Vol. 9, 71-78, January 1998 Cell Growth & Differentiation 71

Constitutive Activation of Fibroblast Growth Factor Receptor3 by Mutations Responsible for the Lethal SkeletalDysplasia Thanatophoric Dysplasia Type 11

Patricia Y. d’Avis,2 Scott C. RobertsonApril N. Meyer, Wendy M. Bardwell,Melanie K. Webster, and Daniel J. Donoghue3Department of Chemistry and Biochemistry, Center for MolecularGenetics, University of California at San Diego, La JOlla, California92093-0367

AbstractThanatophoric dysplasia type I (TDI) is a neonatal lethalskeletal dysplasia caused by several mutations in theextracellular domain of fibroblast growth factorreceptor 3. These mutations occur either In the lg2-1g3linker domain or in the extracellular juxtamembranedomain, and all involve mutation of the wild-typeresidue to Cys. In all cases, the presence of the mutantCys residue allows the receptor to dimerizeabnormally, resulting in ligand-independent activation.This Is also manifested by increased biological

signaling, increased tyrosine phosphorylatlon, and Invitm kinase activity associated wfth dimeric receptors.These results suggest that TDI is caused by Cys-mediated intermolecular disulfide bonding, leading toconstftutive receptor activation as a resuft of thesemutations. Mutations causing TDI are discussed withrespect to activating mutations in other receptors thatare implicated in human disease.

IntroductionTD4 is a neonatal lethal dwarfism that results from pointmutations in FGFR3 (1-4). Affected individuals exhibit se-

verely shortened limbs, a narrow thorax with short ribs, andan enlarged head and generally die within a few hours of birthdue to respiratory failure (5). Two distinct types of TD exist,TDI and TDII, which are distinguished clinically by the pres-ence of straight femurs and severe cloverleaf skull in TOll,compared to curved short femurs and a slight or no clover-leaf skull in TDI. Distinct point mutations in FGFR3 occur inindividuals afflicted with both ThI and TOIl. All reportedcases of TOll result from a single point mutation, K650E, in

ReCeiVed 8/5,’97; revised 11/21/97;accepted 11/24/97.The costs of publication of this article were defrayed it� part by thepayment of page charges. This article must therefore be hereby markedadvertisement In accordance with 18 U.S.C. Section 1734 solely to mdi-cate this fact.1 Supported by NIH Grant DE 12581.2 Present address: Telios Pharmaceuticals, Inc., I 1045 RosalIe Street,

Suite A, San Diego, CA 92121-1299.3 To whom requests for reprints should be addressed. Phone: (619) 534-2167, Fax: (619) 534-7481 , E-mall: ddonoghueOucsd.edu.4 The abbreviations used are: TD, thanatophoric dysplasia; FGFR, fibro-blast growth factor receptor� nt, nucleotide; AIPA, radioimmunoprecipita-tion assay.

the activation loop of the tyrosine kinase domain of FGFR3

(1), whereas mutations in several domains of FGFR3 are

implicated in ThI. The majority of TDI cases involve muta-tions of single amino acids to Cys in the extracellular domain(1 , 2, 4), although TDI has also been associated with muta-

tions in the stop codon, creating a carboxyl-terminal exten-sion of 141 amino acids (3). The Cys mutations cluster in tworegions of FGFR3, as shown in Fig. 1A: (a) the linker regionbetween the 1g2 and lg3 domains (R248C, S249C); and (b)the juxtamembrane region (G370C, S371 C, and Y373C). Ofthese, the mutation R248C occurs in a large majority of TDIpatients, whereas the mutations S249C and Y373C occurless frequently. The mutations G370C and S371 C have been

observed in only single patients (1 , 2, 4).The lg2-lg3 linker domain, a region of about 45 residues

separating the 192 and lg3 domains, is highly conserved in

the FGFR family of receptor tyrosine kinases and containsthe invariant tripeptide Arg-Ser-Pro. Point mutations in thisregion in three of the four known FGFR family members havebeen shown to result in a number of related craniosynostosissyndromes, including Crouzon, Pfeiffer, and Apert syn-dromes, which are characterized by premature closure of thecranial sutures in addition to other developmental abnormal-ities. Within this conserved tnpeptide, mutation of Pro-+Arg

in FGFR1 causes Pfeiffer syndrome (6), whereas mutation of

Ser-+Trp or of Pro-�Arg in FGFR2 results in Apert syndrome(7). In FGFR3, mutations of Arg-+Cys or Ser-�Cys result inTDI, as discussed previously, and mutation of Pro-*Arg re-suIts in a nonsyndromic craniosynostosis (8).

The extracellular juxtamembrane region of FGFRs, wherethree of the TDI mutations occur in FGFR3, has also beenshown to be a sensitive region for mutations that result in a

variety of diseases. Two similar mutations in the same region

of FGFR2, S372C and Y375C, have been shown to cause

Beare-Stevenson cutis gyrata syndrome, a lethal skeletaland skin disorder (9). Mutations in thejuxtamembrane regionof other receptor tyrosine kinases also lead to inappropriateactivation. For example, mutations involving Cys residues inthe juxtamembrane domain of the RET receptor are respon-sible for multiple endocrine neoplasia type 2A and seem to

result in ligand-independent receptor dimerization and acti-

vation (10, 11).

Recently, work from several laboratories has demon-strated that a variety of chondrodysplasias, including achon-droplasia, TOIl, and Crouzon syndrome, arise from autoso-mal dominant mutations that cause constitutive activation ofFGFRs (reviewed in Ref. 12). Whereas these different muta-tions share the common theme of ligand-independent recep-tor activation, they differ in both the extent of the resultingactivation and in the underlying biochemical mechanism. For

instance, our laboratory has previously demonstrated ligand-

72 FGFR3 Activation by TDI Mutations

A. FGFRI3 constructs

I� FGFR3

Ig.1 Ig-2 Ig-3

,- - (�,b?’� “ I’ � � - -,

‘LE11PH’ SEA�flVTAG

cc cc CA

B. FGFft3/Neu chimeric constructs

I� FGFR3 �l( Neu >1

Ig-1 Ig.2 Ig-38P (-=ThAB(�Th(=Th ThI. ‘8 V��’8 .44. .� -

I- � �, LEflPH’

Fig. 1. Structure of FGFR3 constructs. (A) The structure of full-lengthFGFA3 is shown schematically, including the signal peptide (SP), threeimmunoglobulin-like domains (Ig), the acid box (AB), the transmembranedomain (TM), and the split tyrosine kinase domain. The TDI and controlmutations in the Ig2-1g3 linker region and in thejuxtamembrane region areindicated. (B) Chimenc FGFA3/Neu receptors used in this study areshown, consisting of the extracellular domain from FGFA3, and the trans-membrane and intracellular region of Neu.

independent activation of FGFR3 by the transmembrane do-main mutation causing achondroplasia (G380R) and by the

kinase domain mutation responsible for TDII (K650E in theactivation loop; Refs. 1 3 and 1 4). Additionally, one of the ThI

mutations, R248C, occurring in the lg2-lg3 linker domain ofFGFR3, was previously examined and shown to form ligand-

independent receptor dimers (1 5). Several of the mutations inFGFR2 causing Crouzon syndrome have also been shown to

result in constitutive receptor dimerization (1 6-1 8). Nonethe-less, none of the juxtamembrane mutations of FGFR3 havebeen analyzed to date.

Interestingly, Chesi et aL (1 9) recently demonstrated that

dysregulation of FGFR3 is associated with human multiple

myeloma. In these studies, a translocation that juxtaposesthe FGFR3 locus near the lgH switch region was observed inabout 25% of multiple myeloma tumors and cell lines, re-

suiting in high-level expression of FGFR3. Moreover, two celllines and one primary tumor with this t(4;14) translocationselectively overexpressed FGFR3 mutant alleles previouslyidentified in conjunction with severe skeletal abnormalities,

including the Y373C mutation found in TDI (4). Thus, insightsinto the role of FGFR3 mutation in causing TDI may also berelevant to understanding the role of FGFR3 activation inhuman cancers such as multiple myeloma.

In this work, we have examined each of the reportedjuxtamembrane IDI mutations and compared them with the

101 mutations that lie within the 1g2-1g3 linker domain. Foreach of the five mutants examined, the presence of themutant Cys residue allows the receptor to dimerize abnor-mally, resulting in ligand-independent activation. This is alsomanifested by increased biological signaling, increased ty-rosine phosphorylation, and in vitro kinase activity associ-ated with dimeric receptors. As a control, we also comparedactivating mutation R248C with control mutation R248A andshow that this latter mutation does not result in FGFR3activation. This demonstrates that it is the creation of a new

Cys residue, rather than loss of a wild-type residue, that iscritical for activation. In summary, these results show that

TDI is caused by Cys-mediated intermolecular disulfidebonding, leading to constitutive receptor activation as a re-

suIt of mutations in either the juxtamembrane domain or the

lg2-1g3 linker region of FGFR3.

ResultsTranscription Assays. Five different mutations in the extra-

cellular domain of FGFR3 have been associated with the

skeletal disorder known as TDI. These mutations areclustered in two regions, the lg2-lg3 linker domain and the

amino-terminal juxtamembrane domain. All TDI mutationsare single point mutations resulting in the creation of a Cysresidue. Fig. 1A shows the FGFR3 mutants examined in thisstudy, including control mutation R248A. The expression of

each of the mutants was confirmed by indirect immunofluo-rescence in NIH3T3 cells, as shown in Fig. 2.

To examine the effects ofTDI mutations on FGFR3 activity,we exploited a fos-luciferase reporter to examine transcrip-tional activation in response to FGFR3 signaling. We previ-ously used this approach to assess the activation of variousderivatives of the K650E FGFR3 mutant responsible for TOll(20). Full-length FGFR3 constructs encoding each of themutations, shown in Fig. 1A, were transfected into NIH3T3cells along with a fos-luciferase reporter, and the induction ofluciferase activity was measured. As shown in Fig. 3, the1g2-1g3 linker mutants, R248C and S249C, showed approx-imately a 4-fold increase in luciferase activity compared tothat of the wild-type receptor. We also examined controlmutation R248A in this assay and found that it does notsignificantly induce fos-luciferase activity. The juxtamem-

brane mutants also showed activity in this assay and exhib-ited between 8- and 1 5-fold activation over that of the wild-

type FGFR3. For comparison, we assayed the K650Emutation, which causes TDII, and found that it exhibited asimilar level of activation as the juxtamembrane mutants.

Transforming Activity of FGFR3INeu Chimeric Recap-tore. To further examine the effect of the 1g2-1g3 linker mu-tations on receptor signaling, we constructed chimeric re-

ceptors containing the extracellular domain of FGFR3 fusedto the transmembrane and intracellular domains of Neu, asshown in Fig. lB. This approach was undertaken after initialexperiments in which we found that all of the ThI mutants,

expressed as full-length FGFR3 constructs, were inactive in

NIH3T3 transformation assays (data not shown). This resultwas consistent with earlier experiments in which we found

D. R248C E. S249C

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,

. I.

F. G370C

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I. K650E (TDII)G. S371C H. Y373C

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Cell Growth & Differentiation 73

A. Mock B. Wild-type C. R248A

Fig. 2. Localization of TDI mutant proteins by indirect immunotluorescence. NIH3T3 cells were transtected with FGFR3 containing the different TDImutations. Two days after transfection, the cells were fixed, permeabilized and subjected to indirect immunofluorescence using FGFR3 C-terminalantiserum. Panels shown are as follow: (A) mock transtected cells; (B) wild-type FGFR3; (C) R248A; (D) R248C; (E) S249C; (F) G370C; (G) S371C; (H) Y373C;(I) the TDII mutant, K650E.

that other activated mutants of FGFR3 are incapable of

transforming NIH3T3 cells. Recently, we demonstrated that

the K650E mutation responsible for TDII, which exhibits very

strongly activated kinase activity, becomes transforming

only after truncation of the extracellular and transmembrane

domains, together with localization of the active kinase do-

main to the plasma membrane by an amino-terminal myri-

stylation signal (20).

We have previously demonstrated that focus formation in

NIH3T3 cells expressing FGFR/Neu chimeric constructs

serves as a sensitive and reliable indicator for receptor

dimerization and activation (13, 16). As shown in Fig. 4,

expression of constructs containing the two 1g2-lg3 TDI mu-

tations results in morphological transformation, indicating

that these mutations activate signaling of the chimeric re-

ceptor. A control mutation, R248A, did not induce foci, mdi-

cating that the creation of a free Cys residue rather than the

removal of the existing residue is responsible for activation.

Furthermore, these results demonstrate that the TDI muta-

tions act as gain-of-function mutations, resulting in en-

hanced signaling due to constitutive activation of the

receptor.

Dimerization Assays. To observe whether the free cys-

teines created in FGFR3 by each of the TDI mutations re-

suited in the formation of disulfide-bonded dimers, cells were

transfected with full-length FGFR3 expression constructs,

labeled with [35S]Cys and [35S]Met, and lysates were pre-

pared. After immunoprecipitation, samples were analyzed on

reducing and nonreducing gradient gels, and FGFR3 was

detected by fluorography. As shown in Fig. 5A, each of the

I-< 000 00=> � Co 0) 0 ‘- C’) �

,�. ‘e’ ‘� N. �= F- I-C%J C�J C’J (V) (1) C’)� r�CO(D U)>-

74 FGFR3 Activation by TDI Mutations

C

0

(a>t

�00

U-

Fig. 3. Induction of the c-fos promoter by TDI mutants of FGFR3.NIH3T3 cells were cotransfected with either wild-type or mutant FGFR3constructs together with the pFL700 reporter containing the c-tos promoterdriving expression of the luciferase gene. Transfected cells were lysed andanalyzed for luciferase activity. Data shown above are the average of dupli-cate transtections from a single representative experiment.

TDI mutants formed dimers in the absence of ligand, which

were not observed in either the wild-type or the control

R248A constructs. Additionally, the TDII mutant, K650E, was

not observed to form covalent dimers. Equivalent samples

were also electrophoresed under reducing conditions and

visualized by immunoblotting (Fig. 5B), confirming equal ex-

pression for each of the constructs. These results suggest

that the creation of a free Cys residue in either the 1g2-lg3

linker domain or the juxtamembrane domain of FGFR3 is

able to cause constitutive dimerization of the receptor. Ad-

ditionally, interlinker mutants R248C and S249C show a

higher level of disulfide bond formation than juxtamembrane

mutants G370C, S371 C, and Y373C.

In Vitro and in Vivo Tyrosine Kinase Activity Assays. Toassess the kinase activity of each TDI mutant, constructs

were transfected into COS cells and, after incubation in

serum-deficient media, receptors were immunoprecipitated

and subjected to in vitro kinase assays in the presence of

radiolabeled ATP. As shown in Fig. 6B, there is little differ-

ence in the in vitro kinase activity associated with the mo-

nomeric form of the control and TDI mutants. Nonetheless,

the dimeric form of each of the TDI mutants undergoes

autophosphorylation in the absence of ligand (Fig. 6A), sug-

gesting that this dimeric form is the active form of the re-

ceptor. When assayed for in vitro kinase activity, the G370C

mutant exhibited the greatest activity, followed by interlinker

mutants R248C and S249C, with the remaining two jux-

tamembrane mutants, S371C and Y373C, exhibiting the

least activity. These results are in contrast to the dimerization

analysis presented earlier, in which the R248C and S249C

mutants exhibited the greatest dimerization. These results

will be discussed further below.

Fig. 6C demonstrates equivalent levels of receptor in each

kinase assay sample. Cell lysates were also immunoprecipi-

tated with phosphotyrosine-specific antisera, and the ex-

pression of in vivo phosphorylated FGFR3 mutants was ex-

amined by immunoblotting with anti-FGFR3 antisera. As

shown in Fig. 6D, the dimeric form of each of the TDI mutant

receptors exhibited phosphotyrosine, consistent with the Ii-

gand-independent dimerization and activation of these mu-

tant FGFR3 receptors.

When mutant receptors were immunoprecipitated from

cells after stimulation with FGF-1 , no significant increase was

observed in receptor dimerization, kinase activity, or phos-

photyrosine incorporation in comparison with samples from

non-FGF-stimulated cells (data not shown). Although we

cannot eliminate the possibility that there exists a subtle

difference in ligand responsiveness that contributes to TDI,

our data suggest that the primary consequence of the TDI

mutations examined here is a partial activation of receptors

that is constitutive and independent of ligand.

DiscussionTDI is a severe skeletal disorder that results from a variety of

mutations that create a Cys residue in the extracellular do-

main of FGFR3. One group of mutations, G370C, S371C,

and Y373C (1 ,2, 4), is clustered in the extracellular jux-

tamembrane region, a region that has been shown to be

sensitive to activating mutations in other receptor tyrosine

kinases, as discussed below. Another group of mutations,

R248C and S249C (1 , 2), lies within the interlinker region

between the second and third Ig domains, a region that is

also mutated in FGFR1 , leading to Pfeiffer syndrome, and in

FGFR2, leading to Apert syndrome (6, 7). In this report, we

show that the different TDI mutations in both the juxtamem-

brane and interlinker regions of FGFR3 result in activation of

the receptor. When assayed as full-length FGFR3 proteins,

all of the TDI mutants showed an increased ability to induce

transcription from the c-fos promoter. Additionally, the

R248C and S249C TDI mutants exhibited the ability to cause

morphological transformation when expressed as FGFR3/

Neu chimeric receptors.

We also show that activation of these receptors correlates

with their ability to form dimeric complexes. Each of the TDI

mutations creates a free Cys residue, and this correlates with

significant dimerization, whereas a control mutation, R248A,

is unable to dimerize and also is inactive in the biological

assays discussed above. The dimers formed are reducible,

indicating that they result from intermolecular disulfide bond-

ing. Activation of receptor signaling through abnormal disul-

fide bonding has been observed previously with FGFR2 in

our laboratory and by others (1 6-18). In FGFR2, mutations in

the third Ig loop of the extracellular domain result in Crouzon

syndrome and other related craniosynostosis disorders. Ad-

ditionally, mutations involving Cys residues in other recep-

tors such as RET, the erythropoietin receptor, and the epi-

dermal growth factor receptor have all been shown to result

in constitutive activation and disulfide-linked dimerization

(10, 11, 21-23).

Our data demonstrate that there is increased fos-lucifer-

ase transcriptional activity, increased dimerization, and in-

creased in vitro kinase activity for all five of the TDI mutant

receptors that we examined. However, the quantitative dif-

ferences do not always seem to be completely consistent.

For instance, the juxtamembrane mutants G370C, S371C,

and Y373C all induced fos-luciferase to a greater extent

(7-15-fold) than did the lg-2-Ig-3 linker domain mutants (3-

4-fold activation). Furthermore, the Ig-2-lg-3 linker domain

. I #{149}: #{149} “� � ‘

. ‘ � . . . . �. . ‘‘�‘1�#{149}� � T�, .�

E. S249C F. Neu

Cell Growth & Differentiation 75

A. Mock

D. R248C

B. Wild-type C. R248A

Fig. 4. Focus formation assay. Chimeric FGFA3/Neu constructs containing interiinker region mutations were transiently transfected into NIH3T3 cells,which were subsequently scored for focus formation. Panels are as follow: (A) mock-transtected cells (nontransformed); (B) wild type FGFA3; (C) A248A;(D) A248C; E, S249C; F, activated Neu containing the mutation V664E. When normalized with respect to activated Neu (1 00%), the transformationefficiencies of the R248C and S249C mutants were approximately 18 ± 5 and 28 ± 9%, respectively.

mutants, R248C and S249C, exhibited the greatest dimer-

ization, whereas the G370C mutant exhibited the greatest

stimulation of in vitro kinase activity. These results suggest

that physical dimerization of receptors may not strictly cor-

relate with receptor activation. This may not be completely

surprising, because recent work on p1 B5neu has demon-

strated that dimerization of the pl85neu transmembrane

domain is necessary but not sufficient for transformation (24).

To date, no similarly detailed studies have been published

concerning FGFR3 dimerization versus activation. It should

also be noted that other biochemical regulatory mechanisms

may modulate the extent of receptor activation for the TDI

mutants, and that these could include altered routes of re-

ceptor trafficking or altered interactions between the acti-

vated receptor and downstream signaling intermediates.

Such differences might lead to quantitative differences in the

extent of receptor activation, although, qualitatively, all the

mutant receptors are activated, as we have demonstrated in

this work.

The abundance of Cys mutations in the extracellular jux-

tamembrane region of FGFR3 that induce dimer formation

and subsequent receptor activation highlights the impor-

tance of this region to human disease. In addition to muta-

tions at residues 370, 371 , and 373 leading to TDI, a G375C

substitution results in a less severe form of dwarfism known

as achondroplasia (25-27). Although it is surprising that Cys

mutations two residues apart result in such different pheno-

types, it is possible that the difference between the pheno-

types is due to their relative proximity to the membrane.

Residue 375 lies at the beginning of the predicted transmem-

brane domain and may potentially be within the membrane

itself, rendering it unavailable for disulfide bonding. The jux-

tamembrane region of FGFR2 has also been shown to be

susceptible to Cys mutations, as exemplified by two different

mutations in the FGFR2 juxtamembrane domain that have

been identified in patients with the craniosynostosis syn-

drome Beare-Stevenson cutis gyrata syndrome (9). Due to

the similarity of these mutations to the TDI mutations in

FGFR3, it is likely that these mutations in FGFR2 cause the

formation of intermolecular disulfide-bonded dimers, thereby

activating signal transduction through this receptor.

205 - � :i DIMER

116 :i MONOMER

66 -

B NON-REDUCING

��III�I�:: :i MONOMER

REDUCING

Fig. 5. Ligand-independent dimerization of TDI. Lysates from cells ha-beled with [35S]Cys and [�5S]Met were Immunoprecipitated, and equlva-lent ahiquots were electrophoresed under nonreducing and reducing con-ditions through 4-12% SOS-PAGE gradient gels. Proteins weretransferred to nitrocellulose and immunoblotted using FGFA3 antiserum.A, nonreducing gel. The positions of dimeric and monomeric forms ofFGFR3 are indicated. B, reducing gel. The position ot monomeric FGFR3is shown, indicating equivalent expression of all constructs.

205 J DIMER

116 ] MONOMER

KINASE (non-reducing)

B 12345678

116 .i,4�II J MONOMER

KINASE (reducing)

C � 2 3 4 5 6 78

l-.I.I116 � t J MONOMER

76 FGFR3 Activation by TDI Mutations

A 4�W�

*

C,

A Q�c�’C? q�’b4��1 2345678

Recently, several somatic mutations of FGFR3 were iden-

tified in association with human multiple myeloma (19), in-

cluding the Y373C mutation characterized here. This sug-

gests that mutations in the juxtamembrane region of FGFR3

likely contribute to the development of human cancers

through constitutive activation of signaling in relevant cell

types. Mutations involving Cys residues in a similar region of

the RET receptor have also been identified in the familial

cancer syndrome multiple endocrine neoplasia type 2A. The

majority of patients with this disease have a mutation that

results in the replacement of one of five juxtamembrane Cys

residues with another residue. This replacement leads to the

formation of disulfide-bonded homodimers and results in

ligand-independent stimulation of the tyrosine kinase (10,

1 1). Based on these two examples, it is likely that oncogenic

activation through Cys mutations in the juxtamembrane re-

gion will be an emerging theme in human carcinomas.

Materials and MethodsConstruction of FGFR3 and FGFR3/Neu Mutants. The parental FGFA3clone used was described previously (13, 14) and contains the codingregion of human FGFR3, clone 17B (28). The TDI mutants were con-structed by subcloning the unique Hindlll/XhoI fragment of FGFA3 Into apSP64-derived vector. The R248A, R248C, and S249C mutants were

created by inserting complimentary oligonucleotides encoding each of

IP: a-FGFR3 I IB: a-FGFR3

12345678

205J DIMER

IP: a-P-tyr I IB: oc-FGFR3(non-reducing)

Fig. 6. In vitro tyrosine kinase activity of TDI mutants of FGFR3. A and B,incorporation from y-r2P]ATP into FGFA3 is shown, analyzed under non-reducing and reducing conditions, respectively, as described in “Materialsand Methods.” Monomeric and dimeric forms of FGFA3 are indicated. C,equivalent expression of FGFA3 constructs is demonstrated by immuno-blotting using FGFR3 antisera. D, the presence of phosphotyrosine onFGFR3 dimers is demonstrated by immunoprecipitation of lysates usingphosphotyrosine antisera, which were analyzed by nonreducing SOS-PAGE and detected by immunoblothng using FGFR3-specific antisera.

these substitutions between an Rs,1I site (nt 701 , in the sequence of Ret.28) and an Sse8387l site (nt 800).

To make the mutations at amino acids 370, 371 , and 373, the region

between Bfal (nt 1000) and Stul (nt 1 251) was reconstructed using threesynthetic restriction fragments created from pairs of complimentary oh-

Cell Growth & Differentiation 77

gonucleotides. The first synthetic fragment was substituted between theBfaI and Stul sites, replacing approximately half of the original fragmentand also introduced two silent sites that did not aiter the predicted amino

acid sequence: SpeI (ACTAGT in place of GCTGGT, nt 1 125-1 130) and

Afill (CTTAAG instead of CCTCAG, nt 1167-1 172). The second syntheticrestriction fragment was inserted between the AflII and Stul sites andrestored the wild-type coding region. The third synthetic restriction frag-

ment was inserted between the new silent sites Spel and AflIl. Themutants were then readily constructed by inserting synthetic Spel/Aflllfragments encoding the desired mutations, G370C, S371C, and Y373C.All constructs were confirmed by dideoxynucleotide sequencing and then

recloned back into pcDNA3-FGFA3.The wild-type FGFA3/Neu chimeric construct was made by replacing

the extracellular region of Neu with the extracellular region of FGFA3 fromHindlll to Bfal. Complimentary oligonucleotides were ligated between theBfaI site in FGFR3 and the Nhel site in pSV2Neu (NheI/SacI; Aet. 13).Additional FGFA3/Neu chimeric constructs containing mutations in thelg2-lg3 linker domain were made by substituting appropriate restriction

fragments from mutant FGFR3 constructs into the FGFA3/Neu chimericconstruct.

Transformation Assays. NIH3T3 cells were used in transformationassays as described previously (13). Data reported for focus assays arethe average of three independent experiments using the FGFR3/Neuchimeric constructs. Foci were scored after 14 days. Transfection effi-

ciency was determined by cotranstection with 50 ng of pASV2neo andselection in media containing G41 8 (29). For each construct, the numberof foci was normalized with respect to the number of Nec-resistant col-onies and then expressed as a percentage of the transformation efficiencyobtained with pSV2neuNT, encoding pl8sneu with the mutation V664E

(30).Dimerization Assays. COS cells were transfected with 10 �ig of DNA

for each full-length FGFR3 construct using a modified calcium phosphatemethod (31). Approximately 24 h after refeeding, cells were incubated inDMEM lacking Cys and Met for 1 h. The cells were labeled with 110 �Cieach of [�C]Cys and r5S]Met for 5 h. The cells were rinsed twice withPBS and lysed on ice with RIPA buffer[10 m�i sodium phosphate(pH 7.0),1 % Triton X-100, 0.1 % SOS, 1 % sodium deoxycholate, 150 m� NaCI, 2

mM EDTA, 50 mM NaF, 1 % aprotinin, 1 mp�i sodium orthovanadate, 100 �i

phenylmethylsulfonyl fluoride, and 10 m�i iodoacetamide]. Lysates wereimmunoprecipitated with antiserum specific for the carboxyl terminus ofFGFR3 (Santa Cruz Biotechnology). Samples were boiled in reducing ornonreducing sample buffer and resolved by SDS-PAGE gradient gels(4-12%) as described previously (32). Gels were fluorographed, dried,

and exposed.Immunobletting, Immuneprecipitatlen, and Kinase Assays. To ob-

tam cell lysates for immunoblothng analysis, cells were starved for 24 h in

serum-tree media before lysing in AIPA buffer as described above. Sam-pIes were boiled in either reducing buffer or nonreducing buffer andresolved by SDS-PAGE (4-12%). Proteins were then transferred to nitro-cellulose and incubated with FGFR3 antisera (Santa Cruz Biotechnology)followed by horseradish peroxidase-conjugated donkey antirabbit IgGand developed by enhanced chemiluminescence (Amersham) accordingto the manufacturer’s instructions. For antiphosphotyrosine immunopre-

cipitation, samples were lysed as described above and incubated with 2ILg of 4G1 0 antisera at 4#{176}Covernight. Protein A-Sepharose was thenadded for 1-2 h. Samples were washed with AIPA buffer three times and

then boiled in nonreducing sample buffer. Samples were run on gradientgels (4-12%), and proteins were then transferred to nitrocellulose, andimmunoblotting with FGFR3 carboxyl-terminal antisera was performed asdescribed above. FGFR3 kinase assays were performed as describedpreviously, including preparation of lysates from transfected cells, anti-FGFR3 immunoprecipitation, and in vitro kinase assay (13, 14).

IndIrect Immunofluorescence. Two days after transtection, NIH3T3cells were fixed with 3% paratormaldehyde in PBS, permeabilized with0.5% Triton X-100 in PBS, and subjected to indirect immunofluorescenceusing FGFA3 carboxyl-terminal antisera (Santa Cruz Biotechnology) tol-

lowed by a fluorescemn-conjugated goat antirabbit secondary antibody(Boehringer Mannheim).

Transcription Assays. NIH3T3 cells were transtected with 2 �g of thepFL700 reporter containing the upstream 700 nt from the c-fos promoterfused to the luciferase gene (33) together with 8 ig of each FGFR3expression construct. After refeeding, cells were starved for 48 h in

medium containing 0.5% calf serum. Luciferase assays were performedusing the Luciferase Assay System (Promega) according to the manufac-turer’s instructions. The data shown are the average of duplicate trans-factions from a single representative experiment, one of three performed

with each construct.

AcknowledgmentsWe thank Laura Castrejon for excellent editorial assistance and all labo-

ratory members for many valuable comments and suggestions concem-ing the experimental design and preparation of the manuscript.

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