A number of immune relevant genes have been studied

A number of immune-relevant genes have been studied to explore the immunologic mechanisms in large yellow croakers; however, the chemokine receptor CXCR family has never been studied in this organism. Chemokines are a large superfamily of chemotactic cytokines that are utilized to direct the trafficking and migration of leukocytes within the immune system [2]. Chemokines mediate their activity through a large family of G-protein coupled receptors known as chemokine receptors. Much attention has been paid to one particular member of the chemokine receptor family, CXCR4. This is primarily due to the crucial part this receptor plays in HIV infection of permissive NAD/NADH Quantitation Colorimetric Kit and its important role in lymphocyte trafficking and haematopoiesis [3]. In recent years, CXCR4 has been characterized with respect to its innate immune role in a few fish species [4], [5], [6], [7], [8], [9]. CXCR2 is a key regulator of acute inflammatory responses, mediating neutrophil infiltration into inflammatory sites and the activation of neutrophil functions [10]. In addition to its role as a mediator of inflammation, it has been shown to play important roles in angiogenesis, haematopoiesis, and cancer [11]. Although chemokine and chemokine receptors have been widely studied in mammals, particularly in humans [12] with regard to the involvement of the chemokine receptor system in different diseases [7], the chemokine and chemokine receptor biology of teleost fish and its involvement in fish immunity remain ill-defined. To date, CXCR1 and/or CXCR2 have been identified and characterized from only a few fish species [13], [14], [15], [16], [17]. CXCR3 is a chemokine receptor that is highly expressed in effector T cells and plays an important role in T-cell trafficking and function [1], [18]. Even though CXCR3 counterparts have been identified in several fish species, including Danio rerio [19], Scophthalmus maximus [20], Ctenopharyngodon idella [21], and Cyprinus carpio L. [22], and previous studies have shown that CXCR3 expression in fish may be extensively modulated by external stimuli [20], [23], detailed information regarding the CXCR3 immune responses in fish remains scarce. In addition, most receptors have been reported to interact with multiple ligands, and most ligands are known to interact with more than one receptor [24]. CXCR2 binds to CXCL1–3 and CXCL5–8, whilst CXCR3 binds to CXCL4 and CXCL9–11. A notable exception is the stromal cell-derived factor-1 (SDF-1, also called CXCL12)/CXCR4 interaction [1], [3], [25]. Because chemokines and chemokine receptors play an important role in the immune system [26], they have been extensively studied in mammals. For example, Matsuo [27] reported that the CXC-type chemokine/CXCR2 biological axis promotes angiogenesis in vitro and in vivo in pancreatic cancer. Zou [28] researched the function of the chemokine receptor CXCR4 in haematopoiesis and cerebellar development. In recent years, chemokines and chemokine receptors have been increasingly reported in fish. For example, Xu [29] performed molecular cloning and expression analysis of CXCR5 in grass carp, representing the first report of a non-mammalian CXCR5 in a teleost fish. Dixon [30] identified and characterized three novel chemokine receptors in rainbow trout, bringing the number of known CC-type chemokine receptors in this species to five, one of which is teleost-specific. Umasuthan [31] reported two CXC-type chemokine receptors, OfCXCR1 and OfCXCR2, in rock bream fish and characterized these at the molecular, structural, genomic, and transcriptional levels. In this study, we describe the identification and characterization of CXCR2 (LycCXCR2), CXCR3 (LycCXCR3), and CXCR4 (LycCXCR4) in large yellow croaker. Each protein consists of seven transmembrane domains connected by three intracellular loops (ICLs) and three extracellular loops (ECLs). In addition, these proteins have conserved DRY amino acid motifs within the second ICL, which are involved in coupling to G-proteins, and they share a high identity to homologs from other teleost species according to sequence analysis. We report that CXCR3 and CXCR4 are evolving neutrally according to PAML analyses. In addition, these chemokine receptors are involved in the immune system and exhibit high constitutive expression in the liver, kidney, and spleen. The data provided here contribute to a deeper understanding of CXCR signalling in fish.