Elsevier

Cellular Signalling

Volume 20, Issue 8, August 2008, Pages 1471-1477
Cellular Signalling

Sprouty 2 disturbs FGFR3 degradation in thanatophoric dysplasia type II: A severe form of human achondroplasia

https://doi.org/10.1016/j.cellsig.2008.04.001Get rights and content

Abstract

Thanatophoric dysplasia is a member of the achondroplasia family of human skeletal dysplasias, which result from FGFR3 mutations that exaggerate this receptor's inhibitory influence on chondrocyte proliferation and differentiation in the skeletal growth plate. We have previously reported that defective lysosomal degradation of activated receptor contributes to the gain-of-function of the mutant FGFR3. We now provide evidence that this disturbance is mediated by the receptor's kinase activity and involves constitutive induction and activation of Spry2. Our findings suggest that activated Spry2 may interfere with c-Cbl-mediated ubiquitination of FGFR3 by sequestering c-Cbl. They provide novel insight into the pathogenesis of this group of human skeletal dysplasias and identify a mechanism that potentially could be targeted therapeutically.

Introduction

Achondroplasia (ACH) is the prototype of a group of human chondrodysplasias that range in severity from lethal thanatophoric dysplasia types I and II (TDI and TDII) to relatively mild hypochondroplasia [1], [2]. These disorders result from dominantly inherited mutations of fibroblast growth factor receptor 3 (FGFR3), a transmembrane receptor tyrosine kinase (RTK) that serves as a major negative regulator of linear bone growth. The mutations are gain-of-function, i.e., activating mutations associated with increased tyrosine kinase activity and augmentation of FGFR3 signals [3], [4], [5]. These inhibitory signals are propagated through STAT, MAP kinase and other downstream pathways to diminish proliferation and terminal differentiation of growth plate chondrocytes [6], [7], [8], [9].

Several mutation-specific mechanisms have been proposed to explain how different disease-related mutations lead to gain of receptor function. They include stabilization and induction of FGFR3 dimerization, which is necessary for receptor activation, by the transmembrane and cysteine substitution mutations of ACH and TDI, respectively, and kinase domain mutations of TDII that constitutively activate the receptor's intrinsic kinase activity [4], [10], [11]. We have recently uncovered a mechanism that may be common to ACH, TD and other activating FGFR3 mutations. Our findings suggest that mutant receptors are degraded more slowly than their wild type (WT) counterparts allowing for accumulation of actively signaling receptors and an increase in overall FGFR3 signal strength [12]. Our results point to a disturbance in c-Cbl-mediated ubiquitination of the mutant receptors as the underlying defect. Normally, receptor ubiquitination serves as a targeting signal for trafficking activated RTKs to lysosomes for degradation; consequently, defective ubiquitination slows turnover [13]. Similar disturbances have been reported in tumors due to accumulation of mitogenic RTKs [14], [15], [16].

The RTK trafficking defects in some cancers reflect somatic, loss of function mutations of c-Cbl or mutations that disrupt binding of c-Cbl to its RTK substrates, both of which impair receptor ubiquitination [14], [15], [16]. Neither of these mechanisms seems likely in the ACH group of disorders in which mutations in different regions of FGFR3 lead to the ubiquitination disturbance. More plausible is that the ubiquitination defect shared by the different FGFR3 mutations reflects the increased tyrosine kinase activity that they share [1], [3]. Indeed, constitutive kinase activation has been proposed to influence maturation and trafficking of RTKs, including FGFR3 [17], [18]. Also consistent with this notion is that the disturbance we previously observed in FGFR3 ubiquitination correlates with the extent of the increase in kinase activity associated with the specific mutation, i.e., moderate increase in both kinase activity and ubiquitination defect in ACH and marked increase in both kinase activity and ubiquitination defect in TD [3], [12].

In this paper, we examine the possibility that accumulation of mutant FGFR3 is mediated by excessive kinase activity acting through induction and constitutive activation of Sprouty 2 (Spry2). Originally discovered in Drosophila, four Sprys have been identified in mammals [19], [20]. They are classically induced by and antagonize MAP kinase signaling in response to several growth factors including FGF, EGF (epidermal growth factor), VEGF (vascular endothelial growth factor), PDGF (platelet-derived growth factor) and SCF (stem cell factor/kit ligand) [20], [21], [22], [23], [24]. Spry2 has been implicated as an FGF antagonist during lung morphogenesis and limb development and outgrowth [25], [26], [27]. Its postnatal function is less well defined, but it expressed in many adult tissues [27].

In contrast to its better known role as an inducible RTK inhibitor, Spry2 has been shown to promote RTK signaling in certain contexts through its ability to bind and sequester c-Cbl [20], [28], [29], [30], [31], [32], [33], [34], [35]. Our observations based on comparison of WT to constitutively active TDII mutant FGFR3 in cultured cells, suggest that this alternate mechanism involving Spry2 sequestration of c-Cbl may be relevant to the pathogenesis of TDII and potentially other members of the ACH group of disorders.

Section snippets

Cell lines and tissues

Cell culture experiments were carried out in both COS-7 and TRex-293 (Invitrogen) cells maintained in DMEM supplemented with 10% FCS in a humidified incubator with 5% CO2 at 37 °C. COS-7 cells stably expressing WT or TDII FGFR3-GFP were prepared by retroviral transduction under conditions designed to yield one copy of vector per infected cell as reported previously [12]. COS-7 cells stably expressing WT kinase dead (kd) or TDII kd FGFR3-GFP were prepared by lentiviral transduction using the

Kinase activity influences FGFR3 turnover

To determine if the increased tyrosine kinase activity associated with the TDII FGFR3 mutation contributes to the accumulation of mutant receptors, a separate mutation that abolishes kinase activity (K502A <K508A in humans> designated kinase dead, (kd) was introduced into WT FGFR3-V5-His and TDII FGFR3-V5-His [37]. After transient transfection, COS-7 cells were extracted and immunoblotted for receptor using a V5 antibody. The loss of kinase activity leads to a decrease in the amount of both WT

Discussion

We have recently reported that FGFR3 receptors bearing mutations associated with ACH and related disorders turn over more slowly than normal, that this delay results from a disturbance of c-Cbl-mediated ubiquitination of activated FGFR3 and that the delay may contribute to the gain of receptor function associated with these disorders [12]. From in vitro experiments involving the constitutively active TDII FGFR3, we now provide evidence that the disturbance reflects the increased kinase activity

Acknowledgements

We wish to thank Dafna Bar-Sagi, Dirk Bohman, Gerhard Christofori and Brian Druker for generously providing reagents and Chuxia Deng for sharing the K644E FGFR3 knock-in mouse strain. The work was funded by research grants from the Shriners Research Program, Numbers 8540 and 8610 (WAH) and a research grant from the Arthritis Foundation (WAH).

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    Present address: Department of Neurology, Harvard Medical School, Children Hospital, Fegan 11, 300 Longwood Ave, Boston, MA 02115, USA.

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