the regions that have previously been reported to regulate arousal and anxiolytic-like behavior. Previous studies have also suggested that different allergens and extracellular matrix proteins modulate asthmatic airway epithelial and smooth muscle cell proliferation (15–17). The GPRA-A signaling pathway thus becomes a candidate for mediating such effects.

Our data revealed that GPRA has a complex transcript structure and in addition to full-length splice variants A and B, it expresses different truncated splice variants. The full-length protein isoforms of GPRA-A and -B successfully translocate to the plasma membrane, representing functional membrane receptors. In contrast, our study showed that shorter GPRA variants are located in the intracellular compartments, perhaps associated with cellular membranes, because the truncated isoforms still possess hydrophobic regions. Furthermore, in contrast to some other GPCRs (10, 11), shorter GPRA isoforms did not seem to affect the membrane translocation of full-length GPRA-A or -B. Whether the truncated isoforms have any functional roles remains to be determined.

Taken together, our data indicated that truncated GPRA isoforms do not have a regulatory role on the transport of the receptor to the cell surface. Our data revealed that NPS has a potential inhibitory effect on cell growth via a GPRA-A–mediated mechanism. In the present study, we demonstrated that GPRA-A and -B isoforms are plasma membrane receptors expressed in relevant tissues with respect to asthma and allergic diseases, and that they are distinctly regulated in tissues. The distinct expression patterns were documented both in different cell lines representing epithelia and smooth muscle as well as in tissue sections. Factors controlling alternative splicing are presently unknown, but may involve polymorphic sites in the genomic sequence of GPRA, as suggested for the AAA1 gene (2).

	Acknowledgments

 
The authors thank Siv Knaappila, Tuula Lahtinen, Virpi Päivinen, Riitta Känkänen, and Morag Dixon for their skilful laboratory work, Satu Kuure for technical advice, and Paula Salmikangas for providing skeletal muscle cells.

	Footnotes

 
This study has been supported by Finnish National Technology Agency Tekes, Academy of Finland, Sigrid Juselius Foundation, GeneOS Ltd, Ida Montin's Foundation, the Finnish Anti-Tuberculosis Association Foundation, Emil Aaltonen's Foundation; and Väinö and Laina Kivi's Foundation.

Conflict of Interest Statement: T.L. is a stake holder in and the employee of GeneOS Ltd (Helsinki, Finland), which has submitted a patent on GPRA; J.K. is a stake holder, the member of the Board of Directors in GeneOS Ltd and an inventor in a patent application filed by GeneOS Ltd; M.R. is the employee of GeneOS Ltd; A.P. is the employee of GeneOS Ltd. V.P. is an inventor in a patent application filed by GeneOS Ltd; J.V. is as an inventor in a patent application filed by GeneOS Ltd; A.R.-S. does not have a financial relationship with a commercial entity that has an interest in the subject of this manuscript; A.L. does not have a financial relationship with a commercial entity that has an interest in the subject of this manuscript; L.A.L. is a stake holder, the member of the Board of Directors in GeneOS Ltd and an inventor in a patent application filed by GeneOS Ltd.

Received in original form December 15, 2004

Received in final form May 4, 2005

	References

Top Abstract Introduction MATERIALS AND METHODS RESULTS DISCUSSION References

 

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