Comparison of the receptor FGFRL1 from sea urchins and humans illustrates evolution of a zinc binding motif in the intracellular domain

Abstract

Background: FGFRL1, the gene for the fifth member of the fibroblast growth factor receptor (FGFR) family, is found in all vertebrates from fish to man and in the cephalochordate amphioxus. Since it does not occur in more distantly related invertebrates such as insects and nematodes, we have speculated that FGFRL1 might have evolved just before branching of the vertebrate lineage from the other invertebrates (Beyeler and Trueb, 2006).

Results: We identified the gene for FGFRL1 also in the sea urchin Strongylocentrotus purpuratus and cloned its mRNA. The deduced amino acid sequence shares 62% sequence similarity with the human protein and shows conservation of all disulfides and N-linked carbohydrate attachment sites. Similar to the human protein, the S. purpuratus protein contains a histidine-rich motif at the C-terminus, but this motif is much shorter than the human counterpart. To analyze the function of the novel motif, recombinant fusion proteins were prepared in a bacterial expression system. The human fusion protein bound to nickel and zinc affinity columns, whereas the sea urchin protein barely interacted with such columns. Direct determination of metal ions by atomic absorption revealed 2.6 mole zinc/mole protein for human FGFRL1 and 1.7 mole zinc/mole protein for sea urchin FGFRL1.

Conclusion: The FGFRL1 gene has evolved much earlier than previously assumed. A comparison of the intracellular domain between sea urchin and human FGFRL1 provides interesting insights into the shaping of a novel zinc binding domain.

Figure 1

Alignment of the FGFRL1 sequences from eight different species. Identical residues are boxed. The putative signal peptidase cleavage site is shown by an arrow. The three Ig-like domains are marked by brackets. The transmembrane domain is given by a stippled box. Conserved glycosylation sites (NXT) are indicated by asterisks. The relative positions of introns in the corresponding genes are shown by triangles. The sequence that was used for the preparation of the zinc-binding GST fusion protein is underlined. The accession numbers are: FishA (Takifugu rubripes A) BN000669, FishB (Takifugu rubripes B) BN000670, Chicken AJ535114, Frog (Silurana tropicalis) AJ616852, Human AJ277437, Mouse AJ293947, Lancelet (Branchiostoma floridae) AJ888866, Sea Urchin (Strongylocentrotus purpuratus) FN252817, Sea Squirt (Ciona intestinalis) XP_002125836. From the sea squirt sequence only the conserved domain (residues 112-456) is included.

Figure 2

Phylogenetic analysis of the FGFRL1 sequences. An unrooted tree was built by the neighbour joining method. Bootstrap values from 1000 random replicates are indicated at the nodes. The length of the branches inversely correlates with the degree of similarity. Only the sequences from the extracellular domains without signal peptides and transmembrane domains were used.

Figure 3

Expression of the FGFRL1 gene in different tissues from S. purpuratus. A radioactively labelled cDNA probe corresponding to the sequence for amino acid residues 1-374 was hybridized to a Northern blot containing 7.5 μg RNA from five different tissues as indicated. The 26S ribosomal RNA stained with ethidium bromide is included as a loading control.

Figure 4

Interaction of the C-terminal domain from FGFRL1 with zinc and nickel ions. GST fusion proteins were mixed with nickel or zinc beads, washed and eluted with 1.2 M imidazole. The eluted proteins were resolved on a polyacrylamide gel, transferred to a nitrocellulose membrane and stained with antibodies against GST. Specifically eluted proteins are compared to the starting material (Input). The fusion proteins comprised amino acid residues 400-471 (Human U), 472-504 (Human L) and 492-532 (Sea Urchin L).

Figure 5

Comparison of the C-terminal FGFRL1 sequences from humans, lancelet and sea urchin. Histidine residues are highlighted in bold. Atomic absorption spectroscopy showed that the human protein bound 2.6 mole zinc/mole protein, while the sea urchin protein bound 1.7 mole zinc/mole protein. The standard deviation from three different measurements was < 2%. The zinc content of the lancelet protein was not determined (n.d.), but this sequence was included for reasons of comparison.