Effect of Transmembrane and Kinase Domain Mutations on Fibroblast Growth Factor Receptor 3 Chimera Signaling in PC12 Cells

Abstract

The effect of six point mutations causing various human skeletal dysplasias, occurring in the transmembrane (TM) and kinase domains (KD) of fibroblast growth factor receptor 3, were introduced into a chimera composed of the extracellular domain of human platelet-derived growth factor ␤ and the TM and intracellular domains of hFGFR3. Stable transfectants in rat PC12 cells showed distinct differences in the two classes of mutations. The cells containing TM mutants displayed normal expression and activation but higher responsiveness to lower doses of ligand. The KD mutants showed significantly altered expression patterns. Normal amounts of a lower M r receptor (p130) reflecting incomplete glycosylation, but only greatly decreased amounts of the mature (p170) form, were observed. However, the latter material showed normal ligand-dependent activation. In contrast, the p130 form, which is regularly observed in the expression of both native and chimeric receptors, exhibits strong ligand-independent tyrosine phosphorylation, particularly with the K650E mutation. Expression of two of the KD mutants (K650M and K650E), under control of an inducible metallothionein promoter, indicated that this receptor was sufficiently autoactivated to produce at least partial differentiation and, in the case of the K650E mutation, to induce ligand-independent neurite outgrowth. A model is presented that suggests that the low M r (p130) KD mutants can, under the right conditions, signal intracellularly, but when they are fully glycosylated and move to the cell surface they adopt a normal, inhibited conformation, in the form of ligand-independent dimers, that neutralizes the effects of the mutations. When ligands bind, these dimeric receptors are activated in a normal manner. This model suggests that unliganded dimers may be a common intermediate in receptor tyrosine kinase signaling.The fibroblast growth factor receptor (FGFR) 1 tyrosine kinase family consists of four (FGFR1-4) members that are implicated in many developmental and regenerative processes, including cell proliferation, differentiation, and angiogenesis (1-3). Signaling by these receptors is mediated by the high affinity binding of the fibroblast growth factors (FGF), a diverse family consisting of at least 10 characterized members, aided by heparan sulfate proteoglycan interactions (4). Further diversity in expression and ligand specificity is achieved by the generation of subforms of the FGFRs mediated by alternative mRNA splicing events (4, 5). The ligand-receptor complexes activate the receptor kinases inducing tyrosine phosphorylation and a cascade of signaling events mediated by target effector proteins including Ras/MAPK and phospholipase C␥ (PLC␥) (6 -8).It is now well known that mutations in three of the four members of the FGFR family (FGFR1-3) cause a variety of autosomal dominant human skeletal dysplasias (for review see Refs. 9 and 10). Among these is the most common genetic form of short-limbed dwarfism, achondroplasia (ACH) (11,12) caused by a point mutation in...

Sequence survey of receptor tyrosine kinases reveals mutations in glioblastomas

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Identification of Phosphopeptides by MALDI Q-TOF MS in Positive and Negative Ion Modes after Methyl Esterification

“…Gain of function mutations in the tyrosine kinase domain of FGFRs are responsible for a number of human skeletal disorders. The degree of clinical severity associated with the mutations correlates with the level of constitutive kinase activity in these mutants (23)(24)(25). A method for rapidly comparing phosphorylation sites on various mutants of FGFRs and then correlating phosphorylation status with receptor activity would be very useful for understanding the molecular basis for receptor gain of function and potentially facilitate development of therapeutic interventions.…”

Role of Receptor Tyrosine Kinase Transmembrane Domains in Cell Signaling and Human Pathologies

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