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Badiali et al. BMC Gastroenterology 2012, 12:134 http://www.biomedcentral.com/1471-230X/12/134 RESEARCH ARTICLE Open Access Adhesion GPCRs are widely expressed throughout the subsections of the gastrointestinal tract Luca Badiali1,2, Jonathan Cedernaes1, Pawel K Olszewski1,3, Olof Nylander1, Anna V Vergoni2 and Helgi B Schiöth1* Abstract Background: G protein-coupled receptors (GPCRs) represent one of the largest families of transmembrane receptors and the most common drug target. The Adhesion subfamily is the second largest one of GPCRs and its several members are known to mediate neural development and immune system functioning through cell-cell and cell-matrix interactions. The distribution of these receptors has not been characterized in detail in the gastrointestinal (GI) tract. Here we present the first comprehensive anatomical profiling of mRNA expression of all 30 Adhesion GPCRs in the rat GI tract divided into twelve subsegments. Methods: Using RT-qPCR, we studied the expression of Adhesion GPCRs in the esophagus, the corpus and antrum of the stomach, the proximal and distal parts of the duodenum, ileum, jejunum and colon, and the cecum. Results: We found that twenty-one Adhesion GPCRs (70%) had a widespread (expressed in five or more segments) or ubiquitous (expressed in eleven or more segments) distribution, seven (23%) were restricted to a few segments of the GI tract and two were not expressed in any segment. Most notably, almost all Group III members were ubiquitously expressed, while the restricted expression was characteristic for the majority of group VII members, hinting at more specific/localized roles for some of these receptors. Conclusions: Overall, the distribution of Adhesion GPCRs points to their important role in GI tract functioning and defines them as a potentially crucial target for pharmacological interventions. Background Adhesion GPCRs, based on the 7TM regions, revealed The superfamily of G protein-coupled receptors (GPCRs) that there are seven groups [3]. is one of the largest families of membrane bound proteins GPCRs are characterized by having seven α-helices that in the human genome [1] comprising about 800 members. span the plasma membrane and form a receptor with a Being involved in a high number of physiological func- binding cavity for a ligand; the extracellular segment may tions, including development, neurotransmission, metab- also be able to bind a ligand. The main feature of the olism, reproduction, immune responses, and behavior, Adhesion family is the long N terminus with complex GPCRs act as receptors for a great number of different domain architecture which is thought to be highly glyco- signals, both endogenous - amines, peptides, proteins, sylated and form a rigid structure in the outer part lipids, nucleotides, neurotransmitters - and sensory, as of the protein. This extracellular portion contains the organic odorants, pheromones, tastes and photons. Accord- GPCR proteolytic site (GPS) and several various domains ing to the phylogenetic analysis of the entire human GPCR that can also be found in other proteins such as lectin, repertoire, five subfamilies make up the GRAFS classifica- epidermal growth factor, olfactomedin, immunoglobulin, tion system: Glutamate, Rhodopsin, Adhesion, Frizzled/ thrombospondin and cadherin domains [3]. The GPS Taste2 and Secretin [2]. The phylogenetic grouping of domain is referred to as an intracellular cleavage motif, pivotal for the protein transport from the endoplasmic reticulum to the membrane [4], while several other N * Correspondence: [email protected] terminal domains play important roles in the receptor- 1Department of Neuroscience, Uppsala University, BMC, Uppsala SE 75124, Sweden ligand binding as well as cell-to-cell and cell-to-matrix Full list of author information is available at the end of the article adhesion [5]. Distinguishing themselves even more from © 2012 Badiali et al.; licensee BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Badiali et al. BMC Gastroenterology 2012, 12:134 Page 2 of 10 http://www.biomedcentral.com/1471-230X/12/134 other GPCRs, Adhesion GPCRs are genomically com- [29]). Using RT-qPCR with a validated range of house- plex, each receptor having many isoforms [6], with mul- keeping genes, we studied the expression in the esopha- tiple alternatively-spliceable introns and large genomic gus, the corpus and the antrum of the stomach, the sizes, which makes them difficult to study [7]. Only a few proximal and distal parts of the duodenum, jejunum, members of Adhesion GPCRs have been demonstrated to ileum and colon, and in the cecum. interact with G proteins [8,9]. Beyond its evident role in digestion and adsorption, Methods the gastrointestinal (GI) tract is involved in a variety of Animal handling and tissue isolation other physiological functions, such as endo- and exocrine Three male Dark Agouti rats (Scanbur AB, Sweden) secretion and immune responses [10-12]. Its autono- weighing approximately 200 grams, were kept under mous neuronal network, referred to as the enteric ner- constant conditions (12 h dark/light cycle) at 21°C. The vous system or ENS, is also intimately linked with the animals were fasted overnight but had free access to brain in the brain-gut axis important for, among others, water before carrying out the experiment. The following food intake regulation [13,14]. GPCRs in the GI tract are morning the animals were intraperitoneally anesthetized known to be involved in nutrient balancing [15-17] and with Na-5-ethyl-1-(1’-methyl-propyl)-2-thiobarbituric acid W regulation of the immune system [18,19]. In some cases (Inactin ; 125 mg/kg b. wt). Body temperature was main- their gross expression patterns in the GI tract have been tained at 37.5 ± 0.5°C through a temperature regulator established, but more subtle proximodistal variations in controlling a heating pad. Thereafter, a tracheotomy was expression and their biological functions have yet to be performed and a cannula (PE-200) was inserted to guar- determined [20]. Furthermore, dysfunction of GPCRs is antee free airways. Following a midline incision to open already known to contribute to certain diseases affecting the abdominal cavity, the following structures, about 5 to the GI tract. For example, GPR49 overexpression has 10 mm in length, were localized and dissected (Figure 1): been related to increased incidence of human colon Distal esophagus (a few mm from the stomach); corpus primary tumors [21], whereas overexpression of the of the stomach; antrum of the stomach; proximal (1 mm Adhesion GPCR CD97 has been correlated to colorec- from the pylorus) and distal duodenum (4 cm from the tal cancer [22], rectal adenocarcinoma recurrence and pylorus); proximal (9 cm from the pylorus) and distal metastasis [23], and gastric carcinoma [24]. jejunum (19 cm from the pylorus); proximal (29 cm from Though about 85% of Adhesion GPCRs are still pylorus) and distal ileum (2.5 cm from the ileocecal orphans or lack biological characterization (details about valve); cecum; proximal (5 cm from ileocecal valve) and known functions are provided in [6]), EMR receptors distal colon (12 cm from the ileocecal valve). The whole have been shown to be involved in immune responses GI tract wall was isolated for the RT-qPCR analysis. [25]: Crohn’s disease and ulcerative colitis are two of the After the operation, an intravenous bolus injection of main inflammatory bowel diseases affecting the GI tract, a saturated KCl solution was used to euthanize the ani- and there is a growing body of evidence indicating that mals. All animal procedures followed the regulations and the onset of these pathologies may be related to an policies outlined by the Swedish Animal Protection Act immune system deregulation by the ENS or gut micro- and were approved by the Uppsala Ethic Committee. biota [11,12]. The characterization of immunologically important Adhesion GPCRs in the GI tract may therefore RNA isolation and cDNA synthesis lead to a greater understanding of these pathological con- RNA isolation and cDNA synthesis were performed as ditions. Profiling of the entire GI tract, accounting for previously reported [30]. Briefly, tissue samples were intra-regional differences is important since proximal- homogenized by sonication in the TRIzol reagent (Invi- distal part of GI tract’s anatomical regions can serve dif- trogen, Sweden) using a Branson sonifier. Chloroform ferent roles or be selectively affected by diseases; e.g. bile was added to the homogenate and centrifuged at 12000 g acids and vitamin B12 adsorption occur in the distal at 4°C for 15 minutes. The aqueous phase was collected ileum, Barrett’s disease affects the distal esophagus [26], and RNA precipitated with isopropanol. The pellets were and gastric cancer occurs either proximally or distally washed with 75% ethanol, air dried and dissolved in [27]. Likewise, Crohn’s disease more frequently affects RNAse-free water. The DNAse treatment was performed the terminal ileum [28]. Knowledge of the proximodistal to remove DNA contamination: DNAse I (Roche Diag- expression pattern may therefore indirectly facilitate pin- nostics, Sweden) was added to the samples and incubated pointing the function of Adhesion GPCR orphans. at 37°C for 4 h, followed by inactivation by heating to We present here the first complete analysis of mRNA 75°C for 15 minutes. Absence of DNA contamination in W expression of all members of the Adhesion GPCRs sub- the RNA samples was confirmed by PCR. Nanodrop family throughout the entire rat GI tract, which was ND-1000 Spectophotometer (NanoDrop Technologies, divided into twelve subsegments (as described previously Delaware, USA) was used to determine RNA concentration. Badiali et al. BMC Gastroenterology 2012, 12:134 Page 3 of 10 http://www.biomedcentral.com/1471-230X/12/134 Figure 1 GI sectioning approximately indicating the different sections of the rat GI tract used for analysis.
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  • Normalized Expression Values *

    Normalized Expression Values *

    Normalized expression values 0.0 0.2 0.4 0.6 0.8 1.0 1.2 PLCG1 * T CD5 MP T B TCF7 cDC UBASH3A BCL11B C5AR1 * MP TLR4 CXCR5 B CD79B VPREB3 XCR1 CADM1 BEND5 * ARGHAP22 * cDC CIITA ZBTB46 FLT3 PLEKHA5 Figure S1. qPCR analysis of the core gene expression signature of cDC, MP, B and T cells in the chicken cell suBsets. RNA from cDC, MP, T and B cells of 2 disUnct pools of 4 chicken spleen was subjected to qPCR detecUon of the core gene expression signatures of immune cell subsets established in Fig. 3 and of transcripts from the mouse and human gene subset selected compendia that could not be detected on the array due to defecUve probes, i.e. ARGHAP22, BEND5, C5AR1, and PLCG1 (labeled by a star on the figure). Data are represented as the mean and SD of relave gene expression levels normalized to GAPDH expression and the maximal expression across the cell types was set to 1 (independent experimental duplicates). B cell T cell cDC Monocyte/MP Chicken Human Mouse Figure S2. Unsupervised hierarchical clustering of orthologous immune response genes across chicken, human and mouse reveals globally conserved clusters of lymphoïd- specific and myeloïd- specific genes. Heatmap of cross-normalized expression profiles for immune response genes present on all three species arrays and regulated at least 2 folds across all cell suBsets, including chicken B cells (c_B), T cells (c_CD3), MP (c_MP) and cDC (c_cDC), human B cells (h_B), T cells (h_CD4_T and h_CD8_T), monocyte-derived MP (h_MoMP), peripheral blood mononucleated cell-derived MP (h_PBMC_MP), non-classical monocytes (h_non- classical_MO), classical monocytes (h_classical_MO), BDCA3+ cDC (h_BDCA3), BDCA1+ cDC (h_BDCA1), murine B cells (m_B), T cells (m_CD4_T and m_CD8_T), peritoneal cavity MP (m_PC_MPII-480HI), lung MP (m_LU_MP), non-classical monocytes (m_non-classical_MO), classical monocytes (m_classical_MO), splenic CD8α+ cDC (m_SP_DC1), suBcutaneous lymph node CD8α+ cDC (m_LN_DC1), splenic CD11B+ cDC (m_SP_DC2), suBcutaneous lymph node CD11B+ cDC (m_LN_DC2).