High Affinity FGF Receptors

The first receptor for FGF was purified and cloned as a high-affinity receptor for FGF-2 from chicken (41). This receptor, FGF receptor-1 (FGFR-1), was highly homologous to the flg tyrosine kinase previously cloned from human endothelial cells. Subsequently, FGFR-1 was shown to bind FGF-1, FGF-2, and FGF-4 (42). FGFR-2 was first identified by screening of a mouse liver expression library with antiphosphotyrosine antibodies, and was called bek (43). Subsequent molecular cloning of human, murine, and chicken homologs to bek revealed that it is a receptor for FGF-1, FGF-2, and FGF-4 (40). FGFR-3 was cloned from human leukemia cell lines (40,44), and turned out to be highly homologous to the previously cloned orphan tyrosine kinase receptor cek-2. FGFR-4 was also cloned from human leukemia cell lines (44), and was shown to bind FGF-1 and FGF-6 with high affinity, FGF-4 with lower affinity, and FGF-2 with an even lower affinity (40,45).

The overall structure of the four members of the FGFR family is identical, and can be summarized from N- to C-terminus as follows: s ignal peptide, two or three extracellular immunoglobulin-like loops (Ig domains), characteristic acidic region between first and second Ig domain, transmembrane domain, cytoplasmic domain with the catalytic tyrosine kinase domain split by a 14-amino acid kinase insert, and a carboxy-terminal tail (40). All four receptors are highly homologous to each other, with 70-80% amino acid identity in the ligand-binding domains (Ig loops II and III), and in the tyrosine kinase domain. Other less conserved regions still exhibit 50-60% homology (44). Complex alternative splicing, combined with alternative polyadenylation, creates a high diversity in receptor isoforms for FGFR-1 to -3, but not for FGFR-4, resulting both in receptors with distinct, and others with redundant, functions, ligand specificities, and signal trans-duction pathways (39,40).

One of the more general splicing variations found in FGFR-1, FGFR-2, and FGFR-3 affects the FGF-binding domain and, thus, the ligand specificity of the receptors. Alternative usage of exons Illb and IIIc, encoding the alternative second halves of the third Ig domain, creates different membrane spanning forms. In comparison to FGFR-1 containing the Ig loop IIIc, use of Ig loop IIIb reduces the affinity to FGF-2 about 50-fold; the affinity to FGF-1 is not affected (42). Use of a polyadenylation site preceding these alternative exons produces a receptor form that lacks transmembrane and cytoplasmic domains, resulting in a soluble receptor (IIIa; 40). This soluble form of FGFR-1 is functional in that it can bind FGF-1 and FGF-2 (40). With FGFR-2, this alternative splicing event creates a dramatic difference in ligand-binding specificity: Use of Ig loop IIIc results in a receptor that binds FGF-1 and FGF-2 with high affinity, but not FGF-7 (KGF). FGFR-2 containing I g loop IIIb binds FGF-7 (KGF) with very a high affinity and FGF-1 and FGF-2 with 50-fold lower affinity. Thus, two growth factor receptors with different ligand specificities are encoded by alternate transcripts from the same gene (40,46).

Many other alternative products have been described. For example, three truncated extracellular domain forms of FGFR-1 have been identified as soluble FGF-binding proteins in blood, and in the extracellular matrix and basement membrane of vascular endothelial cells. Their biological function, however, remains unclear (47). Specific cleavage of FGFR-1 by gelatinase A (MMP-2) yields a soluble ectodomain containing the three Ig domains, and is capable of binding FGF-1 and FGF-2 (48; Fig. 1).

Although the four members of the FGFR family are very similar to each other in activity, and are expressed in partially overlapping patterns during embryonic development, inactivation of one receptor results in a very severe phenotype, indicating that FGFRs function in a nonredundant manner, and exert very specific activities. Recently, unique mutations in FGFRs have been shown to be associated with human skeletal disorders, suggesting an important role for FGFRs in bone development. Data from many experiments suggest that the high-affinity receptor complex is an intimate ternary complex of the transmembrane tyrosine kinase receptor, heparan sulfate glycosaminogly-

High-alllnliy Low-alllnlty rtceplora receptors

High-alllnliy Low-alllnlty rtceplora receptors

Fgf And Tumor
  1. 1. Modulation of FGF-2-receptor binding and activation by metabolic inhibitors of HS synthesis and sulfation, HS-degrading enzymes, and heparin-mimicking compounds. Modulation of FGF-2 binding to low-affinity cell-surface receptor sites can be brought about by enzymes that degrade the HS side chains (e.g., heparanase) or core protein (e.g., plasmin, thrombin, PI-PLC) of HSPG (right), and by soluble primers (|3-D-xylosides) of HS synthesis, and metabolic inhibitors (e.g., chlorate) of sulfation (center). Binding of FGF-2 to high-affinity cell-surface receptor sites can be modulated by heparin-mimicking compounds (i.e., compound RG-13577) that compete with HS, bind the growth factor, and prevent receptor binding and/or dimerization, and by proteolytic enzymes (e.g., MMP2) that cleave the ectodomain of the receptor (left).
  2. 1. Modulation of FGF-2-receptor binding and activation by metabolic inhibitors of HS synthesis and sulfation, HS-degrading enzymes, and heparin-mimicking compounds. Modulation of FGF-2 binding to low-affinity cell-surface receptor sites can be brought about by enzymes that degrade the HS side chains (e.g., heparanase) or core protein (e.g., plasmin, thrombin, PI-PLC) of HSPG (right), and by soluble primers (|3-D-xylosides) of HS synthesis, and metabolic inhibitors (e.g., chlorate) of sulfation (center). Binding of FGF-2 to high-affinity cell-surface receptor sites can be modulated by heparin-mimicking compounds (i.e., compound RG-13577) that compete with HS, bind the growth factor, and prevent receptor binding and/or dimerization, and by proteolytic enzymes (e.g., MMP2) that cleave the ectodomain of the receptor (left).

cans, and FGF ligands. Together, these components define ligand specificity and binding affinity (40,42,49).

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