EXAMINATION OF FISH IMMUNOREGULATORY RECEPTOR-MEDIATED SIGNALING EVENTS
Myron Zwozdesky, University of Alberta
Myron A. Zwozdesky, Chenjie Fei, Hima Gurupalli and James L. Stafford
Department of Biological Sciences, University of Alberta, Edmonton, AB, Canada
Cells sense and respond to their environment through transmembrane receptors that transduce extracellular cues into biochemical signaling. While many mammalian receptor signal transduction events are well characterized, including Fcγ receptor (FcγR)-mediated phagocytosis, information from earlier vertebrate models is limited. The channel catfish (Ictalurus punctatus) leukocyte immune-type receptor (IpLITR) family consists of multiple receptor-types with variable signaling abilities that are dependent on their tyrosine-containing cytoplasmic tail (CYT) regions to control various innate immune cell effector responses. IpLITR 2.6b associates with the immunoreceptor tyrosine-based activation motif-containing adaptor molecule IpFcRγ-L, and when expressed in mammalian cells activates conserved effector responses, including phagocytosis similar to mammalian FcγRs. Conversely, IpLITR 1.1b is a long immunoreceptor tyrosine-based inhibitory motif-containing receptor with multi-functional capabilities. IpLITR 1.1b-mediated inhibition of cellular cytotoxicity is facilitated by a distal CYT region SHP1-dependent and a proximal CYT region Csk-dependent mechanism. Interestingly, IpLITR 1.1b also activates a unique F-actin-dependent phagocytic pathway involving the rapid capture of extracellular targets on the cell surface, however no detailed biochemical data are available. Using imaging flow cytometry and GST pulldown assays, we examined which regions of the IpLITR 1.1b CYT trigger phagocytosis and established a profile of potential intracellular signaling molecule participants. Our results show that in stably transfected AD293 cells, the membrane proximal and distal CYT regions of IpLITR 1.1b independently regulate phagocytic activities. These CYT regions differentially recruit various SH2 domain containing intracellular mediators, providing new information about IpLITR 1.1b signaling versatility. This work sets the stage for investigating IpLITR 1.1b-mediated phagocytic signal transduction to advance our understanding of novel immunoregulatory receptor-mediated signaling events.