Elsevier

Gene

Volume 401, Issues 1–2, 15 October 2007, Pages 154-164
Gene

Structural characteristics of zebrafish orthologs of adaptor molecules that associate with transmembrane immune receptors

https://doi.org/10.1016/j.gene.2007.07.014Get rights and content

Abstract

Transmembrane bound receptors comprised of extracellular immunoglobulin (Ig) or lectin domains play integral roles in a large number of immune functions including inhibitory and activating responses. The function of many of the activating receptors requires a physical interaction with an adaptor protein possessing a cytoplasmic regulatory motif. The partnering of an activating receptor with an adaptor protein relies on complementary charged residues in the two transmembrane domains. The mammalian natural killer (NK) and Fc receptors (FcR) represent two of many receptor families, which possess activating receptors that partner with adaptor proteins for signaling. Zebrafish represent a powerful experimental model for understanding developmental regulation at early stages of embryogenesis and for efficiently generating transgenic animals. In an effort to understand developmental aspects of immune receptor function, we have accessed the partially annotated zebrafish genome to identify six different adaptor molecules: Dap10, Dap12, Cd3ζ, Cd3ζ-like, FcRγ and FcRγ-like that are homologous to those effecting immune function in mammals. Their genomic organizations have been characterized, cDNA transcripts have been recovered, phylogenetic relationships have been defined and their cell lineage-specific expression patterns have been established.

Introduction

The cells of the mammalian immune system rely on an intricate network of signaling pathways in order to differentiate between “self” and “non-self”. The activation or inhibition of these signaling pathways relies on specific membrane receptors on immune cells engaging specific ligands. In general, these receptors can be classified as inhibitory or activating based on the functional outcome of ligand recognition. For example, when an activating natural killer (NK) cell receptor binds its ligand, the NK cell is activated to kill the target cell; in contrast, when an inhibitory NK receptor binds its ligand, NK cell-mediated killing is repressed. Similarly, engagement of T-cell antigen receptor (TCR) or Fc receptor (FcɛRI) with the appropriate ligand (e.g. peptide–MHC complex or IgE, respectively) leads to a direct cell-mediated immune response.

Despite differences in receptor structures, the cytoplasmic signaling utilized by NK receptors, TCR and Fc receptors, is well conserved (Cerwenka and Lanier, 2000, Billadeau and Leibson, 2002, Yokoyama and Kim, 2006). Inhibitory NK and Fc receptors typically possess one or more cytoplasmic immunoreceptor tyrosine-based inhibition motifs (ITIMs). In contrast, activating NK, Fc and other receptors including TCR partner with adaptor proteins, which ultimately transduce signals to the cell nucleus. These receptors physically associate with an adaptor protein via oppositely charged residues within their transmembrane domains, e.g. a positive charge in the transmembrane domain of the activating receptor and a negative charge in the transmembrane domain of the adaptor protein. The majority of activating NK receptors, including most KIRs and Ly49, utilize the adaptor protein, DAP12; the activating NK receptor NKG2D, another lectin-type receptor, utilizes the adaptor DAP10 (Hyka-Nouspikel and Phillips, 2006, Takaki et al., 2006). TCR and FcR along with other immune related activating receptors, including: CD16, NKp30, NKp46, NKR-P1C and KIR2DL4, utilize FcRγ or CD3ζ (Cerwenka and Lanier, 2000, Tassi et al., 2006). The DAP12, FcRγ and CD3ζ adaptors utilize cytoplasmic immunoreceptor tyrosine-based activation motifs (ITAMs: YxxLX6–12YxxL/I) for signaling (Pitcher and van Oers, 2003), whereas DAP10 uses a YxxM motif similar to CD28 (Wu et al., 1999).

The zebrafish is becoming a more broadly recognized model for infection and immunity and is particularly well suited for examining gene function during embryogenesis (Yoder et al., 2002, Traver, 2003a, Trede et al., 2004, Van Der Sar et al., 2004, Phelps and Neely, 2005, Deiters and Yoder, 2006, Lieschke and Currie, 2007). As part of an ongoing effort to characterize immune receptors in zebrafish and to understand the signaling pathways mediated by their immune cells, we have identified and characterized the adaptor molecules: Dap12, Dap10, CD3ζ, CD3ζ-like, FcRγ, and FcRγ-like.

Section snippets

Cloning zebrafish dap10 (hcst) cDNA

A catfish (Ictalurus punctatus) DAP10 cDNA sequence (GenBank: AAZ16504) was used as the query for a tBLASTn search of the NCBI zebrafish sequence database (Wheeler et al., 2007). Zebrafish bacterial artificial chromosome (BAC) clone DKEY-29H14 (GenBank: BX571853) from chromosome 16 encodes a sequence similar to catfish DAP10 (E value = 1e− 10). A sequential RACE strategy was used to clone the full-length open reading frame (ORF) of dap10 (hcst) from zebrafish kidney/spleen RNA (GeneRacer,

Zebrafish encode six adaptor proteins: Dap10, Dap12, FcRγ, FcRγ-like, Cd3ζ and Cd3ζ-like

As a first step in characterizing immune signaling patterns in zebrafish, we data-mined the zebrafish genome and EST databases to identify sequences corresponding to six candidate adaptor proteins. Full-length cDNAs encoding these proteins termed, Dap12 (tyrobp), Dap10 (hcst), FcRγ (fcer1g), FcRγ-like (fcer1gl), Cd3ζ (cd247) and Cd3ζ-like (cd247l), were cloned by RACE or RT-PCR and sequenced. The predicted protein sequences for these zebrafish adaptors are shown aligned to orthologous proteins

Acknowledgements

We thank Melissa Haendel and John Hansen for very helpful discussions about gene nomenclature and Barb Pryor for editorial assistance. Zebrafish genes were named in consultation with the ZFIN Nomenclature Committee. Sequence data from this article have been deposited with the GenBank and ZFIN databases under accession numbers EF158445 and ZDB-GENE-061130-1 (Dap10/hcst); EF158446 and ZDB-GENE-061130-2 (Dap12/tyrobp); EF158447 and ZDB-GENE-061130-3 (FcRγ/fcer1g); EF601085 and ZDB-GENE-070502-4

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