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

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, Cd3zeta, Cd3zeta-like, FcRgamma and FcRgamma-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.

Figure 1. Comparison of zebrafish and mammalian adaptor protein sequences

The predicted sequences of zebrafish adaptor proteins are aligned with orthologous adaptor proteins. Protein symbols are defined in Table 2. Comparisons include members of the (A) DAP12, (B) DAP10, (C) FcRγ, FcRγL, (D) CD3ζ and (E) CD3ζL families. Black shading indicates identical amino acids and gray shading indicates functionally similar amino acids. Peptide leader sequences (L) and transmembrane (TM) domains are indicated above the alignment. A negatively charged residue (D) is present within the transmembrane domain of all adaptor proteins and indicated with asterisks (below) and pink highlighting. Cytoplasmic immunoreceptor tyrosine-based activation motifs (ITAMs; YxxLX_6-12YxxL/I) and ITAM-like sequences (YxxLX_6-12YxxA) which have been defined in mammalian signaling proteins (Pitcher and van Oers 2003; Underhill and Goodridge 2007) and are conserved in other species are indicated with down black arrowheads and yellow highlighting. Conserved cysteines that are utilized for dimerization are indicated by red arrows (Feng et al 2005; Feng et al 2006; Rutledge et al 1992). The tyrosine present in the DAP10 signaling motif (YxxM) is indicated with a black circle (above) and blue highlighting: note that this motif is modified in catfish, zebrafish (YMNV) and Takifugu (YMNT). Amino acid numbering is on the left. Introduced gaps in the sequence alignment are indicated with a dash (-).

Figure 2. Phylogenetic analyses of adaptor proteins

(A) The six zebrafish adaptor proteins (black text boxed in gray) are compared to the orthologous proteins defined in Table 2 and Figure 1. Note that FcRγL and CD3ζL may be restricted to fish species. (B) Zebrafish Dap10 and Dap12 are compared to orthologous proteins defined in Table 2 and Figure 1. Protein groups are identified on the right (white text boxed in gray). Bootstrap values less than 50 are not shown. Branch lengths are measured in terms of amino acid substitutions, with the scale indicated below the tree.

Figure 3. Genomic organization of zebrafish adaptor genes

The cDNA sequences for zebrafish (A) Dap10 (hcst) and Dap12 (tyrobp), (B) FcRγ (fcer1g), (C) FcRγ-like (fcer1gl) and (D) CD3ζ-like (cd247l) were mapped onto their corresponding genomic sequence in order to define their exon-intron organization. The transcriptional orientation of all genes is left to right. All of these genes encode a translational start site within the first exon and a stop codon in the last exon. The identity of the BAC clone or contig used to deduce the exon-intron organization and the chromosome to which each gene maps is listed below each gene. (E) The relative genomic locations of the genes for all six adaptors are shown on each chromosome (chromosome numbers are listed below each figure). Note that only a single exon of CD3ζ (cd247) can be identified in the current release of the zebrafish genome database and is localized to chromosome 1.

Figure 4. Detection of adaptor protein expression

(A) RT-PCR was used to detect transcripts of the six zebrafish adaptor proteins, dap10, dap12, Fcrγ, Fcrγ-like,CD3ζ and CD3ζ-like from embryonic and larval zebrafish including 0 hours post fertilization (hpf) though 6 days post fertilization (dpf) as well as from adult tissues (ovary, liver, kidney, spleen and intestine). (B) Myelomonocytic (gray gate) or lymphocytic (black gate) cells were purified from whole kidney marrow based on light scatter characteristics. The scatter profile of zebrafish whole kidney marrow (left) is shown in comparison to post-myeloid (middle) and post-lymphoid (right) sorts using forward (FSC) and side (SSC) scatter. Myeloid and lymphoid cells, which represent ∼21.5% and ∼13.5% of the whole kidney marrow were purified to ∼95.6% and ∼75.6% purity, respectively, and used for RT-PCR analyses. (C) RT-PCR was used to assess which adaptor proteins are expressed in the zebrafish lymphoid and myeloid lineages. RT-PCR of mpx provides a positive control for myeloid cells and RT-PCR of TCRα provides a positive control for lymphocytes. β-actin is shown as a positive control for all RNA samples.