Chicken Receptor Research Articles

The chicken SLAM family

The signaling lymphocytic activation molecule (SLAM) family of receptors is critically involved in the immune regulation of lymphocytes but has only been detected in mammals, with one member being present in Xenopus. Here, we describe the identification, cloning, and analysis of the chicken homologues to the mammalian SLAMF1 (CD150), SLAMF2 (CD48), and SLAMF4 (CD244, 2B4). Two additional chicken SLAM genes were identified and designated SLAMF3like and SLAM5like in order to stress that those two receptors have no clear mammalian counterpart but share some features with mammalian SLAMF3 and SLAMF5, respectively. Three of the chicken SLAM genes are located on chromosome 25, whereas two are currently not yet assigned. The mammalian and chicken receptors share a common structure with a V-like domain that lacks conserved cysteine residues and a C2-type Ig domain with four cysteines forming two disulfide bonds. Chicken SLAMF2, like its mammalian counterpart, lacks a transmembrane and cytoplasmic domain and thus represents a glycosyl-phosphatidyl-inositol-anchored protein. The cytoplasmic tails of SLAMF1 and SLAMF4 display two and four conserved immunoreceptor tyrosine-based switch motifs (ITSMs), respectively, whereas both chicken SLAMF3like and SLAMF5like have only a single ITSM. We have also identified the chicken homologues of the SLAM-associated protein family of adaptors (SAP), SAP and EAT-2. Chicken SAP shares about 70% identity with mammalian SAP, and chicken EAT-2 is homologous to mouse EAT-2, whereas human EAT-2 is much shorter. The characterization of the chicken SLAM family of receptors and the SAP adaptors demonstrates the phylogenetic conservation of this family, in particular, its signaling capacities.

Functional characterization and evolution of PTH/PTHrP receptors: insights from the chicken

BackgroundThe parathyroid hormone (PTH)-family consists of a group of structurally related factors that regulate calcium and bone homeostasis and are also involved in development of organs such as the heart, mammary gland and immune system. They interact with specific members of family 2 B1 G-protein coupled receptors (GPCRs), which have been characterised in teleosts and mammals. Two PTH/PTHrP receptors, PTH1R and PTH2R exist in mammals and in teleost fish a further receptor PTH3R has also been identified. Recently in chicken, PTH-family members involved in calcium transport were characterized and specific PTHRs are suggested to exist although they have not yet been isolated or functionally characterized. The aim of this study is to further explore the evolution and function of the vertebrate PTH/PTHrP system through the isolation, phylogenetic analysis and functional characterization of the chicken receptors.ResultsTwo PTHRs were isolated in chicken and sequence comparison and phylogenetic analysis indicate that the chicken receptors correspond to PTH1R and PTH3R, which emerged prior to the teleost/tetrapod divergence since they are present in cartilaginous fish. The vertebrate PTH2R receptor and its ligand TIP39 have been lost from bird genomes. Chicken PTH1R and PTH3R have a divergent and widespread tissue expression and are also evident in very early embryonic stages of development. Receptor stimulation studies using HEK293 cells stably expressing the chicken PTH1R and PTH3R and monitoring cAMP production revealed they are activated by chicken 1–34 N-terminal PTH-family peptides in a dose dependent manner. PTH-L and PTHrP were the most effective peptides in activating PTH1R (EC50 = 7.7 nM and EC50 = 22.7 nM, respectively). In contrast, PTH-L (100 nM) produced a small cAMP accumulation on activation of PTH3R but PTHrP and PTH (EC50 = 2.5 nM and EC50 = 22.1 nM, respectively) readily activated the receptor. PTHrP also stimulated intracellular Ca2+ accumulation on activation of PTH1R but not PTH3R.ConclusionTwo PTHR homologues of the vertebrate PTH1R and PTH3R were isolated and functionally characterized in chicken. Their distinct pattern of expression during embryo development and in adult tissues, together with their ligand preference, suggests that they have acquired specific functions, which have contributed to their maintenance in the genome. PTH2R and its activating ligand, TIP39, are absent from bird genomes. Nonetheless identification of putative PTH2R and TIP39 in the genome of an ancient agnathan, lamprey, suggests the PTH/PTHrP ligand and receptor family was already present in an early basal paraphyletic group of vertebrates and during the vertebrate radiation diverged via gene/genome duplication and deletion events. Knowledge of the role PTH/PTHrP system in early vertebrates will help to establish evolution of function.

Two chicken neuromedin U receptors: Characterization of primary structure, biological activity and tissue distribution

Neuromedin U (NMU) is a bioactive peptide that is involved in a variety of physiological functions. Two of its receptors, NMUR1 and NMUR2, have been identified and characterized in mammals. In this study, we performed cDNA cloning of chicken NMUR1 and NMUR2, and characterized their primary structure, biological activity, and expression patterns in chicken tissues. The chicken NMUR1 and NMUR2 cDNAs encoded 438 and 395 amino acid sequences, respectively. Chicken NMUR1 showed 54.8%–56.5% sequence identity with human, rat, and mouse NMUR1, and NMUR2 shared 67.3%–70.1% sequence identity with mammalian orthologs. Both chicken receptors have typical characteristics of G-protein-coupled receptors with seven transmembrane domains and the D/ERY motif. An increase in intracellular Ca 2+ mobilization was observed in HEK293 cells transfected with chicken NMUR1 or NMUR2 cDNA and treated with chicken or rat NMU. Real-time PCR analysis revealed that NMUR1 mRNA was preferentially expressed in the intestinal tissues such as the duodenum, jejunum, ileum, cecum, and colon/rectum, and brain regions such as the midbrain and optic lobe, and the ovary in adult hens. NMUR2 mRNA was exclusively expressed in the brain regions such as the cerebrum and midbrain. These results indicate that NMUR1 and NMUR2 mRNAs, which encode functional receptor proteins, are expressed in chicken tissues with different distribution patterns.

Induction of inflammatory cytokines and toll-like receptors in chickens infected with avian H9N2 influenza virus

H9N2 influenza virus is endemic in many Asian countries and is regarded as a candidate for the next human pandemic. Knowledge of the induction of inflammatory responses and toll-like receptors (TLRs) in chickens infected with H9N2 is limited. Here, we show that H9N2 induces pro-inflammatory cytokines such as transforming growth factor-beta 3; tumor necrosis factor-alpha; interferon-alpha, -beta, and gamma; and TLR 1, 2, 3, 4, 5, 7, and 15 in trachea, lung, and intestine of infected chickens. In the lung, TLR-15 was dominantly induced. Taken together, it seems that H9N2 infections efficiently induce inflammatory cytokines and TLRs in trachea, lung and intestine of chickens.

Xiang-Qi-Tang Increases Avian Pathogenic Escherichia coli-Induced Survival Rate and Regulates Serum Levels of Tumor Necrosis Factor Alpha, Interleukin-1 and Soluble Endothelial Protein C Receptor in Chicken

Xiang-qi-tang (XQT) is a Chinese herbal formula containing rhizoma Cyperi, Andrographis paniculata and Astragalus membranaceus. The present study investigated the effects of XQT on the mortality and inflammatory mediators in a chicken model challenged with avian pathogenic Escherichia coli (APEC). To detect the effect of XQT, the chickens were pretreated with the formula 12 h before being challenged with 10(8) colony forming unit (CFU) of APEC. The results showed that 0.6 g/kg XQT significantly elevated the survival rate of infected chickens. To further investigate the mechanism of decreasing mortality of XQT, we examined plasma inflammatory mediator levels. The levels of tumor necrosis factor alpha (TNF-α), interleukin-1 (IL-1) and soluble endothelial protein C receptor (sEPCR) were significantly increased in chickens challenged with APEC alone, whereas chickens pretreated with 0.6 g/kg XQT showed marked decrease of these inflammatory mediator levels during the death peak. Taken together, this study demonstrates that XQT has protective effects in APEC-treated chickens. The action mechanisms of XQT involve anti-inflammation and antithrombotic activity. These findings may contribute to future research on the action mechanisms of this formula, as well as prevention of or therapy for avian colibacillosis.

The Role of the Magnetite-Based Receptors in the Beak in Pigeon Homing

Magnetite-containing structures in the upper beak of birds have been described as putative magnetoreceptors [1-4], but so far, all positive evidence indicating their influence on behavior has come from laboratory studies using rather unnatural stimuli (e.g., [5-7]). Here, we demonstrate these receptors' possible role in a natural situation: we released pigeons with these receptors deactivated by a local anesthetic within and outside a magnetic anomaly, together with untreated control birds. Within the anomaly, the untreated birds showed unusually long vanishing intervals and scattered bearings, indicating confusion by the anomalous magnetic conditions. Anesthesia of the beak suppressed this adverse effect. Outside the anomaly, in contrast, the treatment had little effect. These findings indicate that the receptors in the beak mediate magnetic "map" information and that this information is normally included in the navigational process yet can be replaced by nonmagnetic factors at most sites.

Mesenchymal Bone Morphogenetic Protein Signaling Is Required for Normal Pancreas Development

OBJECTIVEPancreas organogenesis is orchestrated by interactions between the epithelium and the mesenchyme, but these interactions are not completely understood. Here we investigated a role for bone morphogenetic protein (BMP) signaling within the pancreas mesenchyme and found it to be required for the normal development of the mesenchyme as well as for the pancreatic epithelium.RESEARCH DESIGN AND METHODSWe analyzed active BMP signaling by immunostaining for phospho-Smad1,5,8 and tested whether pancreas development was affected by BMP inhibition after expression of Noggin and dominant negative BMP receptors in chicken and mouse pancreas.RESULTSEndogenous BMP signaling is confined to the mesenchyme in the early pancreas and inhibition of BMP signaling results in severe pancreatic hypoplasia with reduced epithelial branching. Notably, we also observed an excessive endocrine differentiation when mesenchymal BMP signaling is blocked, presumably secondary to defective mesenchyme to epithelium signaling.CONCLUSIONSWe conclude that BMP signaling plays a previously unsuspected role in the mesenchyme, required for normal development of the mesenchyme as well as for the epithelium.

Functional characterization of chicken glucocorticoid and mineralocorticoid receptors

Glucocorticoid (GR) and mineralocorticoid (MR) receptors are ligand-activated transcription factors that belong to the nuclear hormone receptor superfamily. Little is known about the function of GR and MR in avian species. Recently, the chicken homologue of the GR (cGR) gene was cloned, and its tissue-specific expression was characterized, whereas the full-length sequence of the chicken MR (cMR) gene remains unknown. Therefore, the aims of this project were to clone the full-length cMR and to functionally characterize both chicken receptors. Cos-7 cells were transiently transfected with cGR or cMR expression vectors along with a glucocorticoid response element-luciferase (GRE-Luc) reporter construct. Transfected cells were then treated with increasing doses of corticosterone (CORT) or aldosterone (ALDO) alone and with GR or MR antagonists (ZK98299 and spironolactone, respectively). Transactivation of cGR or cMR was evaluated by luciferase assay. CORT and ALDO induced cGR- and cMR-driven transcriptional activity in a dose-dependent manner. Each receptor responded to both steroids, but cMR transcriptional activity was induced by lower levels of CORT and ALDO than cGR. Coexpression of both chicken corticosteroid receptors in Cos-7 cells had no synergistic or additive effect on CORT- or ALDO-induced transcriptional activity. Corticosteroid-dependent transactivation of cGR and cMR was partially blocked by antagonists. ZK98299 showed high specificity to cGR, while spironolactone had agonist properties toward both receptors. Immunocytochemistry was used to assess the cellular localization of both receptors. Corticosteroids induced translocation of both receptors into the nucleus. The functional properties of cGR and cMR determined in this study will be helpful in defining the physiological roles of GR and MR in avian species.

Biological indications of a novel "short" µ opiate receptor in domestic chicken.

Previous work from our laboratory has established that cellular signaling processes of endogenous morphine are mediated by cognate G protein coupled receptor (GPCR) proteins, designated µ3 and µ4 opiate receptors. µ3 and µ4 opiate receptors are structurally unique “short” 6 transmembrane helical (TMH) domain GPCRs that are selectively responsive to endogenous morphine, not to families of endogenous opioid peptides, and are uniquely coupled to activation of constitutive nitric oxide synthase (cNOS). Based on high resolution predictive measures, it appears likely that domestic poultry express a µ opiate receptor mRNA encoding potentially two novel GPCRs with similar biochemical characteristics as described for µ3 and µ4 opiate receptors as well as traditional µ1 opioid receptors. The biological indications of these novel µ opiate receptors are discussed within the context of this short review.

Cloning, expression and functional characterization of chicken CCR6 and its ligand CCL20

Chemokines are key molecules that drive migration of lymphoid and myeloid cells toward organs in basal as well as inflammatory conditions. By recruiting immature dendritic cells to the mucosal surfaces, CCL20 acts in the very early events leading to the development of a specific immune response. In order to characterize dendritic cells in birds and better understand their role in the initiation of immune responses against pathogens of economic as well as human health relevance, we have cloned and expressed chicken CCL20 (chCCL20) and its specific receptor chCCR6. chCCL20 has 51% identity (60% similarity) with human CCL20, while the chicken receptor and its human counterpart display nearly 55% identity (and up to 70% similarity). chCCL20 and its specific receptor chCCR6 mRNAs are mainly expressed in bone marrow, secondary lymphoid organs and in the mucosal surfaces, in particular lungs and intestine. Both receptor and chemokine are functionally active when expressed as genuine or tagged proteins in mammalian expression systems, that is chCCR6 is mainly located at the cell surface within lipid rafts like its human counterpart. And secondly, both human and chicken chemokines were able to drive the migration of either chicken or human CCR6-transfected cells.

The role of central endogenous histamine and H1, H2 and H3 receptors on food intake in broiler chickens

Summary The role of endogenous histamine and H1, H2 and H3 central receptors on food intake in broiler chickens was investigated. For this purpose, a probe was used to manipulate the concentration of endogenous histamine by intracerebroventricular (ICV) injection of thioperamide, an H3 receptor antagonist, and R-α- methylhistamine, an H3 receptor agonist and subsequently the effects of brain histaminergic system on food intake was assessed. Moreover, to determine the receptors involved in histamine-induced feeding behaviour changes, H1 and H2 blockers were administered to thioperamide-treated chickens. Injection of thioperamide (600 and 300 nmol) decreased food intake dose-dependently (P<0.05). On the contrary, ICV injection of R-α- methylhistamine (400 and 200 nmol) increased food intake (P<0.05). Chlorpheniramine (128 and 256 nmol), a H1 receptor antagonist, increased food intake (P<0.05). Famotidine, a H2 receptor antagonist at 74 or 148 nmol had no effect on food intake but at 296 nmol significantly decreased food intake (P<0.05). Pretreatment with chlorpheniramine (256 nmol) significantly attenuated thioperamide effects (600 nmol) on food intake (P<0.05). In conclusion, the results of the present study demonstrated that histamine exerts anorexigenic effects through H1 but not H2 receptors in broiler chickens. Furthermore, it was shown that thioperamide through stimulation of synthesis and release of endogenous neuronal histamine can decrease food intake in broiler chickens.

Differences in influenza virus receptors in chickens and ducks: Implications for interspecies transmission

Avian influenza viruses are considered to be key contributors to the emergence of human influenza pandemics. A major determinant of infection is the presence of virus receptors on susceptible cells to which the viral haemagglutinin is able to bind. Avian viruses preferentially bind to sialic acid alpha2,3-galactose (SAalpha2,3-Gal) linked receptors, whereas human strains bind to sialic acid alpha2,6-galactose (SAalpha2,6-Gal) linked receptors. While ducks are the major reservoir for influenza viruses, they are typically resistant to the effects of viral infection, in contrast to the frequently severe disease observed in chickens. In order to understand whether differences in receptors might contribute to this observation, we studied the distribution of influenza receptors in organs of ducks and chickens using lectin histochemistry with linkage specific lectins and receptor binding assays with swine and avian influenza viruses. Although the intestinal epithelial cells of both species expressed only SAalpha2,3-Gal receptors, we found widespread presence of both SAalpha2,6-Gal and SAalpha2,3-Gal receptors in many organs of both chickens and ducks. Co-expression of both receptors may allow infection of cells with both avian and human viruses and so present a route to genetic reassortment. There was a marked difference in the primary receptor type in the trachea of chickens and ducks. In chicken trachea, SAalpha2,6-Gal was the dominant receptor type whereas in ducks SAalpha2,3-Gal receptors were most abundant. This suggests that chickens could be more important as an intermediate host for the generation of influenza viruses with increased ability to bind to SAalpha2,6-Gal receptors and thus greater potential for infection of humans. Chicken tracheal and intestinal epithelial cells also expressed a broader range of SAalpha2,3-Gal receptors (both beta(1-4)GlcNAc and beta(1-3)GalNAc subtypes) in contrast to ducks, which suggests that they may be able to support infection with a broader range of avian influenza viruses.

Unfolding the codes of short-term feed appetence in farm and companion animals. A comparative oronasal nutrient sensing biology review

The evolution of the chemical senses has resulted in a sensory apparatus for high taste and smell acuity in mammals and birds to ensure self-nourishment. Such peripheral chemosensory systems function as a code to unfold the nutritional value of feedstuffs. Food ingestion simultaneously evokes odor, taste and thermo-mechanical (somatosensing) sensations. Olfaction represents the capacity to identify feed volatiles that are predominantly derived from essential nutrients in plants. Comparative biology of olfaction shows that primates and chickens have a smaller olfactory epithelium and fewer olfactory receptor (OR) genes than non-primate mammals studied to date including farm and companion animals, such as the pig, the cow, the dog, the cat and the horse. A significant proportion of the total OR genes in mammals and birds have lost their functionality (pseudogenes) in a process that seems to reflect a decrease in the animal’s reliance on the sense of smell, particularly in humans and cows. The taste system allows animals to recognize a diverse repertoire of nutrient (sugars, amino acids, salts, acids and fats) or toxic related chemical entities that provide valuable information about the quality of food. Taste senses non-volatile molecules in the oral cavity through taste receptors (TR). The TR are expressed in the sensory cells forming the taste buds of the tongue’s papillae. Taste cells are linked to a network of solitary chemosensory cells diffused through many non-taste tissues involved in metabolic homeostasis. The number of functional taste receptor genes (TASR) in humans is equivalent to that in other mammals and superior to that in chickens. The TASR family 1 (TAS1R coding for umami and sweet TR) is conserved, in number and type, across the species evaluated, with the exception of the sweet receptor in chicken and feline species. The TASR family 2 (TAS2R coding for bitter TR) shows a strong adaptive capacity to dietary sources and digestive physiology across vertebrates. Pseudogenization (loss of gene functionality) in the TAS2R family seems to be a frequent strategy. The implications of oronasal nutrient sensing related to comparative animal feeding strategies and behaviors such as neophobia, feed refusal and hedonic preferences are discussed. Feed palatability and appetence might be one of the main driving forces in short-term feed consumption. Finally, practical applications relevant to animal production are outlined. Key words: Nutrient sensing, taste, olfaction, somatosensing, feed intake, farm, companion animals

Cloning, expression and bioactivity of chicken receptor activator of NF-κB ligand (chRANKL)

AbstractUsing reverse transcription polymerase chain reaction (RT-PCR) and gene splicing by overlap extension (SOE-PCR), the DNA sequence encoding the chicken receptor activator of NF-κB ligand (RANKL) was amplified, cloned into the expressing plasmid pET-32a(+) with His-tag, and highly expressed in Escherichia coli. The purified protein was then added to primary cultures of chicken osteoclasts to observe its bioactivity. The results showed that the size of the PCR product was 1200 bp, which was consistent with the expected size, and the relative molecular weight of the induced protein was 64 kDa. Western blotting indicated that induced protein could react with anti-His antibody. It was also found that the induced protein can stimulate mature chicken osteoclasts to resorb bone.

Chicken Receptor Activator of Nuclear Factor-κB Ligand Induces Formation of Chicken Osteoclasts from Bone Marrow Cells and also Directly Activates Mature Osteoclasts

Receptor activator of nuclear factor-κB ligand (RANKL), which functions as a major determinant of osteoclast differentiation and activation, is a type II transmembrane protein and is expressed in osteoblasts-stromal cells. The aim of this study was to clarify the role of chicken RANKL (chRANKL) in chicken osteoclast differentiation and to determine its effect on mature chicken osteoclasts. In the present study, chRANKL protein was first cloned and expressed in Escherichia coli. We then treated chicken bone marrow cells with chRANKL protein and found that it induced the formation of chicken osteoclast-like multinucleated cells in a dose-dependent manner in the presence of human macrophage colony-stimulating factor. Moreover, the addition of chicken osteoprotegerin could block the effect of chRANKL with regard to osteoclast-like multi-nucleated cell formation and bone resorption. Using primary cultures of chicken osteoclasts on bone slices, we also found that bone resorption pits per cell increased with chRANKL concentration in a dose-dependent manner. The chRANKL-treated hens exhibited increased blood Ca++ levels within 2 h after injection, showing that chRANKL also activates osteoclasts in vivo. These results clearly indicate that the expressed protein is functional and may also be a critical factor for chicken osteoclastogenesis and bone resorption.

Molecular cloning, expression and functional characterization of the chicken cation dependent mannose 6-phosphate receptor protein

Mammalian mannose 6-phosphate (M6P) receptors function in transport of lysosomal enzymes. To understand the structural and functional significance of the chicken cation dependent mannose 6-phosphate receptor (MPR) (Mr 46 kDa), a full-length cDNA for the chicken protein was cloned and expressed in mpr (−/−) MEF cells devoid of both the receptors. The stably transfected cells express the receptor that could be affinity purified by phosphomannan chromatography. The authenticity of the receptor was confirmed by its immuno-reactivity with mammalian MPR 46 antibodies and its ability to sort cathepsin D in transfected cells (92.3%) as compared to mock transfected cells (50.2%), establishing a functional role for the chicken receptor.

The BAFF-Interacting receptors of chickens

The TNF superfamily cytokine BAFF has crucial roles in homoeostatic regulation of B cell populations in mammals. Similar effects on peripheral B cells have been reported for chicken as for mammalian BAFF. Unlike mammalian BAFF, chicken BAFF is produced by B cells, implying an autocrine loop and consequent differences in regulation of B cell homoeostasis. Understanding of these mechanisms requires investigation of BAFF-binding receptors in chickens. We identified and characterised chicken receptors BAFFR and TACI, but found that the gene encoding the third BAFF-binding receptor, BCMA, was disrupted, implying differences in mechanisms for maintenance of long-lived antibody responses. A BAFFR-Ig fusion protein expressed in vivo lowered B cell numbers, showing that it was functional under physiological conditions. We found changes in the ratio of BAFFR and TACI mRNAs in the bursa after hatch that may account for the altered requirements for B cell survival at this stage of development.

The Central Leucine-Rich Repeat Region of Chicken TLR16 Dictates Unique Ligand Specificity and Species-Specific Interaction with TLR2

The ligand specificity of human TLR (hTLR) 2 is determined through the formation of functional heterodimers with either hTLR1 or hTLR6. The chicken carries two TLR (chTLR) 2 isoforms, type 1 and type 2 (chTLR2t1 and chTLR2t2), and one putative TLR1/6/10 homologue (chTLR16) of unknown function. In this study, we report that transfection of HeLa cells with the various chicken receptors yields potent NF-kappaB activation for the receptor combination of chTLR2t2 and chTLR16 only. The sensitivity of this complex was strongly enhanced by human CD14. The functional chTLR16/chTLR2t2 complex responded toward both the hTLR2/6-specific diacylated peptide S-(2,3-bispalmitoyloxypropyl)-Cys-Gly-Asp-Pro-Lys-His-Pro-Lys-Ser-Phe (FSL-1) and the hTLR2/1 specific triacylated peptide tripalmitoyl-S-(bis(palmitoyloxy)propyl)-Cys-Ser-(Lys)(3)-Lys (Pam(3)CSK(4)), indicating that chTLR16 covers the functions of both mammalian TLR1 and TLR6. Dissection of the species specificity of TLR2 and its coreceptors showed functional chTLR16 complex formation with chTLR2t2 but not hTLR2. Conversely, chTLR2t2 did not function in combination with hTLR1 or hTLR6. The use of constructed chimeric receptors in which the defined domains of chTLR16 and hTLR1 or hTLR6 had been exchanged revealed that the transfer of leucine-rich repeats (LRR) 6-16 of chTLR16 into hTLR6 was sufficient to confer dual ligand specificity to the human receptor and to establish species-specific interaction with chTLR2t2. Collectively, our data indicate that diversification of the central LRR region of the TLR2 coreceptors during evolution has put constraints on both their ligand specificity and their ability to form functional complexes with TLR2.

Chicken Toll-like Receptor 3 Recognizes Its Cognate Ligand When Ectopically Expressed in Human Cells

Recognition of pathogens by toll-like receptors (TLRs) causes activation of signaling cascades that trigger cytokine secretion and, ultimately, innate immunity. Genes encoding proteins with substantial homology to mammalian TLR1, TLR2, TLR3, TLR4, TLR5, and TLR7 are present in the chicken genome, whereas orthologs of TLR8, TLR9, and TLR10 seem to be defective or missing. Except for chicken TLR2 (ChTLR2), which was previously shown to recognize lipopeptides and lipopolysaccharides (LPS), the ligand specificity of ChTLRs had not been determined. We found that polyI:C, LPS, R848, S-28463, and ODN2006, which are specifically recognized by TLR3, TLR4, TLR7/8, and TLR9 in mammals, induced substantial amounts of type I interferon (IFN) and interleukin-6 (IL-6) in freshly prepared chicken splenocytes. To determine the ligand specificity of ChTLR3 and ChTLR7, we used a standard reporter assay frequently employed for analysis of mammalian TLRs. Neither S-28463 nor any other TLR ligand induced reporter activity in human 293 cells expressing ChTLR7. However, human 293 cells expressing ChTLR3 strongly and specifically responded to polyI:C, demonstrating that this chicken receptor represents a true ortholog of mammalian TLR3.

Structure and Tissue Distribution of Cholecystokinin-1 Receptor in Chicken

Chicken cholecystokinin-1 receptor (chCCK1R) cDNA was isolated from a chicken brain and tissue distribution of the chCCK1R mRNA was determined by RT-PCR. The chCCK1R is composed of 429 amino acid residues and showed approximately 73% and 50% identity with those of mammalian CCK1R and CCK-2 receptor (CCK2R) at the amino acid level, respectively. In phylogenetical analysis, the chCCK1R belonged to a cluster of CCK1R and CCK-CHR, another CCK receptor cloned from chicken, was placed into a cluster of CCK2R. By RT-PCR analysis, whilst both chCCK1R and CCK-CHR mRNAs were widely expressed in the chicken tissues, the expression pattern for each receptor was slightly different. Abundant chCCK1R mRNA was observed in multiple tissues, while CCK-CHR mRNA expression was dominantly observed in the brain and proventriculus in the chicken. Taken together, chCCK1R is a potent peripheral CCKR and CCK-CHR cloned and characterized previously is classified as CCK2R in the chicken. Furthermore, mRNA expression for chicken CCKRs is possible to be regulated by tissue specific manner and that may be associated with the diverse roles of CCK and gastrin in the chicken.