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

Zhuang L, Karotki AV, Bruecker P, Trueb B - BMC Biochem. (2009)

Bottom Line: 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).A comparison of the intracellular domain between sea urchin and human FGFRL1 provides interesting insights into the shaping of a novel zinc binding domain.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Clinical Research, University of Bern, 3010 Bern, Switzerland. lei.zhuang@dkf.unibe.ch

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.

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Related in: MedlinePlus

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).
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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).

Mentions: To address this question, we expressed three fragments from the intracellular domain of FGFRL1 as GST-fusion proteins in a bacterial expression system, namely an upper human fragment of 72 residues covering amino acids 400-471, a lower human fragment of 33 residues covering amino acids 472-504 and a sea urchin fragment of 41 residues covering amino acids 492-532. On a polyacrylamide gel, the purified fusion proteins migrated with electrophoretic mobilities that are consistent with the calculated molecular weights (Fig. 4). To demonstrate an interaction with metal ions, the fragments were loaded onto nickel columns, washed with loading buffer and eluted specifically with imidazole. Under our experimental conditions, the lower human fragment bound specifically to the column, whereas the upper human fragment as well as the sea urchin fragment barely interacted with the column matrix (Fig. 4). The binding experiments were repeated with a column matrix for which the nickel ions had been exchanged by zinc ions. Again, the lower human fragment bound to the zinc column and could specifically be eluted with 1.2 M imidazole, while the sea urchin fragment as well as the upper human fragment did not bind to the column. These results suggest that the sea urchin protein has considerably lower affinity for zinc ions than the human homologue.


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

Zhuang L, Karotki AV, Bruecker P, Trueb B - BMC Biochem. (2009)

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).
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC2806250&req=5

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).
Mentions: To address this question, we expressed three fragments from the intracellular domain of FGFRL1 as GST-fusion proteins in a bacterial expression system, namely an upper human fragment of 72 residues covering amino acids 400-471, a lower human fragment of 33 residues covering amino acids 472-504 and a sea urchin fragment of 41 residues covering amino acids 492-532. On a polyacrylamide gel, the purified fusion proteins migrated with electrophoretic mobilities that are consistent with the calculated molecular weights (Fig. 4). To demonstrate an interaction with metal ions, the fragments were loaded onto nickel columns, washed with loading buffer and eluted specifically with imidazole. Under our experimental conditions, the lower human fragment bound specifically to the column, whereas the upper human fragment as well as the sea urchin fragment barely interacted with the column matrix (Fig. 4). The binding experiments were repeated with a column matrix for which the nickel ions had been exchanged by zinc ions. Again, the lower human fragment bound to the zinc column and could specifically be eluted with 1.2 M imidazole, while the sea urchin fragment as well as the upper human fragment did not bind to the column. These results suggest that the sea urchin protein has considerably lower affinity for zinc ions than the human homologue.

Bottom Line: 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).A comparison of the intracellular domain between sea urchin and human FGFRL1 provides interesting insights into the shaping of a novel zinc binding domain.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Clinical Research, University of Bern, 3010 Bern, Switzerland. lei.zhuang@dkf.unibe.ch

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.

Show MeSH
Related in: MedlinePlus