A novel tilapia prolactin receptor is functionally distinct from its paralog

A novel tilapia prolactin (PRL) receptor (OmPRLR2) was identified based on its induction during hyperosmotic stress. OmPRLR2 protein shows 28% identity to tilapia OmPRLR1 and 26% identity to human PRLR. Comparison of OmPRLR1 and OmPRLR2 revealed conserved features of cytokine class I receptors (CKR1): a WS domain and transmembrane domain, two pairs of cysteines and N-glycosylation motifs in the extracellular region, CKR1 boxes I and II, and three tyrosines in the intracellular region. However, OmPRLR2 lacked the ubiquitin ligase and 14-3-3 binding motifs. OmPRLR2 mRNA was present in all tissues analyzed, with highest expression in gills, intestine, kidney and muscle, similar to OmPRLR1. Transfer of fish from fresh water to sea water transiently increased gill OmPRLR2 mRNA levels within 4h but decreased its protein abundance in the long term. OmPRLR2 is expressed in part as a truncated splice variant of 35kDa in addition to the 55kDa full-length protein. Cloning of the mRNA encoding the 35kDa variant revealed that it lacks the extracellular region. It is expressed at significantly higher levels in males than in females. In stably transfected HEK293 cells over-expressing tetracycline-inducible OmPRLR1 and OmPRLR2, activation of these receptors by tilapia PRL177 and PRL188 triggered different downstream signaling pathways. Moreover, OmPRLR2 significantly increased HEK293 salinity tolerance. Our data reveal that tilapia has two PRLR genes whose protein products respond uniquely to PRL and activate different downstream pathways. Expression of a short PRLR2 variant may serve to inhibit PRL binding during osmotic stress and in male tissues.Fil: Fiol, Diego Fernando. University of California at Davis; Estados Unidos. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Mar del Plata. Instituto de Investigaciones Biológicas. Universidad Nacional de Mar del Plata. Facultad de Ciencias Exactas y Naturales. Instituto de Investigaciones Biológicas; ArgentinaFil: Sanmarti, Enio. University of California at Davis; Estados UnidosFil: Sacchi, Romina. University of California at Davis; Estados UnidosFil: Kultz, Dietmar. University of California at Davis; Estados Unido

Establishment and Characterization of an Anoxia-tolerant Cell Line, PSU-AL-WS4ONE, Derived from an Embryo of the Annual Killifish Austrofundulus Limnaeus

Most animal cells rely on aerobic metabolism for survival and are damaged or die within minutes without oxygen. Embryos of the annual killifish Austrofundulus limnaeus, however, survive months without oxygen. Determining how their cells survive without oxygen has the potential to revolutionize our understanding of the cellular mechanisms supporting vertebrate anoxia tolerance and the evolution of such tolerance. Therefore, we aimed to establish and characterize an anoxia-tolerant cell line from A. limnaeus for investigating mechanisms of vertebrate anoxia tolerance. The PSU-AL-WS40NE cell line of neuroepithelial identity was established from embryonic tissue of A. limnaeus using a tissue explant. The cells can survive for at least 49 d without oxygen or replenishment of growth medium, compared to only 3 d of anoxic survival for two mammalian cell lines. PSU-AL-WS40NE cells accumulate lactate during anoxia, indicating use of common metabolic pathways for anaerobic metabolism. Additionally, they express many of the same small noncoding RNAs that are stress-responsive in whole embryos of A. limnaeus and mammalian cells, as well as anoxia-responsive small noncoding RNAs derived from the mitochondrial genome (mitosRNAs). The establishment of the cell line provides a unique tool for investigating cellular mechanisms of vertebrate anoxia tolerance, and has the potential to transform our understanding of the role of oxidative metabolism in cell biology