Identification and functional characterization of interleukin-12 receptor beta 1 and 2 in grass carp (Ctenopharyngodon idella)

Interleukin 12 (IL-12) binds its receptor complex of IL-12 receptor beta 1 (IL-12R beta 1) and IL-12R beta 2 to transduce cellular signaling in mammals. In teleosts, the function of Il-12 is drawing increasing attention, but molecular and functional features of Il-12 receptors remain obscure. Especially, the existence of multiple Il-12 isoforms in some fish species elicits the requirement to clarify their receptors. In this study, we isolated three cDNA sequences as Il-12 receptor candidates from grass carp, entitled as grass carp Il-12r beta 1 (gcIl-12r beta 1), gcIl-12r beta 2a and gcIl-12r beta 2b. In silico analysis showed that gcIl-12r beta 1 and gcIl-12r beta 2a shared the conserved gene locus and similar structure characteristics with their orthologues of zebrafish, frog, chicken, mouse and human, respectively. However, the Il-12r beta 2b of grass carp and zebrafish was similar to IL-27Ra in non-fish species. Further locally installed BLAST and gene synteny analysis uncovered three gcIl-12 receptors being single copied genes. Tissue distribution assay revealed that gcil12r beta 1 and gcil12r beta 2a transcripts were predominantly expressed in head kidney, differing from the even distribution of gcil12r beta 2b transcripts in all detected tissues. Subsequently, the binding ability and antagonistic effects of recombinant extracellular region of gcIl-12r beta 1 with recombinant grass carp Il-12 (rgcIl-12) isoforms were explored, providing functional evidence of the newly cloned gcIl-12r beta 1 being genuine orthologues of mammalian IL-12R beta 1. Moreover, our data showed that gcIl-12r beta 1 and gcIl-12r beta 2a but not gcIl-12r beta 1 and gcIl-12r beta 2b mediated the effects of rgcIl-12 isoforms on ifn-gamma promoter activity, thereby revealing Il-12 receptor signaling in fish. These results identified grass carp Il-12 receptors, thereby advancing our understanding of Il-12 isoform signaling in fish.