Orphan receptor GPR110, an oncogene overexpressed in lung and prostate cancer
- Amy M Lum†1,
- Bruce B Wang†1,
- Gabriele B Beck-Engeser2,
- Lauri Li1,
- Namitha Channa1 and
- Matthias Wabl2Email author
© Lum et al; licensee BioMed Central Ltd. 2010
Received: 23 September 2008
Accepted: 11 February 2010
Published: 11 February 2010
GPR110 is an orphan G protein-coupled receptor--a receptor without a known ligand, a known signaling pathway, or a known function. Despite the lack of information, one can assume that orphan receptors have important biological roles. In a retroviral insertion mutagenesis screen in the mouse, we identified GPR110 as an oncogene. This prompted us to study the potential isoforms that can be gleaned from known GPR110 transcripts, and the expression of these isoforms in normal and transformed human tissues.
Various epitope-tagged isoforms of GPR110 were expressed in cell lines and assayed by western blotting to determine cleavage, surface localization, and secretion patterns. GPR110 transcript and protein levels were measured in lung and prostate cancer cell lines and clinical samples, respectively, by quantitative PCR and immunohistochemistry.
We found four potential splice variants of GPR110. Of these variants, we confirmed three as being expressed as proteins on the cell surface. Isoform 1 is the canonical form, with a molecular mass of about 100 kD. Isoforms 2 and 3 are truncated products of isoform 1, and are 25 and 23 kD, respectively. These truncated isoforms lack the seven-span transmembrane domain characteristic of GPR proteins and thus are not likely to be membrane anchored; indeed, isoform 2 can be secreted. Compared with the median gene expression of ~200 selected genes, GPR110 expression was low in most tissues. However, it had higher than average gene expression in normal kidney tissue and in prostate tissues originating from older donors. Although identified as an oncogene in murine T lymphomas, GPR110 is greatly overexpressed in human lung and prostate cancers. As detected by immunohistochemistry, GPR110 was overexpressed in 20 of 27 (74%) lung adenocarcinoma tissue cores and in 17 of 29 (59%) prostate adenocarcinoma tissue cores. Additionally, staining with a GPR110 antibody enabled us to differentiate between benign prostate hyperplasia and potential incipient malignancy.
Our work suggests a role for GPR110 in tumor physiology and supports it as a potential therapeutic candidate and disease marker for both lung and prostate cancer.
GPCRs are seven transmembrane receptors that vary extensively in their biological functions. Upon ligand binding, these receptors transduce a signal via a G protein. This fact has been used extensively in pharmacology to select inhibitors of biological pathways. A large fraction of all drugs currently on the market target GPCRs. Drugs targeting members of this integral membrane protein superfamily represent the core of modern medicine .
There are many so-called orphan receptors--receptors without a known ligand, a known signaling pathway, or a known function. Despite the lack of information, one can assume that orphan receptors have important biological roles. One of these orphan receptors is GPR110, about which little is known other than its gene structure and potential isoforms that can be inferred from published transcript data. In a large murine retroviral mutagenesis screen, we identified GPR110 as an oncogene.
The GPR110 protein contains two protein domains where cleavage can potentially occur: the SEA domain and the GPS domain. Self-cleavage has been reported for the SEA domain in human MUC1  and in rat Muc3 . According to these reports, the cleaved SEA product reassociates with the membrane-bound protein by noncovalent interactions. Cleavage at the GPS domain was first demonstrated in the GPCR latrophilin . Cleaved products of an overexpressed GPCR might be found in the blood, which could serve as an easily accessible clinical marker. Furthermore, alternatively spliced isoforms that are not membrane anchored may instead be potentially secreted and also be found in the blood. The rich possibility of GPR110 as a therapeutic candidate and diagnostic marker led us to study the synthesis of its various isoforms and to survey human cancers for its overexpression.
Cloning and tagging of GPR110 isoforms
GPR110 isoforms 1 and 2 were amplified from PC-3 cDNA using a set of primers designed to their common 5' UTR and their respective 3' UTR regions. Forward primer 5'-CACCAGTCACAGACTATGC-3' and reverse primer 5'-ACCCGATCGAATACTGAGC-3' (isoform 1, 3' UTR) and reverse primer 5'-CAGGGGAATCTCTTGAACCCG-3' (isoform 2, 3' UTR). Products from the first PCR reactions were used as templates in a nested PCR with the following primers: forward primer 5'-TTCGGTACCACCATGAAAGTTGGAGTGC-3' (110_F_Kpn), reverse primer 5'-CCCTCTAGATTATTCATTTGAGACAAACTG-3' (isoform 1, with stop codon) and reverse primer 5'-CCTTCTAGAGATTGTGCCATTGCACTC-3' (isoform 2, no stop codon). The PCR products were then cloned into pcDNA3.1(+) (Invitrogen) using KpnI and XbaI restriction sites to make constructs pcDNA/Iso1 and pcDNA/Iso2. Sequences of these clones matched published RefSeq sequences on NCBI. GPR110 isoform 3 with no stop codon was amplified from pcDNA/Iso1 using the primers 110_F_Kpn and reverse primer 5'-CCCTCTAGACCGAAATTGGGTGACC-3'. A version of isoform 1 with no stop codon was amplified from the pcDNA/Iso1 construct using primer 110_F_Kpn and reverse primer 5'-CCCTCTAGATTCATTTGAGACAAACTGAG-3'. Isoforms 1-3 containing no stop codons were then cloned into a version of pcDNA3.1(+) containing the HA epitope between restriction sites XbaI and ApaIon the pcDNA3.1(+) vector creating constructs Iso1-HA, Iso2-HA, and Iso3-HA.
Three additional HA-tagged versions of isoform 1 were made using pcDNA/Iso1 as a template with the QuikChangeII Site-Directed Mutagenesis Kit (Stratagene). The following primers were used for the three constructs: HA466: 5'-TTAGAATTATCAGAGCAAAGTACCCATACGATGTTCCAGATTACGCTACCACAGACTGCAACAG-3' and 5'-CTGTTGCAGTCTGTGGTAGCGTAATCTGGAACATCGTATGGGTACTTTGCTCTGATAATTCTAA-3'; HA1036: 5'-CCTGCAGCAGTGGCTACCCATACGATGTTCCAGATTACGCTAGGGGAAACATCACAGC-3' and 5'-GCTGTGATGTTTCCCCTAGCGTAATCTGGAACATCGTATGGGTAGCCACTGCTGCAGG-3'; and HA1393: 5'-GTCTTACTGCGGGAAGAAAAGTACCCATACGATGTTCCAGATTATGCCAGCTCACG-3' and 5'-CGTGAGCTGGCATAATCTGGAACATCGTATGGGTACTTTTCTTCCCGCAGTAAGAC-3'. Construct HA466 has a HA tag located N-terminal to the SEA domain whereas HA1036 and HA1393 contain tags located between the SEA and GPS domains.
Transfections, immunoblotting, cell surface detection, and immunoprecipitation
All cell lines were seeded in 6-well plates at a cell density of 4 × 105 cells per well. Each well was transfected with 4 μg of DNA and 10 μl of Lipofectamine 2000 (Invitrogen) according to the manufacturer's instructions. Cells were incubated in lysis buffer (0.5% Triton X-100, 50 mM Tris pH7.4, 5 mM EDTA, and 150 mM NaCl). Cell lysates were run on 4-12% Bis-Tris gels in NuPAGE MOPS buffer (Invitrogen) and transferred to nitrocellulose membranes. Blots were blocked in 5% nonfat dry milk (wt/vol) in TBST (20 mM Tris, 150 mM NaCl, 0.1% Tween-20) and followed by incubation with primary antibody (anti-HA, HA.11 from Covance). After washing with TBST, blots were incubated with an anti-mouse Ig secondary antibody (Southern Biotech #1010-04), and developed with 1 Step NBT/BCIP (Pierce). For deglycosylation reactions, samples denatured in protein loading buffer were treated with 500 units of PNGase F (NEB) in 1× G7 Reaction Buffer and 1% NP40 for 1.5 hours at 37°C.
For cell surface detection, two 10 cm dishes of HEK293 were transfected with Lipofectamine 2000 for each construct according to manufacturer's instructions. Approximately 24 hr post transfection, surface proteins were isolated using the Cell Surface Protein Isolation Kit (Pierce). Purification fractions were assayed by immunoblotting as described above using an anti-GAPDH antibody (Ambion), an anti-β1 integrin antibody (MAB2000, Chemicon), and HA.11 (Covance).
Immunoprecipitation of media samples was done using Protein-G agarose (Invitrogen). 200 μl of Protein-G agarose (50% slurry) was washed in lysis buffer and incubated with 5 μg of HA.11 antibody for 1 hr at 4°C. The conjugated beads were washed 3 times with lysis buffer to remove any excess antibody. 15 μl aliquots of beads were incubated with 1 mL of media from transfections of various GPR110 isoforms overnight at 4°C with rocking. Beads were washed in lysis buffer and then boiled for 10 min in SDS Loading Buffer. Samples were then assayed by immunoblotting as described above.
Cell lines HEK293T/17, HeLa, PC-3, LNCaP, DU145, A549, NCI-H460, and NCI-H23 were obtained from the American Type Culture Collection. With the exception of LNCaP, all human cell lines used are part of the NCI-60 panel of reference cell lines, for which extensive expression analysis and significant chemical compound screening assays have been done. In addition, these cell lines have been used in xenograft cancer models. All cultures were grown in media supplemented with 10% fetal bovine serum (Hyclone), 100 U/mL penicillin, and 100 μg/mL streptomycin. HEK293T/17 cells were maintained in DMEM (Cellgro); PC-3 and A549 in F12K (Hyclone) with 2 mM glutamine; LNCaP, NCI-H460, and NCI-H23 in RPMI with 2 mM glutamine (Hyclone) and supplemented with 10 mM HEPES, 4.5 g/L glucose, 1 mM sodium pyruvate, and 1.5 g/L sodium bicarbonate; DU145 and HeLa in MEM/EBSS (Hyclone) with 2 mM glutamine, 1 mM sodium pyruvate, 1.5 g/L sodium bicarbonate, and 0.1 mM nonessential amino acids.
RNA and quantitative PCR
The FirstChoice Human Total RNA Survey Panel (Ambion) was used to screen GPR110 expression in normal human tissues. The Human Lung Cancer TissueScan Real Time Expression Panel (Origene) containing cDNA from 40 lung tumor and 8 normal lung samples was used to screen GPR110 expression in lung tumors. Additional human lung adenocarcinoma RNAs were from Asterand, and normal lung RNAs from Ambion and Biochain. RNA was extracted from frozen mouse spleen and thymus tumor samples and from human cell lines with the RNeasy Mini Kit (Qiagen). Mouse RNA samples were treated with rDNase (Ambion) prior to reverse transcription. For all cDNA synthesis, 500 ng of RNA was reverse transcribed using the SuperScript First-Strand Synthesis System III (Invitrogen).
Quantitative PCR (qPCR) was done on the Stratagene MX3000P. All cDNA samples were assayed in triplicates except the Origene Lung Expression Panels, which were run in duplicates for each gene probe. Mouse tumor samples were assayed with the Mouse ACTB-VIC/MGB probe (ABI) as an endogenous control and GPR110 probe Mm00505409_m1 (ABI). Tumor samples containing no integration sites in the GPR110 locus were used as control tumors. Relative expression values (2-ΔΔCt ) were calculated using normal mouse spleen cDNA. GUSB (Hs99999908_m1, ABI) was used as an endogenous control for all human samples. ABI Taqman probe Hs00228100_m1 (spanning exons 2 and 3) was used to detect human GPR110. The following SYBR primers were used to detect the three GPR110 isoforms in GPR110 positive cell lines: Isoform 1 - 5'-CTTTCTGTCATCATTCGGCAAAAC-3' and 5'-TTGGTTACTGAGGCTGAATTAAGG-3', Isoform 2 - 5'-GAATTCATCTTCTGCTATATACTCC-3' and 5'-CCAACTTGGGCGACAAGAGTGAC-3', Isoform 3 - 5'-CAACAACCTCAGCCAGAGTGT-3' and 5'-ATGCCCTTCTAAGACCATTGTGT-3', and endogenous control GUSB - 5'-CACTGAAGAGTACCAGAAAAGTC-3' and 5'-TCTCTGCCGAGTGAAGATCC-3'.
Human tissue arrays containing formalin-fixed paraffin-embedded tissues (Cybrdi) were processed according to standard procedures. LS-A2021 and LS-A2019 (MBL International), or control rabbit IgG antibodies (Invitrogen) were incubated with the arrays for 1 hour at room temperature. For detection of bound antibody, the arrays were then processed using the SuperPicture kit (Invitrogen) according to the manufacturer's instructions.
Identification of provirus integration sites
The genomic locations of the proviral integrations were determined using the splinkerette-based PCR method . This method recovers genomic DNA directly flanking the 5' LTR of the integrated provirus. Genomic DNA was isolated from tumors using the DNeasy Tissue kit (Qiagen) and digested using restriction enzymes BstYI or NspI. A double-stranded splinkerette adapter molecule  containing the appropriate restriction site was ligated to the digested genomic DNA using the Quick Ligation kit (New England Biolabs). These ligation products were then digested with EcoRV to prevent subsequent amplification of internal viral fragments. The resulting mixture was purified using QIAquick PCR purification kits (Qiagen), and subject to three rounds of PCR using nested PCR primers that had homology to the adapter DNA and to the 5' LTR sequence of the SL3-3 virus. After resolving the PCR products by gel electrophoresis, the desired bands were purified using QIAquick Gel Extraction kits (Qiagen) and subject to standard DNA sequencing.
Results and discussion
Structure and mRNA expression of the GPR110 gene
Not only is the ligand(s) of GPR110 unknown, but also very little is known about the putative transcripts that direct protein synthesis. To date, all published research on GPR110 has dealt with its sequence identification and analysis [7–9]. Public genome databases give some information about GPR110, and there is one publication that analyzes potential GPR110 splice variants in silico . According to these sources, human GPR110 is located on chromosome 6. Two mRNAs are reported for this gene: isoform 1 (NM_153840.2), which encodes the full-length protein containing the characteristic seven-span transmembrane region of G-protein-coupled receptors, and isoform 2 (NM_025048.2), a truncated version of isoform 1.
Expression of GPR110 polypeptides in cell lines
Of the three isoforms, isoform 1 must be the canonical form, with a predicted molecular mass of about 100 kD. Isoforms 2 and 3--truncated products of isoform 1--are 25 and 23 kD, respectively. These isoforms lack the seven-span transmembrane domain and thus are not likely to be membrane anchored. However, the presence of a signal peptide suggests isoforms 2 and 3 may be potentially secreted. Figure 1C shows the various domains of the full-length protein. Apart from the signal peptide, the figure depicts three protein domains: (1) an SEA domain, (2) a GPS domain, and (3) a 7TM (seven transmembrane domain). The SEA domain is a conserved protein domain with an unknown function that was first observed in Sea urchin sperm protein, Enterokinase, and Agrin. Self-cleavage was reported for this domain in human MUC1  and in rat Muc3 . The cleaved SEA product has been shown to reassociate with the membrane-bound protein by noncovalent interactions . The predicted GPR110 SEA domain contains the sequence GSIVA, ~24 kD downstream of the N terminus, which is consistent with the reported SEA consensus cleavage site G^SVVV. Cleavage in the GPS domain was first shown in the GPCR latrophilin . GPR110 contains the consensus GPS cleavage site H^LT, ~63 kD downstream of the N terminus.
Because GPR110 contains 19 predicted N-linked glycosylation sites, the bands running at 100 and 80 kD fall short of the expected size. However, the banding pattern of the tagged GPR110 proteins does not indicate the presence of another cleavage site. To estimate the sizes of the non-glycosylated protein, cell lysates from transfected HEK cells were treated with the glycosidase PNGaseF. This treatment reduced the four bands of isoform 2 to a single band of the expected molecular weight of 26 kD (molecular weight plus tag), and isoform 3 to ~25 kD (MW plus tag) (Figure 2B). Thus, both isoforms 2 and 3 are produced at the expected molecular weight in multiple cell lines and are present as multiple glycoforms within the cell. Glycosidase treated isoform 1 reduces the two major bands of 80 and 100 kD to 60 and 75 kD, respectively (Figure 2C, arrows). The pair of bands in HA466 at 30 kD also reduces to a single band at ~25 kD, which agrees with the size of an SEA cleavage product (Figure 2C, arrow).
Expression of isoforms on the cell surface
Endogenous expression in normal tissue
GPR110 is a proto-oncogene
GPR110 overexpression in human lung cancer
GPR110 overexpression in prostate cancer
As before with the lung cancer tissue cores, we also performed immunohistochemistry on prostate tissues. On these, we hypothesized that GPR110 expression may enable us to differentiate between benign prostate hyperplasia (BPH) and potential incipient malignancy. On BPH cores, 94% (33/35) were positive for PSA (Figure 10B, right panels, cores 1 and 2 shown), and 26% (9/35) were positive with antibody LS-A2021 (Figure 10B, left panels). In these tissues, however, only the few cells that are PSA negative are positive for the epitope stained by LS-A2021 (Figure 10B, arrows); most of the tissue core is negative for LS-A2021 staining. Because antibody to this epitope stains prostate adenocarcinoma (see below), we interpret this to possibly mean that among the hyperplastic non-cancerous epithelial cells, adenocarcinoma cells slowly begin to develop. Here it is not clear whether the carcinoma cells are derived from the BPH cells, although it is intriguing to note that expression of the epitopes of GPR110 and PSA are mutually exclusive. In prostate adenocarcinomas, LS-A2021 stained over 50% (17/29) of tissue cores while LS-A2019 stained over ~30% (10/29) (Figure 10C). Most of the cores with LS-A2019 staining also tested positive with LS-A2021, but the two antibodies displayed differential staining in these overlapping cores. This differential staining may be due to various splice forms of GPR110 or to different post-transcriptional processing of GPR110 in these prostate cancers.
In a large retroviral mutagenesis screen, we identified Gpr110 as an oncogene. As an orphan G protein-coupled receptor, this protein has not been subject to experimental study. We identified three isoforms of GPR110 at the protein level by their production in multiple human cell lines. Isoform 1 is cleaved within the SEA domain and isoform 2 is secreted into the culture medium. However, all isoforms are glycosylated, and all are present on the cell surface. At the transcript level, GPR110 is overexpressed in some lung tumors as well as highly expressed in a commonly used prostate cancer cell line. In lung and prostate tumor tissue cores, GPR110 protein is overexpressed, and it may differentiate between benign prostate hyperplasia and prostate carcinoma. This work supports GPR110 as a potential therapeutic candidate and disease marker for both lung and prostate cancer.
Supported by NIH grant R01AI041570. We thank Gabor Bartha for help with tag recovery and identification; and Finn Pedersen for advice and discussion.
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