Identification and Characterization of a FcR Homolog in an Ectothermic Vertebrate, the Channel Catfish (Ictalurus punctatus) 1,2 James L. Stafford,* Melanie Wilson,* Deepak Nayak,* Sylvie M. Quiniou, † L. W. Clem,* Norman W. Miller,* and Eva Bengte ´n 3 * An FcR homolog (IpFcRI), representing the first such receptor from an ectothermic vertebrate, has been identified in the channel catfish (Ictalurus punctatus). Mining of the catfish expressed sequence tag databases using mammalian FcR sequences for CD16, CD32, and CD64 resulted in the identification of a teleost Ig-binding receptor. IpFcRI is encoded by a single-copy gene containing three Ig C2-like domains, but lacking a transmembrane segment and cytoplasmic tail. The encoded Ig domains of IpFcRI are phylogenetically and structurally related to mammalian FcR and the presence of a putative Fc-binding region appears to be conserved. IpFcRI-related genomic sequences are also present in both pufferfish and rainbow trout, indicating the likely presence of a soluble FcR in other fish species. Northern blot and qualitative PCR analyses demonstrated that IpFcRI is primarily expressed in IgM-negative leukocytes derived from the lymphoid kidney tissues and PBL. Significantly lower levels of IpFcRI expression were detected in catfish clonal leukocyte cell lines. Using the native leader, IpFcRI was secreted when transfected into insect cells and importantly the native IpFcRI glycoprotein was detected in catfish plasma using a polyclonal Ab. Recombinant IpFcRI binds catfish IgM as assessed by both coimmunoprecipation and cell transfection studies and it is presumed that it functions as a secreted FcR akin to the soluble FcR found in mammals. The identification of an FcR homolog in an ectothermic vertebrate is an important first step toward understanding the evolutionary history and functional importance of vertebrate Ig-binding receptors. The Journal of Immunology, 2006, 177: 2505–2517. R eceptors specific for the Fc portion of Ig (i.e., FcR) are expressed by a wide variety of mammalian cells of he- mopoietic origin. In general, FcR participate in activation or inhibition of immune responses following the recognition of monomeric Ig or Ig in the form of immune complexes. Since early reports describing the presence of FcR on macrophages (1) and lymphocytes (2– 4), more recent studies have expanded the under- standing of the genomics and functional significance of these im- portant innate immune receptors. In humans, eight genes found on chromosome 1q21-23 encode for the IgG FcR family (FcR) (5–7). These include the high- affinity FcRI (RIA, RIB, and RIC) as well as the low-affinity FcRII (RIIA, RIIB, and RIIC) and FcRIII (RIIIA and RIIIB). Comparatively, only three genes, split between two different chro- mosomes, encode for the mouse FcR (5, 8). Recently, a murine IgG FcR (FcRIV) with preferential specificity for IgG2a and IgG2b has been described, and it appears to be conserved in all mammalian species (9). Receptors for IgE (FcR) are present in both humans and mice (10, 11), and a novel FcR that binds to both IgM and IgA isotypes (Fc/R) has also been reported in mam- mals (12, 13). In most instances, the classical mammalian FcR genes encode for immune receptors consisting of extracellular C2 Ig domains (D), 4 a transmembrane (TM) segment, and a cytoplasmic tail (CYT) that may contain signaling motifs. The membrane distal D1 and D2 Ig domains of FcRI are structurally related to the two Ig domains of the low-affinity receptors, albeit the acquisition of a third membrane proximal domain (D3) is necessary for the high- affinity Ig binding exhibited by FcRI (14 –17). Depending on the type of FcR engaged, cellular activation or inhibition occurs through association with adaptor molecules or signaling motifs present within the CYT. In addition to the positive and negative regulation of cellular responses, FcR also participate in the uptake and clearing of immune complexes as well as the transport of Ig (18). Alternative splicing and proteolytic cleavage of membrane-as- sociated FcR have been shown to generate soluble forms of these receptors (sFcR) (19). For example, alternative splicing results in the generation of a TM-deleted FcRIIb2 mRNA (termed FcRIIb3), providing a third isoform encoded by the single murine FcRII gene (8, 20). This FcRIIb3 is a secreted protein and has been identified in culture supernatants of macrophage cell lines *Department of Microbiology, University of Mississippi Medical Center, Jackson, MS 39216; and † U. S. Department of Agriculture/Agricultural Research Service, Cat- fish Genetics Research Unit, Stoneville, MS 38701 Received for publication November 17, 2005. Accepted for publication June 6, 2006. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. 1 This work was supported by grants from the National Institutes of Health (RO1AI- 19530), National Science Foundation (MCB-0211785), and the U.S. Department of Agriculture (2002–35204-12211), and a Natural Sciences and Engineering Research Council of Canada Post Doctoral Fellowship. 2 The sequences presented in this article have been submitted to GenBank under the accession numbers DQ286289 (IpFcRI cDNA) and DQ286289 (IpFcRI gene). 3 Address correspondence and reprint requests to Dr. Eva Bengte ´n, Department Mi- crobiology, University of Mississippi Medical Center, 2500 North State Street, Jack- son, MS 39216-4505. E-mail: ebengten@microbio.umsmed.edu 4 Abbreviations used in this paper: D, domain; TM, transmembrane; CYT, cytoplas- mic tail; FCRL, FcR-related protein; XFL, Xenopus leukocyte FcR-like protein; Ip, Ictalurus punctatus; LITR, leukocyte immune-type receptor; sFcR, soluble FcR; EST, expressed sequence tag; LRC, leukocyte receptor complex; CFS, catfish serum; UT, untranslated; qPCR, quantitative PCR; PDGFR, platelet-derived growth factor recep- tor; BLAST, basic local alignment search tool; BAC, bacterial artificial chromosome. The Journal of Immunology Copyright © 2006 by The American Association of Immunologists, Inc. 0022-1767/06/$02.00