title: Examination of the stimulatory signaling potential of a channel catfish leukocyte immune-type receptor and associated adaptor.
abstract: Expressed by various subsets of myeloid and lymphoid immune cells, channel catfish (Ictalurus punctatus) leukocyte immune-type receptors (IpLITRs) are predicted to play a key role in the initiation and termination of teleost cellular effector responses. These type I transmembrane proteins belong to the immunoglobulin superfamily and display features of immunoregulatory receptors with inhibitory and/or stimulatory signaling potential. Expanding on our previous work, which demonstrated that putative stimulatory IpLITR-types associated with the catfish adaptor proteins IpFcRγ and FcRγ-L, this study focuses on the functional significance of this immune receptor-adaptor signaling complex. Specifically, we generated an epitope-tagged chimeric receptor construct by fusing the extracellular domain of IpLITR 2.6b with the transmembrane region and cytoplasmic tail of IpFcRγ-L. This chimera was stably expressed in a rat basophilic leukemia (RBL) cell line, RBL-2H3, and following cross-linking of the surface receptor with an anti-hemagglutinin monoclonal antibody or opsonized microspheres, the chimeric teleost receptor induced cellular degranulation and phagocytic responses, respectively. Site-directed mutagenesis of the immunoreceptor tyrosine-based activation motif encoded within the cytoplasmic tail of the chimera confirmed that these functional responses were dependent on the phosphorylated tyrosines within this motif. Using a combination of phospho-specific antibodies and pharmacological inhibitors, we also demonstrate that the IpLITR/IpFcRγ-L-induced degranulation response requires the activity of Src homology 2 domain containing protein tyrosine phosphatases, phosphatidylinositol 3-kinase, protein kinase C, and mitogen-activated protein kinases but appears independent of the c-Jun N-terminal kinase and p38 MAP kinase pathways. In addition to this first look at stimulatory IpLITR-mediated signaling and its influence on cellular effector responses, the advantage of generating RBL-2H3 cells stably expressing a functional IpLITR-adaptor chimera will be discussed.
authors.affiliation: Department of Biological Sciences, University of Alberta, Edmonton, Alberta, Canada.
authors.forename: Herman D
authors.initials: HD
authors.lastname: Cortes
authors.forename: Benjamin C
authors.initials: BC
authors.lastname: Montgomery
authors.forename: Karlijn
authors.initials: K
authors.lastname: Verheijen
authors.forename: Erick
authors.initials: E
authors.lastname: García-García
authors.forename: James L
authors.initials: JL
authors.lastname: Stafford
mesh_terms: D048868:Adaptor Proteins, Signal Transducing / Q000235:genetics / Q000378:metabolism*; D000818:Animals; D000911:Antibodies, Monoclonal / Q000494:pharmacology; D001491:Basophils / Q000187:drug effects / Q000276:immunology / Q000378:metabolism* / Q000473:pathology; D015550:Cell Degranulation / Q000187:drug effects; D045744:Cell Line, Tumor; D029941:Fish Proteins / Q000235:genetics / Q000378:metabolism; D007059:Ictaluridae; D007111:Immunity, Cellular; D016297:Mutagenesis, Site-Directed; D010587:Phagocytosis; D010766:Phosphorylation; D017434:Protein Structure, Tertiary / Q000235:genetics; D051381:Rats; D017452:Receptors, IgG / Q000235:genetics / Q000378:metabolism; D011971:Receptors, Immunologic / Q000235:genetics / Q000378:metabolism*; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism*; D015398:Signal Transduction; D019076:Transgenes / Q000235:genetics; D018909:src Homology Domains / Q000235:genetics
chemical_list: D048868:Adaptor Proteins, Signal Transducing; D000911:Antibodies, Monoclonal; D029941:Fish Proteins; D017452:Receptors, IgG; D011971:Receptors, Immunologic; D011993:Recombinant Fusion Proteins