The Kinase Activity of Fibroblast Growth Factor Receptor 3 with Activation Loop Mutations Affects Receptor Trafficking and Signaling

Abstract

Amino acid substitutions at the Lys-650 codon within the activation loop kinase domain of fibroblast growth factor receptor 3 (FGFR3) result in graded constitutive phosphorylation of the receptor. Accordingly, the Lys-650 mutants are associated with dwarfisms with graded clinical severity. To assess the importance of the phosphorylation level on FGFR3 maturation along the secretory pathway, hemagglutinin A-tagged derivatives were studied. The highly activated SADDAN (severe achondroplasia with developmental delay and acanthosis nigricans) mutant accumulates in its immature and phosphorylated form in the endoplasmic reticulum (ER), which fails to be degraded. Furthermore, the Janus kinase (Jak)/STAT pathway is activated from the ER by direct recruitment of Jak1. Abolishing the autocatalytic property of the mutated FGFR3 by replacing the critical Tyr-718 reestablishes the receptor full maturation and inhibits signaling. Differently, the low activated hypochondroplasia mutant is present as a mature phosphorylated form on the plasma membrane, although with a delayed transition in the ER, and is completely processed. Signaling does not occur in the presence of brefeldin A; instead, STAT1 is activated when protein secretion is blocked with monensin, suggesting that the hypochondroplasia receptor signals at the exit from the ER. Our results suggest that kinase activity affects FGFR3 trafficking and determines the spatial segregation of signaling pathways. Consequently, the defect in down-regulation of the highly activated receptors results in the increased signaling capacity from the intracellular compartments, and this may determine the severity of the diseases.Protein tyrosine kinase receptors play a fundamental role in the control of a variety of cellular processes during embryonic development; furthermore, they regulate many metabolic and physiological processes in a variety of tissues and organs (1-3). Indeed, aberrant receptor kinase activation results in severe diseases such as cancer, diabetes, cardiovascular diseases, and many others (4). Fibroblast growth factor receptors (FGFRs) 1 belong to the tyrosine kinase receptor (RTK) family. FGFRs are glycosylated transmembrane proteins that, upon binding with fibroblast growth factor ligands and heparin, dimerize and undergo interchain autophosphorylation of key tyrosine residues, thus transmitting signals into the cells (5-8). Tyrosine autophosphorylation sites in the cytoplasmic domain of the receptor serve as docking sites for the Src homology 2 domain of signaling proteins that are recruited and activated upon growth factor stimulation (9). FGFR3 is a member of the FGFR family and plays a pivotal role in skeletal development as a negative regulator of bone growth, because targeted disruption of the mouse FGFR3 gene causes a skeletal overgrowth (10, 11). In addition, mutations in FGFR3 are frequently involved in a variety of human skeletal disorders and cancer (12, 13).Particular attention has been paid to the Lys-650 codon of FGFR3 located within a critical regi...

Novel FGFR3 mutations creating cysteine residues in the extracellular domain of the receptor cause achondroplasia or severe forms of hypochondroplasia

“…Correct glycosylation of the receptor is required for its normal processing to the plasma membrane and occurs both in the endoplasmic reticulum (ER) and the Golgi system. 23,24 Defective glyco- Novel FGFR3 mutations S Heuertz et al has been reported with the K650N mutation causing HCH. 24 Another mutation (N328I) affecting a putative glycosylation site has been reported previously 10 and identified in one of our patients; both mutations were associated with typical HCH phenotype in the affected individuals.…”

“…23,24 Defective glyco- Novel FGFR3 mutations S Heuertz et al has been reported with the K650N mutation causing HCH. 24 Another mutation (N328I) affecting a putative glycosylation site has been reported previously 10 and identified in one of our patients; both mutations were associated with typical HCH phenotype in the affected individuals. Amino acids forming the Ig I loop are poorly conserved among the different members of the FGFR family.…”

Suppressors of cytokine signaling (SOCS) 1 and SOCS3 interact with and modulate fibroblast growth factor receptor signaling

“…In the absence of ligand, the GFP-tagged FGFR3 localized to the cell membrane ( Fig. 3A) (Lievens et al, 2004). Addition of FGF induced internalization of the receptor, as can be seen by its localization in the cytosol in diffused and granular form, and its clustering prominently in the perinuclear region (Fig.…”

Hypochondroplasia and acanthosis nigricans: a new syndrome due to the p.Lys650Thr mutation in the fibroblast growth factor receptor 3 gene?

“…Alternatively, different mutations in the Lys650 residue could affect different intracellular localizations, which is now believed to have a role in chondrocyte defects observed in skeletal dysplasias (31). It should be noted that FGFR3 can be expressed as different isoforms, and while chondrocytes express the isoform IIIc, epithelial cells present the isoform IIIb.…”